人巨细胞病毒UL49 ORF表达及产物功能的初步研究
|
摘要
背景与目的
人巨细胞病毒UL49开放阅读框(ORF)为疱疹病毒保守基因。目前,除Walter Dunn等利用细菌人工重组染色质(BAC)技术发现UL49缺失突变病毒不能再包装,间接证实UL49为人巨细胞病毒生长的必须基因外,对UL49相应功能研究均局限于生物信息学分析与推测。为了进一步证实UL49在病毒生物学方面的功能,有必要首先阐明以下问题：UL49的mRNA表达及全长cDNA精确序列；能否检测到UL49编码的蛋白(pUL49)、确定pUL49表达时相及蛋白特性(病毒结构蛋白、还是非病毒结构蛋白?)等；在此基础上,对以下问题进行实验论证：pUL49在病毒感染宿主细胞中的定位以及在病毒感染、复制中的生物学功能；pUL49可能与宿主细胞哪些蛋白分子相互作用等。
方法
将HCMVA接种生长良好的人胚肺成纤维细胞(HELF),抽提病变细胞中的病毒RNA;以SMART RACE(Rapid Amplification of cDNA Ends, RACE)技术扩增UL49的5’和3’末端,TA克隆两类RACE产物,测序；用RT—PCR扩增UL49全长cDNA序列。
获取Genebank公布的UL49编码氨基酸序列,用在线软件对其Hopp&Woods亲水性、可及性、极性及柔韧性、Welling抗原性和二级结构等参数法进行分析,用吴玉章等建立的B细胞表位预测法综合评价；以化学方法对预测片断进行多肽合成、纯化、与载体血兰蛋白(KLH)联接；将多肽-血兰蛋白化合物(免疫原)免疫新西兰大白兔,获得兔免疫血清后,用ELISA测定效价；构建HCMV UL49大片断(aa3-246)原核表达载体pGEX-4T-3-UL49A,诱导表达pUL49A-GST融合蛋白；构建HCMV UL49全基因真核表达载体pCDNA3.1-UL49-myc,瞬时表达pUL49-myc融合蛋白；以包装成功的HCMV-Towne病毒株感染HFF细胞,收集7天后的培养上清液,以anti-HCMV pUL49为一抗进行上述三种样本Western blotting实验。
以RV-Towne感染HFF,收集3、6、12、24、48、72、96、120hr后细胞裂解液,以anti-HCMV pUL49、anti-IE (CMVpp72/86)、anti-E (CMVgB/CH28)、anti-LA (CMVpp28/CH19)分别对细胞裂解上清液进行WB；收集接近100%细胞病变的HFF[RV-Towne]培养上清液(病毒颗粒),于58750×g、4℃离心纯化病毒,分别用Triton X-100、Trypsin预处理并4℃、100000g离心,以可溶混合上清液、不溶蛋白部分分别进行Western blotting。
用试剂盒抽提、纯化重组人巨细胞病毒Towne基因组(Towne-BAC)、delUL49 Towne基因组(delUL49 Towne-BAC),构建pcDNA3.1(+)-UL82表达质粒；将Towne-BAC、delUL49 Towne分别与pcDNA3.1 (+)-UL82共转HFF,将Towne-BAC、delUL49 Towne分别与pcDNA3.1 (+)-UL82、pcDNA3.1 (+)-UL49共转HFF,培养病毒、观察细胞病变；以RV-Towne感染HFF,收集3、6、12、24、48、72、96、120hr后细胞,分别用Hochest33258、anti-HCMVpUL49、anti-E、anti-LA进行细胞免疫影像实验,荧光显微镜下观察、影像合成定位分析；将纯化的重组病毒与anti-pUL49抗体经37℃预处理0.5hr后,以1 PFU／cell量感染HFF细胞,收集1-7天的病毒细胞上清夜,再观察其生长状况；将上述预处理RV-Towne以1 PFU／cell量感染HFF细胞,收集1、3、6hr的病毒感染细胞,抽提总染色质DNA,以连续稀释后的总染色质DNA为模板,PCR扩增RV-Towne特异基因IE1序列,半定量分析RV侵入细胞的量。
扩增UL49全序列,克隆入pGBKT7,将pGBKT7-UL49转化到酵母细胞AH109,用蛋白印迹法分析诱饵蛋白的表达,检测诱饵蛋白有无毒性和自激活效应；将人胚肾细胞cDNA文库与pGBKT7-UL49共转AH109,于营养缺陷型培养基中筛选阳性克隆,按文献将阳性克隆酵母作β-半乳糖苷酶活性测定；将酵母质粒电转DH5α,抽提质粒,设计文库插入片段上游引物,对双杂交阳性且β-半乳糖苷酶阳性细菌质粒的AD插入片段进行PCR；将非重复克隆细菌质粒的AD插入片段PCR扩增产物进行HaeⅢ酶切,将非重复克隆质粒进行测序,通过Genebank同源性搜索和序列比对分析；选取同源性搜索和序列比对分析后的基因重复酵母双杂交及其β-半乳糖苷酶活性实验；扩增CYB5D2、C11ORF17、COL3A1、UCHL3因片断,构建其pGEX4T-3的表达质粒,构建pCDNA3.1(+)-UL49真核表达质粒；用IPTG诱导细菌表达CYB5D2、C11ORF17、COL3A1、UCHL3的GST融合蛋白,瞬时表达pUL49蛋白,按文献将pUL49分别与CYB5D2、C11ORF17、COL3A1、UCHL3的GST融合蛋白进行GST-pull down；构建pCDNA3.1(+)-COL3A1、pCDNA3.1(+)-C11ORF17真核表达质粒,分别瞬时转染HFF细胞,以1.0PFU/cell量感染上述两组细胞,收集感染1-7后的细胞培养上清夜进行病毒生长实验。
结果
HCMVA在体外成功感染HELF,感染的HELF中有HCMV的IE1和LA标志性基因；用SMART RACE技术从病毒RNA中获得UL49基因的5’和3'UTR,分别克隆后测序,UL49基因5'UTR长90bp、3'UTR长315bp；RT-PCR扩增获得2118bp的UL49基因全长cDNA序列,其开放读码框编码570个氨基酸。
多种预测法重复了人巨细胞病毒pUL49蛋白的N端第228～243位氨基酸区域内或附近,该区域含有β转角和无规卷曲结构；化学方法合成得到了该区域氨基酸序列KRFDARADLAVY-KLH的免疫原；获得免疫兔血清,ELISA测定效价达1：8000以上；以此anti-HCMV pUL49为一抗得WB实验显示pUL49A-GST融合蛋白、pUL49A-myc融合蛋白、HFF[RV-Towne]细胞蛋白裂解液均显示蛋白条带,其分子量与理论值吻合。
RV-Towne感染HFF在12hr后,pUL49开始出现表达,至RV-Towne感染96hr达峰值,与典型的HCMV时相基因pp72／86、gB、pp28的表达对照,类似gB蛋白时相；用细胞表面活性剂TX-100以及蛋白胰酶对纯化病毒进行不同处理后,pUL49与pp28的WB实验结果一致,而与gB存在明显差异。
成功构建pcDNA3.1(+)一UL82.pcDNA3.1(+)-UL49；Towne-BAC与pcDNA3.1(+)-UL82共转15天后可见绿色荧光,delUL49 Towne-BAC与pcDNA3.1(+)-UL82共转30天后仍未见绿色荧光；Towne-BAC与pcDNA3.1(+)-UL82、pcDNA3.1(+)-UL49共转15天后也可见绿色荧光,delUL49 Towne-BAC与pcDNA3.1(+)-UL82共转20天后方见绿色荧光,至30天后荧光强度仍较Towne-BAC的结果相距甚远；病毒感染HFF细胞过程中,pUL49出现时间与蛋白表达出现时间一致,均出现于12hr,影像显示pUL49定位于细胞质而非细胞核,且在细胞感染24hr内显现均匀散布状,而后渐成聚集状,定位于核外边界某一点状区域、且量逐渐增多；以早期表达蛋白gB、晚期表达蛋白pp28为对照,pUL49与pp28在72、96hr存在共定位,而与gB虽然在24、72、96hr均出现表达影像,但未发现两者之间的共定位；经anti-pUL49预处理的重组病毒在感染前2天与未经处理的病毒出现近1个数量级的生长差异,两者在其后的差异均在1个数量级内,差异并不明显；anti-pUL49预处理的病毒在感染侵入阶段(1-6hr),RV-Towne特异基因IE1序列PCR扩增产物与未经anti-pUL49预处理的病毒差异不显著。
克隆成功pGBKT7-UL49并成功转化到AH109,AH109[pGBKT7-UL49]表达诱饵蛋白pUL49,pUL49对转化细胞无细胞毒性、无自激活；酵母双杂交筛选得到30个SD／-Trp-Leu-His-Ade阳性且β-半乳糖苷酶阳性克隆,PCR、HaeⅢ酶切鉴定结果显示11个非重复克隆质粒；其中4个基因重复酵母双杂交及其β-半乳糖苷酶活性,显示C11ORF17、COL3A1表达蛋白与pUL49间的相互作用相对较强；诱导细菌表达pGEX4T-3-CYB5D2、pGEX4T-3-C110RF17、pGEX4T-3-COL3A1、pGEX4T-3-UCHL3的GST融合蛋白与pUL49瞬时表达蛋白相互对应的GST-pull down,结果显示C11ORF17、COL3A1出现条带呈现阳性结果,而CYB5D2、UCHL3未出现条带；pC11ORF17表达的宿主细胞实验组重组病毒生长受到一定抑制,而pCOL3A1表达的宿主细胞实验组重组病毒生长与未处理组无显著差异。
结论
在体外HCMVA感染过程中,UL49的mRNA存在表达,其cDNA序列全长共2118bp,其中5'UTR长90bp,3'UTR长315bp,UL49开放阅读框编码570个氨基酸。
多种预测法重复了pUL49蛋白的N端第228-243位氨基酸区域内或附近为B细胞识别区；用化学合成得到KRFDARADLAVY-KLH免疫原,获得效价高、特异性强的兔anti-HCMV pUL49抗血清。
人巨细胞病毒UL49开放阅读框编码蛋白在病毒感染过程中存在表达,且pUL49为早期表达蛋白；pUL49与pp28对TX-100、蛋白胰酶的耐受相近,暗示两者定位于病毒颗粒同一区域(皮层)
pUL49为Towne的生长必需基因；在表达pUL49的HFF细胞中,缺失UL49的Towne能部分得到回复生长；病毒感染过程pUL49定位于细胞质而非细胞核,随感染时间的持续有被招募于宿主细胞特定区域的趋势；pUL49与pp28在72、96hr存在共定位,暗示pUL49在感染晚期定位于细胞内质网；在病毒增殖过程中,anti-pUL49影响了病毒生长速度,但在感染过程中,未影响病毒的侵入环节。
成功构建了酵母诱饵表达载体pGBKT7-UL49,经双杂交系统筛选,成功获得11个非重复克隆质粒；其中C110RF17、COL3A1表达蛋白与pUL49间的相互作用相对较强；GST-Pull down验证了该两个蛋白与pUL49的体外相互作用；pUL49与pC110RF17的相互作用可能影响病毒的增殖与生长,而pUL49与pCOL3A1的相互作用对病毒的增殖与生长基本无影响。
Background and objectives
UL49 is a herpes virus conserved Open Reading Frame (ORF).Up to now, besides that Walter Dunn and his team found UL49 was a essential gene for HCMV growth when they recovered the bacterial artificial chomosome containing human cytomegalovirus genome deleted UL49 ORF, the study of UL49 gene function limited only to the biophysical information analysis. For studying the biophysical function of UL49 gene during HCMV infection, it is necessary to unclose firstly the question as below:weather is the mRNA expression and how are the cDNA sequences? Can detect the UL49 ORF encoding protein? When do the protein express and where do the protein locate in virion or host cells? After answering the above question, we will observe pUL49 location in host cells infected HCMV and the biophysical function of pUL49 during HCMV infection and production, and also observed the target proteins interacting with the bait protein pUL49.
Methods
HCMV AD 169 (HCMVA) was inoculated to well growth primary human embryonic lung fibroblasts, the HELFs were collected while the cytopathic effect appearing, and HCMVA RNA was extracted from the collected cells.5'RACE and 3'RACE of cDNA of HCMV UL49 were amplified by SMART rapid amplification of cDNA ends technology. The full lengh cDNA of HCMV UL49 was amplified by RT-PCR, and cloned the cDNA into pMD18-T simple vector and sequenced subsequently. The secondary structure and transmembrane domain were predicted by SOPMA and TMHMM respectively. Hydrophilicity, accessibility, polarity, flexibility, surface probability and antigenicity index predicted by methods of Kyte&Doolittle, Emini, Zimmerman and average flexibility. EMBOSS and Bopped Wu'method were combined and the possible B cell epitopes of pUL49 protein were predicted. The B cell epitome of pUL49 protein was synthesized and purified using chemical method then linked it with KLH and condensed with EDC to form immunogen KRFDARADLAVY-KLH. When New Zealand Rabbits were immuned by the immunogen. After collected serum from the immuned rabbits, the serum titer was detected using ELISA. pGEX-4T-3-UL49A from HCMV UL49 (aa3-246) was contracted and pUL49A-GST was induced using IPTG, pCDNA3.1-UL49A-myc from HCMV UL49 was contracted and the pUL49A-myc protein was obtained using instantaneous transfection method. Cell-free supernatants were collected from the medium infected HCMV seven days later. The pUL49 was finally detected from above three samples using Western blotting.
Human foreskin fibroblasts (HFFs), after infected RV-Towne 3,6,12,24,48,72,96 and 120 hours, were collected and cracking, samples were confirmed by Western blotting using the monoclonal antibody anti-IE (CMVpp72/86,CH160:sc-69748, Santa Cruz Biotechnology, Inc.), anti-E (CMV gB, CH28, sc-69742, Santa Cruz Biotechnology, Inc.), anti-LA (CMVpp28, CH19, sc-69749, Santa Cruz Biotechnology, Inc.) and anti-HCMV pUL49. HCMV virion were collected from the medium HFFs appearing near 100% cytopathic effect, and purified using 58,750xg at 4℃.The purified virion were pretreated with triton X-100 or trypsin, then treated with 100000 g at 4℃. The pUL49 from above samples were finally detected using WB.
HCMV BACs were extracted and purified by using a Nucleobond AX kit. The fragment of UL82 was amplified by PCR and cloned into the vector pcDNA3.1 (+). The vector pcDNA3.1 (+)-UL82 or pcDNA3.1 (+)-UL82 and pcDNA3.1 (+)-UL49 together with BACs were co transformed into HFFs respectively. Confluent cytopathic effect was observed from the infected cells by microscope. HFFs, after infected RV-Towne 3,6,12,24,48,72,96 and 120 hours, were collected and confirmed by immunohistochemistry using the monoclonal antibody anti-E, anti-LA, hochest33258 and anti-HCMV pUL49. The cells images were observed by fluorescence microscope. The purified RV were treated with anti-pUL49 at 37℃for 30 minutes, then infected HFFs with 1 PFU/cell. The cell-free supernatants after infected 1-7 days were collected to observe the growth curve. As above, the cells after infected 1-6 hours were collected and extracted total DNA from these cells, then amplified RV-Towne special gene IE1 and analyzed the product titer.
The fragments of UL49 was amplified by PCR, and then cloned into the bait expression vector pGBKT7. The bait vector pGBKT7-UL49, being verified by sequencing, was transformed into AH 109 yeast cells. Then Western Blotting analyzed the bait protein pUL49. Toxicity and self-activation of the bait protein were detected by cultured in different SD cultures. Human embryonic kidney cells CDNA library together with pGBKT7-UL49 were co transformed into yeast cells AH109. The positive clones were selected in different medium deleted nutrient, and detected by filter assay. The positive vectors were transformed to DH5 a and extracted plasmid.Designed PCR primer of cDNA library vector, and the positive gene sequences were amplified, the PCR products also digested by HaeⅢ. The positive sequences were identified by DNA sequence analyzer. With Genebank BLAST and other analysis software, the positive genes were recovered by hybrid and filter assay. Amplified the genes CYB5D2, C11ORF17, COL3A1 and UCHL3, the four fragments were cloned into the vectors pGEX4T-3, pCDNA3.1 (+)-UL49 was constructed meantime. The fusion proteins of CYB5D2, C11ORF17, COL3A1 and UCHL3 with GST were induced by IPTG respectively, pUL49 was expressed also. According to reference, pUL49-3Xflag together with the GST fusion CYB5D2, C11ORF17, COL3A1 and UCHL3 proteins were done GST-pull down test respectively. pCDNA3.1(+)-COL3A1 and pCDNA3.1(+)-C11ORF17 were constructed, and they were transformed into HFFs respectively,24 hours later the cells were infected RV with 1.OPFU/cell. The cell-free supernatants after infected 1-7 days were collected to observe the RV growth curve. The cell-free supernatants after infected 1-7 days were collected to observe the RV growth curve.
Results
We infected HCMVA successfully to well growth HELFs. and HCMV typical genes IE1 and LA were identified using PCR. Using Smart assay, we obtained and cloned the 3'and 5'termination of UL49 gene. Sequencing results suggested its 3'- untranslated region and 5'-untranslated regions were 315bp and 90bp respectively. The full-length cDNA of UL49 gene,2118bp, was amplified using RT-PCR.
The pUL49 protein N-terminal amino acid residues locating at 228-243 regions were predicted by all prediction methods, and this regions containedβsheet and coils structure, which regions or their surrounding regions were the predominant epitomes. The B cell epitomes of pUL49 protein were synthesized and purified, linked with KLH and condensed successfully with EDC. Immunogen KRFDARADLAVY-KLH was formed finally. After immuned, immuned rabbit serum was collected and then detected the titer being 1:8000. pGEX-4T-3-UL49A (3-246) was contracted and the pUL49A-GST was induced, pCDNA3.1-UL49 was contracted and pUL49-myc protein was obtained, the pUL49 from above three samples were detected.
pUL49 protein began to express after HFF infected RV-Towne 12 hours and the maxim titer appeared after infected RV-Towne 96 hours. pUL49 expression kinetics was related with the type HCMV gene gB.pUL49 WB image was related with the type HCMV gene pp28 after the samples treated with TX-100 and trypsin.
We succeed in constructed the pcDNA3.1(+)-UL82 and pcDNA3.1(+)-UL49, the HFFs co transformed with pcDNA3.1(+)-UL82 or together with pcDNA3.1(+)-UL49 and Towne-BACs were observed green fluorescence 15 days later. pcDNA3.1(+)-UL82 together only with pcDNA3.1(+)-UL49 and delTowne-BACs were observed green fluorescence 20 days later. pUL49 appeared red fluorescence in infected cells 12 hours later and located in cytoplasm, and pUL49 appeared from spreading uniformly before infected 24 hours to condense somewhere region. pUL49 was also collocated with pp28 at infected 72-96 hours. The growth curve of RV treated with anti-HCMV pUL49 reduced 10 times than untreated with anti-HCMV pUL49. The DNA of RV treated with anti-HCMV pUL49 was not different to untreated with anti-HCMV pUL49 during virus invasive period.
UL49 was amplified and cloned into pGBKT7 successfully. The vector pGBKT7-UL49 was transformed into AH 109 as well and those cells exhibited neither toxicity nor self-activation. Western Blotting detected the expression of the bait protein pUL49. Human embryonic kidney cells CDNA library together with pGBKT7-UL49 were co transformed into yeast cells AH 109.30 positive clones were selected in different medium deleted nutrient and filter assay.11 none repeated positive vectors were identified by PCR and HaeⅢdigestion.4 positive genes were recovered hybrid and filter assay, gene C11ORF17 and COL3A1 appeared stronger ability to interact with the bait protein pUL49. The vectors pGEX4T-3 with above four fragments were constructed, pCDNA3.1 (+)-UL49 was constructed meantime. The fusion GST proteins, pUL49 protein were expressed. pC11ORF17 and pCOL3A1 proteins appeared positive pull down test result. pCDNA3.1(+)-COL3A1 and pCDNA3.1(+)-C11ORF17 were constructed. The HFFs expressed pCDNA3.1(+)-C11ORF17 appeared lower virus growth than normal HFFs, but the pCDNA3.1(+)-COL3A1 appeared no affection virus growth to normal HFFs.
Conclusion
We observed the mRNA expression of HCMV UL49. The cDNA sequence were 2118bp in size, and its 5'-untranslated region were 90bp, while its 3'-untranslated region were 315bp. HCMV UL49 open reading frame were 1710bp and capable of encoding 570 amino acids.
Prediction of the secondary structure and B cell epitopes of pUL49 protein laid the foundation for studying the characteristics of the protein and developing the epitope-based the monoclonal antibody against pUL49 protein. Immunogen
KRFDARADLAVY-KLH was synthesized to the B cell epitopes of pUL49 protein. We obtained high titer and strong specific rabbit serum anti-HCMV pUL49. pUL49, HCMV UL49 ORF encoding protein, expressed in the host cells infected HCMV, and its expression kinetic was early period. That pUL49 endured TX-100 and trypsin was near to pp28, these two proteins maybe located one region in virion. We succeed in recovering the recombinant HCMV (RV) from the recovered BACs containing HCMV genome. pUL49 was the essential gene for Towne growth. In HFFs expressed pUL49. delTowne-BACs only recovered partly. pUL49 was located at cytoplasm in infected cells and was recruited to somewhere region during infected later period. pUL49 appeared collocated in endoplasmic reticulum with pp28 at 72-96 hours.Anti-pUL49 affected the growth of RV, but did not affect virion entry to its host cells.
The bait expression vector of UL49 was constructed successfully, which laid the foundation for screening target proteins interacting with the bait protein pUL49 using the yeast two-hybrid technique. With hybrid test, we obtained 11 none repeated positive vectors. From 4 positive genes, gene C11ORF17 and COL3A1 appeared stronger ability to interact with the bait protein pUL49. Which result was reinsured by GST pull down test. The interaction between pUL49 with pC11ORF17 maybe affects growth and proliferation of HCMV in HFFs, but the interaction between pUL49 with pCOL3A1 maybe not affects growth and proliferation of HCMV in HFFs.
人巨细胞病毒UL49开放阅读框(ORF)为疱疹病毒保守基因。目前,除Walter Dunn等利用细菌人工重组染色质(BAC)技术发现UL49缺失突变病毒不能再包装,间接证实UL49为人巨细胞病毒生长的必须基因外,对UL49相应功能研究均局限于生物信息学分析与推测。为了进一步证实UL49在病毒生物学方面的功能,有必要首先阐明以下问题：UL49的mRNA表达及全长cDNA精确序列；能否检测到UL49编码的蛋白(pUL49)、确定pUL49表达时相及蛋白特性(病毒结构蛋白、还是非病毒结构蛋白?)等；在此基础上,对以下问题进行实验论证：pUL49在病毒感染宿主细胞中的定位以及在病毒感染、复制中的生物学功能；pUL49可能与宿主细胞哪些蛋白分子相互作用等。
方法
将HCMVA接种生长良好的人胚肺成纤维细胞(HELF),抽提病变细胞中的病毒RNA;以SMART RACE(Rapid Amplification of cDNA Ends, RACE)技术扩增UL49的5’和3’末端,TA克隆两类RACE产物,测序；用RT—PCR扩增UL49全长cDNA序列。
获取Genebank公布的UL49编码氨基酸序列,用在线软件对其Hopp&Woods亲水性、可及性、极性及柔韧性、Welling抗原性和二级结构等参数法进行分析,用吴玉章等建立的B细胞表位预测法综合评价；以化学方法对预测片断进行多肽合成、纯化、与载体血兰蛋白(KLH)联接；将多肽-血兰蛋白化合物(免疫原)免疫新西兰大白兔,获得兔免疫血清后,用ELISA测定效价；构建HCMV UL49大片断(aa3-246)原核表达载体pGEX-4T-3-UL49A,诱导表达pUL49A-GST融合蛋白；构建HCMV UL49全基因真核表达载体pCDNA3.1-UL49-myc,瞬时表达pUL49-myc融合蛋白；以包装成功的HCMV-Towne病毒株感染HFF细胞,收集7天后的培养上清液,以anti-HCMV pUL49为一抗进行上述三种样本Western blotting实验。
以RV-Towne感染HFF,收集3、6、12、24、48、72、96、120hr后细胞裂解液,以anti-HCMV pUL49、anti-IE (CMVpp72/86)、anti-E (CMVgB/CH28)、anti-LA (CMVpp28/CH19)分别对细胞裂解上清液进行WB；收集接近100%细胞病变的HFF[RV-Towne]培养上清液(病毒颗粒),于58750×g、4℃离心纯化病毒,分别用Triton X-100、Trypsin预处理并4℃、100000g离心,以可溶混合上清液、不溶蛋白部分分别进行Western blotting。
用试剂盒抽提、纯化重组人巨细胞病毒Towne基因组(Towne-BAC)、delUL49 Towne基因组(delUL49 Towne-BAC),构建pcDNA3.1(+)-UL82表达质粒；将Towne-BAC、delUL49 Towne分别与pcDNA3.1 (+)-UL82共转HFF,将Towne-BAC、delUL49 Towne分别与pcDNA3.1 (+)-UL82、pcDNA3.1 (+)-UL49共转HFF,培养病毒、观察细胞病变；以RV-Towne感染HFF,收集3、6、12、24、48、72、96、120hr后细胞,分别用Hochest33258、anti-HCMVpUL49、anti-E、anti-LA进行细胞免疫影像实验,荧光显微镜下观察、影像合成定位分析；将纯化的重组病毒与anti-pUL49抗体经37℃预处理0.5hr后,以1 PFU／cell量感染HFF细胞,收集1-7天的病毒细胞上清夜,再观察其生长状况；将上述预处理RV-Towne以1 PFU／cell量感染HFF细胞,收集1、3、6hr的病毒感染细胞,抽提总染色质DNA,以连续稀释后的总染色质DNA为模板,PCR扩增RV-Towne特异基因IE1序列,半定量分析RV侵入细胞的量。
扩增UL49全序列,克隆入pGBKT7,将pGBKT7-UL49转化到酵母细胞AH109,用蛋白印迹法分析诱饵蛋白的表达,检测诱饵蛋白有无毒性和自激活效应；将人胚肾细胞cDNA文库与pGBKT7-UL49共转AH109,于营养缺陷型培养基中筛选阳性克隆,按文献将阳性克隆酵母作β-半乳糖苷酶活性测定；将酵母质粒电转DH5α,抽提质粒,设计文库插入片段上游引物,对双杂交阳性且β-半乳糖苷酶阳性细菌质粒的AD插入片段进行PCR；将非重复克隆细菌质粒的AD插入片段PCR扩增产物进行HaeⅢ酶切,将非重复克隆质粒进行测序,通过Genebank同源性搜索和序列比对分析；选取同源性搜索和序列比对分析后的基因重复酵母双杂交及其β-半乳糖苷酶活性实验；扩增CYB5D2、C11ORF17、COL3A1、UCHL3因片断,构建其pGEX4T-3的表达质粒,构建pCDNA3.1(+)-UL49真核表达质粒；用IPTG诱导细菌表达CYB5D2、C11ORF17、COL3A1、UCHL3的GST融合蛋白,瞬时表达pUL49蛋白,按文献将pUL49分别与CYB5D2、C11ORF17、COL3A1、UCHL3的GST融合蛋白进行GST-pull down；构建pCDNA3.1(+)-COL3A1、pCDNA3.1(+)-C11ORF17真核表达质粒,分别瞬时转染HFF细胞,以1.0PFU/cell量感染上述两组细胞,收集感染1-7后的细胞培养上清夜进行病毒生长实验。
结果
HCMVA在体外成功感染HELF,感染的HELF中有HCMV的IE1和LA标志性基因；用SMART RACE技术从病毒RNA中获得UL49基因的5’和3'UTR,分别克隆后测序,UL49基因5'UTR长90bp、3'UTR长315bp；RT-PCR扩增获得2118bp的UL49基因全长cDNA序列,其开放读码框编码570个氨基酸。
多种预测法重复了人巨细胞病毒pUL49蛋白的N端第228～243位氨基酸区域内或附近,该区域含有β转角和无规卷曲结构；化学方法合成得到了该区域氨基酸序列KRFDARADLAVY-KLH的免疫原；获得免疫兔血清,ELISA测定效价达1：8000以上；以此anti-HCMV pUL49为一抗得WB实验显示pUL49A-GST融合蛋白、pUL49A-myc融合蛋白、HFF[RV-Towne]细胞蛋白裂解液均显示蛋白条带,其分子量与理论值吻合。
RV-Towne感染HFF在12hr后,pUL49开始出现表达,至RV-Towne感染96hr达峰值,与典型的HCMV时相基因pp72／86、gB、pp28的表达对照,类似gB蛋白时相；用细胞表面活性剂TX-100以及蛋白胰酶对纯化病毒进行不同处理后,pUL49与pp28的WB实验结果一致,而与gB存在明显差异。
成功构建pcDNA3.1(+)一UL82.pcDNA3.1(+)-UL49；Towne-BAC与pcDNA3.1(+)-UL82共转15天后可见绿色荧光,delUL49 Towne-BAC与pcDNA3.1(+)-UL82共转30天后仍未见绿色荧光；Towne-BAC与pcDNA3.1(+)-UL82、pcDNA3.1(+)-UL49共转15天后也可见绿色荧光,delUL49 Towne-BAC与pcDNA3.1(+)-UL82共转20天后方见绿色荧光,至30天后荧光强度仍较Towne-BAC的结果相距甚远；病毒感染HFF细胞过程中,pUL49出现时间与蛋白表达出现时间一致,均出现于12hr,影像显示pUL49定位于细胞质而非细胞核,且在细胞感染24hr内显现均匀散布状,而后渐成聚集状,定位于核外边界某一点状区域、且量逐渐增多；以早期表达蛋白gB、晚期表达蛋白pp28为对照,pUL49与pp28在72、96hr存在共定位,而与gB虽然在24、72、96hr均出现表达影像,但未发现两者之间的共定位；经anti-pUL49预处理的重组病毒在感染前2天与未经处理的病毒出现近1个数量级的生长差异,两者在其后的差异均在1个数量级内,差异并不明显；anti-pUL49预处理的病毒在感染侵入阶段(1-6hr),RV-Towne特异基因IE1序列PCR扩增产物与未经anti-pUL49预处理的病毒差异不显著。
克隆成功pGBKT7-UL49并成功转化到AH109,AH109[pGBKT7-UL49]表达诱饵蛋白pUL49,pUL49对转化细胞无细胞毒性、无自激活；酵母双杂交筛选得到30个SD／-Trp-Leu-His-Ade阳性且β-半乳糖苷酶阳性克隆,PCR、HaeⅢ酶切鉴定结果显示11个非重复克隆质粒；其中4个基因重复酵母双杂交及其β-半乳糖苷酶活性,显示C11ORF17、COL3A1表达蛋白与pUL49间的相互作用相对较强；诱导细菌表达pGEX4T-3-CYB5D2、pGEX4T-3-C110RF17、pGEX4T-3-COL3A1、pGEX4T-3-UCHL3的GST融合蛋白与pUL49瞬时表达蛋白相互对应的GST-pull down,结果显示C11ORF17、COL3A1出现条带呈现阳性结果,而CYB5D2、UCHL3未出现条带；pC11ORF17表达的宿主细胞实验组重组病毒生长受到一定抑制,而pCOL3A1表达的宿主细胞实验组重组病毒生长与未处理组无显著差异。
结论
在体外HCMVA感染过程中,UL49的mRNA存在表达,其cDNA序列全长共2118bp,其中5'UTR长90bp,3'UTR长315bp,UL49开放阅读框编码570个氨基酸。
多种预测法重复了pUL49蛋白的N端第228-243位氨基酸区域内或附近为B细胞识别区；用化学合成得到KRFDARADLAVY-KLH免疫原,获得效价高、特异性强的兔anti-HCMV pUL49抗血清。
人巨细胞病毒UL49开放阅读框编码蛋白在病毒感染过程中存在表达,且pUL49为早期表达蛋白；pUL49与pp28对TX-100、蛋白胰酶的耐受相近,暗示两者定位于病毒颗粒同一区域(皮层)
pUL49为Towne的生长必需基因；在表达pUL49的HFF细胞中,缺失UL49的Towne能部分得到回复生长；病毒感染过程pUL49定位于细胞质而非细胞核,随感染时间的持续有被招募于宿主细胞特定区域的趋势；pUL49与pp28在72、96hr存在共定位,暗示pUL49在感染晚期定位于细胞内质网；在病毒增殖过程中,anti-pUL49影响了病毒生长速度,但在感染过程中,未影响病毒的侵入环节。
成功构建了酵母诱饵表达载体pGBKT7-UL49,经双杂交系统筛选,成功获得11个非重复克隆质粒；其中C110RF17、COL3A1表达蛋白与pUL49间的相互作用相对较强；GST-Pull down验证了该两个蛋白与pUL49的体外相互作用；pUL49与pC110RF17的相互作用可能影响病毒的增殖与生长,而pUL49与pCOL3A1的相互作用对病毒的增殖与生长基本无影响。
Background and objectives
UL49 is a herpes virus conserved Open Reading Frame (ORF).Up to now, besides that Walter Dunn and his team found UL49 was a essential gene for HCMV growth when they recovered the bacterial artificial chomosome containing human cytomegalovirus genome deleted UL49 ORF, the study of UL49 gene function limited only to the biophysical information analysis. For studying the biophysical function of UL49 gene during HCMV infection, it is necessary to unclose firstly the question as below:weather is the mRNA expression and how are the cDNA sequences? Can detect the UL49 ORF encoding protein? When do the protein express and where do the protein locate in virion or host cells? After answering the above question, we will observe pUL49 location in host cells infected HCMV and the biophysical function of pUL49 during HCMV infection and production, and also observed the target proteins interacting with the bait protein pUL49.
Methods
HCMV AD 169 (HCMVA) was inoculated to well growth primary human embryonic lung fibroblasts, the HELFs were collected while the cytopathic effect appearing, and HCMVA RNA was extracted from the collected cells.5'RACE and 3'RACE of cDNA of HCMV UL49 were amplified by SMART rapid amplification of cDNA ends technology. The full lengh cDNA of HCMV UL49 was amplified by RT-PCR, and cloned the cDNA into pMD18-T simple vector and sequenced subsequently. The secondary structure and transmembrane domain were predicted by SOPMA and TMHMM respectively. Hydrophilicity, accessibility, polarity, flexibility, surface probability and antigenicity index predicted by methods of Kyte&Doolittle, Emini, Zimmerman and average flexibility. EMBOSS and Bopped Wu'method were combined and the possible B cell epitopes of pUL49 protein were predicted. The B cell epitome of pUL49 protein was synthesized and purified using chemical method then linked it with KLH and condensed with EDC to form immunogen KRFDARADLAVY-KLH. When New Zealand Rabbits were immuned by the immunogen. After collected serum from the immuned rabbits, the serum titer was detected using ELISA. pGEX-4T-3-UL49A from HCMV UL49 (aa3-246) was contracted and pUL49A-GST was induced using IPTG, pCDNA3.1-UL49A-myc from HCMV UL49 was contracted and the pUL49A-myc protein was obtained using instantaneous transfection method. Cell-free supernatants were collected from the medium infected HCMV seven days later. The pUL49 was finally detected from above three samples using Western blotting.
Human foreskin fibroblasts (HFFs), after infected RV-Towne 3,6,12,24,48,72,96 and 120 hours, were collected and cracking, samples were confirmed by Western blotting using the monoclonal antibody anti-IE (CMVpp72/86,CH160:sc-69748, Santa Cruz Biotechnology, Inc.), anti-E (CMV gB, CH28, sc-69742, Santa Cruz Biotechnology, Inc.), anti-LA (CMVpp28, CH19, sc-69749, Santa Cruz Biotechnology, Inc.) and anti-HCMV pUL49. HCMV virion were collected from the medium HFFs appearing near 100% cytopathic effect, and purified using 58,750xg at 4℃.The purified virion were pretreated with triton X-100 or trypsin, then treated with 100000 g at 4℃. The pUL49 from above samples were finally detected using WB.
HCMV BACs were extracted and purified by using a Nucleobond AX kit. The fragment of UL82 was amplified by PCR and cloned into the vector pcDNA3.1 (+). The vector pcDNA3.1 (+)-UL82 or pcDNA3.1 (+)-UL82 and pcDNA3.1 (+)-UL49 together with BACs were co transformed into HFFs respectively. Confluent cytopathic effect was observed from the infected cells by microscope. HFFs, after infected RV-Towne 3,6,12,24,48,72,96 and 120 hours, were collected and confirmed by immunohistochemistry using the monoclonal antibody anti-E, anti-LA, hochest33258 and anti-HCMV pUL49. The cells images were observed by fluorescence microscope. The purified RV were treated with anti-pUL49 at 37℃for 30 minutes, then infected HFFs with 1 PFU/cell. The cell-free supernatants after infected 1-7 days were collected to observe the growth curve. As above, the cells after infected 1-6 hours were collected and extracted total DNA from these cells, then amplified RV-Towne special gene IE1 and analyzed the product titer.
The fragments of UL49 was amplified by PCR, and then cloned into the bait expression vector pGBKT7. The bait vector pGBKT7-UL49, being verified by sequencing, was transformed into AH 109 yeast cells. Then Western Blotting analyzed the bait protein pUL49. Toxicity and self-activation of the bait protein were detected by cultured in different SD cultures. Human embryonic kidney cells CDNA library together with pGBKT7-UL49 were co transformed into yeast cells AH109. The positive clones were selected in different medium deleted nutrient, and detected by filter assay. The positive vectors were transformed to DH5 a and extracted plasmid.Designed PCR primer of cDNA library vector, and the positive gene sequences were amplified, the PCR products also digested by HaeⅢ. The positive sequences were identified by DNA sequence analyzer. With Genebank BLAST and other analysis software, the positive genes were recovered by hybrid and filter assay. Amplified the genes CYB5D2, C11ORF17, COL3A1 and UCHL3, the four fragments were cloned into the vectors pGEX4T-3, pCDNA3.1 (+)-UL49 was constructed meantime. The fusion proteins of CYB5D2, C11ORF17, COL3A1 and UCHL3 with GST were induced by IPTG respectively, pUL49 was expressed also. According to reference, pUL49-3Xflag together with the GST fusion CYB5D2, C11ORF17, COL3A1 and UCHL3 proteins were done GST-pull down test respectively. pCDNA3.1(+)-COL3A1 and pCDNA3.1(+)-C11ORF17 were constructed, and they were transformed into HFFs respectively,24 hours later the cells were infected RV with 1.OPFU/cell. The cell-free supernatants after infected 1-7 days were collected to observe the RV growth curve. The cell-free supernatants after infected 1-7 days were collected to observe the RV growth curve.
Results
We infected HCMVA successfully to well growth HELFs. and HCMV typical genes IE1 and LA were identified using PCR. Using Smart assay, we obtained and cloned the 3'and 5'termination of UL49 gene. Sequencing results suggested its 3'- untranslated region and 5'-untranslated regions were 315bp and 90bp respectively. The full-length cDNA of UL49 gene,2118bp, was amplified using RT-PCR.
The pUL49 protein N-terminal amino acid residues locating at 228-243 regions were predicted by all prediction methods, and this regions containedβsheet and coils structure, which regions or their surrounding regions were the predominant epitomes. The B cell epitomes of pUL49 protein were synthesized and purified, linked with KLH and condensed successfully with EDC. Immunogen KRFDARADLAVY-KLH was formed finally. After immuned, immuned rabbit serum was collected and then detected the titer being 1:8000. pGEX-4T-3-UL49A (3-246) was contracted and the pUL49A-GST was induced, pCDNA3.1-UL49 was contracted and pUL49-myc protein was obtained, the pUL49 from above three samples were detected.
pUL49 protein began to express after HFF infected RV-Towne 12 hours and the maxim titer appeared after infected RV-Towne 96 hours. pUL49 expression kinetics was related with the type HCMV gene gB.pUL49 WB image was related with the type HCMV gene pp28 after the samples treated with TX-100 and trypsin.
We succeed in constructed the pcDNA3.1(+)-UL82 and pcDNA3.1(+)-UL49, the HFFs co transformed with pcDNA3.1(+)-UL82 or together with pcDNA3.1(+)-UL49 and Towne-BACs were observed green fluorescence 15 days later. pcDNA3.1(+)-UL82 together only with pcDNA3.1(+)-UL49 and delTowne-BACs were observed green fluorescence 20 days later. pUL49 appeared red fluorescence in infected cells 12 hours later and located in cytoplasm, and pUL49 appeared from spreading uniformly before infected 24 hours to condense somewhere region. pUL49 was also collocated with pp28 at infected 72-96 hours. The growth curve of RV treated with anti-HCMV pUL49 reduced 10 times than untreated with anti-HCMV pUL49. The DNA of RV treated with anti-HCMV pUL49 was not different to untreated with anti-HCMV pUL49 during virus invasive period.
UL49 was amplified and cloned into pGBKT7 successfully. The vector pGBKT7-UL49 was transformed into AH 109 as well and those cells exhibited neither toxicity nor self-activation. Western Blotting detected the expression of the bait protein pUL49. Human embryonic kidney cells CDNA library together with pGBKT7-UL49 were co transformed into yeast cells AH 109.30 positive clones were selected in different medium deleted nutrient and filter assay.11 none repeated positive vectors were identified by PCR and HaeⅢdigestion.4 positive genes were recovered hybrid and filter assay, gene C11ORF17 and COL3A1 appeared stronger ability to interact with the bait protein pUL49. The vectors pGEX4T-3 with above four fragments were constructed, pCDNA3.1 (+)-UL49 was constructed meantime. The fusion GST proteins, pUL49 protein were expressed. pC11ORF17 and pCOL3A1 proteins appeared positive pull down test result. pCDNA3.1(+)-COL3A1 and pCDNA3.1(+)-C11ORF17 were constructed. The HFFs expressed pCDNA3.1(+)-C11ORF17 appeared lower virus growth than normal HFFs, but the pCDNA3.1(+)-COL3A1 appeared no affection virus growth to normal HFFs.
Conclusion
We observed the mRNA expression of HCMV UL49. The cDNA sequence were 2118bp in size, and its 5'-untranslated region were 90bp, while its 3'-untranslated region were 315bp. HCMV UL49 open reading frame were 1710bp and capable of encoding 570 amino acids.
Prediction of the secondary structure and B cell epitopes of pUL49 protein laid the foundation for studying the characteristics of the protein and developing the epitope-based the monoclonal antibody against pUL49 protein. Immunogen
KRFDARADLAVY-KLH was synthesized to the B cell epitopes of pUL49 protein. We obtained high titer and strong specific rabbit serum anti-HCMV pUL49. pUL49, HCMV UL49 ORF encoding protein, expressed in the host cells infected HCMV, and its expression kinetic was early period. That pUL49 endured TX-100 and trypsin was near to pp28, these two proteins maybe located one region in virion. We succeed in recovering the recombinant HCMV (RV) from the recovered BACs containing HCMV genome. pUL49 was the essential gene for Towne growth. In HFFs expressed pUL49. delTowne-BACs only recovered partly. pUL49 was located at cytoplasm in infected cells and was recruited to somewhere region during infected later period. pUL49 appeared collocated in endoplasmic reticulum with pp28 at 72-96 hours.Anti-pUL49 affected the growth of RV, but did not affect virion entry to its host cells.
The bait expression vector of UL49 was constructed successfully, which laid the foundation for screening target proteins interacting with the bait protein pUL49 using the yeast two-hybrid technique. With hybrid test, we obtained 11 none repeated positive vectors. From 4 positive genes, gene C11ORF17 and COL3A1 appeared stronger ability to interact with the bait protein pUL49. Which result was reinsured by GST pull down test. The interaction between pUL49 with pC11ORF17 maybe affects growth and proliferation of HCMV in HFFs, but the interaction between pUL49 with pCOL3A1 maybe not affects growth and proliferation of HCMV in HFFs.
引文
1.Griffiths, P. D. (2000). Cytomegalovirus. In A. J. Zuckerman, J. E. Banatvala,& J. R. Pattison (Eds.), Principles and Practice of Clinical Virology(pp.79-116). London:John Wiley and Sons.
2. Pass, R. F. (2001). Cytomegalovirus.In D. Knipe,& P. Howley (Eds.),Fields Virology (pp. 2675-2705). Philadelphia:Lippincott, Williams and Wilkins.
3 Mocarski, E. S., T. Shenk, and R. F. Pass.2007. Cytomegaloviruses, p.2701-2772. In D. M. Knipe and P. M. Howley (ed.), Fields virology,5th ed. Lippincott Williams & Wilkins, Philadelphia, PA.
4.Mocarski, E. S.,& Courcelle, C. T. (2001). Cytomegalovirus and their replication. In D. Knipe, & P. Howley (Eds.), Fields Virology (pp.2629-2673). Philadelphia:Lippincott, Williams and Wilkins.
5.Britt, W. J.,& Mach, M. (1996). Human cytomegalovirus glycoproteins.Intervirology 39,401-412.
6 Gretch, D. R., Kari, B., Rasmussen, L., Gehrz, R. C.,& Stinski, M. F.(1988). Identification and characterization of three distinct families of glycoprotein complexes in the envelopes of human cytomegalovirus.J Virol 62,875-881.
7 Theiler, R. N.,& Compton, T. (2001). Characterization of the signal peptide processing and membrane association of human cytomegalovirus glycoprotein O. J Biol Chem 276,39226-39231.
8 Gonczol, E.,& Plotkin, S. (2001). Development of a cytomegalovirus vaccine:lessons from recent clinical trials. Expert Opin Biol Ther 1,401-412.
9 Huber, M. T.,& Compton, T. (1998). The human cytomegalovirus UL74 gene encodes the third component of the glycoprotein H-glycoprotein L-containing envelope complex. J Virol 72,8191-8197.
10 Kari, B., Li, W., Cooper, J., Goertz, R.,& Radeke, B. (1994). The human cytomegalovirus UL100 gene encodes the gC-11 glycoproteins recognized by group 2 monoclonal antibodies. J Gen Virol 75,3081-3086.
11 Baldanti, F., Underwood, M. R., Stanat, S.C., Biron, K. K., Chou, S.,Sarasini, A., Silini, E., & Gerna, G. (1996). Single amino acid changes in the DNA polymerase confer foscarnet resistance and slow-growth phenotype, while mutations in the UL97-encoded phosphotransferase confer ganciclovir resistance in three double-resistant human cytomegalovirus strains recovered from patients with AIDS.J Virol 70,1390-1395.
12 Lu, M.,& Shenk, T. (1999). Human cytomegalovirus UL69 protein induces cells to accumulate in G1 phase of the cell cycle. J Virol 73,676-683.
13 Hayashi, M. L., Blankenship, C.,& Shenk, T. (2000). Human cytomegalovirus UL69 protein is required for efficient accumulation of infected cells in the G1 phase of the cell cycle. Proc Natl Acad Sci USA 97,2692-2696.
14 Liu, B.,& Stinski, M. F. (1992). Human cytomegalovirus contains a tegument protein that enhances transcription from promoters with upstream ATF and AP-1 cis-acting elements. J Virol 66,4434-4444.
15 Baldick, C. J., Marchini, A., Patterson, C. E.,& Shenk, T. (1997). Human cytomegalovirus tegument protein pp71 (ppUL82) enhances the infectivity of viral DNA and accelerates the infectious cycle. J Virol 71,4400-4408.
16 Bresnahan, W. A.,& Shenk, T. (2000b). UL82 virion protein activates expression of immediate early viral genes in human cytomegalovirusinfected cells. Proc Natl Acad Sci USA 97,14506-14511.
17 Romanowski, M. J., Garrido-Guerrero, E.,& Shenk, T. (1997). pIRSl and pTRS1 are present in human cytomegalovirus virions. J Virol 71,5703-5705.
18 Bresnahan, W. A.,& Shenk, T. (2000a). A subset of viral transcripts packaged within human cytomegalovirus particles. Science 288,2373-2376.
19 Chee, M. S., Bankier, A. T., Beck, S., Bohni, R., Brown, C. M., Cerny, R.,Horsnell, T., Hutchison Ⅲ, C. A., Kouzarides, T., Martignetti, J. A.,Preddie, E., Satchwell, S. C., Tomlinson, P., Weston, K. M.,& Barrell,B. G. (1990a). Analysis of the protein-coding content of human cytomegalovirus strain AD169. Curr Top Microbiol Immunol 154,125-169
20 Novotny, J., Rigoutsos, I., Coleman, D.,& Shenk, T. (2001). In silico structural and functional analysis of the human cytomegalovirus(HHV5) genome. J Mol Biol 310,1151-1166.
21 Cha, T. A., Tom, E., Kemble, G. W., Duke, G. M., Mocarski, E. S.,&Spaete, R. R. (1996). Human cytomegalovirus clinical isolates carry at least 19 genes not found in laboratory strains. J Virol 70.78-83.
22 Karlin, S., Mocarski, E. S.,& Schachtel, G. A. (1994). Molecular evolution of herpesviruses: genomic and protein sequence comparisons. J Virol68,1886-1902.
23 Murphy, E. A., Streblow, D. N., Nelson, J. A.,& Stinski, M. F. (2000). The human cytomegalovirus IE86 protein can block cell cycle progression after inducing transition into the S phase of permissive cells. J Virol 74,7108-7118.
24 Smith, I. L., Cherrington, J. M., Jiles, R. E., Fuller, M. D., Freeman, W. R.,& Spector, S. A. (1997). High-level resistance of cytomegalovirus to ganciclovir is associated with alterations in both the UL97 and DNA polymerase genes. J Infect Dis 176,69-77.
25 Tomasec, P., Braud, V. M., Rickards, C., Powell, M. B., McSharry, B. P.,Gadola, S., Cerundolo, V., Borysiewicz, L. K., McMichael, A. J.,&Wilkinson, G. W. (2000). Surface expression of HLA E an inhibitor of natural killer cells, enhanced by human cytomegalovirus gpUL40. Science287,1031-1033.
26 Human cytomegalovirus virion protein complex required for epithelial and endothelial cell tropism. Proc Natl Acad Sci USA 102:18153-18158
27 Bissinger, A. L., Singzer, C., Kaiserling, E.,& Jahn, G. (2002). Human cytomegalovirus as a direct pathogen:correlation of multiorgan involvement and cell distribution with clinical and pathological findings in a case of congenital inclusion disease. J Med Virol 67,200-206.
28 Plachter, B., Sinzger, C.,& Jahn, G. (1996). Cell types involved in replication and distribution of human cytomegalovirus. Adv Virus Res 46,195-261.
29 Sinzger, C.,& Jahn, G. (1996). Human cytomegalovirus cell tropism and pathogenesis. Intervirology 39,302-319.
30 Sinzger, C., Plachter, B., Grefte, A., The, T. H.,& Jahn, G. (1996). Tissue macrophages are infected by human cytomegalovirus. J Infect Dis 173,240-245. 31 Kahl, M., Siegel-Axel, D., Stenglein, S., Jahn, G.,& Sinzger, C. (2000).Efficient lytic infection of human arterial endothelial cells by human cytomegalovirus strains. J Virol 74,7628-7635.
32 Kondo, K., Kaneshima, H.,& Mocarski, E. S. (1994). Human cytomegalovirus latent infection of granulocyte-macrophage progenitors. Proc Natl Acad Sci USA 91,11879-11883.
33 Soderberg-Naucler, C., Fish, K.N.,& Nelson, J. A. (1997). Reactivation of latent human cytomegalovirus by allogeneic stimulation of blood cells from healthy donors. Cell 91,119-126.
34 Sinzger, C., Kahl, M., Laib, K., Klingel, K., Rieger, P., Plachter, B.,& Jahn,G. (2000). Tropism of human cytomegalovirus for endothelial cells is determined by a post-entry step dependent on efficient translocation to the nucleus. J Gen Virol 81,3021-3035.
35 Compton, T., Nowlin, D. M.,& Cooper, N. R. (1993). Initiation of human cytomegalovirus infection requires initial interaction with cell surface heparan sulfate. Virology 193,834-841.
36 Browne, E. P., Wing, B., Coleman, D.,& Shenk, T. (2001). Altered cellular mRNA levels in human cytomegalovirus infected fibroblasts:viral block to the accumulation of antiviral mRNAs. J Virol 75,12319-12330.
37 Boyle, K. A., Pietropaolo, R. L.,& Compton, T. (1999). Engagement of the cellular receptor for glycoprotein B of human cytomegalovirus activates the interferon-responsive pathway. Mol Cell Biol 19,3607-3613.
38 Simmen, K. A., Singh, J., Luukkonen, B. G. M., Lopper, M., Bittner, A.,Miller, N. E., Jackson, M. R., Compton, T.,& Fruh, K. (2001). Global modulation of cellular transcription by human cytomegalovirus is initiated by viral glycoprotein B. Proc Natl Acad Sci USA 98,7140-7145.
39 Yurochko, A. D., Mayo, M. W., Poma, E. E., Baldwin Jr., A. S.,& Huang,E.-S. (1997). Induction of the transcription factor Spl during human cytomegalovirus infection mediates upregulation of the p65 and p105/p50 NF-kB promoters. J Virol 71,4638-4648.
40 Bodaghi, B., Jones, T. R., Zipeto, D., Vita, C., Sun, L., Laurent, L.,Arenzana-Seisdedos, F., Virelizier, J. L.,& Michelson, S. (1998).Chemokine sequestration by viral chemoreceptors as a novel viral.
41 Varnum SM, Streblow DN, Monroe ME, Smith P, Auberry KJ, Pasa-Tolic L, Wang D, Camp DG 2nd, Rodland K, Wiley S, Britt W, Shenk T, Smith RD, Nelson JA (2004) Identification of proteins in human cytomegalovirus (HCMV) particles:the HCMV proteome. J Virol 78:10960-10966
42 Feire AL, Koss H, Compton T (2004) Cellular integrins function as entry receptors for human cytomegalovirus via a highly conserved disintegrin-like domain. Proc Natl Acad Sci USA 101:15470-15475
43 Wang X, Huang DY, Huong SM, Huang ES (2005) Integrin alphavbeta3 is a coreceptor for human cytomegalovirus. Nat Med 11:515-521
44 English EP, Chumanov RS, Gellman SH, Compton T (2006) Rational development of beta-peptide inhibitors of human cytomegalovirus entry. J Biol Chem 281:2661-2667
45 Boyle, K. A., Pietropaolo, R. L.,& Compton, T. (1999). Engagement of the cellular receptor for glycoprotein B of human cytomegalovirus activates the interferon-responsive pathway. Mol Cell Biol 19,3607-3613.
46 Fortunato, E. A.,& Spector, D. H. (1999). Regulation of human cytomegalovirus gene expression. Adv Virus Res 54,61-128.
47 Nelson, J. A., Gnann, J. W.,& Ghazal, P. (1990). Regulation and tissuespecific expression of human cytomegalovirus. Curr Top Microbiol Immunol 154,75-100.
48 Meier, J. L.,& Stinski, M. F. (1997). Effect of a modulator deletion on transcription of the human cytomegalovirus major immediate-early genes in infected undifferentiated and differentiated cells. J Virol 71,1246-1255.
49 Boshart, M., Weber, F., Jahn, G., Dorsch-Hasler, K., Fleckenstein, B.,& Schaffner, W. (1985). A very strong enhancer is located upstream of an immediate early gene of human cytomegalovirus. Cell 41,521-530.
50 Mocarski, E. S. (1996). Cytomegalovirus and their replication. In B. N. Fields, D. M. Knipe, & P. M. Howley (Eds.), Virology (pp.2447-2492). Philadelphia:Lippincott-Raven. 51 Hagemeier, C., Walker, S., Caswell, R., Kouzarides, T.,& Sinclair, J. H.(1992a). The 72K IE1 and 80K IE2 proteins of human cytomegalovirus independently trans-activate the c-fos, c-myc and hsp70 promoter via basal promoter elements. J Gen Virol 73,2385-2393.
52 Wade, M., Kowalik, T. F., Mudryj, M., Huang, E.-S.,& Azizkhan, J. C.(1992). E2F mediates dihydrofolate reducatse promoter activation and multiprotein complex formation in human cytomegalovirus infection.Mol Cell Biol 12,4364-4374.
53 Hayhurst, G. P., Bryant, L. A., Caswell, R. C., Walker, S. M.,& Sinclair,J. H. (1995). CCAAT box-dependent activation of the TATA-less human DNA polymerase a promoter by the human cytomegalovirus 72-kilodalton major immediate-early protein. J Virol 69,182-188.
54 Yurochko, A. D., Kowalik, T. F., Huong, S. M.,& Huang, E.-S. (1995).Human cytomegalovirus upregulates NF-kB activity by transactivating the NF-kB p105/p50 and p65 promoter. J Virol 69,5391-5400.
55 Gribaudo, G., Riera, L., Rudge, T. L., Caposio, P., Johnson, L. F.,& Landolfo,S. (2002). Human cytomegalovirus infection induces cellular thymidylate synthase gene expression in quiescent fibroblasts. J Gen Virol83,2983-2993.
56 Poma, E. E., Kowalik, T. F., Zhu, L., Sinclair, J. H.,& Huang, E.-S. (1996).The human cytomegalovirus IE 1-72 protein interacts with the cellular p107 protein and relieves p107-mediated transcriptional repression of an E2F-responsive promoter. J Virol 70,7867-7877.
57 Johnson, R. A., Yurochko, A. D., Poma, E.E., Zhu, L.,& Huang, E.-S.(1999). Domain mapping of the human cytomegalovirus IE1-72 and cellular p107 protein-protein interaction and the possible functional consequences. J Gen Virol 80,1293-1303.
58 Pajovic, S., Wong, E. L., Black, A. R.,& Azizkhan, C. J. (1997). Identification of a viral kinase that phosphorylates specific E2Fs and pocket proteins. Mol Cell Biol 17,6459-6464.
59 Marchini, A., Liu, H.,& Zhu, H. (2001). Human cytomegalovirus with IE2 (UL122) deleted fails to express early lytic genes. J Virol 75,1870-1878.
60 Song, Y.,& Stinski, M. F. (2002). Effect of the human cytomegalovirus IE86 protein on expression of E2F-responsive genes:a DNA microarray analysis. Proc Natl Acad Sci USA 99, 2836-2841.
61 Caswell, R., Hagemeier, C., Chiou, C.-J., Hayward, G., Kouzarides, T.,& Sinclair, J.H. (1993). The human cytomegalovirus 86K immediate early (IE2) protein requires the basic region of the TATA-box binding protein (TBP) for binding, and interacts with TBP and transcription factor TFIIB via region of IE2 required for transcriptional regulation.J Gen Virol 74,2691-2698.
62 Jupp, R., Hoffmann, S., Stenberg, R. M., Nelson, J. A.,& Ghazal, P.(1993). Human cytomegalovirus IE86 protein interacts with promoterbound TATA-binding protein via a specific region distinct from the autorepression domain. J Virol 67,7539-7546.
63 Lukac, D. M., Harel, N. Y., Tanese, N.,& Alwine, J. C. (1997). TAF-like functions of human cytomegalovirus immediate-early proteins. J Virol 71,7227-7239.
64 Speir, E., Modali, R.:, Huang, E.-S., Leon, M. B., Shawl, F., Finkel, T.,& Epstein, S. E. (1993). Potential role of human cytomegalovirus and p53 interaction in coronary restenosis. Science 265, 391-394.
65 Hagemeier, C., Caswell, R., Hayhurst, G., Sinclair, J. H.,& Kouzarides, T.(1994). Functional interaction between the HCMV IE2 transactivator and the retinoblastoma protein. EMBO J 13, 2897-2903.
66 Sommer, M. H., Scully, A. L.,& Spector, D. H. (1994). Transactivation by the human cytomegalovirus IE286-kilodalton protein requires a domain that binds to both the TATA box-binding protein and the retinoblastoma protein. J Virol 68,6223-6231.
67 Fortunato, E. A., Sommer, M. H., Yoder, K.,& Spector, D. H. (1997).Identification of domains within the human cytomegalovirus major immediate-early 86-kilodalton protein and the retinoblastoma protein required for physical and functional interaction with each other. J Virol71, 8176-8185.
68 Pizzorno, M. C.,& Hayward, G. S. (1990). The IE2 gene products of human cytomegalovirus specifically down-regulate expression from the major immediate-early promoter through a target sequence located near the cap site. J Virol 64,6154-6165.
69 Cherrington, J. M., Khoury, E. L.,& Mocarski, E. S. (1991). Human cytomegalovirus IE2 negatively regulates a gene expression via a short target sequence near the transcription start site. J Virol 65,887-896.
70 Zhu, H., Shen, Y.,& Shenk, T. (1995). Human cytomegalovirus IE1 and IE2 proteins block apoptosis. J Virol 69,7960-7970.
71 Kalejta, R. F.,& Shenk, T. (2002). Manipulation of the cell cycle by human cytomegalovirus. Front Biosc 7,295-306.
72 Wiebush, L.,& Hagemeier, C. (1999). Human cytomegalovirus 86-kilodalton 1E2 protein blocks cell cycle progression in G1. J Virol 73,9274-9283.Wiebush, L.,& Hagemeier, C. (2001). The human cytomegalovirus immediate early 2 protein dissociates cellular DNA synthesis from cyclindependent kinase activation. EMBO J 20,1086-1098.
73 Murphy, E. A., Streblow, D. N., Nelson, J. A.,& Stinski, M. F. (2000). The human cytomegalovirus 1E86 protein can block cell cycle progression after inducing transition into the S phase of permissive cells. J Virol 74,7108-7118.
74 Jault, F. M., Jault, J. M., Ruchti, F., Fortunato, E. A., Clark, C., Corbeil, J.,Richman, D. D.,& Spector, D. H. (1995). Cytomegalovirus infection induces high levels of cyclins, phosphorylated Rb, and p53, leading to cell cycle arrest. J Virol 69,6697-6704.
75 Lu, M.,& Shenk, T. (1996). Human cytomegalovirus infection inhibits cell cycle progression at multiple points, including the transition from G1 to S. J Virol 70,8850-8857.
76 Bresnahan, W. A., Thompson, E. A.,& Albrecht, T. (1997). Human cytomegalovirus infection results in altered Cdk2 subcellular localization.J Gen Virol 78,1993-1997.
77 Dittmer, D.,& Mocarski, E. S. (1997). Human cytomegalovirus infection inhibits Gl/S transition. J Virol 71,1629-1634.
78 Salvant, B. S., Fortunato, E. A.,& Spector, D. H. (1998). Cell cycle dysregulation by human cytomegalovirus:influence of the cell cycle phase at the time of infection and effects on cyclin transcription. J Virol72,3729-3741.
79 Fortunato, E. A., McElroy, A. K., Sanchez, V.,& Spector, D. H. (2000).Exploitation of cellular signalling and regulatory pathways by human cytomegalovirus. Trends Microbiol 8,111-119.
80 Stasiak, P. C.,& Mocarski, E. S. (1992). Transactivation of the human cytomegalovirus ICP36 gene promoter requires the a gene product TRS1 in addition to the IE1 and IE2. J Virol 66, 1050-1058.
81 Jones, T. R., Wiertz, E. J., Sun, L., Fish, K. N., Nelson, J. A.,& Ploegh, H. L. (1996). Human cytomegalovirus US3 impairs transport and maturation of major histocompatibility complex class I heavy chains. Proc Natl Acad Sci USA 93,11327-11333.
82 Skaletskaya, A., Bartle, L. M., Chittenden, T., McCormick, A. L., Mocarski,E. S.,& Goldmacher, V. S. (2001). A cytomegalovirus-encoded inhibitor of apoptosis that suppresses caspase-8 activation. Proc Natl Acad Sci USA 98,7829-7834.
83 Goldmacher, V. S. (2002). vMIA, a viral inhibitor of apoptosis targeting mitochondria. Biochimie 84,177-185.
84 Chambers, J., Angulo, A., Amaratunga, D., Guo, H., Jiang, Y., Wan, J. S.,Bittner, A., Frueh, K., Jackson, M. R., Peterson, P. A., Erlander, M. G.,& Ghazal, P. (1999). DNA microarrays of the complex human cytomegalovirus genome:profiling kinetic class with drug sensitivity of viral gene expression. J Virol 73,5757-5766.
85 Shamu, C. E., Story, C.M., Rapoport, T. A.,& Ploegh, H. L. (1999). The pathway of US 11-dependent degradation of MHC class Ⅰ heavy chains involves a ubiquitin-conjugated intermediate. J Cell Biol 147,45-58. Shellam, G. R., Allen, J. E., Papdimitriou, J. M.,& Bancroft, J. M. (1981).
86 Story, C. M., Furman, M. H.,& Ploegh, H. L. (1999). The cytosolic tail of class I MHC heavy chain is required for its dislocation by the human cytomegalovirus US2 and US11 gene products. Proc Natl Acad Sci USA 96,8516-8521.
87 Gao, J. L.,& Murphy, P. M. (1994). Human cytomegalovirus open reading frame US28 encodes a functional b chemokine receptor. J Biol Chem 269,28539-28542.
88 Hobom, U., Brune, W., Messerle, M., Hahn, G.,& Koszinowski, U. H.(2000). Fast screening procedures for random transposon libraries of cloned herpesvirus genomes:mutational analysis of human cytomegalovirus envelope glycoproteins genes. J Virol 74,7720-7729.
89 Margolis, M. J., Pajovic, S.,Wong, E. L.,Wade, M., Jupp, R., Nelson, J. A.,& Clifford Azizkhan, J. (1995). Interaction of the 72-kilodalton human cytomegalovirus IE1 gene product with E2F1 coincides with E2F-dependent activation of dihydrofolate reductase transcription. J Virol 69,7759-7767.
90 Wiebush, L.,& Hagemeier, C. (2001). The human cytomegalovirus immediate early 2 protein dissociates cellular DNA synthesis from cyclindependent kinase activation. EMBO J 20, 1086-1098.
91 Song and Stinski 2005, chapter by M.F. Stinski and D.T. Petrik.
92 Wiebush, L.,& Hagemeier, C. (1999). Human cytomegalovirus 86-kilodalton IE2 protein blocks cell cycle progression in G1. J Virol 73,9274-9283.
93 Petrik DT, Schmitt KP, Stinski MF (2006) Inhibition of cellular DNA synthesis by the human cytomegalovirus IE86 protein is necessary for efficient virus replication. J Virol80:3872-3883
94 Chen Z, Knutson E, Kurosky A, Albrecht T (2001) Degradation of p21cipl in cells productively infected with human cytomegalovirus. J Virol 75:3613-3625
95 Hsu C-H, Chang MDT, Tai K-Y, Yang Y-T, Wang P-S, Chen C-J, Wang Y-H, Lee S-C, Wu C-W,Juan L-J (2004) HCMV IE2-mediated inhibition of HAT activity downregulates p53 function.EMBO J 23:2269-2280 [159], [159],
96 Estes, J. E.,& Huang, E.-S. (1977). Stimulation of cellular thymidine kinase by human cytomegalovirus. J Virol 24,13-21.
97 Isom, H. C. (1979). Stimulation of ornithine decarboxylase by human cytomegalovirus. J Gen Virol 42,265-278.
98 Boldogh I, AbuBakar S, Deng CZ, Albrecht T (1991) Transcriptional activation of cellular oncogenes fos, jun and myc by human cytomegalovirus. J Virol 65:1568-1571
99 Bresnahan WA, Albrecht T, Thompson EA (1998) The cyclin E promoter is activated by human cytomegalovirus 86-kDa immediate early protein. J Biol Chem 273:22075-22082
100 McElroy AK, Dwarakanath RS, Spector DH (2000) Dysregulation of cyclin E gene expression in human cytomegalovirus-infected cells requires viral early gene expression and is associated with changes in the Rb-related protein p130. J Virol 74:4192-4206
101 Tessari MA, Gostissa M, Altamura S, Sgarra R, Rustighi A, Salvagno C, Caretti G, Imbriano C,Mantovani R, Sal GD, Giancotti V, Manfioletti G (2003) Transcriptional activation of the cyclin A gene by the architectural transcription factor HMGA2. Mol Cell Biol 23:9104-9116
102 Shlapobersky M, Sanders R, Clark C, Spector DH (2006) Repression of HMGA2 gene expression by human cytomegalovirus involves the IE2 86-kilodalton protein and is necessary for efficient viral replication and inhibition of cyclin A transcription. J Virol 80:9951-9961
103 Greaves RF, Mocarski ES (1998) Defective growth correlates with reduced accumulation of a viral DNA replication protein after low-multiplicity infection by a human cytomegalovirus ielmutant. J Virol 72:366-379
104 Sanchez V, Clark CL, Yen JY, Dwarakanath R, Spector DH (2002) Viable human cytomegalovirus recombinant virus with an internal deletion of the IE286 gene affects late stages of viral replication.J Virol 76:2973-2989
105 Ferguson M, Henry PA, Currie RA (2003) Histone deacetylase inhibition is associated with transcriptional repression of the Hmga2 gene. Nucleic Acids Res 31:3123-3133
106 Hu J, Colburn NH (2005) Histone deacetylase inhibition down-regulates cyclin D1 transcription by inhibiting nuclear factor-kappaB/p65 DNA binding. Cancer Res 3:100-109
107 Benson, J. D.,& Huang, E.-S. (1990). Human cytomegalovirus induces expression of cellular topoisomerase Ⅱ. J Virol 64,9-15.
108 Lembo, D., Gribaudo, G., Cavallo, R., Riera, L., Angeretti, A., Hertel, L.,& Landolfo, S. (1999). Human cytomegalovirus stimulates cellular dihydrofolate reductase activity in quiescent cells. Intervirology 42,30-36.
109 Cavallo, R., Lembo, D., Gribaudo, G.,& Landolfo, S. (2001). Murine cytomegalovirus infection induces cellular folylpolyglutamate synthetase activity in quiescent cells. Intervirology 44,224-226.
110 Lembo, D., Gribaudo, G., Hofer, A., Riera, L., Cornaglia, M., Mondo, A.,Angeretti, A., Gariglio, M., Thelander, L.,& Landolfo, S. (2000). Expression of an altered ribonucleotide reductase activity associated with the replication of murine cytomegalovirus in quiescent fibroblasts.J Virol 74,11557-11565.
111 Penfold, M. E.,& Mocarski, E. S. (1997). Formation of cytomegalovirus DNA replication compartments defined by localization of viral proteins and DNA synthesis. Virology 239,46-61.
112 Ahn, J. H., Jang, W. J.,& Hayward, G. S. (1999). The human cytomegalovirus IE2 and UL112-113 proteins accumulate in viral DNA replication compartments that initiate from the periphery of promyelocytic leukemia protein associated nuclear bodies (PODs or ND10). J Virol 73,10471-10548.
113. Courcelle, C. T., Courcelle, J., Prichard, M. N.,& Mocarski, E. S. (2001).Requirement for uracil-DNA glycosylase during the transition to latephase cytomegalovirus DNA replication. J Virol 75,7592-7601.
114 McVoy, M. A.,& Adler, S. P. (1994). Human cytomegalovirus DNA replicates after early circularization by concatemer formation, and inversion occurs within concatemer. J Virol 68, 1040-1051.
115 Wood LJ, Baxter MK, Plafker SM, Gibson W (1997) Human cytomegalovirus capsid assembly protein precursor (pUL80.5) interacts with itself and with the major capsid protein (pUL86)through two different domains. J Virol 71:179-190
116 Plafker SM, Gibson W (1998) Cytomegalovirus assembly protein precursor and proteinase precursor contain two nuclear localization signals that mediate their own nuclear translocation and that of the major capsid protein. J Virol 72:7722-7732
117 Loveland AN, Nguyen NL, Brignole EJ, Gibson W (2007) The amino-conserved domain of human cytomegalovirus UL80a proteins is required for key interactions during early stages of capsid formation and virus production. J Virol 81:620-628
118 Nguyen NL, Loveland AN, Gibson W (2008) Nuclear localization sequences in cytomegalovirus capsid assembly proteins (UL80 proteins) are required for virus production: inactivating NLS1, NLS2, or both affects replication to strikingly different extents. J Virol, in press
119 Baxter MK, Gibson W (1997) The putative human cytomegalovirus triplex proteins, minor capsid protein (mCP) and mCP-binding protein (mC-BP), form a heterotrimeric complex that localizes to the cell nucleus in the absence of other viral proteins. In:22nd International HerpesvirusWorkshop, La Jolla, CA
120 Spencer JV, Newcomb WW, Thomsen DR, Homa FL, Brown JC (1998) Assembly of the herpes simplex virus capsid:preformed triplexes bind to the nascent capsid. J Virol 72:3944-3951
121 Singer GP, Newcomb WW, Thomsen DR, Homa FL, Brown JC (2005) Identification of a region in the herpes simplex virus scaffolding protein required for interaction with the portal. J Virol79:132-139
122 Patel AH, MacLean JB (1995) The product of the UL6 gene of herpes simplex virus type 1 associated with virus capsids.206:465-478
123 Patel AH, Rixon FJ, Cunningham C, Davison AJ (1996) Isolation and characterization of herpes simplex virus type 1 mutants defective in the UL6 gene.217:111-123
124 Newcomb WW, Homa FL, Thomsen DR, Trus BL, Cheng N, Steven A, Booy F, Brown JC (1999)Assembly of the herpes simplex virus procapsid from purified components and identification of small complexes containing the major capsid and scaffolding proteins. J Virol73:4239-4250
125 Rixon FJ, McNab D (1999) Packaging-competent capsids of a herpes simplex virus temperaturesensitive mutant have properties similar to those of in vitro-assembled procapsids. J Virol73:5714-5721
126 Tatman JD, Preston VG, Nicholson P, Elliott RM, Rixon FJ (1994) Assembly of herpes simplex virus type 1 capsids using a panel of recombinant baculoviruses. J Gen. Virol75:1101-1113
127 Thomsen DR, Roof LL, Homa FL (1994) Assembly of herpes simplex virus (HSV) intermediate capsids in insect cells infected with recombinant baculoviruses expressing HSV capsid proteins.J Virol 68:2442-2457
128 Brignole EJ, Gibson W (2007) Enzymatic activities of human cytomegalovirus maturational protease assemblin and its precursor (pPR, pUL80a) are comparable:maximal activity of pPR requires self-interaction through its scaffolding domain. J Virol 81:4091-4103
129 Chen P, Tsuge H, Almassy RJ, Gribskov CL, Katoh S, Vanderpool DL, Margosiak SA, Pinko C,Matthews DA, Kan C-C (1996) Structure of the human cytomegalovirus protease catalytic domain reveals a novel serine protease fold and catalytic triad. Cell 86:835-843
130 Cole JL (1996) Characterization of human cytomegalovirus protease dimerization by analytical centrifugation. Biochemistry 35:15601-15610
131 Darke PL, Cole JL, Waxman L, Hall DL, Sardana MK, Kuo LC (1996) Active human cytomegalovirus protease is a dimer. J Biol Chem 271:7445-7449
132 McCartney SA, Brignole EJ, Kolegraff KN, Loveland AN, Ussin LM, Gibson W (2005) Chemical rescue of I-site cleavage in living cells and in vitro discriminates between the cytomegalovirus protease, assemblin, and its precursor, pUL80a. J Biol Chem 280:33206-33212
133 Margolis, M. J., Pajovic, S.,Wong, E. L.,Wade, M., Jupp, R., Nelson, J. A.,& Clifford Azizkhan, J. (1995). Interaction of the 72-kilodalton human cytomegalovirus IE1 gene product with E2F1 coincides with E2F-dependent activation of dihydrofolate reductase transcription. J Virol 69,7759-7767.
134 Buisson M, Hernandez JF, Lascoux D, Schoehn G, Forest E, Arlaud G, Seigneurin JM, Ruigrok RW, Burmeister WP (2002) The crystal structure of the Epstein-Barr virus protease shows rearrangement of the processed C terminus. J Mol Biol 324:89-103
135 Chan CK, Brignole EJ, Gibson W (2002) Cytomegalovirus assemblin (pUL80a):cleavage at internal site not essential for virus growth; proteinase absent from virions. J Virol76:8667-8674
136 Loveland AN, Chan CK, Brignole EJ, Gibson W (2005) Cleavage of human cytomegalovirus protease pUL80a at internal and cryptic sites is not essential but enhances infectivity. J Virol79:12961-12968
137 Casaday RJ, Bailey JR, Kalb SR, Brignole EJ, Loveland AN, Cotter RJ, Gibson W (2004)Assembly protein precursor (pUL80.5 Homolog) of simian cytomegalovirus is phosphorylated at a glycogen synthase kinase 3 site and its downstream "priming" site: phosphorylation affects interactions of protein with itself and with major capsid protein. J Virol 78:13501-13511
138 Gibson W (2006) Assembly and maturation of the capsid. In:Reddehase MJ (ed) Cytomegaloviruses:molecular biology and immunology. Caister Academic Press, pp 231-244
139 Church GA, Wilson DW (1997) Study of herpes simplex virus maturation during a synchronous wave of assembly. J Virol 71:3603-3612
140 Newcomb WW, Juhas RM, Thomsen DR, Homa FL, Burch AD, Weller SK, Brown JC (2001) The UL6 gene product forms the portal for entry of DNA into the herpes simplex virus capsid.J Virol 75:10923-10932
141 Trus BL, Cheng N, Newcomb WW, Homa FL, Brown JC, Steven AC (2004) Structure and polymorphism of the UL6 portal protein of herpes simplex virus type 1. J Virol 78:12668-12671
142 Dittmer A, Bogner E (2005) Analysis of the quaternary structure of the putative HCMV portal protein PUL104. Biochemistry 44:759-765
143 Bogner E, Radsak K, Stinski MF (1998) The gene product of human cytomegalovirus open reading frame UL56 binds the pac motif and has specific nuclease activity. J Virol 72:2259-2264
144 Holzenburg A, Bogner E (2002) From concatemeric DNA into unit-length genomes-a miracle or clever genes? In:Holzenburg A, Bogner E (eds) Structure-function relationships of human pathogenic viruses, vol 1. Kluwer Academic, New York, pp 155-173
145 Scheffczik H, Savva CG, Holzenburg A, Kolesnikova L, Bogner E (2002) The terminase subunits pUL56 and pUL89 of human cytomegalovirus are DNA-metabolizing proteins with toroidal structure. Nucleic Acids Res 30:1695-1703
146 White CA, Stow ND, Patel AH, Hughes M, Preston VG (2003) Herpes simplex virus type 1 portal protein UL6 interacts with the putative terminase subunits UL15 and UL28. J Virol77:6351-6358
147 Newcomb WW, Homa FL, Brown JC (2006) Herpes simplex virus capsid structure:DNA packaging protein UL25 is located on the external surface of the capsid near the vertices. J Virol 80:6286-6294
148 Eickmann M, Gicklhorn D, Radsak K (2006) Glycoprotein trafficking in virion morphogenesis.In:Reddehase MJ (ed) Cytomegaloviruses molecular biology and immunology. Caister Academic Press, Norfolk, UK, pp 245-264
149 Herpesvirus assembly and egress. J Virol 76:1537-1547
150 Leuzinger H, Ziegler U, Schraner EM, Fraefel C, Glauser DL, Heid I, Ackermann M, Mueller M,Wild P (2005) Herpes simplex virus 1 envelopment follows two diverse pathways. J Virol79:13047-13059
151 Klupp BG, Granzow H, Mettenleiter TC (2000) Primary envelopment of pseudorabies virus at the nuclear membrane requires the UL34 gene product.25th International Herpesvirus Workshop:Abstract 7.04
152 Roller RJ, Zhou Y, Schnetzer R, Ferguson J, DeSalvo D (2000) Herpes simplex virus type 1 U(L)34 gene product is required for viral envelopment. J Virol 74:117-129
153 Reynolds AE, Wills EG, Roller RJ, Ryckman BJ, Baines JD (2002) Ultrastructural localization of the herpes simplex virus type 1 UL31, UL34, and US3 proteins suggests specific roles in primary envelopment and egress of nucleocapsids. J Virol 76:8939-8952
154 Fuchs W, Granzow H, Klupp BG, Kopp M, Mettenleiter TC (2002) The UL48 tegument protein of pseudorabies virus is critical for intracytoplasmic assembly of infectious virions. J Virol76:6729-6742
155 Purves FC, Spector D, Roizman B (1992) UL34, the target of the herpes simplex virus U(S)3 protein kinase, is a membrane protein which in its unphosphorylated state associates with novel phosphoproteins. J Virol 66:4295-4303
156 Murphy, E. A., Streblow, D. N., Nelson, J. A.,& Stinski, M. F. (2000). The human cytomegalovirus IE86 protein can block cell cycle progression after inducing transition into the S phase of permissive cells. J Virol 74,7108-7118.
157 Bjerke SL, Cowan JM, Kerr JK, Reynolds AE, Baines JD, Roller RJ (2003) Effects of charged cluster mutations on the function of herpes simplex virus type 1 UL34 protein. J Virol77:7601-7610
158 Hensel G, Meyer H, Gartner S, Brand G, Kern HF (1995) Nuclear localization of the human cytomegalovirus tegument protein pp150 (ppUL32). J Gen Virol 76:1591-1601
159 Nii S, Uno F, Yoshida M, Akatsuka K (1998) Structure and assembly of human beta herpesviruses.Nippon Rinsho 56:22-28
160 J. Wang and W. Gibson, unpublished data from studies using mutant viruses encoding CysCysProGlyCysCys-tagged pUL32 detected in live, infected cells with the biarsenical dye FIAsH
161 Silva MC, Yu A-C, Enquist L, Shenk T (2003) Human cytomegalovirus UL99-encoded pp28 is required for the cytoplasmic envelopment of tegument-associated capsids. J Virol77:10594-10605
162 Nogalski MT, Podduturi JP, Demeritt IB, Milford LE, Yurochko AD (2007) The human cytomegalovirus virion possesses an activated casein kinase ii that allows for the rapid phosphorylation of the inhibitor of NF-{kappa}B, I{kappa}B{alpha}. J Virol 81:5305-5314
163 Kattenhorn LM, Korbel GA, Kessler BM, Spooner E, Ploegh HL (2005) A deubiquitinating enzyme encoded by HSV-1 belongs to a family of cysteine proteases that is conserved across the family Herpesviridae. Mol Cell 19:547-557
164 Schlieker C, Korbel GA, Kattenhorn LM, Ploegh HL (2005) A deubiquitinating activity is conserved in the large tegument protein of the herpesviridae. J Virol 79:15582-15585
165 Wang J, Loveland AN, Kattenhorn LM, Ploegh HL, Gibson W (2006) High-molecular-weight protein (pUL48) of human cytomegalovirus is a competent deubiquitinating protease:mutant viruses altered in its active-site cysteine or histidine are viable. J Virol 80:6003-6012
166 Anders DG, Kacica MA, Pari G, Punturieri SM (1992) Boundaries and structure of human cytomegalovirus oriLyt, a complex origin for lytic-phase DNA replication. J Virol66:3373-3384
167 Masse, M. J., Karlin, S., Schachtel, G. A.,& Mocarski, E. S. (1992).Human cytomegalovirus origin of DNA replication (oriLyt) resides within a highly complex repetitive region. Proc Natl Acad Sci USA89,5246-5250.
168 Borst EM, Messerle. (2005) Analysis of human cytomegalovirus oriLyt sequence requirements in the context of the viral genome. J Virol 79:3615-3626
169 Sarisky, R. T.,& Hayward, G. S. (1996). Evidence that the UL84 gene product of human cytomegalovirus is essential for promoting oriLytdependent DNA replication and formation of replication compartments in cotransfection assays. J Virol 70,7398-7413.
170 Xu Y, Cei SA, Rodriguez Huete A, Colletti KS, Pari GS (2004b) Human cytomegalovirus DNA replication requires transcriptional activation via an IE2-and UL84-responsive bidirectional promoter element within oriLyt. J Virol 78:11664-11677
171 Gibson, W. (1996). Structure and assembly of the virion. Intervirology 39,389-400.
172 Butcher, S. J., Aitken, J., Mitchell, J., Gowen, B.,& Dargan, D. J. (1998).Structure of the human cytomegalovirus B capsid by electron microscopy and image reconstruction. J Struct Biol 124,70-76.
173 Sanchez, V., Sztul, E.,& Britt, W.(2000). Accumulation of virion tegument and envelope proteins in a stable cytoplasmatic compartment during human cytomegalovirus replication: characterization of a potential site of virus assembly. J Virol 74,975-986.
174 Andrew J. Davison,l Aidan Dolan,l Parvis Akter,1 Clare Addison,l Derrick j. Dargan,l Donald J. Alcendor,2 Duncan J. McGeochl and Gary S. Hayward2 (2003).The human cytomegalovirus genome revisited:comparison with the chimpanzee cytomegalovirus genome Journal of General Virology,84,17-28
175 Mocarski and Courcelle in Field's Virology,4th ed., "Cytomegaloviruses and Their Replication" (6/27/2000
176 Udo B,Gholamreza D(2001).Analysis and Characterization of the Complete Genome of Tupaia (Tree Shrew) Herpesvirus.. Journal of Virology, p.4854-4870
177 Chambers J, Angulo A, Amaratunga D,etal.(1999) DNA Microarrays of the Complex Human Cytomegalovirus Genome:Profiling Kinetic Class with Drug Sensitivity of Viral Gene Expression. Journal of Virology, p.5757-5766.
178 Murphy E, Rigoutsos I, Shibuya T, etal.(2003).Reevaluation of human cytomegalovirus coding potential.(?).PNAS vol.100 (23) 13585-13590
179 Dunn W, Chou C, Li H, etal (2003).Functional profiling of a human cytomegalovirus genome.PNAS,vol.100(24) 14223-14228.
180 Frohman M A, Dush M K, Martin G R. Rapid production of full-length cDNAs from rare amplificatio nusing a single gene-specific oligo nucleotide primer. Proc. Natl. Acad. Sci. USA., 1988;85:8998-9002
181潘鸿春,宋大祥,周开亚.悦目金蛛丝腺SMART RACE cDNA文库的构建与鉴定.蛛形学报.2005,14(2)：65-69
182 Krogh A,Larsson B,Heijne G,E.LL.Sonnhammer2001.Predicting transmembrane protein topology with a hidden Markov model:Application to complete genomes[J].Journal of Molecular Biology,305(3):567-580.
183 Kyte JJ, Doolittle RF1982.A simple method for displaying the hydrophobic character of a protein[J].J.Mol.Biol,,157:105-132.
184 Janin J.1979.Surface and inside volumes in globular proteins[J].Nature,277:491-492.
185 Zimmerman JM,Eliezer N, Simha R.1968.The characteriza-tion of amino acid sequences in proteins by statistical methods[J].J Theor Biol,21:170-201.
186 Bhaskaran.R,Ponnuswamy.PK.1988.Positional flexibilities of amino acid residues in globular proteins[J].Int. J.Pept Prot. Res.,32:242-255.
187吴玉章,朱锡华.一种病毒蛋白B细胞表位预测方法的建立[J].科学通报,1994,39(24)：2275～22279.
188万涛,孙涛,吴家金.蛋白顺序性抗原决定簇的多参数综合预测[J].中国免疫学杂志,1997,13(6)：329～333.
189 Patrone M, Percivalle E, Secchi M,, etal 5(2003).The human cytomegalovirus UL45 gene product is a late, virion-associated protein and influences virus growth at low multiplicities of infection..Journal of General Virology,84,3359-3370
190 Landini, M. P.,Ripalti A..1982. A DNA-nicking activity associated with the nucleocapsid of human cytomegalovirus. Arch. Virol.73:351-356.
191 Patrone M, Percivalle E, Secchi, etal.(2003).The human cytomegalovirus UL45 gene product is a late, virion-associated protein and influences virus growth at low multiplicities of infection. Journal of General Virology,84,3359-3370
192 Adair R,Elaine R. Douglas,Jean B, etal (2002).The products of human cytomegalovirus genes UL49、 UL24,UL43 and US22 are tegument components. Journal of General Virology,83 1315-1324.
193 Baker A, Cotton M.1997.Methods of performing homologous recombination based modification of nucleic acids in recombination deficient cells and use of the modified nucleic acid products thereof[J]. Nucleic Acids Research,25:1950-1965
194 Borst E M, Ulrich G, Koszinowski, et al.1999.Cloning of the Human Cytomegalovirus (HCMV) Genome as an Infectious Bacterial Artificial Chromosome in Escherichia coli:a New Approach for Construction of HCMV Mutants[J]. JOURNAL OF VIROLOG Y,73(10):8320-8329
195 Jones, T R. Muzithras V P.1992.A cluster of dispensable genes within the human cytomegalovirus genome short component:IRS1, US1 through US5, and the US6 family[J].J Virol,66:2541-2546.
196 Dunn W, Chou C, Li H, et al.2003.Functional profiling of a human cytomegalovirus genome[J]. PNAS November,100 (24):14223-14228.
197 Hensel G M, Meyer H H., Buchmann I, et al.1996.Intracellular localization and expression of the human cytomegalovirus matrix phosphoprotein pp71 (ppUL82):Evidence for its translocation into the nucleus[J]. J Gen. Virol,77:3087-3097.
198 Scholz M, Doerr H W, Cinatl J.2001.Inhibition of cytomegalovirus immediate early gene expression:a therapeutic option? [J]. Antiviral Res,49:129-145.
199 Irmiere, A., and W. Gibson.1983. Isolation and characterization of a noninfectious virion-like particle released from cells infected with human strains of cytomegalovirus. Virology 130:118-133.
200 Revello, M. G., E. Percivalle, A. Di Matteo, F. Morini, and G. Gerna.1992.Nuclear expression of the lower matrix protein of human cytomegalovirus in peripheral blood leukocytes of immunocompromised viraemic patients.J. Gen. Virol.73:437-442.
201 Yao, Z. Q., G. Gallez-Hawkins, N. A. Lomeli, X. Li, K. M. Molinder, D. J.Diamond, and J. A. Zaia.2001. Site-directed mutation in a conserved kinase domain of human cytomegalovirus-pp65 with preservation of cytotoxic T lymphocyte targeting. Vaccine 19:1628-1635.
202 Gallina, A., L. Simoncini, S. Garbelli, E. Percivalle, G. Pedrali-Noy, K. S.Lee, R. L. Erikson, B. Plachter, G. Gerna, and G. Milaneshi.1999. Polo-like kinase 1 as a target for human cytomegalovirus pp65 lower matrix protein.J. Virol.73:1468-1478.
203 Schmolke, S., H. F. Kern, P. Drescher, G. Jahn, and B. Plachter.1995. The dominant phosphoprotein pp65 (UL83) of human cytomegalovirus is dispensable for growth in cell culture. J. Virol.69:5959-5968.
204 Wills, M. R., A. J. Carmichael, K. Mynard, X. Jin, M. P. Weeks, B. Plachter,and J. G. P. Sissons.1996. The human cytotoxic T-lymphocyte (CTL)response to cytomegalovirus is dominated by structural protein pp65:frequency,specificity, and T-cell receptor usage of pp65-specific CTL. J. Virol.70:7569-7579.
205 Yu, D., M. C. Silva, and T. Shenk.2003. Functional map of human cytomegalovirus AD169 defined by global mutagenesis analysis. Proc. Natl.Acad. Sci. USA 100:12396-12401.
206 Baxter, M. K., and W. Gibson.2001. Cytomegalovirus basic phosphoprotein (pUL32) binds to capsids in vitro through its amino one-third. J. Virol.75:6865-6873.
207 AuCoin, D. P., G. B. Smith, C. D. Meiering, and E. S. Mocarski. 2006.Betaherpesvirus-conserved cytomegalovirus tegument protein ppUL32(pp150) controls cytoplasmic events during virion maturation. J. Virol.80:8199-8210.
208 Bechtel, J. T., and T. Shenk.2002. Human cytomegalovirus UL47 tegument protein functions after entry and before immediate-early gene expression.J. Virol.76:1043-1050
209 Trgovcich, J., C. Cebulla, P. Zimmerman, and D. D. Sedmak.2006. Human cytomegalovirus protein pp71 disrupts major histocompatibility complex class Ⅰ cell surface expression. J. Virol. 80:951-963.
210 Cantrell, S. R., and W. A. Bresnahan.2006. Human cytomegalovirus (HCMV) UL82 gene product (pp71) relieves hDaxx-mediated repression of HCMV replication. J. Virol.80:6188-6191.
211 Saffert, R. T., and R. F. Kalejta.2006. Inactivating a cellular intrinsic immune defense mediated by Daxx is the mechanism through which the human cytomegalovirus pp71 protein stimulates viral immediate early gene expression. J. Virol.80:3863-3871.
212 Terhune, S. S., J. Schroer, and T. Shenk.2004. RNAs are packaged into human cytomegalovirus virions in proportion to their intracellular concentration.J. Virol.78:10390-10398.
213 Maiti S, Doskow J, Li S, Nhim RP, Lindsey JS,Wilkinson MF. The Pem homeobox gene.Androgen-dependent and-independent promoters and tissue-specific alternative RNAsplicing [J]. J Biol Chem,1996,271(29):17536-46.
214罗星,张峪涵,于娜,等.神经突起因子酵母双杂交诱饵质粒构建和转化[J].中国公共卫生,2006,22(4)：444-445.
2. Pass, R. F. (2001). Cytomegalovirus.In D. Knipe,& P. Howley (Eds.),Fields Virology (pp. 2675-2705). Philadelphia:Lippincott, Williams and Wilkins.
3 Mocarski, E. S., T. Shenk, and R. F. Pass.2007. Cytomegaloviruses, p.2701-2772. In D. M. Knipe and P. M. Howley (ed.), Fields virology,5th ed. Lippincott Williams & Wilkins, Philadelphia, PA.
4.Mocarski, E. S.,& Courcelle, C. T. (2001). Cytomegalovirus and their replication. In D. Knipe, & P. Howley (Eds.), Fields Virology (pp.2629-2673). Philadelphia:Lippincott, Williams and Wilkins.
5.Britt, W. J.,& Mach, M. (1996). Human cytomegalovirus glycoproteins.Intervirology 39,401-412.
6 Gretch, D. R., Kari, B., Rasmussen, L., Gehrz, R. C.,& Stinski, M. F.(1988). Identification and characterization of three distinct families of glycoprotein complexes in the envelopes of human cytomegalovirus.J Virol 62,875-881.
7 Theiler, R. N.,& Compton, T. (2001). Characterization of the signal peptide processing and membrane association of human cytomegalovirus glycoprotein O. J Biol Chem 276,39226-39231.
8 Gonczol, E.,& Plotkin, S. (2001). Development of a cytomegalovirus vaccine:lessons from recent clinical trials. Expert Opin Biol Ther 1,401-412.
9 Huber, M. T.,& Compton, T. (1998). The human cytomegalovirus UL74 gene encodes the third component of the glycoprotein H-glycoprotein L-containing envelope complex. J Virol 72,8191-8197.
10 Kari, B., Li, W., Cooper, J., Goertz, R.,& Radeke, B. (1994). The human cytomegalovirus UL100 gene encodes the gC-11 glycoproteins recognized by group 2 monoclonal antibodies. J Gen Virol 75,3081-3086.
11 Baldanti, F., Underwood, M. R., Stanat, S.C., Biron, K. K., Chou, S.,Sarasini, A., Silini, E., & Gerna, G. (1996). Single amino acid changes in the DNA polymerase confer foscarnet resistance and slow-growth phenotype, while mutations in the UL97-encoded phosphotransferase confer ganciclovir resistance in three double-resistant human cytomegalovirus strains recovered from patients with AIDS.J Virol 70,1390-1395.
12 Lu, M.,& Shenk, T. (1999). Human cytomegalovirus UL69 protein induces cells to accumulate in G1 phase of the cell cycle. J Virol 73,676-683.
13 Hayashi, M. L., Blankenship, C.,& Shenk, T. (2000). Human cytomegalovirus UL69 protein is required for efficient accumulation of infected cells in the G1 phase of the cell cycle. Proc Natl Acad Sci USA 97,2692-2696.
14 Liu, B.,& Stinski, M. F. (1992). Human cytomegalovirus contains a tegument protein that enhances transcription from promoters with upstream ATF and AP-1 cis-acting elements. J Virol 66,4434-4444.
15 Baldick, C. J., Marchini, A., Patterson, C. E.,& Shenk, T. (1997). Human cytomegalovirus tegument protein pp71 (ppUL82) enhances the infectivity of viral DNA and accelerates the infectious cycle. J Virol 71,4400-4408.
16 Bresnahan, W. A.,& Shenk, T. (2000b). UL82 virion protein activates expression of immediate early viral genes in human cytomegalovirusinfected cells. Proc Natl Acad Sci USA 97,14506-14511.
17 Romanowski, M. J., Garrido-Guerrero, E.,& Shenk, T. (1997). pIRSl and pTRS1 are present in human cytomegalovirus virions. J Virol 71,5703-5705.
18 Bresnahan, W. A.,& Shenk, T. (2000a). A subset of viral transcripts packaged within human cytomegalovirus particles. Science 288,2373-2376.
19 Chee, M. S., Bankier, A. T., Beck, S., Bohni, R., Brown, C. M., Cerny, R.,Horsnell, T., Hutchison Ⅲ, C. A., Kouzarides, T., Martignetti, J. A.,Preddie, E., Satchwell, S. C., Tomlinson, P., Weston, K. M.,& Barrell,B. G. (1990a). Analysis of the protein-coding content of human cytomegalovirus strain AD169. Curr Top Microbiol Immunol 154,125-169
20 Novotny, J., Rigoutsos, I., Coleman, D.,& Shenk, T. (2001). In silico structural and functional analysis of the human cytomegalovirus(HHV5) genome. J Mol Biol 310,1151-1166.
21 Cha, T. A., Tom, E., Kemble, G. W., Duke, G. M., Mocarski, E. S.,&Spaete, R. R. (1996). Human cytomegalovirus clinical isolates carry at least 19 genes not found in laboratory strains. J Virol 70.78-83.
22 Karlin, S., Mocarski, E. S.,& Schachtel, G. A. (1994). Molecular evolution of herpesviruses: genomic and protein sequence comparisons. J Virol68,1886-1902.
23 Murphy, E. A., Streblow, D. N., Nelson, J. A.,& Stinski, M. F. (2000). The human cytomegalovirus IE86 protein can block cell cycle progression after inducing transition into the S phase of permissive cells. J Virol 74,7108-7118.
24 Smith, I. L., Cherrington, J. M., Jiles, R. E., Fuller, M. D., Freeman, W. R.,& Spector, S. A. (1997). High-level resistance of cytomegalovirus to ganciclovir is associated with alterations in both the UL97 and DNA polymerase genes. J Infect Dis 176,69-77.
25 Tomasec, P., Braud, V. M., Rickards, C., Powell, M. B., McSharry, B. P.,Gadola, S., Cerundolo, V., Borysiewicz, L. K., McMichael, A. J.,&Wilkinson, G. W. (2000). Surface expression of HLA E an inhibitor of natural killer cells, enhanced by human cytomegalovirus gpUL40. Science287,1031-1033.
26 Human cytomegalovirus virion protein complex required for epithelial and endothelial cell tropism. Proc Natl Acad Sci USA 102:18153-18158
27 Bissinger, A. L., Singzer, C., Kaiserling, E.,& Jahn, G. (2002). Human cytomegalovirus as a direct pathogen:correlation of multiorgan involvement and cell distribution with clinical and pathological findings in a case of congenital inclusion disease. J Med Virol 67,200-206.
28 Plachter, B., Sinzger, C.,& Jahn, G. (1996). Cell types involved in replication and distribution of human cytomegalovirus. Adv Virus Res 46,195-261.
29 Sinzger, C.,& Jahn, G. (1996). Human cytomegalovirus cell tropism and pathogenesis. Intervirology 39,302-319.
30 Sinzger, C., Plachter, B., Grefte, A., The, T. H.,& Jahn, G. (1996). Tissue macrophages are infected by human cytomegalovirus. J Infect Dis 173,240-245. 31 Kahl, M., Siegel-Axel, D., Stenglein, S., Jahn, G.,& Sinzger, C. (2000).Efficient lytic infection of human arterial endothelial cells by human cytomegalovirus strains. J Virol 74,7628-7635.
32 Kondo, K., Kaneshima, H.,& Mocarski, E. S. (1994). Human cytomegalovirus latent infection of granulocyte-macrophage progenitors. Proc Natl Acad Sci USA 91,11879-11883.
33 Soderberg-Naucler, C., Fish, K.N.,& Nelson, J. A. (1997). Reactivation of latent human cytomegalovirus by allogeneic stimulation of blood cells from healthy donors. Cell 91,119-126.
34 Sinzger, C., Kahl, M., Laib, K., Klingel, K., Rieger, P., Plachter, B.,& Jahn,G. (2000). Tropism of human cytomegalovirus for endothelial cells is determined by a post-entry step dependent on efficient translocation to the nucleus. J Gen Virol 81,3021-3035.
35 Compton, T., Nowlin, D. M.,& Cooper, N. R. (1993). Initiation of human cytomegalovirus infection requires initial interaction with cell surface heparan sulfate. Virology 193,834-841.
36 Browne, E. P., Wing, B., Coleman, D.,& Shenk, T. (2001). Altered cellular mRNA levels in human cytomegalovirus infected fibroblasts:viral block to the accumulation of antiviral mRNAs. J Virol 75,12319-12330.
37 Boyle, K. A., Pietropaolo, R. L.,& Compton, T. (1999). Engagement of the cellular receptor for glycoprotein B of human cytomegalovirus activates the interferon-responsive pathway. Mol Cell Biol 19,3607-3613.
38 Simmen, K. A., Singh, J., Luukkonen, B. G. M., Lopper, M., Bittner, A.,Miller, N. E., Jackson, M. R., Compton, T.,& Fruh, K. (2001). Global modulation of cellular transcription by human cytomegalovirus is initiated by viral glycoprotein B. Proc Natl Acad Sci USA 98,7140-7145.
39 Yurochko, A. D., Mayo, M. W., Poma, E. E., Baldwin Jr., A. S.,& Huang,E.-S. (1997). Induction of the transcription factor Spl during human cytomegalovirus infection mediates upregulation of the p65 and p105/p50 NF-kB promoters. J Virol 71,4638-4648.
40 Bodaghi, B., Jones, T. R., Zipeto, D., Vita, C., Sun, L., Laurent, L.,Arenzana-Seisdedos, F., Virelizier, J. L.,& Michelson, S. (1998).Chemokine sequestration by viral chemoreceptors as a novel viral.
41 Varnum SM, Streblow DN, Monroe ME, Smith P, Auberry KJ, Pasa-Tolic L, Wang D, Camp DG 2nd, Rodland K, Wiley S, Britt W, Shenk T, Smith RD, Nelson JA (2004) Identification of proteins in human cytomegalovirus (HCMV) particles:the HCMV proteome. J Virol 78:10960-10966
42 Feire AL, Koss H, Compton T (2004) Cellular integrins function as entry receptors for human cytomegalovirus via a highly conserved disintegrin-like domain. Proc Natl Acad Sci USA 101:15470-15475
43 Wang X, Huang DY, Huong SM, Huang ES (2005) Integrin alphavbeta3 is a coreceptor for human cytomegalovirus. Nat Med 11:515-521
44 English EP, Chumanov RS, Gellman SH, Compton T (2006) Rational development of beta-peptide inhibitors of human cytomegalovirus entry. J Biol Chem 281:2661-2667
45 Boyle, K. A., Pietropaolo, R. L.,& Compton, T. (1999). Engagement of the cellular receptor for glycoprotein B of human cytomegalovirus activates the interferon-responsive pathway. Mol Cell Biol 19,3607-3613.
46 Fortunato, E. A.,& Spector, D. H. (1999). Regulation of human cytomegalovirus gene expression. Adv Virus Res 54,61-128.
47 Nelson, J. A., Gnann, J. W.,& Ghazal, P. (1990). Regulation and tissuespecific expression of human cytomegalovirus. Curr Top Microbiol Immunol 154,75-100.
48 Meier, J. L.,& Stinski, M. F. (1997). Effect of a modulator deletion on transcription of the human cytomegalovirus major immediate-early genes in infected undifferentiated and differentiated cells. J Virol 71,1246-1255.
49 Boshart, M., Weber, F., Jahn, G., Dorsch-Hasler, K., Fleckenstein, B.,& Schaffner, W. (1985). A very strong enhancer is located upstream of an immediate early gene of human cytomegalovirus. Cell 41,521-530.
50 Mocarski, E. S. (1996). Cytomegalovirus and their replication. In B. N. Fields, D. M. Knipe, & P. M. Howley (Eds.), Virology (pp.2447-2492). Philadelphia:Lippincott-Raven. 51 Hagemeier, C., Walker, S., Caswell, R., Kouzarides, T.,& Sinclair, J. H.(1992a). The 72K IE1 and 80K IE2 proteins of human cytomegalovirus independently trans-activate the c-fos, c-myc and hsp70 promoter via basal promoter elements. J Gen Virol 73,2385-2393.
52 Wade, M., Kowalik, T. F., Mudryj, M., Huang, E.-S.,& Azizkhan, J. C.(1992). E2F mediates dihydrofolate reducatse promoter activation and multiprotein complex formation in human cytomegalovirus infection.Mol Cell Biol 12,4364-4374.
53 Hayhurst, G. P., Bryant, L. A., Caswell, R. C., Walker, S. M.,& Sinclair,J. H. (1995). CCAAT box-dependent activation of the TATA-less human DNA polymerase a promoter by the human cytomegalovirus 72-kilodalton major immediate-early protein. J Virol 69,182-188.
54 Yurochko, A. D., Kowalik, T. F., Huong, S. M.,& Huang, E.-S. (1995).Human cytomegalovirus upregulates NF-kB activity by transactivating the NF-kB p105/p50 and p65 promoter. J Virol 69,5391-5400.
55 Gribaudo, G., Riera, L., Rudge, T. L., Caposio, P., Johnson, L. F.,& Landolfo,S. (2002). Human cytomegalovirus infection induces cellular thymidylate synthase gene expression in quiescent fibroblasts. J Gen Virol83,2983-2993.
56 Poma, E. E., Kowalik, T. F., Zhu, L., Sinclair, J. H.,& Huang, E.-S. (1996).The human cytomegalovirus IE 1-72 protein interacts with the cellular p107 protein and relieves p107-mediated transcriptional repression of an E2F-responsive promoter. J Virol 70,7867-7877.
57 Johnson, R. A., Yurochko, A. D., Poma, E.E., Zhu, L.,& Huang, E.-S.(1999). Domain mapping of the human cytomegalovirus IE1-72 and cellular p107 protein-protein interaction and the possible functional consequences. J Gen Virol 80,1293-1303.
58 Pajovic, S., Wong, E. L., Black, A. R.,& Azizkhan, C. J. (1997). Identification of a viral kinase that phosphorylates specific E2Fs and pocket proteins. Mol Cell Biol 17,6459-6464.
59 Marchini, A., Liu, H.,& Zhu, H. (2001). Human cytomegalovirus with IE2 (UL122) deleted fails to express early lytic genes. J Virol 75,1870-1878.
60 Song, Y.,& Stinski, M. F. (2002). Effect of the human cytomegalovirus IE86 protein on expression of E2F-responsive genes:a DNA microarray analysis. Proc Natl Acad Sci USA 99, 2836-2841.
61 Caswell, R., Hagemeier, C., Chiou, C.-J., Hayward, G., Kouzarides, T.,& Sinclair, J.H. (1993). The human cytomegalovirus 86K immediate early (IE2) protein requires the basic region of the TATA-box binding protein (TBP) for binding, and interacts with TBP and transcription factor TFIIB via region of IE2 required for transcriptional regulation.J Gen Virol 74,2691-2698.
62 Jupp, R., Hoffmann, S., Stenberg, R. M., Nelson, J. A.,& Ghazal, P.(1993). Human cytomegalovirus IE86 protein interacts with promoterbound TATA-binding protein via a specific region distinct from the autorepression domain. J Virol 67,7539-7546.
63 Lukac, D. M., Harel, N. Y., Tanese, N.,& Alwine, J. C. (1997). TAF-like functions of human cytomegalovirus immediate-early proteins. J Virol 71,7227-7239.
64 Speir, E., Modali, R.:, Huang, E.-S., Leon, M. B., Shawl, F., Finkel, T.,& Epstein, S. E. (1993). Potential role of human cytomegalovirus and p53 interaction in coronary restenosis. Science 265, 391-394.
65 Hagemeier, C., Caswell, R., Hayhurst, G., Sinclair, J. H.,& Kouzarides, T.(1994). Functional interaction between the HCMV IE2 transactivator and the retinoblastoma protein. EMBO J 13, 2897-2903.
66 Sommer, M. H., Scully, A. L.,& Spector, D. H. (1994). Transactivation by the human cytomegalovirus IE286-kilodalton protein requires a domain that binds to both the TATA box-binding protein and the retinoblastoma protein. J Virol 68,6223-6231.
67 Fortunato, E. A., Sommer, M. H., Yoder, K.,& Spector, D. H. (1997).Identification of domains within the human cytomegalovirus major immediate-early 86-kilodalton protein and the retinoblastoma protein required for physical and functional interaction with each other. J Virol71, 8176-8185.
68 Pizzorno, M. C.,& Hayward, G. S. (1990). The IE2 gene products of human cytomegalovirus specifically down-regulate expression from the major immediate-early promoter through a target sequence located near the cap site. J Virol 64,6154-6165.
69 Cherrington, J. M., Khoury, E. L.,& Mocarski, E. S. (1991). Human cytomegalovirus IE2 negatively regulates a gene expression via a short target sequence near the transcription start site. J Virol 65,887-896.
70 Zhu, H., Shen, Y.,& Shenk, T. (1995). Human cytomegalovirus IE1 and IE2 proteins block apoptosis. J Virol 69,7960-7970.
71 Kalejta, R. F.,& Shenk, T. (2002). Manipulation of the cell cycle by human cytomegalovirus. Front Biosc 7,295-306.
72 Wiebush, L.,& Hagemeier, C. (1999). Human cytomegalovirus 86-kilodalton 1E2 protein blocks cell cycle progression in G1. J Virol 73,9274-9283.Wiebush, L.,& Hagemeier, C. (2001). The human cytomegalovirus immediate early 2 protein dissociates cellular DNA synthesis from cyclindependent kinase activation. EMBO J 20,1086-1098.
73 Murphy, E. A., Streblow, D. N., Nelson, J. A.,& Stinski, M. F. (2000). The human cytomegalovirus 1E86 protein can block cell cycle progression after inducing transition into the S phase of permissive cells. J Virol 74,7108-7118.
74 Jault, F. M., Jault, J. M., Ruchti, F., Fortunato, E. A., Clark, C., Corbeil, J.,Richman, D. D.,& Spector, D. H. (1995). Cytomegalovirus infection induces high levels of cyclins, phosphorylated Rb, and p53, leading to cell cycle arrest. J Virol 69,6697-6704.
75 Lu, M.,& Shenk, T. (1996). Human cytomegalovirus infection inhibits cell cycle progression at multiple points, including the transition from G1 to S. J Virol 70,8850-8857.
76 Bresnahan, W. A., Thompson, E. A.,& Albrecht, T. (1997). Human cytomegalovirus infection results in altered Cdk2 subcellular localization.J Gen Virol 78,1993-1997.
77 Dittmer, D.,& Mocarski, E. S. (1997). Human cytomegalovirus infection inhibits Gl/S transition. J Virol 71,1629-1634.
78 Salvant, B. S., Fortunato, E. A.,& Spector, D. H. (1998). Cell cycle dysregulation by human cytomegalovirus:influence of the cell cycle phase at the time of infection and effects on cyclin transcription. J Virol72,3729-3741.
79 Fortunato, E. A., McElroy, A. K., Sanchez, V.,& Spector, D. H. (2000).Exploitation of cellular signalling and regulatory pathways by human cytomegalovirus. Trends Microbiol 8,111-119.
80 Stasiak, P. C.,& Mocarski, E. S. (1992). Transactivation of the human cytomegalovirus ICP36 gene promoter requires the a gene product TRS1 in addition to the IE1 and IE2. J Virol 66, 1050-1058.
81 Jones, T. R., Wiertz, E. J., Sun, L., Fish, K. N., Nelson, J. A.,& Ploegh, H. L. (1996). Human cytomegalovirus US3 impairs transport and maturation of major histocompatibility complex class I heavy chains. Proc Natl Acad Sci USA 93,11327-11333.
82 Skaletskaya, A., Bartle, L. M., Chittenden, T., McCormick, A. L., Mocarski,E. S.,& Goldmacher, V. S. (2001). A cytomegalovirus-encoded inhibitor of apoptosis that suppresses caspase-8 activation. Proc Natl Acad Sci USA 98,7829-7834.
83 Goldmacher, V. S. (2002). vMIA, a viral inhibitor of apoptosis targeting mitochondria. Biochimie 84,177-185.
84 Chambers, J., Angulo, A., Amaratunga, D., Guo, H., Jiang, Y., Wan, J. S.,Bittner, A., Frueh, K., Jackson, M. R., Peterson, P. A., Erlander, M. G.,& Ghazal, P. (1999). DNA microarrays of the complex human cytomegalovirus genome:profiling kinetic class with drug sensitivity of viral gene expression. J Virol 73,5757-5766.
85 Shamu, C. E., Story, C.M., Rapoport, T. A.,& Ploegh, H. L. (1999). The pathway of US 11-dependent degradation of MHC class Ⅰ heavy chains involves a ubiquitin-conjugated intermediate. J Cell Biol 147,45-58. Shellam, G. R., Allen, J. E., Papdimitriou, J. M.,& Bancroft, J. M. (1981).
86 Story, C. M., Furman, M. H.,& Ploegh, H. L. (1999). The cytosolic tail of class I MHC heavy chain is required for its dislocation by the human cytomegalovirus US2 and US11 gene products. Proc Natl Acad Sci USA 96,8516-8521.
87 Gao, J. L.,& Murphy, P. M. (1994). Human cytomegalovirus open reading frame US28 encodes a functional b chemokine receptor. J Biol Chem 269,28539-28542.
88 Hobom, U., Brune, W., Messerle, M., Hahn, G.,& Koszinowski, U. H.(2000). Fast screening procedures for random transposon libraries of cloned herpesvirus genomes:mutational analysis of human cytomegalovirus envelope glycoproteins genes. J Virol 74,7720-7729.
89 Margolis, M. J., Pajovic, S.,Wong, E. L.,Wade, M., Jupp, R., Nelson, J. A.,& Clifford Azizkhan, J. (1995). Interaction of the 72-kilodalton human cytomegalovirus IE1 gene product with E2F1 coincides with E2F-dependent activation of dihydrofolate reductase transcription. J Virol 69,7759-7767.
90 Wiebush, L.,& Hagemeier, C. (2001). The human cytomegalovirus immediate early 2 protein dissociates cellular DNA synthesis from cyclindependent kinase activation. EMBO J 20, 1086-1098.
91 Song and Stinski 2005, chapter by M.F. Stinski and D.T. Petrik.
92 Wiebush, L.,& Hagemeier, C. (1999). Human cytomegalovirus 86-kilodalton IE2 protein blocks cell cycle progression in G1. J Virol 73,9274-9283.
93 Petrik DT, Schmitt KP, Stinski MF (2006) Inhibition of cellular DNA synthesis by the human cytomegalovirus IE86 protein is necessary for efficient virus replication. J Virol80:3872-3883
94 Chen Z, Knutson E, Kurosky A, Albrecht T (2001) Degradation of p21cipl in cells productively infected with human cytomegalovirus. J Virol 75:3613-3625
95 Hsu C-H, Chang MDT, Tai K-Y, Yang Y-T, Wang P-S, Chen C-J, Wang Y-H, Lee S-C, Wu C-W,Juan L-J (2004) HCMV IE2-mediated inhibition of HAT activity downregulates p53 function.EMBO J 23:2269-2280 [159], [159],
96 Estes, J. E.,& Huang, E.-S. (1977). Stimulation of cellular thymidine kinase by human cytomegalovirus. J Virol 24,13-21.
97 Isom, H. C. (1979). Stimulation of ornithine decarboxylase by human cytomegalovirus. J Gen Virol 42,265-278.
98 Boldogh I, AbuBakar S, Deng CZ, Albrecht T (1991) Transcriptional activation of cellular oncogenes fos, jun and myc by human cytomegalovirus. J Virol 65:1568-1571
99 Bresnahan WA, Albrecht T, Thompson EA (1998) The cyclin E promoter is activated by human cytomegalovirus 86-kDa immediate early protein. J Biol Chem 273:22075-22082
100 McElroy AK, Dwarakanath RS, Spector DH (2000) Dysregulation of cyclin E gene expression in human cytomegalovirus-infected cells requires viral early gene expression and is associated with changes in the Rb-related protein p130. J Virol 74:4192-4206
101 Tessari MA, Gostissa M, Altamura S, Sgarra R, Rustighi A, Salvagno C, Caretti G, Imbriano C,Mantovani R, Sal GD, Giancotti V, Manfioletti G (2003) Transcriptional activation of the cyclin A gene by the architectural transcription factor HMGA2. Mol Cell Biol 23:9104-9116
102 Shlapobersky M, Sanders R, Clark C, Spector DH (2006) Repression of HMGA2 gene expression by human cytomegalovirus involves the IE2 86-kilodalton protein and is necessary for efficient viral replication and inhibition of cyclin A transcription. J Virol 80:9951-9961
103 Greaves RF, Mocarski ES (1998) Defective growth correlates with reduced accumulation of a viral DNA replication protein after low-multiplicity infection by a human cytomegalovirus ielmutant. J Virol 72:366-379
104 Sanchez V, Clark CL, Yen JY, Dwarakanath R, Spector DH (2002) Viable human cytomegalovirus recombinant virus with an internal deletion of the IE286 gene affects late stages of viral replication.J Virol 76:2973-2989
105 Ferguson M, Henry PA, Currie RA (2003) Histone deacetylase inhibition is associated with transcriptional repression of the Hmga2 gene. Nucleic Acids Res 31:3123-3133
106 Hu J, Colburn NH (2005) Histone deacetylase inhibition down-regulates cyclin D1 transcription by inhibiting nuclear factor-kappaB/p65 DNA binding. Cancer Res 3:100-109
107 Benson, J. D.,& Huang, E.-S. (1990). Human cytomegalovirus induces expression of cellular topoisomerase Ⅱ. J Virol 64,9-15.
108 Lembo, D., Gribaudo, G., Cavallo, R., Riera, L., Angeretti, A., Hertel, L.,& Landolfo, S. (1999). Human cytomegalovirus stimulates cellular dihydrofolate reductase activity in quiescent cells. Intervirology 42,30-36.
109 Cavallo, R., Lembo, D., Gribaudo, G.,& Landolfo, S. (2001). Murine cytomegalovirus infection induces cellular folylpolyglutamate synthetase activity in quiescent cells. Intervirology 44,224-226.
110 Lembo, D., Gribaudo, G., Hofer, A., Riera, L., Cornaglia, M., Mondo, A.,Angeretti, A., Gariglio, M., Thelander, L.,& Landolfo, S. (2000). Expression of an altered ribonucleotide reductase activity associated with the replication of murine cytomegalovirus in quiescent fibroblasts.J Virol 74,11557-11565.
111 Penfold, M. E.,& Mocarski, E. S. (1997). Formation of cytomegalovirus DNA replication compartments defined by localization of viral proteins and DNA synthesis. Virology 239,46-61.
112 Ahn, J. H., Jang, W. J.,& Hayward, G. S. (1999). The human cytomegalovirus IE2 and UL112-113 proteins accumulate in viral DNA replication compartments that initiate from the periphery of promyelocytic leukemia protein associated nuclear bodies (PODs or ND10). J Virol 73,10471-10548.
113. Courcelle, C. T., Courcelle, J., Prichard, M. N.,& Mocarski, E. S. (2001).Requirement for uracil-DNA glycosylase during the transition to latephase cytomegalovirus DNA replication. J Virol 75,7592-7601.
114 McVoy, M. A.,& Adler, S. P. (1994). Human cytomegalovirus DNA replicates after early circularization by concatemer formation, and inversion occurs within concatemer. J Virol 68, 1040-1051.
115 Wood LJ, Baxter MK, Plafker SM, Gibson W (1997) Human cytomegalovirus capsid assembly protein precursor (pUL80.5) interacts with itself and with the major capsid protein (pUL86)through two different domains. J Virol 71:179-190
116 Plafker SM, Gibson W (1998) Cytomegalovirus assembly protein precursor and proteinase precursor contain two nuclear localization signals that mediate their own nuclear translocation and that of the major capsid protein. J Virol 72:7722-7732
117 Loveland AN, Nguyen NL, Brignole EJ, Gibson W (2007) The amino-conserved domain of human cytomegalovirus UL80a proteins is required for key interactions during early stages of capsid formation and virus production. J Virol 81:620-628
118 Nguyen NL, Loveland AN, Gibson W (2008) Nuclear localization sequences in cytomegalovirus capsid assembly proteins (UL80 proteins) are required for virus production: inactivating NLS1, NLS2, or both affects replication to strikingly different extents. J Virol, in press
119 Baxter MK, Gibson W (1997) The putative human cytomegalovirus triplex proteins, minor capsid protein (mCP) and mCP-binding protein (mC-BP), form a heterotrimeric complex that localizes to the cell nucleus in the absence of other viral proteins. In:22nd International HerpesvirusWorkshop, La Jolla, CA
120 Spencer JV, Newcomb WW, Thomsen DR, Homa FL, Brown JC (1998) Assembly of the herpes simplex virus capsid:preformed triplexes bind to the nascent capsid. J Virol 72:3944-3951
121 Singer GP, Newcomb WW, Thomsen DR, Homa FL, Brown JC (2005) Identification of a region in the herpes simplex virus scaffolding protein required for interaction with the portal. J Virol79:132-139
122 Patel AH, MacLean JB (1995) The product of the UL6 gene of herpes simplex virus type 1 associated with virus capsids.206:465-478
123 Patel AH, Rixon FJ, Cunningham C, Davison AJ (1996) Isolation and characterization of herpes simplex virus type 1 mutants defective in the UL6 gene.217:111-123
124 Newcomb WW, Homa FL, Thomsen DR, Trus BL, Cheng N, Steven A, Booy F, Brown JC (1999)Assembly of the herpes simplex virus procapsid from purified components and identification of small complexes containing the major capsid and scaffolding proteins. J Virol73:4239-4250
125 Rixon FJ, McNab D (1999) Packaging-competent capsids of a herpes simplex virus temperaturesensitive mutant have properties similar to those of in vitro-assembled procapsids. J Virol73:5714-5721
126 Tatman JD, Preston VG, Nicholson P, Elliott RM, Rixon FJ (1994) Assembly of herpes simplex virus type 1 capsids using a panel of recombinant baculoviruses. J Gen. Virol75:1101-1113
127 Thomsen DR, Roof LL, Homa FL (1994) Assembly of herpes simplex virus (HSV) intermediate capsids in insect cells infected with recombinant baculoviruses expressing HSV capsid proteins.J Virol 68:2442-2457
128 Brignole EJ, Gibson W (2007) Enzymatic activities of human cytomegalovirus maturational protease assemblin and its precursor (pPR, pUL80a) are comparable:maximal activity of pPR requires self-interaction through its scaffolding domain. J Virol 81:4091-4103
129 Chen P, Tsuge H, Almassy RJ, Gribskov CL, Katoh S, Vanderpool DL, Margosiak SA, Pinko C,Matthews DA, Kan C-C (1996) Structure of the human cytomegalovirus protease catalytic domain reveals a novel serine protease fold and catalytic triad. Cell 86:835-843
130 Cole JL (1996) Characterization of human cytomegalovirus protease dimerization by analytical centrifugation. Biochemistry 35:15601-15610
131 Darke PL, Cole JL, Waxman L, Hall DL, Sardana MK, Kuo LC (1996) Active human cytomegalovirus protease is a dimer. J Biol Chem 271:7445-7449
132 McCartney SA, Brignole EJ, Kolegraff KN, Loveland AN, Ussin LM, Gibson W (2005) Chemical rescue of I-site cleavage in living cells and in vitro discriminates between the cytomegalovirus protease, assemblin, and its precursor, pUL80a. J Biol Chem 280:33206-33212
133 Margolis, M. J., Pajovic, S.,Wong, E. L.,Wade, M., Jupp, R., Nelson, J. A.,& Clifford Azizkhan, J. (1995). Interaction of the 72-kilodalton human cytomegalovirus IE1 gene product with E2F1 coincides with E2F-dependent activation of dihydrofolate reductase transcription. J Virol 69,7759-7767.
134 Buisson M, Hernandez JF, Lascoux D, Schoehn G, Forest E, Arlaud G, Seigneurin JM, Ruigrok RW, Burmeister WP (2002) The crystal structure of the Epstein-Barr virus protease shows rearrangement of the processed C terminus. J Mol Biol 324:89-103
135 Chan CK, Brignole EJ, Gibson W (2002) Cytomegalovirus assemblin (pUL80a):cleavage at internal site not essential for virus growth; proteinase absent from virions. J Virol76:8667-8674
136 Loveland AN, Chan CK, Brignole EJ, Gibson W (2005) Cleavage of human cytomegalovirus protease pUL80a at internal and cryptic sites is not essential but enhances infectivity. J Virol79:12961-12968
137 Casaday RJ, Bailey JR, Kalb SR, Brignole EJ, Loveland AN, Cotter RJ, Gibson W (2004)Assembly protein precursor (pUL80.5 Homolog) of simian cytomegalovirus is phosphorylated at a glycogen synthase kinase 3 site and its downstream "priming" site: phosphorylation affects interactions of protein with itself and with major capsid protein. J Virol 78:13501-13511
138 Gibson W (2006) Assembly and maturation of the capsid. In:Reddehase MJ (ed) Cytomegaloviruses:molecular biology and immunology. Caister Academic Press, pp 231-244
139 Church GA, Wilson DW (1997) Study of herpes simplex virus maturation during a synchronous wave of assembly. J Virol 71:3603-3612
140 Newcomb WW, Juhas RM, Thomsen DR, Homa FL, Burch AD, Weller SK, Brown JC (2001) The UL6 gene product forms the portal for entry of DNA into the herpes simplex virus capsid.J Virol 75:10923-10932
141 Trus BL, Cheng N, Newcomb WW, Homa FL, Brown JC, Steven AC (2004) Structure and polymorphism of the UL6 portal protein of herpes simplex virus type 1. J Virol 78:12668-12671
142 Dittmer A, Bogner E (2005) Analysis of the quaternary structure of the putative HCMV portal protein PUL104. Biochemistry 44:759-765
143 Bogner E, Radsak K, Stinski MF (1998) The gene product of human cytomegalovirus open reading frame UL56 binds the pac motif and has specific nuclease activity. J Virol 72:2259-2264
144 Holzenburg A, Bogner E (2002) From concatemeric DNA into unit-length genomes-a miracle or clever genes? In:Holzenburg A, Bogner E (eds) Structure-function relationships of human pathogenic viruses, vol 1. Kluwer Academic, New York, pp 155-173
145 Scheffczik H, Savva CG, Holzenburg A, Kolesnikova L, Bogner E (2002) The terminase subunits pUL56 and pUL89 of human cytomegalovirus are DNA-metabolizing proteins with toroidal structure. Nucleic Acids Res 30:1695-1703
146 White CA, Stow ND, Patel AH, Hughes M, Preston VG (2003) Herpes simplex virus type 1 portal protein UL6 interacts with the putative terminase subunits UL15 and UL28. J Virol77:6351-6358
147 Newcomb WW, Homa FL, Brown JC (2006) Herpes simplex virus capsid structure:DNA packaging protein UL25 is located on the external surface of the capsid near the vertices. J Virol 80:6286-6294
148 Eickmann M, Gicklhorn D, Radsak K (2006) Glycoprotein trafficking in virion morphogenesis.In:Reddehase MJ (ed) Cytomegaloviruses molecular biology and immunology. Caister Academic Press, Norfolk, UK, pp 245-264
149 Herpesvirus assembly and egress. J Virol 76:1537-1547
150 Leuzinger H, Ziegler U, Schraner EM, Fraefel C, Glauser DL, Heid I, Ackermann M, Mueller M,Wild P (2005) Herpes simplex virus 1 envelopment follows two diverse pathways. J Virol79:13047-13059
151 Klupp BG, Granzow H, Mettenleiter TC (2000) Primary envelopment of pseudorabies virus at the nuclear membrane requires the UL34 gene product.25th International Herpesvirus Workshop:Abstract 7.04
152 Roller RJ, Zhou Y, Schnetzer R, Ferguson J, DeSalvo D (2000) Herpes simplex virus type 1 U(L)34 gene product is required for viral envelopment. J Virol 74:117-129
153 Reynolds AE, Wills EG, Roller RJ, Ryckman BJ, Baines JD (2002) Ultrastructural localization of the herpes simplex virus type 1 UL31, UL34, and US3 proteins suggests specific roles in primary envelopment and egress of nucleocapsids. J Virol 76:8939-8952
154 Fuchs W, Granzow H, Klupp BG, Kopp M, Mettenleiter TC (2002) The UL48 tegument protein of pseudorabies virus is critical for intracytoplasmic assembly of infectious virions. J Virol76:6729-6742
155 Purves FC, Spector D, Roizman B (1992) UL34, the target of the herpes simplex virus U(S)3 protein kinase, is a membrane protein which in its unphosphorylated state associates with novel phosphoproteins. J Virol 66:4295-4303
156 Murphy, E. A., Streblow, D. N., Nelson, J. A.,& Stinski, M. F. (2000). The human cytomegalovirus IE86 protein can block cell cycle progression after inducing transition into the S phase of permissive cells. J Virol 74,7108-7118.
157 Bjerke SL, Cowan JM, Kerr JK, Reynolds AE, Baines JD, Roller RJ (2003) Effects of charged cluster mutations on the function of herpes simplex virus type 1 UL34 protein. J Virol77:7601-7610
158 Hensel G, Meyer H, Gartner S, Brand G, Kern HF (1995) Nuclear localization of the human cytomegalovirus tegument protein pp150 (ppUL32). J Gen Virol 76:1591-1601
159 Nii S, Uno F, Yoshida M, Akatsuka K (1998) Structure and assembly of human beta herpesviruses.Nippon Rinsho 56:22-28
160 J. Wang and W. Gibson, unpublished data from studies using mutant viruses encoding CysCysProGlyCysCys-tagged pUL32 detected in live, infected cells with the biarsenical dye FIAsH
161 Silva MC, Yu A-C, Enquist L, Shenk T (2003) Human cytomegalovirus UL99-encoded pp28 is required for the cytoplasmic envelopment of tegument-associated capsids. J Virol77:10594-10605
162 Nogalski MT, Podduturi JP, Demeritt IB, Milford LE, Yurochko AD (2007) The human cytomegalovirus virion possesses an activated casein kinase ii that allows for the rapid phosphorylation of the inhibitor of NF-{kappa}B, I{kappa}B{alpha}. J Virol 81:5305-5314
163 Kattenhorn LM, Korbel GA, Kessler BM, Spooner E, Ploegh HL (2005) A deubiquitinating enzyme encoded by HSV-1 belongs to a family of cysteine proteases that is conserved across the family Herpesviridae. Mol Cell 19:547-557
164 Schlieker C, Korbel GA, Kattenhorn LM, Ploegh HL (2005) A deubiquitinating activity is conserved in the large tegument protein of the herpesviridae. J Virol 79:15582-15585
165 Wang J, Loveland AN, Kattenhorn LM, Ploegh HL, Gibson W (2006) High-molecular-weight protein (pUL48) of human cytomegalovirus is a competent deubiquitinating protease:mutant viruses altered in its active-site cysteine or histidine are viable. J Virol 80:6003-6012
166 Anders DG, Kacica MA, Pari G, Punturieri SM (1992) Boundaries and structure of human cytomegalovirus oriLyt, a complex origin for lytic-phase DNA replication. J Virol66:3373-3384
167 Masse, M. J., Karlin, S., Schachtel, G. A.,& Mocarski, E. S. (1992).Human cytomegalovirus origin of DNA replication (oriLyt) resides within a highly complex repetitive region. Proc Natl Acad Sci USA89,5246-5250.
168 Borst EM, Messerle. (2005) Analysis of human cytomegalovirus oriLyt sequence requirements in the context of the viral genome. J Virol 79:3615-3626
169 Sarisky, R. T.,& Hayward, G. S. (1996). Evidence that the UL84 gene product of human cytomegalovirus is essential for promoting oriLytdependent DNA replication and formation of replication compartments in cotransfection assays. J Virol 70,7398-7413.
170 Xu Y, Cei SA, Rodriguez Huete A, Colletti KS, Pari GS (2004b) Human cytomegalovirus DNA replication requires transcriptional activation via an IE2-and UL84-responsive bidirectional promoter element within oriLyt. J Virol 78:11664-11677
171 Gibson, W. (1996). Structure and assembly of the virion. Intervirology 39,389-400.
172 Butcher, S. J., Aitken, J., Mitchell, J., Gowen, B.,& Dargan, D. J. (1998).Structure of the human cytomegalovirus B capsid by electron microscopy and image reconstruction. J Struct Biol 124,70-76.
173 Sanchez, V., Sztul, E.,& Britt, W.(2000). Accumulation of virion tegument and envelope proteins in a stable cytoplasmatic compartment during human cytomegalovirus replication: characterization of a potential site of virus assembly. J Virol 74,975-986.
174 Andrew J. Davison,l Aidan Dolan,l Parvis Akter,1 Clare Addison,l Derrick j. Dargan,l Donald J. Alcendor,2 Duncan J. McGeochl and Gary S. Hayward2 (2003).The human cytomegalovirus genome revisited:comparison with the chimpanzee cytomegalovirus genome Journal of General Virology,84,17-28
175 Mocarski and Courcelle in Field's Virology,4th ed., "Cytomegaloviruses and Their Replication" (6/27/2000
176 Udo B,Gholamreza D(2001).Analysis and Characterization of the Complete Genome of Tupaia (Tree Shrew) Herpesvirus.. Journal of Virology, p.4854-4870
177 Chambers J, Angulo A, Amaratunga D,etal.(1999) DNA Microarrays of the Complex Human Cytomegalovirus Genome:Profiling Kinetic Class with Drug Sensitivity of Viral Gene Expression. Journal of Virology, p.5757-5766.
178 Murphy E, Rigoutsos I, Shibuya T, etal.(2003).Reevaluation of human cytomegalovirus coding potential.(?).PNAS vol.100 (23) 13585-13590
179 Dunn W, Chou C, Li H, etal (2003).Functional profiling of a human cytomegalovirus genome.PNAS,vol.100(24) 14223-14228.
180 Frohman M A, Dush M K, Martin G R. Rapid production of full-length cDNAs from rare amplificatio nusing a single gene-specific oligo nucleotide primer. Proc. Natl. Acad. Sci. USA., 1988;85:8998-9002
181潘鸿春,宋大祥,周开亚.悦目金蛛丝腺SMART RACE cDNA文库的构建与鉴定.蛛形学报.2005,14(2)：65-69
182 Krogh A,Larsson B,Heijne G,E.LL.Sonnhammer2001.Predicting transmembrane protein topology with a hidden Markov model:Application to complete genomes[J].Journal of Molecular Biology,305(3):567-580.
183 Kyte JJ, Doolittle RF1982.A simple method for displaying the hydrophobic character of a protein[J].J.Mol.Biol,,157:105-132.
184 Janin J.1979.Surface and inside volumes in globular proteins[J].Nature,277:491-492.
185 Zimmerman JM,Eliezer N, Simha R.1968.The characteriza-tion of amino acid sequences in proteins by statistical methods[J].J Theor Biol,21:170-201.
186 Bhaskaran.R,Ponnuswamy.PK.1988.Positional flexibilities of amino acid residues in globular proteins[J].Int. J.Pept Prot. Res.,32:242-255.
187吴玉章,朱锡华.一种病毒蛋白B细胞表位预测方法的建立[J].科学通报,1994,39(24)：2275～22279.
188万涛,孙涛,吴家金.蛋白顺序性抗原决定簇的多参数综合预测[J].中国免疫学杂志,1997,13(6)：329～333.
189 Patrone M, Percivalle E, Secchi M,, etal 5(2003).The human cytomegalovirus UL45 gene product is a late, virion-associated protein and influences virus growth at low multiplicities of infection..Journal of General Virology,84,3359-3370
190 Landini, M. P.,Ripalti A..1982. A DNA-nicking activity associated with the nucleocapsid of human cytomegalovirus. Arch. Virol.73:351-356.
191 Patrone M, Percivalle E, Secchi, etal.(2003).The human cytomegalovirus UL45 gene product is a late, virion-associated protein and influences virus growth at low multiplicities of infection. Journal of General Virology,84,3359-3370
192 Adair R,Elaine R. Douglas,Jean B, etal (2002).The products of human cytomegalovirus genes UL49、 UL24,UL43 and US22 are tegument components. Journal of General Virology,83 1315-1324.
193 Baker A, Cotton M.1997.Methods of performing homologous recombination based modification of nucleic acids in recombination deficient cells and use of the modified nucleic acid products thereof[J]. Nucleic Acids Research,25:1950-1965
194 Borst E M, Ulrich G, Koszinowski, et al.1999.Cloning of the Human Cytomegalovirus (HCMV) Genome as an Infectious Bacterial Artificial Chromosome in Escherichia coli:a New Approach for Construction of HCMV Mutants[J]. JOURNAL OF VIROLOG Y,73(10):8320-8329
195 Jones, T R. Muzithras V P.1992.A cluster of dispensable genes within the human cytomegalovirus genome short component:IRS1, US1 through US5, and the US6 family[J].J Virol,66:2541-2546.
196 Dunn W, Chou C, Li H, et al.2003.Functional profiling of a human cytomegalovirus genome[J]. PNAS November,100 (24):14223-14228.
197 Hensel G M, Meyer H H., Buchmann I, et al.1996.Intracellular localization and expression of the human cytomegalovirus matrix phosphoprotein pp71 (ppUL82):Evidence for its translocation into the nucleus[J]. J Gen. Virol,77:3087-3097.
198 Scholz M, Doerr H W, Cinatl J.2001.Inhibition of cytomegalovirus immediate early gene expression:a therapeutic option? [J]. Antiviral Res,49:129-145.
199 Irmiere, A., and W. Gibson.1983. Isolation and characterization of a noninfectious virion-like particle released from cells infected with human strains of cytomegalovirus. Virology 130:118-133.
200 Revello, M. G., E. Percivalle, A. Di Matteo, F. Morini, and G. Gerna.1992.Nuclear expression of the lower matrix protein of human cytomegalovirus in peripheral blood leukocytes of immunocompromised viraemic patients.J. Gen. Virol.73:437-442.
201 Yao, Z. Q., G. Gallez-Hawkins, N. A. Lomeli, X. Li, K. M. Molinder, D. J.Diamond, and J. A. Zaia.2001. Site-directed mutation in a conserved kinase domain of human cytomegalovirus-pp65 with preservation of cytotoxic T lymphocyte targeting. Vaccine 19:1628-1635.
202 Gallina, A., L. Simoncini, S. Garbelli, E. Percivalle, G. Pedrali-Noy, K. S.Lee, R. L. Erikson, B. Plachter, G. Gerna, and G. Milaneshi.1999. Polo-like kinase 1 as a target for human cytomegalovirus pp65 lower matrix protein.J. Virol.73:1468-1478.
203 Schmolke, S., H. F. Kern, P. Drescher, G. Jahn, and B. Plachter.1995. The dominant phosphoprotein pp65 (UL83) of human cytomegalovirus is dispensable for growth in cell culture. J. Virol.69:5959-5968.
204 Wills, M. R., A. J. Carmichael, K. Mynard, X. Jin, M. P. Weeks, B. Plachter,and J. G. P. Sissons.1996. The human cytotoxic T-lymphocyte (CTL)response to cytomegalovirus is dominated by structural protein pp65:frequency,specificity, and T-cell receptor usage of pp65-specific CTL. J. Virol.70:7569-7579.
205 Yu, D., M. C. Silva, and T. Shenk.2003. Functional map of human cytomegalovirus AD169 defined by global mutagenesis analysis. Proc. Natl.Acad. Sci. USA 100:12396-12401.
206 Baxter, M. K., and W. Gibson.2001. Cytomegalovirus basic phosphoprotein (pUL32) binds to capsids in vitro through its amino one-third. J. Virol.75:6865-6873.
207 AuCoin, D. P., G. B. Smith, C. D. Meiering, and E. S. Mocarski. 2006.Betaherpesvirus-conserved cytomegalovirus tegument protein ppUL32(pp150) controls cytoplasmic events during virion maturation. J. Virol.80:8199-8210.
208 Bechtel, J. T., and T. Shenk.2002. Human cytomegalovirus UL47 tegument protein functions after entry and before immediate-early gene expression.J. Virol.76:1043-1050
209 Trgovcich, J., C. Cebulla, P. Zimmerman, and D. D. Sedmak.2006. Human cytomegalovirus protein pp71 disrupts major histocompatibility complex class Ⅰ cell surface expression. J. Virol. 80:951-963.
210 Cantrell, S. R., and W. A. Bresnahan.2006. Human cytomegalovirus (HCMV) UL82 gene product (pp71) relieves hDaxx-mediated repression of HCMV replication. J. Virol.80:6188-6191.
211 Saffert, R. T., and R. F. Kalejta.2006. Inactivating a cellular intrinsic immune defense mediated by Daxx is the mechanism through which the human cytomegalovirus pp71 protein stimulates viral immediate early gene expression. J. Virol.80:3863-3871.
212 Terhune, S. S., J. Schroer, and T. Shenk.2004. RNAs are packaged into human cytomegalovirus virions in proportion to their intracellular concentration.J. Virol.78:10390-10398.
213 Maiti S, Doskow J, Li S, Nhim RP, Lindsey JS,Wilkinson MF. The Pem homeobox gene.Androgen-dependent and-independent promoters and tissue-specific alternative RNAsplicing [J]. J Biol Chem,1996,271(29):17536-46.
214罗星,张峪涵,于娜,等.神经突起因子酵母双杂交诱饵质粒构建和转化[J].中国公共卫生,2006,22(4)：444-445.
© 2004-2018 中国地质图书馆版权所有 京ICP备05064691号 京公网安备11010802017129号 地址：北京市海淀区学院路29号 邮编：100083 电话：办公室：(+86 10)66554848；文献借阅、咨询服务、科技查新：66554700 |