Ⅱ组冠状病毒非结构蛋白1内保守序列LLRKxGxKG的功能研究
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摘要
冠状病毒可引起人和动物的多种疾病,其中人类冠状病毒可引起10%-30%的普通感冒,亦可引起如SARS等严重传染病。我国是冠状病毒的主要分布地之一,因此开展冠状病毒致病机制的研究对于提高我国应对此类疾病的防控能力具有重要意义。
但目前出于生物安全方面的考虑,针对SARS-CoV致病机制的研究受到了严重的制约。在这种情况下,与SARS亲缘关系较近的鼠肝炎冠状病毒(Mouse Hepatitis Virus,MHV)便成为较为理想的研究对象。这是因为:第一,鼠肝炎冠状病毒(Mouse Hepatitis Virus,MHV)与SARS冠状病毒(SARS-CoV)同属于Ⅱ组冠状病毒,两者的亲缘关系较近;第二,MHV是Ⅱ组冠状病毒的代表株,它具有良好的动物模型;第三,MHV对人不致病,易于开展实验研究,其研究成果对人类疾病也更具有指导意义。
研究表明,MHV和SARS-CoV的非结构蛋白1(Non-Structural Protein 1,NSP1)具有抑制宿主抗病毒反应的功能,是冠状病毒的重要致病因子。并且通过配对分析发现,在MHV和SARS-CoV NSP1中存在着一段高度保守的氨基酸序列-LLRKxGxKG。该序列可能是NSP1的重要功能域,与MHV NSP1和SARS-CoV NSP1共同具有的某些功能有关。这值得我们进行深入的研究。
因此,本研究以MHV为研究模式株,从Ⅱ组冠状病毒MHV与SARS-CoV NSP1内高度保守的氨基酸序列-LLRKxGxKG入手,对该保守序列进行缺失突变,分别构建了MHV NSP1突变基因及MHV突变体。通过检测LLRKxGxKG序列缺失前后NSP1蛋白的功能变化,并分析MHV突变体的复制能力,以及对小鼠的致病力和组织嗜性等方面的变化,从分子、细胞和动物三级水平上揭示Ⅱ组冠状病毒NSP1及其保守序列LLRKxGxKG的功能。以期加深我们对SARS-CoV等Ⅱ组冠状病毒致病机制的了解,并为抗病毒药物的设计和新型减毒疫苗的研制提供参考。
一、鼠肝炎冠状病毒非结构蛋白1及其保守序列LLRKxGxKG的体外功能研究
首先,我们分别构建了MHV NSP1的真核表达质粒(pcDNA3.1-NSP1)及LLRKxGxKG序列缺失的突变NSP1真核表达质粒(pcDNA3.1-NSP1-mu)。为检测MHV NSP1对IFN-β生成的影响,我们分别用pcDNA3.1-NSP1和pcDNA3.1-NSP1-mu转染L929细胞,利用新城疫病毒(new-castle disease virus,NDV)刺激诱导后,检测细胞上清中IFN-β的表达水平。结果显示,瞬时表达MHV NSP1或缺失突变NSP1分别使IFN-β的表达水平下降了12.8%和18.4%,两者之间的差异无统计学意义。这表明MHV NSP1对IFN-β的生成具有一定的抑制作用,但LLRKxGxKG序列与此功能无关。
为进一步分析MHV NSP1对IFN-β信号通路的影响,我们用报告质粒(-110- IFNβ)-CAT和ISRE-Luc分别与真核表达质粒pcDNA3.1-NSP1或pcDNA3.1-NSP1-mu进行共转染。通过检测CAT和Luc报告基因表达水平,分析MHV NSP1及保守序列LLRKxGxKG对IFN-β启动子和IFN刺激应答元件活性的影响。结果发现,在进行刺激诱导的情况下,与空载体对照组相比,pcDNA3.1-NSP1和pcDNA3.1-NSP1-mu转染组的报告基因表达水平均显著下降(P<0.01),且两组间差异无统计学意义(P>0.05)。这表明,MHV NSP1可显著抑制IFN-β启动子和IFN刺激应答元件的活性,并且该抑制作用不受LLRKxGxKG序列缺失的影响。
上述实验中我们偶然发现,在不进行刺激诱导的情况下,LLRKxGxKG序列缺失前后MHV NSP1对报告基因的抑制作用有显著变化。根据文献报道,SARS-CoV NSP1具有抑制宿主基因表达的功能。为检验MHV NSP1是否具有相同功能,我们另外选择了三种报告质粒(pRLuc-CMV、pGL3-basic和pGL3-control)分别与pcDNA3.1-NSP1或pcDNA3.1-NSP1-mu共转染,并检测报告基因的表达水平。结果发现,与空载体对照组相比,pcDNA3.1-NSP1和pcDNA3.1-NSP1-mu转染组的报告基因表达水平均显著下降(P<0.01),但当LLRKxGxKG序列缺失后,该抑制作用显著降低(P<0.01)。该结果表明,MHV NSP1具有抑制共转染基因表达的功能,LLRKxGxKG序列在此过程中发挥重要作用。这提示我们,LLRKxGxKG序列是MHV NSP1的重要功能域,它对宿主基因的表达可能具有同样的抑制作用,值得进行深入研究。
二、LLRKxGxKG序列缺失的鼠肝炎冠状病毒突变体的拯救
为深入了解MHV NSP1内保守序列LLRKxGxKG与病毒致病性的关系,我们利用以痘苗病毒为载体的MHV反向遗传学系统,构建了LLRKxGxKG序列缺失的MHV突变体,以便对LLRKxGxKG序列的功能进行研究。
首先,利用含鼠肝炎冠状病毒基因组全长cDNA的重组痘苗病毒(Vaccinia Virus-inf-1(V.V.-inf-1)介导的同源重组,以大肠杆菌gpt(guanine-phosphoribosyl transferase)基因作为双向筛选标记,通过GPT-in和GPT-out两轮重组,用NSP1突变基因替换掉原MHV基因组的NSP1基因,经过筛选、纯化,获得含突变MHV基因组的重组痘苗病毒(V.V.-MHV-NSP1 mu)。然后,扩增V.V.-MHV-NSP1 mu并提取病毒DNA,经酶切获得突变的MHV基因组全长cDNA,并以此为模板通过体外转录,得到突变的MHV基因组全长RNA,进而转染BHK-21细胞。经对转染后得到重组MHV的鉴定,证明成功地拯救到了LLRKxGxKG序列缺失的MHV突变体,命名为MHV-NSP1⊿ 27。通过测定病毒生长曲线发现,MHV-NSP1⊿ 27在体外仍可高效复制,其病毒滴度可达8×106PFU/ml,略低于野生型MHV(2×107PFU/ml)。
三、LLRKxGxKG序列缺失的鼠肝炎冠状病毒突变体的毒力分析
为进一步了解LLRKxGxKG序列与病毒致病性的关系,我们选择了3-4周龄雄性C57BL/6小鼠,分别开展野生型MHV与LLRKxGxKG序列缺失的MHV突变体的感染实验。结果显示,腹腔感染(5×106PFU)时,野生型MHV的致死率为87.5%,而MHV-NSP1⊿ 27的致死率为37.5%;脑内感染(2×104PFU)时,野生型MHV的致死率为100%,而MHV-NSP1⊿ 27对小鼠完全不致死。由此得出结论:LLRKxGxKG序列缺失后,MHV突变体对C57BL/6小鼠的致病力大大降低。经过检测发现,MHV-NSP1⊿ 27感染后小鼠肝和脾的病毒滴度显著低于野生型MHV ,并且MHV-NSP1⊿ 27很快就被机体清除,2天后已检测不到病毒。同时,感染小鼠的血清ALT检测结果显示,MHV-NSP1⊿ 27致小鼠肝脏损伤的程度远远低于野生型MHV。攻毒实验结果表明,MHV-NSP1⊿ 27保留了较好的免疫原性,对野生型MHV感染具有良好的保护效果。
本研究表明,LLRKxGxKG序列是MHV的一个重要毒力位点,该序列缺失后,MHV突变体对小鼠的致病力显著降低,同时可有效诱导机体产生免疫保护,因而LLRKxGxKG序列缺失的冠状病毒突变体可作为一个很好的减毒疫苗候选株。鉴于MHV与SARS-CoV NSP1内LLRKxGxKG序列的高度保守性,本研究结果对于SARS-CoV等Ⅱ组冠状病毒致病机制的研究以及减毒疫苗的研制都具有重要的参考价值。但目前,LLRKxGxKG序列作用的具体机制尚不清楚,仍需进一步的研究。
另外,本研究应用以痘苗病毒为载体的冠状病毒反向遗传学系统,在国内首次成功拯救得到了重组冠状病毒。这为我们开展冠状病毒的相关研究提供了良好的技术平台,同时该系统对于其他病毒反向遗传学系统的构建也具有重要的借鉴意义。
Coronavirus can cause severe disease in livestock animals and lead thereby to high economic losses. In humans, almost 10%-30% mild respiratory tract disease (common cold) is caused by coronavirus infections. In 2002-2003, the appearance of severe acute respiratory syndrome (SARS) in our country,caused by a formerly unknown coronavirus (SARS-CoV), exemplified the potential of coronaviruss to seriously affect human health. Therefore, it is of the utmost importance to reveal the pathogenic mechanism of coronavirus.
But SARS-CoV related study is severely restricted for bio-security reasons. Under this circumstances, mouse hepatitis virus A59 (MHV A59) is the most convenient and valuable type strain for study. That’s because first, both MHV and SARS-CoV belong to groupⅡcoronavirus. And they are homologous to each other. So the study of MHV can give us important indications for SARS. Second, MHV is harmless to human beings. And there is well developed animal model for MHV study. So it is easy to carry out the research.
Some reports showed that in MHV and SARS-CoV, non-structural protein 1 (NSP1) is an important pathogenic factor and it can suppress the production of IFN-β. Sequence alignment revealed that there is a highly conserved region (LLRKxGxKG) in MHV and SARS-CoV NSP1. It may be an important functional domain, and plays a key role in some identical functions between MHV NSP1 and SARS-CoV NSP1. It deserves a further study. For this goal, we constructed MHV NSP1 mutant gene with LLRKxGxKG region deletion, and also rescued mutant MHV. By detecting the changes of IFN-βregulation function, replication capacity, virulence and tissue tropism, we hope to characterize the function of the conserved LLRKxGxKG region in NSP1 of coronavirus groupⅡ.
1. In vitro functional study of MHV NSP1 and conserved region LLRKxGxKG
In order to reveal the effect of MHV NSP1 and the LLRKxGxKG region on IFN-βproduction, we constructed the eukaryotic plasmids pcDNA3.1-NSP1 which can express MHV NSP1 and pcDNA3.1-NSP1-mu which can express the LLRKxGxKG deleted NSP1. Then they were transfected into L929 cells. After stimulation, the expression level of IFN-βin the culture supernatant was measured by ELISA. Results showed that MHV NSP1 can reduce IFN-βexpression by 12.8%, and the mutant one by 18.4%. While the LLRKxGxKG region seems to have no effect on this.
To further investigate the effect of MHV NSP1 on IFN signal pathways, two reporter plasmids (-110-IFNβ)-CAT and ISRE-Luc, respectively, were used to co-transfect with the eukaryotic expressing plasmids. The activity of IFN-βpromoter and Interferon Stimulated Response Element (ISRE) were determined by CAT ELISA assay and Luciferase assay. Results showed that MHV NSP1 can inhibit the activity of IFN-βpromoter and ISRE dramatically (P<0.01). But it makes no difference whether the LLRKxGxKG region was deleted or not.
In former experiments, we also found by accident that without stimulation the inhibiting capacity decreased dramatically after the deletion of the LLRKxGxKG region. And someone else found that SARS-CoV NSP1 can suppress host gene expression. We speculate that MHV NSP1 may have the same function. So in another set of experiments, some other reporter plasmids (pRLuc-CMV、pGL3-basic and pGL3-control) were used to co-transfect with the eukaryotic expressing plasmid. The expressing levels of reporter genes were examined. Results showed that MHV NSP1 can suppress reporter genes expression dramatically (P<0.01). And the suppression decreased dramatically (P<0.01) after the deletion of the LLRKxGxKG region. Our results suggest that MHV NSP1 can suppress host gene expression, and the conserved region LLRKxGxKG plays an important role in this process.
2. Rescue of MHV mutant with LLRKxGxKG region deleted
To deeply understand the relationship between NSP1 conserved region LLRKxGxKG and viral pathogenicity, we used the MHV reverse genetic system to rescue MHV mutant with this region deleted.
The E. coli guanine-phosphoribosyl transferase gene (gpt) was used as both a positive and a negative selection marker. By vaccinia virus-mediated homologous recombination, we replaced NSP1 gene in MHV genome by the mutant one. After GPT selection and plaque purification, we got the vaccinia virus which contains the mutant MHV genome. Then the genomic vMHV-inf-1 DNA was prepared from this virus stocks and used as a template to transcribe, in vitro, a capped RNA corresponding to the MHV genome with bacteriophage T7 RNA polymerase. When this RNA was transfected into BHK-21 cells by lipofection, cytopathic effects indicative of mouse hepatitis virus infection developed throughout the culture after 24-48 hours. A virus, designated MHV-NSP1⊿ 27, was rescued from the culture supernatant, and then was plaque purified and identified. And viral growth and peak titers of MHV-NSP1⊿ 27 in 17CL-1cells were almost the same with that of wide type MHV (MHV wt).
3. Pathogenicity analysis of MHV mutant with LLRKxGxKG region deleted
Our study revealed MHV-NSP1⊿ 27 is trongly attenuated in vivo. Infections with a high dose (5×106pfu, intraperitoneal) of wide type MHV may result in 87.5% fatality. While for MHV-NSP1⊿ 27 the fatility was 37.5%. And mice infected intracranialy with 2×104pfu of MHV wt succumbed to infection. Whereas mice infected with 2×104pfu of MHV-NSP1⊿ 27 all survived. Both MHV-NSP1⊿ 27 and MHV wt could replicate in spleen and liver, whereby MHV-NSP1⊿ 27 titers were consistently lower than MHV wt. Furthermore, MHV-NSP1⊿2 7 was rapidly cleared and not detectable after day 2 p.i.. And liver enzyme values of mice infected with MHV-NSP1⊿ 27 were much lower than that infected with MHV wt. Finaly, MHV-NSP1⊿ 27 can elicit strong immune response and protect mice from homologous viral infections. So it is a good candidate of live attenuated vaccine.
The most remarkable finding of this study is the level of attenuation of the nsp1 mutant MHV. This finding reveals that the conserved region LLRKxGxKG is an important pathogenic site. And MHV mutant reserved good immunogenicity,so it provides good evidence for the study of SARS-CoV pathogenic mechanism and attenuated vaccine. But it needs a further study on the details of LLRKxGxKG region’s function.
And it’s the first time that the recombinant coronavirus was successfully rescued using vaccinia virus vector reverse genetic system in China. This provides us a convenient platform for coronavirus research. And it can be used to some other fields. The use of vaccinia virus vectors also enlight us on the development of reverse genetics of other viruses.
但目前出于生物安全方面的考虑,针对SARS-CoV致病机制的研究受到了严重的制约。在这种情况下,与SARS亲缘关系较近的鼠肝炎冠状病毒(Mouse Hepatitis Virus,MHV)便成为较为理想的研究对象。这是因为:第一,鼠肝炎冠状病毒(Mouse Hepatitis Virus,MHV)与SARS冠状病毒(SARS-CoV)同属于Ⅱ组冠状病毒,两者的亲缘关系较近;第二,MHV是Ⅱ组冠状病毒的代表株,它具有良好的动物模型;第三,MHV对人不致病,易于开展实验研究,其研究成果对人类疾病也更具有指导意义。
研究表明,MHV和SARS-CoV的非结构蛋白1(Non-Structural Protein 1,NSP1)具有抑制宿主抗病毒反应的功能,是冠状病毒的重要致病因子。并且通过配对分析发现,在MHV和SARS-CoV NSP1中存在着一段高度保守的氨基酸序列-LLRKxGxKG。该序列可能是NSP1的重要功能域,与MHV NSP1和SARS-CoV NSP1共同具有的某些功能有关。这值得我们进行深入的研究。
因此,本研究以MHV为研究模式株,从Ⅱ组冠状病毒MHV与SARS-CoV NSP1内高度保守的氨基酸序列-LLRKxGxKG入手,对该保守序列进行缺失突变,分别构建了MHV NSP1突变基因及MHV突变体。通过检测LLRKxGxKG序列缺失前后NSP1蛋白的功能变化,并分析MHV突变体的复制能力,以及对小鼠的致病力和组织嗜性等方面的变化,从分子、细胞和动物三级水平上揭示Ⅱ组冠状病毒NSP1及其保守序列LLRKxGxKG的功能。以期加深我们对SARS-CoV等Ⅱ组冠状病毒致病机制的了解,并为抗病毒药物的设计和新型减毒疫苗的研制提供参考。
一、鼠肝炎冠状病毒非结构蛋白1及其保守序列LLRKxGxKG的体外功能研究
首先,我们分别构建了MHV NSP1的真核表达质粒(pcDNA3.1-NSP1)及LLRKxGxKG序列缺失的突变NSP1真核表达质粒(pcDNA3.1-NSP1-mu)。为检测MHV NSP1对IFN-β生成的影响,我们分别用pcDNA3.1-NSP1和pcDNA3.1-NSP1-mu转染L929细胞,利用新城疫病毒(new-castle disease virus,NDV)刺激诱导后,检测细胞上清中IFN-β的表达水平。结果显示,瞬时表达MHV NSP1或缺失突变NSP1分别使IFN-β的表达水平下降了12.8%和18.4%,两者之间的差异无统计学意义。这表明MHV NSP1对IFN-β的生成具有一定的抑制作用,但LLRKxGxKG序列与此功能无关。
为进一步分析MHV NSP1对IFN-β信号通路的影响,我们用报告质粒(-110- IFNβ)-CAT和ISRE-Luc分别与真核表达质粒pcDNA3.1-NSP1或pcDNA3.1-NSP1-mu进行共转染。通过检测CAT和Luc报告基因表达水平,分析MHV NSP1及保守序列LLRKxGxKG对IFN-β启动子和IFN刺激应答元件活性的影响。结果发现,在进行刺激诱导的情况下,与空载体对照组相比,pcDNA3.1-NSP1和pcDNA3.1-NSP1-mu转染组的报告基因表达水平均显著下降(P<0.01),且两组间差异无统计学意义(P>0.05)。这表明,MHV NSP1可显著抑制IFN-β启动子和IFN刺激应答元件的活性,并且该抑制作用不受LLRKxGxKG序列缺失的影响。
上述实验中我们偶然发现,在不进行刺激诱导的情况下,LLRKxGxKG序列缺失前后MHV NSP1对报告基因的抑制作用有显著变化。根据文献报道,SARS-CoV NSP1具有抑制宿主基因表达的功能。为检验MHV NSP1是否具有相同功能,我们另外选择了三种报告质粒(pRLuc-CMV、pGL3-basic和pGL3-control)分别与pcDNA3.1-NSP1或pcDNA3.1-NSP1-mu共转染,并检测报告基因的表达水平。结果发现,与空载体对照组相比,pcDNA3.1-NSP1和pcDNA3.1-NSP1-mu转染组的报告基因表达水平均显著下降(P<0.01),但当LLRKxGxKG序列缺失后,该抑制作用显著降低(P<0.01)。该结果表明,MHV NSP1具有抑制共转染基因表达的功能,LLRKxGxKG序列在此过程中发挥重要作用。这提示我们,LLRKxGxKG序列是MHV NSP1的重要功能域,它对宿主基因的表达可能具有同样的抑制作用,值得进行深入研究。
二、LLRKxGxKG序列缺失的鼠肝炎冠状病毒突变体的拯救
为深入了解MHV NSP1内保守序列LLRKxGxKG与病毒致病性的关系,我们利用以痘苗病毒为载体的MHV反向遗传学系统,构建了LLRKxGxKG序列缺失的MHV突变体,以便对LLRKxGxKG序列的功能进行研究。
首先,利用含鼠肝炎冠状病毒基因组全长cDNA的重组痘苗病毒(Vaccinia Virus-inf-1(V.V.-inf-1)介导的同源重组,以大肠杆菌gpt(guanine-phosphoribosyl transferase)基因作为双向筛选标记,通过GPT-in和GPT-out两轮重组,用NSP1突变基因替换掉原MHV基因组的NSP1基因,经过筛选、纯化,获得含突变MHV基因组的重组痘苗病毒(V.V.-MHV-NSP1 mu)。然后,扩增V.V.-MHV-NSP1 mu并提取病毒DNA,经酶切获得突变的MHV基因组全长cDNA,并以此为模板通过体外转录,得到突变的MHV基因组全长RNA,进而转染BHK-21细胞。经对转染后得到重组MHV的鉴定,证明成功地拯救到了LLRKxGxKG序列缺失的MHV突变体,命名为MHV-NSP1⊿ 27。通过测定病毒生长曲线发现,MHV-NSP1⊿ 27在体外仍可高效复制,其病毒滴度可达8×106PFU/ml,略低于野生型MHV(2×107PFU/ml)。
三、LLRKxGxKG序列缺失的鼠肝炎冠状病毒突变体的毒力分析
为进一步了解LLRKxGxKG序列与病毒致病性的关系,我们选择了3-4周龄雄性C57BL/6小鼠,分别开展野生型MHV与LLRKxGxKG序列缺失的MHV突变体的感染实验。结果显示,腹腔感染(5×106PFU)时,野生型MHV的致死率为87.5%,而MHV-NSP1⊿ 27的致死率为37.5%;脑内感染(2×104PFU)时,野生型MHV的致死率为100%,而MHV-NSP1⊿ 27对小鼠完全不致死。由此得出结论:LLRKxGxKG序列缺失后,MHV突变体对C57BL/6小鼠的致病力大大降低。经过检测发现,MHV-NSP1⊿ 27感染后小鼠肝和脾的病毒滴度显著低于野生型MHV ,并且MHV-NSP1⊿ 27很快就被机体清除,2天后已检测不到病毒。同时,感染小鼠的血清ALT检测结果显示,MHV-NSP1⊿ 27致小鼠肝脏损伤的程度远远低于野生型MHV。攻毒实验结果表明,MHV-NSP1⊿ 27保留了较好的免疫原性,对野生型MHV感染具有良好的保护效果。
本研究表明,LLRKxGxKG序列是MHV的一个重要毒力位点,该序列缺失后,MHV突变体对小鼠的致病力显著降低,同时可有效诱导机体产生免疫保护,因而LLRKxGxKG序列缺失的冠状病毒突变体可作为一个很好的减毒疫苗候选株。鉴于MHV与SARS-CoV NSP1内LLRKxGxKG序列的高度保守性,本研究结果对于SARS-CoV等Ⅱ组冠状病毒致病机制的研究以及减毒疫苗的研制都具有重要的参考价值。但目前,LLRKxGxKG序列作用的具体机制尚不清楚,仍需进一步的研究。
另外,本研究应用以痘苗病毒为载体的冠状病毒反向遗传学系统,在国内首次成功拯救得到了重组冠状病毒。这为我们开展冠状病毒的相关研究提供了良好的技术平台,同时该系统对于其他病毒反向遗传学系统的构建也具有重要的借鉴意义。
Coronavirus can cause severe disease in livestock animals and lead thereby to high economic losses. In humans, almost 10%-30% mild respiratory tract disease (common cold) is caused by coronavirus infections. In 2002-2003, the appearance of severe acute respiratory syndrome (SARS) in our country,caused by a formerly unknown coronavirus (SARS-CoV), exemplified the potential of coronaviruss to seriously affect human health. Therefore, it is of the utmost importance to reveal the pathogenic mechanism of coronavirus.
But SARS-CoV related study is severely restricted for bio-security reasons. Under this circumstances, mouse hepatitis virus A59 (MHV A59) is the most convenient and valuable type strain for study. That’s because first, both MHV and SARS-CoV belong to groupⅡcoronavirus. And they are homologous to each other. So the study of MHV can give us important indications for SARS. Second, MHV is harmless to human beings. And there is well developed animal model for MHV study. So it is easy to carry out the research.
Some reports showed that in MHV and SARS-CoV, non-structural protein 1 (NSP1) is an important pathogenic factor and it can suppress the production of IFN-β. Sequence alignment revealed that there is a highly conserved region (LLRKxGxKG) in MHV and SARS-CoV NSP1. It may be an important functional domain, and plays a key role in some identical functions between MHV NSP1 and SARS-CoV NSP1. It deserves a further study. For this goal, we constructed MHV NSP1 mutant gene with LLRKxGxKG region deletion, and also rescued mutant MHV. By detecting the changes of IFN-βregulation function, replication capacity, virulence and tissue tropism, we hope to characterize the function of the conserved LLRKxGxKG region in NSP1 of coronavirus groupⅡ.
1. In vitro functional study of MHV NSP1 and conserved region LLRKxGxKG
In order to reveal the effect of MHV NSP1 and the LLRKxGxKG region on IFN-βproduction, we constructed the eukaryotic plasmids pcDNA3.1-NSP1 which can express MHV NSP1 and pcDNA3.1-NSP1-mu which can express the LLRKxGxKG deleted NSP1. Then they were transfected into L929 cells. After stimulation, the expression level of IFN-βin the culture supernatant was measured by ELISA. Results showed that MHV NSP1 can reduce IFN-βexpression by 12.8%, and the mutant one by 18.4%. While the LLRKxGxKG region seems to have no effect on this.
To further investigate the effect of MHV NSP1 on IFN signal pathways, two reporter plasmids (-110-IFNβ)-CAT and ISRE-Luc, respectively, were used to co-transfect with the eukaryotic expressing plasmids. The activity of IFN-βpromoter and Interferon Stimulated Response Element (ISRE) were determined by CAT ELISA assay and Luciferase assay. Results showed that MHV NSP1 can inhibit the activity of IFN-βpromoter and ISRE dramatically (P<0.01). But it makes no difference whether the LLRKxGxKG region was deleted or not.
In former experiments, we also found by accident that without stimulation the inhibiting capacity decreased dramatically after the deletion of the LLRKxGxKG region. And someone else found that SARS-CoV NSP1 can suppress host gene expression. We speculate that MHV NSP1 may have the same function. So in another set of experiments, some other reporter plasmids (pRLuc-CMV、pGL3-basic and pGL3-control) were used to co-transfect with the eukaryotic expressing plasmid. The expressing levels of reporter genes were examined. Results showed that MHV NSP1 can suppress reporter genes expression dramatically (P<0.01). And the suppression decreased dramatically (P<0.01) after the deletion of the LLRKxGxKG region. Our results suggest that MHV NSP1 can suppress host gene expression, and the conserved region LLRKxGxKG plays an important role in this process.
2. Rescue of MHV mutant with LLRKxGxKG region deleted
To deeply understand the relationship between NSP1 conserved region LLRKxGxKG and viral pathogenicity, we used the MHV reverse genetic system to rescue MHV mutant with this region deleted.
The E. coli guanine-phosphoribosyl transferase gene (gpt) was used as both a positive and a negative selection marker. By vaccinia virus-mediated homologous recombination, we replaced NSP1 gene in MHV genome by the mutant one. After GPT selection and plaque purification, we got the vaccinia virus which contains the mutant MHV genome. Then the genomic vMHV-inf-1 DNA was prepared from this virus stocks and used as a template to transcribe, in vitro, a capped RNA corresponding to the MHV genome with bacteriophage T7 RNA polymerase. When this RNA was transfected into BHK-21 cells by lipofection, cytopathic effects indicative of mouse hepatitis virus infection developed throughout the culture after 24-48 hours. A virus, designated MHV-NSP1⊿ 27, was rescued from the culture supernatant, and then was plaque purified and identified. And viral growth and peak titers of MHV-NSP1⊿ 27 in 17CL-1cells were almost the same with that of wide type MHV (MHV wt).
3. Pathogenicity analysis of MHV mutant with LLRKxGxKG region deleted
Our study revealed MHV-NSP1⊿ 27 is trongly attenuated in vivo. Infections with a high dose (5×106pfu, intraperitoneal) of wide type MHV may result in 87.5% fatality. While for MHV-NSP1⊿ 27 the fatility was 37.5%. And mice infected intracranialy with 2×104pfu of MHV wt succumbed to infection. Whereas mice infected with 2×104pfu of MHV-NSP1⊿ 27 all survived. Both MHV-NSP1⊿ 27 and MHV wt could replicate in spleen and liver, whereby MHV-NSP1⊿ 27 titers were consistently lower than MHV wt. Furthermore, MHV-NSP1⊿2 7 was rapidly cleared and not detectable after day 2 p.i.. And liver enzyme values of mice infected with MHV-NSP1⊿ 27 were much lower than that infected with MHV wt. Finaly, MHV-NSP1⊿ 27 can elicit strong immune response and protect mice from homologous viral infections. So it is a good candidate of live attenuated vaccine.
The most remarkable finding of this study is the level of attenuation of the nsp1 mutant MHV. This finding reveals that the conserved region LLRKxGxKG is an important pathogenic site. And MHV mutant reserved good immunogenicity,so it provides good evidence for the study of SARS-CoV pathogenic mechanism and attenuated vaccine. But it needs a further study on the details of LLRKxGxKG region’s function.
And it’s the first time that the recombinant coronavirus was successfully rescued using vaccinia virus vector reverse genetic system in China. This provides us a convenient platform for coronavirus research. And it can be used to some other fields. The use of vaccinia virus vectors also enlight us on the development of reverse genetics of other viruses.
引文
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[1] Roth-Cross JK, Martínez-Sobrido L, Scott EP, et al. Inhibition of the alpha/beta interferon response by mouse hepatitis virus at multiple levels[J]. J Virol, 2007, 81(13): 7189-7199.
[2] Wathelet MG, Orr M, Frieman MB, et al. Severe Acute Respiratory Syndrome Coronavirus EvadesAntiviral Signaling: Role of NSP1 and Rational Design of an Attenuated Strain[J]. J Virol, 2007, 81(21): 11620–11633
[3] Kopecky-Bromberg SA, Martínez-Sobrido L, Frieman M, et al. SARS coronavirus proteins Orf 3b, Orf 6, and nucleocapsid function as interferon antagonists[J]. J Virol, 2007, 81(2):548–557.
[4] Almeida MS, Johnson MA, Herrmann T, et al. Novel -Barrel Fold in the Nuclear Magnetic Resonance Structure of the Replicase Nonstructural Protein 1 from the Severe Acute Respiratory Syndrome Coronavirus[J]. J Virol, 2007, 81(7): 3151–3161
[5] Roth-Cross JK, Stokes H, Chang G, et al. Organ-specific attenuation of murine hepatitis virus strain A59 by replacement of catalytic residues in the putative viral cyclic phosphodiesterase ns2[J]. J Virol, 2009, 83(8): 3743-3753.
[6]秦成峰,赵慧,秦鄂德等.维甲酸诱导基因Ⅰ信号通路在西尼罗病毒感染中的作用研究[J].解放军医学杂志,2009,34(6):719
[7] Perry AK, Chen G, Zheng D, et al. The host type I interferon response to viral and bacterial infections[J]. Cell Res. 2005,15(6):407–422.
[8] Kamitani W, Huang C, Narayanan K, et al. A two-pronged strategy to suppress host protein synthesis by SARS coronavirus Nsp1 protein[J]. Nat Struct Mol Biol. 2009, 16 (11) : 1134-1140
[9] Kamitani W, Narayanan K, Huang C, et al. Severe acute respiratory syndrome coronavirus NSP1 protein suppresses host gene expression by promoting host mRNA degradation[J]. Proc Natl Acad Sci USA.2006, 103(34):12885–12890
[10] Law AH, Lee DC, Cheung BK,et al. Role for Nonstructural Protein 1 of Severe Acute Respiratory Syndrome Coronavirus in Chemokine Dysregulation[J]. J Virol, 2007, 81(1):416–422
[11] Chen CJ, Sugiyama K, Kubo H, et al. Murine Coronavirus Nonstructural Protein p28 Arrests Cell Cycle in G0/G1 Phase[J].J Virol, 2004, 78(19): 10410–10419
[12] Kr?hling V, Stein DA, Spiegel M, et al. Severe Acute Respiratory Syndrome Coronavirus Triggers Apoptosis via Protein Kinase R but Is Resistant to Its Antiviral Activity[J]. J Virol, 2009, 83(5):2298–2309
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[2] Stadler K, Masignani V, Eickmann M, et al. SARS-beginning to understand a new virus. Nat Rev Microbiol 2003,1: 209–218
[3] Zhu qingyu, Qin E’de, Wang Cui’e, et al. Isolation and Identification of a Novel Coronavirus from Patients with SARS. China Biotechnology 2003, 4(23):106-112
[4] Sarah M. Brockway, Mark R. Denison. Mutagenesis of the murine hepatitis virus NSP1-coding region identifies residues important for protein processing, viral RNA synthesis, and viral replication. Virology 2005, 340: 209– 223
[5] Siddell SG, Ziebuhr J, Snijder EJ. Coronaviruses, toroviruses, and arteriviruses. In: Mahy BWJ, ter Meulen V, editors. 2005 Virology. 10th ed. London: Hodder Arnold. pp. 823–855
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[1] Roth-Cross JK, Martínez-Sobrido L, Scott EP, et al. Inhibition of the alpha/beta interferon response by mouse hepatitis virus at multiple levels[J]. J Virol, 2007, 81(13): 7189-7199.
[2] Wathelet MG, Orr M, Frieman MB, et al. Severe Acute Respiratory Syndrome Coronavirus EvadesAntiviral Signaling: Role of NSP1 and Rational Design of an Attenuated Strain[J]. J Virol, 2007, 81(21): 11620–11633
[3] Kopecky-Bromberg SA, Martínez-Sobrido L, Frieman M, et al. SARS coronavirus proteins Orf 3b, Orf 6, and nucleocapsid function as interferon antagonists[J]. J Virol, 2007, 81(2):548–557.
[4] Almeida MS, Johnson MA, Herrmann T, et al. Novel -Barrel Fold in the Nuclear Magnetic Resonance Structure of the Replicase Nonstructural Protein 1 from the Severe Acute Respiratory Syndrome Coronavirus[J]. J Virol, 2007, 81(7): 3151–3161
[5] Roth-Cross JK, Stokes H, Chang G, et al. Organ-specific attenuation of murine hepatitis virus strain A59 by replacement of catalytic residues in the putative viral cyclic phosphodiesterase ns2[J]. J Virol, 2009, 83(8): 3743-3753.
[6]秦成峰,赵慧,秦鄂德等.维甲酸诱导基因Ⅰ信号通路在西尼罗病毒感染中的作用研究[J].解放军医学杂志,2009,34(6):719
[7] Perry AK, Chen G, Zheng D, et al. The host type I interferon response to viral and bacterial infections[J]. Cell Res. 2005,15(6):407–422.
[8] Kamitani W, Huang C, Narayanan K, et al. A two-pronged strategy to suppress host protein synthesis by SARS coronavirus Nsp1 protein[J]. Nat Struct Mol Biol. 2009, 16 (11) : 1134-1140
[9] Kamitani W, Narayanan K, Huang C, et al. Severe acute respiratory syndrome coronavirus NSP1 protein suppresses host gene expression by promoting host mRNA degradation[J]. Proc Natl Acad Sci USA.2006, 103(34):12885–12890
[10] Law AH, Lee DC, Cheung BK,et al. Role for Nonstructural Protein 1 of Severe Acute Respiratory Syndrome Coronavirus in Chemokine Dysregulation[J]. J Virol, 2007, 81(1):416–422
[11] Chen CJ, Sugiyama K, Kubo H, et al. Murine Coronavirus Nonstructural Protein p28 Arrests Cell Cycle in G0/G1 Phase[J].J Virol, 2004, 78(19): 10410–10419
[12] Kr?hling V, Stein DA, Spiegel M, et al. Severe Acute Respiratory Syndrome Coronavirus Triggers Apoptosis via Protein Kinase R but Is Resistant to Its Antiviral Activity[J]. J Virol, 2009, 83(5):2298–2309
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