N-糖基化位点在流感病毒（H5N1）中的协同进化及对宿主特异性影响
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摘要
流感病毒所造成的人员伤亡和经济损失在世界范围内提高了对其的关注程度,过去半个世纪所积累的相关研究资料为我们对抗流感病毒提供有力武器。流感病毒基因组结构相对简单但变异速率非常快,每年都需要更新相应的预防疫苗,这主要与病毒囊膜表面的两种糖蛋白,血凝素(Hemagglutinin, HA)与神经氨酸酶(Neuraminidase, NA)密切相关。这两种糖蛋白在识别、进入宿主细胞和增殖扩散中扮演了重要的生物学角色。目前研究显示流感病毒发生的糖基化仅为N-糖基化类型。为了研究N-糖基化在流感病毒糖蛋白中的分布规律,位点变化以及不同结构特征的N-糖链对病毒囊膜糖蛋白功能和结合效率影响,本研究采用生物信息学方法展开讨论。
在第一部分研究中,本课题收集并比对已报道的17种HA亚型与10种NA亚型氨基酸序列,通过分析糖基化位点的出现位置与保守率发现随着样本量的增加或跨种间传播的产生,该亚型序列集会产生较多的糖基化位点。例如只含有禽流感H4亚型或H6亚型病毒HA的糖基化位点远远少于含有季节性人流感H1N1或H3N2病毒的相关序列集,而随着294NSS糖基化位点的出现某些H3N2病毒的HA中甚至出现了多达11个糖基化位点数目。本研究根据保守性和出现位置推断流感病毒糖蛋白中存在两类糖基化位点：一类在各亚型序列中具有相同位置且高度保守,另一个则存在于各亚型内部且保守率不一。位于HA0裂解位点附近(如H1序列中的27NNST或H8序列中30NGT)与HA2融膜肽附近的(如H1序列集中的498NGT或H5序列集中500NGS)糖基化位点在各亚型中高度保守,除参与宿主免疫系统逃避外还发挥着保护与调节HA0裂解位点与融膜肽等病毒基本生物学功能。此外位于HA1序列C-端即HA的头茎结合部还存在一个除H7和H15亚型外在各亚型中高度保守的糖基化位点。每种亚型中还存在有3-10个不等的糖基化位点,它们受到了所处不同进化枝的影响,保守率从0.1%(如H3亚型中的294NSS(0.15%))到100%不等。值得一提某些高度保守的糖基化位点距离各亚型高度保守的半胱氨酸残基较为接近,这可能与在稳定新生蛋白折叠时二硫键形成需要N-糖链引导的钙联蛋白／钙网蛋白复合体作用相关。NA茎部通常出现2至4个高度保守的糖化位点,其它保守率不一的糖基化位点通常位于NA头部的各种抗原位点附近。位于NA四聚体头部顶端的146N糖基化位点的在各NA亚型中高度保守,有研究显示其与病毒神经毒力密切相关。如同对HA的描述,由于大量记录的H1N1、H3N2、H5N1、H7N2以及H9N2病毒存在导致了N1与N2序列集中出现了多样的糖基化位点,尤其位于头部区域,它们主要参与了免疫逃避。NA的糖基化位点数目受到茎部特异性序列缺失的影响。茎部序列的缺失有可能降低NA的酶活性并降低对临近未感染细胞表面唾液酸糖链的切除能力从而增强子代病毒对临近细胞的感染。除N4、N8与N9外,每种NA亚型都存在3至24个氨基酸与相应1至4个不等的糖基化位点的缺失。有意思的即使亚型内部特异性序列缺失还与与不同HA组合病毒来源有关。本节结果提供了全面而直观的糖基化位点在流感病毒两种糖蛋白各亚型之间的分布及相互关系。
本课题第二部分切入H5N1病毒,研究糖基化位点在进化过程中发生于糖结合蛋白的HA与外切糖苷酶的NA中的动态平衡关系。根据序列比对以及统计分析发现其HA与NA各含有12个糖基化位点,而最近五年中HA中糖基化位点逐渐增多并复杂化,而在NA中的影响逐渐降低。在2003年香港再次出现高致病性禽流感致人死亡前,大多数H5N1病毒均属于clade0,HA与NA中不同糖基化位点模式共同存在。这个时期NA包含除极偶然出现的341NGT位点外位于茎部四个和头部七个所有目前已知糖基化位点,而HA则除五个高度保守的糖基化位点外还出现了170N与181N两个位点。从2003年开始世卫组织记录到由三波H5N1病毒爆发造成的数百人死亡和巨大的经济损失,但是截至目前在除clade7外所有只感染禽的H5N1病毒进化枝(即clade3、4、5、6、8和9)中其糖基化模式高度保守。2005年H5N1病毒中clade2.2进化枝在青海湖附近造成上百只候鸟死亡后向西传播并导致欧洲等地部分禽类死亡。2006年中,H5N1病毒首次出现在非洲大陆,继而导致上百人死亡与禽鸟的大量捕杀。目前埃及等地最新出现的clade2.2.1.1进化枝中其HA新增一个糖基化位点：88NVS。除clade2.2.1.1外大多数位于clade2.2中病毒普遍由于T172A的突变缺失170N糖基化位点。据猜测170N的缺失可能会增强H5N1病毒HA对人呼吸道中常见唾液酸糖链的亲和力,相比该进化枝中NA糖基化位点迅速减少仅残留3个。然而目前从中国和越南等地clade2.3.4进化枝中病毒的糖基化位点模式在HA中保守,在NA中较为多变。相反另一大常见进化枝clade2.3.2中以170N的缺失和NA中糖基化位点保守为特征。181N糖基化位点位于HA顶部β-折叠,在所有的人感染病毒中保守禽感染病毒中保守率较低。事实上,所有的181N糖基化位点缺失毒株仅从禽类宿主中分离得到,如clade7.1、7.2或2.2.1.1。这样的结论为研究糖基化位点与宿主特异性的关系提供了启示。
在第三部分中本课题通过三维模型构建和分子动力学模拟优化研究糖基化HA与唾液酸糖链的动力学变化特征。HA结合到宿主唾液酸糖链受体是感染过程的首要步骤,不同亚型中结合唾液酸糖链的受体结合域序列高度保守。而170N与181N位点均位于受体结合域附近,也可能参与或影响了受体识别过程。为了探索糖基化HA与SA-α-2,3-Gal/SA-α-2,6-Gal糖链的结合能力,本研究通过对不同程度糖基化HA分子进行16ns分子动力学模拟并采用多种动力学参数分析,发现N-糖链末端单糖残基具有极高的RMSF (root-mean-square fluctuation),意味着N-糖链末端极为活跃。作为具有复杂构象且高分子量的亲水性分子,N-糖链与蛋白中仅由一个共价键相连,这说明N-糖链的存在将影响HA受体结合能力。通过比较唾液酸糖链50ns水溶液分子动力学模拟并间隔5ns进行构象采样将其唾液酸残基进行叠加,发现两类唾液酸具有不同的拓扑学构象,即SA-α-2,3-Gal唾液酸糖链其余部分向左而SA-α-2,6-Gal唾液酸糖链其余部分向右。经过优化的HA和唾液酸糖链体系为随后进行的分子对接试验提供了研究材料。
第四部分中,本课题通过分子对接与三聚体HA-唾液酸糖链复合体分子动力学模拟为判断N-糖链在受体识别过程中的影响提供了直观的结论：RBD附近的170N与181N位点的N-糖链所影响范围极大而相应的唾液酸糖链则主要局限于受体结合域附近。在自由或结合状态下都可以了解SA-α-2,3-Gal唾液酸糖链具有线状朝向HA外侧的拓扑学构象,而SA-α-2,6-Gal唾液酸糖链则出现类似鱼钩状的弯曲并朝向Helix200。利用在模拟过程中的容积分析法可以看出两类唾液酸糖链独特的拓扑构象与HA中不同位点的N-糖链相互影响从而造成HA对不同唾液酸糖链的结合差异：181N糖基化位点的缺失有利于结合线状且朝外的SA-α-2,3-Gal唾液酸糖链,而170N糖基化位点的缺失则利于识别鱼钩状且朝向HA轴心的SA-α-2,6-Gal唾液酸糖链。而当糖基化程度更加复杂时,适应HA受体识别域的唾液酸糖链配体构象将更加受到限制。
本课题的实验方法和结论还可以推广至流感病毒其它亚型HA受体结合域附近类似糖基化位点的研究。因此本实验结论有助于对疫苗设计、防控病毒扩散和病毒毒力与糖基化位点关系的进一步研究。
The experimental results and the datum accumulated in the past50years have provided the powerful tools to investage the dreadful influenza virus (IV). The flu vaccines must be reformulated each year to counter the constantly mutation in IVs, especially the mutations in the envelop glycoproteins. Two envelope glycoproteins, hemagglutinin (HA) and neuraminidase (NA), on the surface of IVs play crucial roles in transfaunation, membrane fusion and the release of progeny virions.
In the first part, our research collected and aligned the amino acid sequences of all the HA and NA subtypes. By analyzing the position of N-glycosylation sites (glycosites) and their conservative rates, one most obvious characteristic of glycosites was that the increasing samples or cross-species reports would lead to more glycosites. For example, the HAs from avian H4or H6subtype has less glycosites than those of the seasonal human H1N1or H3N2viruses and the occurrence of294NSS has even increased the glycosite numbers to11in parts of current HA of H3N2viruses. We conclude that two types of glycosites appear in HAs:one with a high level of conservation in all HA subtypes and another with various conservative rates in different HA subtypes. Two highly conserved glycosites are located near the HAO cleavage site (e.g., the27NNST in H1or the30NGT in H8) and the fusion peptide of the HA2(e.g., the498NGT in H1or500NGS in H5) respectively in all subtypes, and these two glycosites play necessary role in viral life cycle for protecting the HAO cleavage sites and fusion peptide. In addition, another highly conserved glycosite appears at C-terminal part of the HA1sequence, which is near the connection of the global and stalk domains, except for the H7and H15subtypes. Three to ten characteristic glycosites were distinctive in each subtype. Their conservative rates were influenced by different internal evolution branches, ranked from0.1%to100%, distributed mainly in the global domain. It is worth mentioning that some highly conserved glycosites were near to cysteines, which indicated that the formation of disulfide bonds depend on a complex from calreticulin/calnexin and the precursor N-glycans. Two to four highly conserved glycosites are located in the NA stalk domain in each subtype; two conserved glycosites and most middle-low conserved glycosites are mainly located in the global domain, which are near the tip of NA, the connection of the global and stalk domains, or the antigenic sites. The glycosite of146N is conservative in all NA subtypes. It has been shown that the N-glycan at this glycosite affects NA enzymatic activity. Similar to the description of HAs, a large number of H1N1, H3N2, H5N1, H7N2and H9N2viruses have accumulated numerous glycosites in N1and N2, especially in the global domain, mainly participating in immune evasion. The significance of the conserved glycosites in the stalk domain was providing the N-glycans to avoid the cleavage by host enzyme. The variance of the glycosites was closely related to the deletion of the stalk domain. Although the three-dimensional structure of stalk domain has not been determined yet, it is speculated that the presence of an a-helix motif in the uncrystallized structure has also been provided by cryoelectron microscopy. It believed that a longer stalked domain would enhance the replication capacity of the virus, while the deletion of the stem domain would decrease the enzymatic activity of NA. Various subtypes had stalk domain deletions of3to24residues, except for N4, N8and N9. The numbers of deletions were also distinctive across different combinations of IVs and even within one subtype. This part provides the comprehensive results of the distributed regularity of glycosites in all HA and NA subtypes.
Our research has further focused on the H5N1virus and explored the distribution of glycosites in HA and NA during the natural course of viral evolution in the second part. By analyzing the glycosites and statistical analysis, we found both12glycosites in HA and NA and concluded that the glycosites have become more complicated in HA and less influential in NA in the last five years. Most H5N1viruses were grouped into clade0before it appeared in Hong Kong again in2003. Various patterns of the glycosites in HA and NA had co-existed in these original viruses. These original NAs contained the known glycosites, including four highly conserved glycosites in the stalk domain and seven in the global domain (except the occasional341NGT). Since2003, the WHO has recorded a three-wave epidemic of H5N1, which resulted in hundreds of deaths and huge economic losses. Until recently, the glycosite patterns were highly conserved in all the avian clades except for clade7(e.g., clade3,4,5,6,8and9). During this decade, there have been increasing human infections and new glycosite patterns of HA and NA. It has been reported that the H5N1virus of clade2.2was involved in the outbreak that occurred among the migratory bird population near Qinghai Lake in2005. Since then clade2.2spread westward. This resulted in a number of deaths of wild birds in Europe. In2006, the H5N1virus appeared in Africa for the first time, followed by hundreds of mortally infected humans in Egypt, Nigeria and Djibouti. The most recent H5N1virus isolated from North Africa belongs to clade2.2.1.1, along with one novel glycosite88NVS. In addition, most viruses in clade2.2, except for clade2.2.1.1, lack the170N glycosite created by the T172A mutation. It is guessed that the lack of170N would enhance the HA affinity for SA sialoglycans, especially to human sialoglycans, which could be one reason for the propensity of clade2.2.1to infect humans, compared with only three conserved glycosites in the NA of clade2.2.1.1. Parts of the H5N1virus isolated from China and Vietnam belong to clade2.3.4, with conserved glycosites in HA, but not in NA. In contrast, another dominant virus from clade2.3.2featured the loss of170NST in HA and stable glycosites in NA. The181N glycosite, which is located at the apical β-folding of HA, is conserved in all human IVs and unconserved in avian IVs. Actually, all the viruses that had deficiencies of the181N glycosite were only isolated from the avian host, which were concentrated in clades7.1,7.2and2.2.1.1. It indicated that the possible relationship between the170N/181N glycosites and the host specificity.
Our research has constructed a series of HAs and sialoglycans to investigate the dynamics properties during the molecular dynamic simulation in the third part. The binding of HA on the viral surface to host sialoglycans is a requisite step in the infection process. Protein sequence alignment in various subtypes indicates the high conservation in the receptor-binding domains (RBDs) is essential for binding with the sialoglycans. The170N and181N located to the RBD, which may also participate in assisting receptor recognition. To further explore the binding affinity between glycosylated HAs and SA (sialic acid)-a-2,3-Gal (galactose)/SA-a-2,6-Gal sialoglycans, further computational methods were adopted. During the16ns Molecular Dynamic simulations, the energetic and structural properties were monitored for HA systems. The higher RMSFs (root-mean-square fluctuation) in the distal monosaccharide residues reveal their active fluctuation in N-glycans. As a type of hydrophilic long-chain and high-mass molecule, N-glycans have only one covalent bond to the Asn, which discloses that the glycans are rather mobile and flexible. Undoubtedly, the presence of huge N-glycans would impact the binding efficiency of the RBD. The superimpositions of SA residues in sialoglycans during50ns explicitly solvated MD simulation reflect the free SA-a-2,3-Gal and SA-a-2,6-Gal sialoglycans adopted two distinctive topologies and the remainder of all SA-a-2,3-Gal receptors are leftward, compare to the rightward SA-a-2,6-Gal receptors, which result in two orientations. The optimized HAs and sialoglycans prepared the docked materials for next part.
The docking analysis and the complementary MD simulation of trimerical docking complexes provided the visual methods to judge the influence of N-glycans in receptor recognition in the fourth part:three N-glycans on170N and181N were swinging dramatically near one RBD while the sialoglycans constrained in the RBD, especially for the SA residues. Straight and extrorse topologies were observed in the free and docked SA-a-2,3-Gal sialoglycans. Bound SA-a-2,6-Gal sialoglycans adopted tightly folded fishhook conformations and bented toward the Helix200. These two distinctive conformations resulted in different preference of contaction to the N-glycans on170N or181N. Obviously, the loss of the181N glycosite has an advantage in binding the straight and extrorse SA-a-2,3-Gal sialoglycans, whereas the loss of the170N glycosite facilitates the recognition of the fishhook-like and ental SA-a-2,6-Gal sialoglycans. With more complicated glycosylation, the reasonable sialoglycans for docking would be more restricted.
Our conclusion may provide the same description in similar glycosites of different HA subtypes and constructive suggestions for the research into the glycoylation of IVs as well as the design of surveillance and the production of viral vaccines.
在第一部分研究中,本课题收集并比对已报道的17种HA亚型与10种NA亚型氨基酸序列,通过分析糖基化位点的出现位置与保守率发现随着样本量的增加或跨种间传播的产生,该亚型序列集会产生较多的糖基化位点。例如只含有禽流感H4亚型或H6亚型病毒HA的糖基化位点远远少于含有季节性人流感H1N1或H3N2病毒的相关序列集,而随着294NSS糖基化位点的出现某些H3N2病毒的HA中甚至出现了多达11个糖基化位点数目。本研究根据保守性和出现位置推断流感病毒糖蛋白中存在两类糖基化位点：一类在各亚型序列中具有相同位置且高度保守,另一个则存在于各亚型内部且保守率不一。位于HA0裂解位点附近(如H1序列中的27NNST或H8序列中30NGT)与HA2融膜肽附近的(如H1序列集中的498NGT或H5序列集中500NGS)糖基化位点在各亚型中高度保守,除参与宿主免疫系统逃避外还发挥着保护与调节HA0裂解位点与融膜肽等病毒基本生物学功能。此外位于HA1序列C-端即HA的头茎结合部还存在一个除H7和H15亚型外在各亚型中高度保守的糖基化位点。每种亚型中还存在有3-10个不等的糖基化位点,它们受到了所处不同进化枝的影响,保守率从0.1%(如H3亚型中的294NSS(0.15%))到100%不等。值得一提某些高度保守的糖基化位点距离各亚型高度保守的半胱氨酸残基较为接近,这可能与在稳定新生蛋白折叠时二硫键形成需要N-糖链引导的钙联蛋白／钙网蛋白复合体作用相关。NA茎部通常出现2至4个高度保守的糖化位点,其它保守率不一的糖基化位点通常位于NA头部的各种抗原位点附近。位于NA四聚体头部顶端的146N糖基化位点的在各NA亚型中高度保守,有研究显示其与病毒神经毒力密切相关。如同对HA的描述,由于大量记录的H1N1、H3N2、H5N1、H7N2以及H9N2病毒存在导致了N1与N2序列集中出现了多样的糖基化位点,尤其位于头部区域,它们主要参与了免疫逃避。NA的糖基化位点数目受到茎部特异性序列缺失的影响。茎部序列的缺失有可能降低NA的酶活性并降低对临近未感染细胞表面唾液酸糖链的切除能力从而增强子代病毒对临近细胞的感染。除N4、N8与N9外,每种NA亚型都存在3至24个氨基酸与相应1至4个不等的糖基化位点的缺失。有意思的即使亚型内部特异性序列缺失还与与不同HA组合病毒来源有关。本节结果提供了全面而直观的糖基化位点在流感病毒两种糖蛋白各亚型之间的分布及相互关系。
本课题第二部分切入H5N1病毒,研究糖基化位点在进化过程中发生于糖结合蛋白的HA与外切糖苷酶的NA中的动态平衡关系。根据序列比对以及统计分析发现其HA与NA各含有12个糖基化位点,而最近五年中HA中糖基化位点逐渐增多并复杂化,而在NA中的影响逐渐降低。在2003年香港再次出现高致病性禽流感致人死亡前,大多数H5N1病毒均属于clade0,HA与NA中不同糖基化位点模式共同存在。这个时期NA包含除极偶然出现的341NGT位点外位于茎部四个和头部七个所有目前已知糖基化位点,而HA则除五个高度保守的糖基化位点外还出现了170N与181N两个位点。从2003年开始世卫组织记录到由三波H5N1病毒爆发造成的数百人死亡和巨大的经济损失,但是截至目前在除clade7外所有只感染禽的H5N1病毒进化枝(即clade3、4、5、6、8和9)中其糖基化模式高度保守。2005年H5N1病毒中clade2.2进化枝在青海湖附近造成上百只候鸟死亡后向西传播并导致欧洲等地部分禽类死亡。2006年中,H5N1病毒首次出现在非洲大陆,继而导致上百人死亡与禽鸟的大量捕杀。目前埃及等地最新出现的clade2.2.1.1进化枝中其HA新增一个糖基化位点：88NVS。除clade2.2.1.1外大多数位于clade2.2中病毒普遍由于T172A的突变缺失170N糖基化位点。据猜测170N的缺失可能会增强H5N1病毒HA对人呼吸道中常见唾液酸糖链的亲和力,相比该进化枝中NA糖基化位点迅速减少仅残留3个。然而目前从中国和越南等地clade2.3.4进化枝中病毒的糖基化位点模式在HA中保守,在NA中较为多变。相反另一大常见进化枝clade2.3.2中以170N的缺失和NA中糖基化位点保守为特征。181N糖基化位点位于HA顶部β-折叠,在所有的人感染病毒中保守禽感染病毒中保守率较低。事实上,所有的181N糖基化位点缺失毒株仅从禽类宿主中分离得到,如clade7.1、7.2或2.2.1.1。这样的结论为研究糖基化位点与宿主特异性的关系提供了启示。
在第三部分中本课题通过三维模型构建和分子动力学模拟优化研究糖基化HA与唾液酸糖链的动力学变化特征。HA结合到宿主唾液酸糖链受体是感染过程的首要步骤,不同亚型中结合唾液酸糖链的受体结合域序列高度保守。而170N与181N位点均位于受体结合域附近,也可能参与或影响了受体识别过程。为了探索糖基化HA与SA-α-2,3-Gal/SA-α-2,6-Gal糖链的结合能力,本研究通过对不同程度糖基化HA分子进行16ns分子动力学模拟并采用多种动力学参数分析,发现N-糖链末端单糖残基具有极高的RMSF (root-mean-square fluctuation),意味着N-糖链末端极为活跃。作为具有复杂构象且高分子量的亲水性分子,N-糖链与蛋白中仅由一个共价键相连,这说明N-糖链的存在将影响HA受体结合能力。通过比较唾液酸糖链50ns水溶液分子动力学模拟并间隔5ns进行构象采样将其唾液酸残基进行叠加,发现两类唾液酸具有不同的拓扑学构象,即SA-α-2,3-Gal唾液酸糖链其余部分向左而SA-α-2,6-Gal唾液酸糖链其余部分向右。经过优化的HA和唾液酸糖链体系为随后进行的分子对接试验提供了研究材料。
第四部分中,本课题通过分子对接与三聚体HA-唾液酸糖链复合体分子动力学模拟为判断N-糖链在受体识别过程中的影响提供了直观的结论：RBD附近的170N与181N位点的N-糖链所影响范围极大而相应的唾液酸糖链则主要局限于受体结合域附近。在自由或结合状态下都可以了解SA-α-2,3-Gal唾液酸糖链具有线状朝向HA外侧的拓扑学构象,而SA-α-2,6-Gal唾液酸糖链则出现类似鱼钩状的弯曲并朝向Helix200。利用在模拟过程中的容积分析法可以看出两类唾液酸糖链独特的拓扑构象与HA中不同位点的N-糖链相互影响从而造成HA对不同唾液酸糖链的结合差异：181N糖基化位点的缺失有利于结合线状且朝外的SA-α-2,3-Gal唾液酸糖链,而170N糖基化位点的缺失则利于识别鱼钩状且朝向HA轴心的SA-α-2,6-Gal唾液酸糖链。而当糖基化程度更加复杂时,适应HA受体识别域的唾液酸糖链配体构象将更加受到限制。
本课题的实验方法和结论还可以推广至流感病毒其它亚型HA受体结合域附近类似糖基化位点的研究。因此本实验结论有助于对疫苗设计、防控病毒扩散和病毒毒力与糖基化位点关系的进一步研究。
The experimental results and the datum accumulated in the past50years have provided the powerful tools to investage the dreadful influenza virus (IV). The flu vaccines must be reformulated each year to counter the constantly mutation in IVs, especially the mutations in the envelop glycoproteins. Two envelope glycoproteins, hemagglutinin (HA) and neuraminidase (NA), on the surface of IVs play crucial roles in transfaunation, membrane fusion and the release of progeny virions.
In the first part, our research collected and aligned the amino acid sequences of all the HA and NA subtypes. By analyzing the position of N-glycosylation sites (glycosites) and their conservative rates, one most obvious characteristic of glycosites was that the increasing samples or cross-species reports would lead to more glycosites. For example, the HAs from avian H4or H6subtype has less glycosites than those of the seasonal human H1N1or H3N2viruses and the occurrence of294NSS has even increased the glycosite numbers to11in parts of current HA of H3N2viruses. We conclude that two types of glycosites appear in HAs:one with a high level of conservation in all HA subtypes and another with various conservative rates in different HA subtypes. Two highly conserved glycosites are located near the HAO cleavage site (e.g., the27NNST in H1or the30NGT in H8) and the fusion peptide of the HA2(e.g., the498NGT in H1or500NGS in H5) respectively in all subtypes, and these two glycosites play necessary role in viral life cycle for protecting the HAO cleavage sites and fusion peptide. In addition, another highly conserved glycosite appears at C-terminal part of the HA1sequence, which is near the connection of the global and stalk domains, except for the H7and H15subtypes. Three to ten characteristic glycosites were distinctive in each subtype. Their conservative rates were influenced by different internal evolution branches, ranked from0.1%to100%, distributed mainly in the global domain. It is worth mentioning that some highly conserved glycosites were near to cysteines, which indicated that the formation of disulfide bonds depend on a complex from calreticulin/calnexin and the precursor N-glycans. Two to four highly conserved glycosites are located in the NA stalk domain in each subtype; two conserved glycosites and most middle-low conserved glycosites are mainly located in the global domain, which are near the tip of NA, the connection of the global and stalk domains, or the antigenic sites. The glycosite of146N is conservative in all NA subtypes. It has been shown that the N-glycan at this glycosite affects NA enzymatic activity. Similar to the description of HAs, a large number of H1N1, H3N2, H5N1, H7N2and H9N2viruses have accumulated numerous glycosites in N1and N2, especially in the global domain, mainly participating in immune evasion. The significance of the conserved glycosites in the stalk domain was providing the N-glycans to avoid the cleavage by host enzyme. The variance of the glycosites was closely related to the deletion of the stalk domain. Although the three-dimensional structure of stalk domain has not been determined yet, it is speculated that the presence of an a-helix motif in the uncrystallized structure has also been provided by cryoelectron microscopy. It believed that a longer stalked domain would enhance the replication capacity of the virus, while the deletion of the stem domain would decrease the enzymatic activity of NA. Various subtypes had stalk domain deletions of3to24residues, except for N4, N8and N9. The numbers of deletions were also distinctive across different combinations of IVs and even within one subtype. This part provides the comprehensive results of the distributed regularity of glycosites in all HA and NA subtypes.
Our research has further focused on the H5N1virus and explored the distribution of glycosites in HA and NA during the natural course of viral evolution in the second part. By analyzing the glycosites and statistical analysis, we found both12glycosites in HA and NA and concluded that the glycosites have become more complicated in HA and less influential in NA in the last five years. Most H5N1viruses were grouped into clade0before it appeared in Hong Kong again in2003. Various patterns of the glycosites in HA and NA had co-existed in these original viruses. These original NAs contained the known glycosites, including four highly conserved glycosites in the stalk domain and seven in the global domain (except the occasional341NGT). Since2003, the WHO has recorded a three-wave epidemic of H5N1, which resulted in hundreds of deaths and huge economic losses. Until recently, the glycosite patterns were highly conserved in all the avian clades except for clade7(e.g., clade3,4,5,6,8and9). During this decade, there have been increasing human infections and new glycosite patterns of HA and NA. It has been reported that the H5N1virus of clade2.2was involved in the outbreak that occurred among the migratory bird population near Qinghai Lake in2005. Since then clade2.2spread westward. This resulted in a number of deaths of wild birds in Europe. In2006, the H5N1virus appeared in Africa for the first time, followed by hundreds of mortally infected humans in Egypt, Nigeria and Djibouti. The most recent H5N1virus isolated from North Africa belongs to clade2.2.1.1, along with one novel glycosite88NVS. In addition, most viruses in clade2.2, except for clade2.2.1.1, lack the170N glycosite created by the T172A mutation. It is guessed that the lack of170N would enhance the HA affinity for SA sialoglycans, especially to human sialoglycans, which could be one reason for the propensity of clade2.2.1to infect humans, compared with only three conserved glycosites in the NA of clade2.2.1.1. Parts of the H5N1virus isolated from China and Vietnam belong to clade2.3.4, with conserved glycosites in HA, but not in NA. In contrast, another dominant virus from clade2.3.2featured the loss of170NST in HA and stable glycosites in NA. The181N glycosite, which is located at the apical β-folding of HA, is conserved in all human IVs and unconserved in avian IVs. Actually, all the viruses that had deficiencies of the181N glycosite were only isolated from the avian host, which were concentrated in clades7.1,7.2and2.2.1.1. It indicated that the possible relationship between the170N/181N glycosites and the host specificity.
Our research has constructed a series of HAs and sialoglycans to investigate the dynamics properties during the molecular dynamic simulation in the third part. The binding of HA on the viral surface to host sialoglycans is a requisite step in the infection process. Protein sequence alignment in various subtypes indicates the high conservation in the receptor-binding domains (RBDs) is essential for binding with the sialoglycans. The170N and181N located to the RBD, which may also participate in assisting receptor recognition. To further explore the binding affinity between glycosylated HAs and SA (sialic acid)-a-2,3-Gal (galactose)/SA-a-2,6-Gal sialoglycans, further computational methods were adopted. During the16ns Molecular Dynamic simulations, the energetic and structural properties were monitored for HA systems. The higher RMSFs (root-mean-square fluctuation) in the distal monosaccharide residues reveal their active fluctuation in N-glycans. As a type of hydrophilic long-chain and high-mass molecule, N-glycans have only one covalent bond to the Asn, which discloses that the glycans are rather mobile and flexible. Undoubtedly, the presence of huge N-glycans would impact the binding efficiency of the RBD. The superimpositions of SA residues in sialoglycans during50ns explicitly solvated MD simulation reflect the free SA-a-2,3-Gal and SA-a-2,6-Gal sialoglycans adopted two distinctive topologies and the remainder of all SA-a-2,3-Gal receptors are leftward, compare to the rightward SA-a-2,6-Gal receptors, which result in two orientations. The optimized HAs and sialoglycans prepared the docked materials for next part.
The docking analysis and the complementary MD simulation of trimerical docking complexes provided the visual methods to judge the influence of N-glycans in receptor recognition in the fourth part:three N-glycans on170N and181N were swinging dramatically near one RBD while the sialoglycans constrained in the RBD, especially for the SA residues. Straight and extrorse topologies were observed in the free and docked SA-a-2,3-Gal sialoglycans. Bound SA-a-2,6-Gal sialoglycans adopted tightly folded fishhook conformations and bented toward the Helix200. These two distinctive conformations resulted in different preference of contaction to the N-glycans on170N or181N. Obviously, the loss of the181N glycosite has an advantage in binding the straight and extrorse SA-a-2,3-Gal sialoglycans, whereas the loss of the170N glycosite facilitates the recognition of the fishhook-like and ental SA-a-2,6-Gal sialoglycans. With more complicated glycosylation, the reasonable sialoglycans for docking would be more restricted.
Our conclusion may provide the same description in similar glycosites of different HA subtypes and constructive suggestions for the research into the glycoylation of IVs as well as the design of surveillance and the production of viral vaccines.
引文
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