流感病毒基质蛋白DNA疫苗的免疫保护作用研究

流感病毒基质蛋白(matrix protein,M)在病毒复制和毒力方面有重要作用.编码基质蛋白的M1基因和M2基因胞外域序列是A型流感病毒的保守序列,是研究具有交叉保护能力流感疫苗的候选基因.我们构建了真核表达质粒pCAGGSP7/M1和pCAGGSP7/M2,用质粒DNA免疫小鼠以观察其免疫原性.分别在M1DNA免疫2、3、4、5、6次或M2 DNA免疫4、5、6次7 d后,用致死量同源流感病毒A/PR/8攻击小鼠,通过检测小鼠血清抗体滴度、肺部病毒量和小鼠存活率来观察质粒DNA的保护效果.结果表明,随着免疫次数增加,M1 DNA免疫组在病毒攻击后小鼠存活率增高,而M2 DNA免疫组小鼠攻毒后全部死亡.说明M1 DNA多次免疫后能提供抗流感病毒的部分保护,M2 DNA没有免疫保护作用.     

稳定表达流感病毒基质蛋白2的哺乳动物细胞系的建立

本研究构建了稳定表达甲型流感病毒基质蛋白2(M2)的哺乳动物细胞系。应用PCR方法扩增A/PR/8/34(H1N1)株流感病毒M2基因,将其克隆至真核表达载体pcDNA5/FRT(pDF)上,构建出pDF-M2重组质粒。将鉴定正确的pDF-M2与表达Flp重组酶的pOG44质粒共转染Flp-In-CHO细胞,通过体内同源重组使目的基因整合到宿主细胞染色体上。筛选具有Hygromycin B抗性的重组细胞株命名为CHO-M2。以间接免疫荧光法(IFA)和Western blot法检测M2的表达,共获得15株高表达M2蛋白的重组细胞株。这些重组细胞株在连续培养10代后,PCR方法仍可检测到M2基因的存在,IFA也检测到蛋白的稳定表达。本研究成功获得了稳定表达甲型流感病毒M2的哺乳动物细胞系,为M2蛋白的功能研究和非复制型流感病毒疫苗的研制提供了新的工具。     

Defective Influenza Viral Ribonucleoproteins Cause Interference

Ribonucleoproteins (RNPs) isolated from infectious and defective interfering (DI) influenza virus (WSN) contained three major RNP peaks when analyzed in a glycerol gradient. Peak I RNP was predominant in infectious virus but was greatly reduced in DI virus preparations. Conversely, peak III RNP was elevated in DI virus, suggesting a large increase in DI RNA in this fraction. Labeled [(32)P]RNA was isolated from each RNP region and analyzed by electrophoresis on polyacrylamide gels. Peak I RNP contained primarily the polymerase and some HA genes, peak II contained some HA gene but mostly the NP and NA genes, and peak III contained the M and NS genes. In addition, peak III RNP from DI virus also contained the characteristic DI RNA segments. Interference activity of RNP fractions isolated from infectious and DI virus was tested using infectious center reduction assay. RNP peaks (I, II, and III) from infectious virus did not show any interference activity, whereas the peak III DI RNP caused a reduction in the number of infectious centers as compared to controls. Similar interference was not demonstrable with peak I RNP of DI virus nor with any RNP fractions from infectious virus alone. The interference activity of RNP fractions was RNase sensitive, suggesting that the DI RNA contained in DI RNPs was the interfering agent, and dilution experiments supported the conclusion that a single DI RNP could cause interference. The interfering RNPs were heterogeneous, and the majority migrated slower than viral RNPs containing M and NS genes. These results suggest that DI RNP (or DI RNA) is also responsible for interference in segmented, negative-stranded viruses.     

Proton Channel Activity of Influenza A Virus Matrix Protein 2 Contributes to Autophagy Arrest

Influenza A virus infection can arrest autophagy, as evidenced by autophagosome accumulation in infected cells. Here, we report that this autophagosome accumulation can be inhibited by amantadine, an antiviral proton channel inhibitor, in amantadine-sensitive virus infected cells or cells expressing influenza A virus matrix protein 2 (M2). Thus, M2 proton channel activity plays a role in blocking the fusion of autophagosomes with lysosomes, which might be a key mechanism for arresting autophagy.     

Crystal Structures of Influenza A Virus Matrix Protein M1: Variations on a Theme

Matrix protein 1 (M1) of the influenza A virus plays multiple roles in virion assembly and infection. Interest in the pH dependence of M1''s multiple functions led us to study the effect of subtle pH changes on M1 structure, resulting in the elucidation of a unique low-pH crystal structure of the N^1-165-domain of A/WSN/33 (H1N1) M1 that has never been reported. Although the 2.2 Å crystal structure of M1 N-terminus shows a dimer with the two monomers interacting in a face-to-face fashion at low pH as observed earlier, a 44° rotation of the second monomer has led to a significantly different dimer interface that possibly affects dimer stability. More importantly, while one of the monomers is fully defined, the N-terminal half of the second monomer shows considerable disorder that appears inherent in the protein and is potentially physiologically relevant. Such disorder has not been observed in any other previously reported structure at either low or high pH conditions, despite similar crystallization pH conditions. By comparing our novel N^1-165-domain structure with other low-pH or neutral-pH M1 structures, it appears that M1 can energetically access different monomer and dimer conformations, as well as oligomeric states, with varying degree of similarities. The study reported here provides further insights into M1 oligomerization that may be essential for viral propagation and infectivity.     

Toxicity of Influenza A Virus Matrix Protein 2 for Mammalian Cells is Associated with its Intrinsic Proton-Channeling Activity

Molecules of influenza matrix protein 2 (M2) are organized in tetramers that constitute a well-conserved virion component and also form proton channels in the plasma membrane of infected cells. In this report we demonstrate that influenza M2 protein is cytopathic in vitro for mammalian cells. An M2 point-mutant (M2pm) protein was constructed that contained amino acid changes designed to block the proton channel via introduction of large hydrophobic residues. This mutant was significantly less toxic upon transient transfection in vitro than the wild-type M2 (M2wt). To assess the possible correlation between M2 cytotoxicity and its proton channel activity, we monitored changes in mitochondria membrane potential induced by M2wt and M2pm. M2wt rapidly decreased mitochondria membrane potential reflecting the transmembrane proton gradient, while M2pm was markedly less efficient. Thus, M2 is cytotoxic for mammalian cells, likely via its proton channel activity and may therefore contribute to influenza pathogenesis through this previously unknown mechanism.     

Structural Analysis of Influenza A Virus Matrix Protein M1 and Its Self-Assemblies at Low pH

Influenza A virus matrix protein M1 is one of the most important and abundant proteins in the virus particles broadly involved in essential processes of the viral life cycle. The absence of high-resolution data on the full-length M1 makes the structural investigation of the intact protein particularly important. We employed synchrotron small-angle X-ray scattering (SAXS), analytical ultracentrifugation and atomic force microscopy (AFM) to study the structure of M1 at acidic pH. The low-resolution structural models built from the SAXS data reveal a structurally anisotropic M1 molecule consisting of a compact NM-fragment and an extended and partially flexible C-terminal domain. The M1 monomers co-exist in solution with a small fraction of large clusters that have a layered architecture similar to that observed in the authentic influenza virions. AFM analysis on a lipid-like negatively charged surface reveals that M1 forms ordered stripes correlating well with the clusters observed by SAXS. The free NM-domain is monomeric in acidic solution with the overall structure similar to that observed in previously determined crystal structures. The NM-domain does not spontaneously self assemble supporting the key role of the C-terminus of M1 in the formation of supramolecular structures. Our results suggest that the flexibility of the C-terminus is an essential feature, which may be responsible for the multi-functionality of the entire protein. In particular, this flexibility could allow M1 to structurally organise the viral membrane to maintain the integrity and the shape of the intact influenza virus.     

Time-Resolved Visualisation of Nearly-Native Influenza A Virus Progeny Ribonucleoproteins and Their Individual Components in Live Infected Cells

Influenza viruses are a global health concern because of the permanent threat of novel emerging strains potentially capable of causing pandemics. Viral ribonucleoproteins (vRNPs) containing genomic RNA segments, nucleoprotein oligomers, and the viral polymerase, play a central role in the viral replication cycle. Our knowledge about critical events such as vRNP assembly and interactions with other viral and cellular proteins is poor and could be substantially improved by time lapse imaging of the infected cells. However, such studies are limited by the difficulty to achieve live-cell compatible labeling of active vRNPs. Previously we designed the first unimpaired recombinant influenza WSN-PB2-GFP11 virus allowing fluorescent labeling of the PB2 subunit of the viral polymerase (Avilov et al., J.Virol. 2012). Here, we simultaneously labeled the viral PB2 protein using the above-mentioned strategy, and virus-encoded progeny RNPs through spontaneous incorporation of transiently expressed NP-mCherry fusion proteins during RNP assembly in live infected cells. This dual labeling enabled us to visualize progeny vRNPs throughout the infection cycle and to characterize independently the mobility, oligomerization status and interactions of vRNP components in the nuclei of live infected cells.     

A Second CRM1-Dependent Nuclear Export Signal in the Influenza A Virus NS2 Protein Contributes to the Nuclear Export of Viral Ribonucleoproteins

Influenza A virus NS2 protein, also called nuclear export protein (NEP), is crucial for the nuclear export of viral ribonucleoproteins. However, the molecular mechanisms of NEP mediation in this process remain incompletely understood. A leucine-rich nuclear export signal (NES2) in NEP, located at the predicted N2 helix of the N-terminal domain, was identified in the present study. NES2 was demonstrated to be a transferable NES, with its nuclear export activity depending on the nuclear export receptor chromosome region maintenance 1 (CRM1)-mediated pathway. The interaction between NEP and CRM1 is coordinately regulated by both the previously reported NES (NES1) and now the new NES2. Deletion of the NES1 enhances the interaction between NEP and CRM1, and deletion of the NES1 and NES2 motifs completely abolishes this interaction. Moreover, NES2 interacts with CRM1 in the mammalian two-hybrid system. Mutant viruses containing NES2 alterations generated by reversed genetics exhibit reduced viral growth and delay in the nuclear export of viral ribonucleoproteins (vRNPs). The NES2 motif is highly conserved in the influenza A and B viruses. The results demonstrate that leucine-rich NES2 is involved in the nuclear export of vRNPs and contributes to the understanding of nucleocytoplasmic transport of influenza virus vRNPs.     

Aggregation of Influenza A Virus Nuclear Export Protein

Influenza A virus nuclear export protein (NEP) plays an important role in the viral life cycle. Recombinant NEP proteins containing (His)₆-tag at either N-or C-terminus were obtained by heterologous expression in Escherichia coli cells and their high propensity for aggregation was demonstrated. Dynamic light scattering technique was used to study the kinetics and properties of NEP aggregation in solutions under different conditions (pH, ionic strength, presence of low-molecular-weight additives and organic solvents). Using atomic force microscopy, the predominance of spherical aggregates in all examined NEP preparations was shown, with some amyloid-like structures being observed in the case of NEP-C protein. A number of structure prediction programs were used to identify aggregation-prone regions in the NEP structure. All-atom molecular dynamics simulations indicate a high rate of NEP molecule aggregation and reveal the regions preferentially involved in the intermolecular contacts that are located at the edges of the rod-like protein molecule. Our results suggest that NEP aggregation is determined by different types of interactions and represents an intrinsic property of the protein that appears to be necessary for its functioning in vivo.     

Identification of the Protease-Binding Domain in the N-Terminal Region of Influenza Virus A Matrix Protein M1

A region responsible for protease binding by influenza virus A matrix protein M1 was identified. Trypsin binding was observed with the N-proximal 9-kDa fragment obtained by cleaving M1 with formic acid. The binding was inhibited by monoclonal antibodies (mAb) to region 46–70 of M1 and by an antiserum to region 21–45, whereas mAb to the middle and C-terminal regions had no effect. Thus, the protease-binding domain was mapped to the N-terminal part of M1.     

NS1 Protein of Influenza A Virus Down-Regulates Apoptosis

Wild-type (WT) influenza A/PR/8/34 virus and its variant lacking the NS1 gene (delNS1) have been compared for their ability to mediate apoptosis in cultured cells and chicken embryos. Cell morphology, fragmentation of chromatin DNA, and caspase-dependent cleavage of the viral NP protein have been used as markers for apoptosis. Another marker was caspase cleavage of the viral M2 protein, which was also found to occur in an apoptosis-specific manner. In interferon (IFN)-competent host systems, such as MDCK cells, chicken fibroblasts, and 7-day-old chicken embryos, delNS1 virus induced apoptosis more rapidly and more efficiently than WT virus. As a consequence, delNS1 virus was also more lethal for chicken embryos than WT virus. In IFN-deficient Vero cells, however, apoptosis was delayed and developed with similar intensity after infection with both viruses. Taken together, these data indicate that the IFN antagonistic NS1 protein of influenza A viruses has IFN-dependent antiapoptotic potential.     

流感病毒A非结构蛋白功能的研究进展

流感病毒属正粘病毒科(Orthomyxoviridae),是一种带包膜并且分节段的单负链RNA病毒。根据病毒核衣壳蛋白(Nucleocapsid)和基质蛋白(Matrix,M)抗原性的差异,流感病毒可分为甲、乙、丙3个型。甲型流感病毒呈球形或丝状,其RNA基因组总长度在13.6kb左右,分为8个节段,共编码10个蛋白     

禽流感病毒神经氨酸酶蛋白的研究进展

禽流感病毒在世界范围内广泛流行,给养禽业造成了巨大的经济损失,有效的疫苗接种策略可以帮助预防和控制该疾病.抗原转换和漂移是流感病毒发生变异的两种主要机制.NA蛋白是流感病毒的表面纤突之一属于Ⅱ型糖蛋白,在病毒成熟时发挥神经氨酸酶的作用进而有利于病毒的成熟和释放,并在调控受体结合、病毒出芽等方面发挥着重要的作用.抑制NA蛋白活性具有预防和治疗疾病作用的事实证实NA蛋白是一个抗病毒的有效靶点.此外基于NA蛋白设计流感疫苗,具有免疫保护期更持久和保护范围更广的优势.