Apical budding of a recombinant influenza A virus expressing a hemagglutinin protein with a basolateral localization signal

Influenza virions bud preferentially from the apical plasma membrane of infected epithelial cells, by enveloping viral nucleocapsids located in the cytosol with its viral integral membrane proteins, i.e., hemagglutinin (HA), neuraminidase (NA), and M2 proteins, located at the plasma membrane. Because individually expressed HA, NA, and M2 proteins are targeted to the apical surface of the cell, guided by apical sorting signals in their transmembrane or cytoplasmic domains, it has been proposed that the polarized budding of influenza virions depends on the interaction of nucleocapsids and matrix proteins with the cytoplasmic domains of HA, NA, and/or M2 proteins. Since HA is the major protein component of the viral envelope, its polarized surface delivery may be a major force that drives polarized viral budding. We investigated this hypothesis by infecting MDCK cells with a transfectant influenza virus carrying a mutant form of HA (C560Y) with a basolateral sorting signal in its cytoplasmic domain. C560Y HA was expressed nonpolarly on the surface of infected MDCK cells. Interestingly, viral budding remained apical in C560Y virus-infected cells, and so did the location of NP and M1 proteins at late times of infection. These results are consistent with a model in which apical viral budding is a shared function of various viral components rather than a role of the major viral envelope glycoprotein HA.     

Influenza virus assembly: effect of influenza virus glycoproteins on the membrane association of M1 protein

Influenza virus matrix protein (M1), a critical protein required for virus assembly and budding, is presumed to interact with viral glycoproteins on the outer side and viral ribonucleoprotein on the inner side. However, because of the inherent membrane-binding ability of M1 protein, it has been difficult to demonstrate the specific interaction of M1 protein with hemagglutinin (HA) or neuraminidase (NA), the influenza virus envelope glycoproteins. Using Triton X-100 (TX-100) detergent treatment of membrane fractions and floatation in sucrose gradients, we observed that the membrane-bound M1 protein expressed alone or coexpressed with heterologous Sendai virus F was totally TX-100 soluble but the membrane-bound M1 protein expressed in the presence of HA and NA was predominantly detergent resistant and floated to the top of the density gradient. Furthermore, both the cytoplasmic tail and the transmembrane domain of HA facilitated binding of M1 to detergent-resistant membranes. Analysis of the membrane association of M1 in the early and late phases of the influenza virus infectious cycle revealed that the interaction of M1 with mature glycoproteins which associated with the detergent-resistant lipid rafts was responsible for the detergent resistance of membrane-bound M1. Immunofluorescence analysis by confocal microscopy also demonstrated that, in influenza virus-infected cells, a fraction of M1 protein colocalized with HA and associated with the HA in transit to the plasma membrane via the exocytic pathway. Similar results for colocalization were obtained when M1 and HA were coexpressed and HA transport was blocked by monensin treatment. These studies indicate that both HA and NA interact with influenza virus M1 and that HA associates with M1 via its cytoplasmic tail and transmembrane domain.     

Identification of a hydrophobic domain of HA2 essential to morphogenesis of Helicoverpa armigera nucleopolyhedrovirus

The HA2 protein of the Helicoverpa armigera single-nucleocapsid nucleopolyhedrovirus (HearNPV) is a WASP homology protein capable of nucleating branched actin filaments in the presence of the Arp2/3 complex in vitro. To determine the role of ha2 in the HearNPV life cycle, ha2 knockout and ha2 repair bacmids were constructed. Transfection and infection analysis demonstrated that the ha2 null bacmid was unable to produce infectious budded virus (BV), while the repair bacmid rescued the defect. In vitro analysis demonstrated that the WCA domain of HA2 accelerates Arp2/3-mediated actin assembly and is indispensable to the function of HA2. However, analysis of the repaired recombinant with a series of truncated ha2 mutants demonstrated that the WCA domain was essential but not enough to yield infectious virions, and a hydrophobic domain (H domain) consisting of amino acids (aa) 167 to 193 played a pivotal role in the production of BV. Subcellular localization analysis with enhanced green fluorescent protein fusions showed that the H domain functioned as a nuclear localization signal. In addition, deletion of the C terminus of the ha2 product, a phosphatidylinositol 4-kinase homolog, dramatically decreased the viral titer, while deletion of 128 aa from the N terminus did not affect HA2 function.     

Influenza virus hemagglutinin and neuraminidase glycoproteins stimulate the membrane association of the matrix protein.

We have analyzed the mechanism by which the matrix (M1) protein associates with cellular membranes during influenza A virus assembly. Interaction of the M1 protein with the viral hemagglutinin (HA) or neuraminidase (NA) glycoprotein was extensively analyzed by using wild-type and transfectant influenza viruses as well as recombinant vaccinia viruses expressing the M1 protein, HA, or NA. Membrane binding of the M1 protein was significantly stimulated at the late stage of virus infection. Using recombinant vaccinia viruses, we found that a relatively small fraction (20 to 40%) of the cytoplasmic M1 protein associated with cellular membranes in the absence of other viral proteins, while coexpression of the HA and the NA stimulated membrane binding of the M1 protein. The stimulatory effect of the NA (>90%) was significant and higher than that of the HA (>60%). Introduction of mutations into the cytoplasmic tail of the NA interfered with its stimulatory effect. Meanwhile, the HA may complement the defective NA and facilitate virus assembly in cells infected with the NA/TAIL(-) transfectant. In conclusion, the highly conserved cytoplasmic tails of the HA and NA play an important role in virus assembly.     

Comparative Efficacy of Hemagglutinin,Nucleoprotein, and Matrix 2 Protein Gene-Based Vaccination against H5N1 Influenza in Mouse and Ferret

Efforts to develop a broadly protective vaccine against the highly pathogenic avian influenza A (HPAI) H5N1 virus have focused on highly conserved influenza gene products. The viral nucleoprotein (NP) and ion channel matrix protein (M2) are highly conserved among different strains and various influenza A subtypes. Here, we investigate the relative efficacy of NP and M2 compared to HA in protecting against HPAI H5N1 virus. In mice, previous studies have shown that vaccination with NP and M2 in recombinant DNA and/or adenovirus vectors or with adjuvants confers protection against lethal challenge in the absence of HA. However, we find that the protective efficacy of NP and M2 diminishes as the virulence and dose of the challenge virus are increased. To explore this question in a model relevant to human disease, ferrets were immunized with DNA/rAd5 vaccines encoding NP, M2, HA, NP+M2 or HA+NP+M2. Only HA or HA+NP+M2 vaccination conferred protection against a stringent virus challenge. Therefore, while gene-based vaccination with NP and M2 may provide moderate levels of protection against low challenge doses, it is insufficient to confer protective immunity against high challenge doses of H5N1 in ferrets. These immunogens may require combinatorial vaccination with HA, which confers protection even against very high doses of lethal viral challenge.     

Inhibition of Influenza A Virus Replication by Compounds Interfering with the Fusogenic Function of the Viral Hemagglutinin

Several compounds that specifically inhibited replication of the H1 and H2 subtypes of influenza virus type A were identified by screening a chemical library for antiviral activity. In single-cycle infections, the compounds inhibited virus-specific protein synthesis when added before or immediately after infection but were ineffective when added 30 min later, suggesting that an uncoating step was blocked. Sequencing of hemagglutinin (HA) genes of several independent mutant viruses resistant to the compounds revealed single amino acid changes that clustered in the stem region of the HA trimer in and near the HA2 fusion peptide. One of the compounds, an N-substituted piperidine, could be docked in a pocket in this region by computer-assisted molecular modeling. This compound blocked the fusogenic activity of HA, as evidenced by its inhibition of low-pH-induced cell-cell fusion in infected cell monolayers. An analog which was more effective than the parent compound in inhibiting virus replication was synthesized. It was also more effective in blocking other manifestations of the low-pH-induced conformational change in HA, including virus inactivation, virus-induced hemolysis of erythrocytes, and susceptibility of the HA to proteolytic degradation. Both compounds inhibited viral protein synthesis and replication more effectively in cells infected with a virus mutated in its M2 protein than with wild-type virus. The possible functional relationship between M2 and HA suggested by these results is discussed.     

Involvement of an Arginine Triplet in M1 Matrix Protein Interaction with Membranes and in M1 Recruitment into Virus-Like Particles of the Influenza A(H1N1)pdm09 Virus

The influenza A(H1N1)pdm09 virus caused the first influenza pandemic of the 21st century. In this study, we wanted to decipher the role of conserved basic residues of the viral M1 matrix protein in virus assembly and release. M1 plays many roles in the influenza virus replication cycle. Specifically, it participates in viral particle assembly, can associate with the viral ribonucleoprotein complexes and can bind to the cell plasma membrane and/or the cytoplasmic tail of viral transmembrane proteins. M1 contains an N-terminal domain of 164 amino acids with two basic domains: the nuclear localization signal on helix 6 and an arginine triplet (R76/77/78) on helix 5. To investigate the role of these two M1 basic domains in influenza A(H1N1)pdm09 virus molecular assembly, we analyzed M1 attachment to membranes, virus-like particle (VLP) production and virus infectivity. In vitro, M1 binding to large unilamellar vesicles (LUVs), which contain negatively charged lipids, decreased significantly when the M1 R76/77/78 motif was mutated. In cells, M1 alone was mainly observed in the nucleus (47%) and in the cytosol (42%). Conversely, when co-expressed with the viral proteins NS1/NEP and M2, M1 was relocated to the cell membranes (55%), as shown by subcellular fractionation experiments. This minimal system allowed the production of M1 containing-VLPs. However, M1 with mutations in the arginine triplet accumulated in intracellular clusters and its incorporation in VLPs was strongly diminished. M2 over-expression was essential for M1 membrane localization and VLP production, whereas the viral trans-membrane proteins HA and NA seemed dispensable. These results suggest that the M1 arginine triplet participates in M1 interaction with membranes. This R76/77/78 motif is essential for M1 incorporation in virus particles and the importance of this motif was confirmed by reverse genetic demonstrating that its mutation is lethal for the virus. These results highlight the molecular mechanism of M1-membrane interaction during the formation of influenza A(H1N1)pdm09 virus particles which is essential for infectivity.     

Influenza A M2 recruits M1 to the plasma membrane: A fluorescence fluctuation microscopy study

Influenza A virus (IAV) is a respiratory pathogen that causes seasonal epidemics with significant mortality. One of the most abundant proteins in IAV particles is the matrix protein 1 (M1), which is essential for the virus structural stability. M1 organizes virion assembly and budding at the plasma membrane (PM), where it interacts with other viral components. The recruitment of M1 to the PM as well as its interaction with the other viral envelope proteins (hemagglutinin [HA], neuraminidase, matrix protein 2 [M2]) is controversially discussed in previous studies. Therefore, we used fluorescence fluctuation microscopy techniques (i.e., scanning fluorescence cross-correlation spectroscopy and number and brightness) to quantify the oligomeric state of M1 and its interactions with other viral proteins in co-transfected as well as infected cells. Our results indicate that M1 is recruited to the PM by M2, as a consequence of the strong interaction between the two proteins. In contrast, only a weak interaction between M1 and HA was observed. M1-HA interaction occurred only in the event that M1 was already bound to the PM. We therefore conclude that M2 initiates the assembly of IAV by recruiting M1 to the PM, possibly allowing its further interaction with other viral proteins.     

Acylation and cholesterol binding are not required for targeting of influenza A virus M2 protein to the hemagglutinin-defined budozone

Influenza virus assembles in the budozone, a cholesterol-/sphingolipid-enriched (“raft”) domain at the apical plasma membrane, organized by hemagglutinin (HA). The viral protein M2 localizes to the budozone edge for virus particle scission. This was proposed to depend on acylation and cholesterol binding. We show that M2–GFP without these motifs is still transported apically in polarized cells. Employing FRET, we determined that clustering between HA and M2 is reduced upon disruption of HA’s raft-association features (acylation, transmembranous VIL motif), but remains unchanged with M2 lacking acylation and/or cholesterol-binding sites. The motifs are thus irrelevant for M2 targeting in cells.     

Structural vaccinology: structure-based design of influenza A virus hemagglutinin subtype-specific subunit vaccines

There is a great need for new vaccine development against influenza A viruses due to the drawbacks of traditional vaccines that are mainly prepared using embryonated eggs. The main component of the current split influenza A virus vaccine is viral hemagglutinin (HA) which induces a strong antibody-mediated immune response. To develop a modern vaccine against influenza A viruses, the current research has been focused on the universal vaccines targeting viral M2, NP and HA proteins. Crystallographic studies have shown that HA forms a trimer embedded on the viral envelope surface, and each monomer consists of a globular head (HA1) and a “rod-like” stalk region (HA2), the latter being more conserved among different HA subtypes and being the primary target for universal vaccines. In this study, we rationally designed the HA head based on the crystal structure of the 2009-pandemic influenza A (H1N1) virus HA as a model, tested its immunogenicity in mice, solved its crystal structure and further examined its immunological characteristics. The results show that the HA globular head can be easily prepared by in vitro refolding in an E. coli expression system, which maintains its intact structure and allows for the stimulation of a strong immune response. Together with recent reports on some similar HA globular head preparations we conclude that structure-based rational design of the HA globular head can be used for subtype-specific vaccines against influenza viruses.     

流感病毒在脂筏上组装与出芽

流感病毒造成的季节性流行性疾病给全世界带来沉重的健康负担.近年来,甲型流感病毒的变种H5N1、H7N9给各国带来了很大危害.流感病毒属于正黏附病毒科,它的遗传物质由多个节段的负链RNA组成,其组装和出芽剪切生殖是一个涉及到多种病毒因子,多步骤、复杂的生化过程.流感病毒会使用宿主的细胞膜上的"脂筏"区域作为病毒出芽位点.首先病毒的两种糖蛋白NA蛋白、HA蛋白会在脂筏区域聚集,造成脂筏区膜变形弯曲,并且发动出芽的过程.接着,流感病毒基质蛋白M1的C端与HA、NA结合,其自身在脂筏区域开始多聚化并使膜向外弯曲形成原始病毒体的内部结构,接着招募病毒的核糖核蛋白复合物(VRNP)与M2蛋白,使组装的过程进一步完成.最后,M2蛋白会富集在原始病毒体的底部,完成膜的剪切和病毒体的释放.     

Influenza virus hemagglutinin (H3 subtype) requires palmitoylation of its cytoplasmic tail for assembly: M1 proteins of two subtypes differ in their ability to support assembly

The influenza A virus hemagglutinin (HA) transmembrane domain boundary region and the cytoplasmic tail contain three cysteines (residues 555, 562, and 565 for the H3 HA subtype) that are highly conserved among the 16 HA subtypes and which are each modified by the covalent addition of palmitic acid. Previous analysis of the role of these conserved cysteine residues led to differing data, suggesting either no role for HA palmitoylation or an important role for HA palmitoylation. To reexamine the role of these residues in the influenza virus life cycle, a series of cysteine-to-serine mutations were introduced into the HA gene of influenza virus A/Udorn/72 (Ud) (H3N2) by using a highly efficient reverse genetics system. Mutant viruses containing HA-C562S and HA-C565S mutations had reduced growth and failed to form plaques in MDCK cells but formed wild-type-like plaques in an MDCK cell line expressing wild-type HA. In cell-cell fusion assays, nonpalmitoylated H3 HA, in both cDNA-transfected and virus-infected cells, was fully competent for HA-mediated membrane fusion. When the HA cytoplasmic tail cysteine mutants were examined for lipid raft association, using as the criterion Triton X-100 insolubility, loss of raft association did not show a direct correlation with a reduction in virus replication. However, mutant virus assembly was reduced in parallel with reduced virus replication. Additionally, a reassortant of strain A/WSN/33 (WSN), containing the Ud HA gene with mutations C555S, C562S, and C565S, produced virus that could form plaques on regular MDCK cells and had only moderately decreased replication, suggesting differences in the interactions between Ud and WSN HA and internal viral proteins. Analysis of M1 mutants containing substitutions in the six residues that differ between the Ud and WSN M1 proteins indicated that a constellation of residues are responsible for the difference between the M1 proteins in their ability to support virus assembly with nonpalmitoylated H3 HA.     

Oligosaccharide composition of an influenza virus hemagglutinin with host-determined binding properties

We have previously reported that the binding properties of the hemagglutinin (HA) of the WSN-F strain of influenza A are affected by the cells in which the virus is grown (Crecelius, D. M., Deom, C. M., and Schulze, I.T. (1984) Virology 139, 164-177); at 37 degrees C chick embryo fibroblast-grown F virus has a greater affinity for host cells than does the same virus grown in Madin-Darby bovine kidney (MDBK) cells. In an attempt to explain this host-determined property, we have characterized the carbohydrate put onto the viral HA by these two cells. Experiments using tunicamycin indicate that the HA made by MDBK cells contains about 4000 daltons of carbohydrate in excess of that on the HA from chick embryo fibroblast. Serial lectin affinity chromatography of the asparagine-linked oligosaccharides on the HA subunits, HA1 and HA2, detected a number of host-dependent differences in the complex oligosaccharides. Both HA1 and HA2 from MDBK cells contained more highly branched (i.e. tri- and tetraantennary) complex oligosaccharides than did the subunits from chick embryo fibroblasts. In addition, the HA subunits from the two sources differed in the amount of galactose-containing "bisected" complex oligosaccharides and in the presence of certain fucosylated triantennary oligosaccharides. Profiles of the asparagine-linked oligosaccharides from the host cells did not show these differences, indicating that the HA subunit profiles were not necessarily representative of the structures found on the cellular glycoproteins. The data support the conclusion that bulky oligosaccharides on the MDBK-HA subunits of WSN-F reduce the affinity of the virus for cellular receptors.     

Influenza virus hemagglutinin and neuraminidase, but not the matrix protein, are required for assembly and budding of plasmid-derived virus-like particles

For influenza virus, we developed an efficient, noncytotoxic, plasmid-based virus-like particle (VLP) system to reflect authentic virus particles. This system was characterized biochemically by analysis of VLP protein composition, morphologically by electron microscopy, and functionally with a VLP infectivity assay. The VLP system was used to address the identity of the minimal set of viral proteins required for budding. Combinations of viral proteins were expressed in cells, and the polypeptide composition of the particles released into the culture media was analyzed. Contrary to previous findings in which matrix (M1) protein was considered to be the driving force of budding because M1 was found to be released copiously into the culture medium when M1 was expressed by using the vaccinia virus T7 RNA polymerase-driven overexpression system, in our noncytotoxic VLP system M1 was not released efficiently into the culture medium. Additionally, hemagglutinin (HA), when treated with exogenous neuraminidase (NA) or coexpressed with viral NA, could be released from cells independently of M1. Incorporation of M1 into VLPs required HA expression, although when M1 was omitted from VLPs, particles with morphologies similar to those of wild-type VLPs or viruses were observed. Furthermore, when HA and NA cytoplasmic tail mutants were included in the VLPs, M1 failed to be efficiently incorporated into VLPs, consistent with a model in which the glycoproteins control virus budding by sorting to lipid raft microdomains and recruiting the internal viral core components. VLP formation also occurred independently of the function of Vps4 in the multivesicular body pathway, as dominant-negative Vps4 proteins failed to inhibit influenza VLP budding.     

Putative phosphorylation sites on WCA domain of HA2 is essential for <Emphasis Type="Italic">Helicoverpa armigera</Emphasis> single nucleopolyhedrovirus replication

Protein phosphorylation is one of the most common post-translational modification processes that play an essential role in regulating protein functionality.The Helicoverpa armigera single nucleopolyhedrovirus (HearNPv) orf2-encoded nucleocapsid protein HA2 participates in orchestration of virus-induced actin polymerization through its WCA domain,in which phosphorylation status are supposed to be critical in respect to actin polymerization.In the present study,two putative phosphorylation sites (232Thr and 250Ser) and a highly conserved Serine (245Ser) on the WCA domain of HA2 were mutated,and their phenotypes were characterized by reintroducing the mutated HA2 into the HearNPV genome.Viral infectivity assays demonstrated that only the recombinant HearNPV bearing HA2 mutation at 245Ser can produce infectious virions,both 232Tbr and 250Ser mutations were lethal to the virus.However,actin polymerization assay demonstrated that all the three viruses bearing HA2 mutations were still capable of initiating actin polymerization in the host nucleus,which indicated the putative phosphorylation sites on HA2 may contribute to HearNPV replication through another unidentified pathway.     

The influenza virus M2 protein cytoplasmic tail interacts with the M1 protein and influences virus assembly at the site of virus budding

The cytoplasmic tail of the influenza A virus M2 proton-selective ion channel has been shown to be important for virus replication. Previous analysis of M2 cytoplasmic tail truncation mutants demonstrated a defect in incorporation of viral RNA (vRNA) into virions, suggesting a role for M2 in the recruitment of M1-vRNA complexes. To further characterize the effect of the M2 cytoplasmic tail mutations on virus assembly and budding, we constructed a series of alanine substitution mutants of M2 with mutations in the cytoplasmic tail, from residues 71 to 97. Mutant proteins M2-Mut1 and M2-Mut2, with mutations of residues 71 to 73 and 74 to 76, respectively, appeared to have the greatest effect on virus-like particle and virus budding, showing a defect in M1 incorporation. Mutant viruses containing M2-Mut1 and M2-Mut2 failed to replicate in multistep growth analyses on wild-type (wt) MDCK cells and were able to form plaques only on MDCK cells stably expressing wt M2 protein. Compared to wt M2 protein, M2-Mut1 and M2-Mut2 were unable to efficiently coimmunoprecipitate with M1. Furthermore, statistical analysis of planar sheets of membrane from cells infected by virus containing M2-Mut1 revealed a reduction in M1-hemagglutinin (HA) and M2-HA clustering as well as a severe loss of clustering between M1 and M2. These results suggest an essential, direct interaction between the cytoplasmic tail of M2 and M1 that promotes the recruitment of the internal viral proteins and vRNA to the plasma membrane for efficient virus assembly to occur.     

Enhanced pathogenicity and transmissibility of H9N2 avian influenza virus in mammals by hemagglutinin mutations combined with PB2-627K

H9N2 avian influenza viruses (AIVs) circulate globally in poultry and have become the dominant AIV subtype in China in recent years. Previously, we demonstrated that the H9N2 virus (A/chicken/Eastern China/SDKD1/2015) naturally harbors a mammalian-adaptive molecular factor (627K) in the PB2 protein and is weakly pathogenic in mice. Here, we focused on new markers for virulence in mammals. A mouse-adapted H9N2 virus was serially passaged in mice by infecting their lungs. As expected, infected mice showed clinical symptoms and died at passage six. A comparison between the wild-type and mouse-adapted virus sequences identified amino acid substitutions in the hemagglutinin (HA) protein. H9N2 viruses with the T187P ?+ ?M227L double mutation exhibited an increased affinity to human-type (SAα2,6Gal) receptors and significantly enhanced viral attachment to mouse lung tissues, which contributed to enhancing viral replication and virulence in mice. Additionally, HA with the T187P ?+ ?M227L mutation enabled H9N2 viral transmission in guinea pigs via direct contact. AIV pathogenicity in mice is a polygenic trait. Our results demonstrated that these HA mutations might be combined with PB2-627K to significantly increase H9N2 virulence in mice, and this enhanced virulence was achieved in other H9N2 AIVs by generating the same combination of mutations. In summary, our study identified novel key elements in the HA protein that are required for H9N2 pathogenicity in mice and provided valuable insights into pandemic preparedness against emerging H9N2 strains.     

人高致病性禽流感病毒H5N1安徽株HA、M2蛋白双顺反子表达载体的构建及表达研究

将我国分离的人H5N1亚型禽流感病毒A/Anhui/1/2005作为研究对象,扩增其HA和M2基因片段并克隆至DNA疫苗表达载体pVRC中,构建成真核表达质粒。为提高HA的表达量,按照人偏爱密码子将HA基因进行优化改造,经全基因合成后插入真核表达载体pVRC,以β-actin蛋白为内参比证明了优化后的HA蛋白表达效果明显提高。将M2基因和优化后的HA基因共同克隆入双顺反子表达载体pIRES中,获得同时表达HA或M2的双顺反子真核表达质粒;通过Western blot和间接免疫荧光检测方法,确认构建的重组质粒在真核细胞中成功地表达了目的蛋白HA和M2。通过上述结果为进一步开展人高致病性禽流感病毒安徽株HA和M2基因的功能与致病性研究及使用表达HA和M2蛋白进行新型人用禽流感双价疫苗研发奠定基础。