Inhibition of influenza virus A/WSN replication by a trypsin inhibitor, 6-amidino-2-naphthyl p-guanidinobenzoate

A trypsin inhibitor, 6-amidino-2-naphthyl p-guanidinobenzoate (FUTHAN) reduced both the number and size of plaques of influenza virus A/WSN/33 (H1N1) that can grow without trypsin treatment in MDCK cells. The resulting virus particles with uncleaved hemagglutinin (HA) in the presence of FUTHAN was activated to produce infectious virions by trypsin treatment. Uncleaved HA of WSN virus grown in the presence of FUTHAN was found to be accumulated by protein analysis of WSN virus labeled biosynthetically with [35S]-methionine. It was strongly suggested that FUTHAN inhibited viral replication by preventing proteolytic cleavage of HA.     

Palmitylation of the influenza virus hemagglutinin (H3) is not essential for virus assembly or infectivity.

The C terminus of the influenza virus hemagglutinin (HA) contains three cysteine residues that are highly conserved among HA subtypes, two in the cytoplasmic tail and one in the transmembrane domain. All of these C-terminal cysteine residues are modified by the covalent addition of palmitic acid through a thio-ether linkage. To investigate the role of HA palmitylation in virus assembly, we used reverse genetics technique to introduce substitutions and deletions that affected the three conserved cysteine residues into the H3 subtype HA. The rescued viruses contained the HA of subtype H3 (A/Udorn/72) in a subtype H1 helper virus (A/WSN/33) background. Rescued viruses which do not contain a site for palmitylation (by residue substitution or substitution combined with deletion of the cytoplasmic tail) were obtained. Rescued virions had a normal polypeptide composition. Analysis of the kinetics of HA low-pH-induced fusion of the mutants showed no major change from that of virus with wild-type (wt) HA. The PFU/HA ratio of the rescued viruses grown in eggs ranged from that of virus with wt HA to 16-fold lower levels, whereas the PFU/HA ratio of the rescued viruses grown in MDCK cells varied only 2-fold from that of virus with wt HA. However, except for one rescued mutant virus (CAC), the mutant viruses were attenuated in mice, as indicated by a > or = 400-fold increase in the 50% lethal dose. Interestingly, except for one mutant virus (CAC), all of the rescued mutant viruses were restricted for replication in the upper respiratory tract but much less restricted in the lungs. Thus, the HA cytoplasmic tail may play a very important role in the generation of virus that can replicate in multiple cell types.     

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.     

Virulence factors of influenza A viruses: WSN virus neuraminidase required for plaque production in MDBK cells.

The genetic basis for the distinctive capacity of influenza A/WSN/33 (H0N1) virus (WSN virus) to produce plaques on bovine kidney (MDBK) cells was found to be related to virus neuraminidase. Recombinant viruses that derived only the neuraminidase of WSN virus were capable of producing plaques, whereas recombinant viruses identical to WSN except for neuraminidase did not produce plaques. With viruses that do not contain WSN neuraminidase, infectivity of virus yields from MDBK cells was increased approximately 1,000-fold after in vitro treatment with trypsin. In contrast, no significant increase in infectivity was observed after trypsin treatment of viruses containing WSN neuraminidase. In addition, polyacrylamide gel analysis of proteins of WSN virus obtained after infection of MDBK cells demonstrated that hemagglutinin was present in the cleaved form (HA1 + HA2), whereas only uncleaved hemagglutinin was obtained with a recombinant virus that derived all of its genes from WSN virus except its neuraminidase. These data are in accord with the hypothesis that neuraminidase may facilitate production of infectious particles by removing sialic acid residues and exposing appropriate cleavage sites on hemagglutinin.     

Chimeric influenza A viruses with a functional influenza B virus neuraminidase or hemagglutinin

Reassortment of influenza A and B viruses has never been observed in vivo or in vitro. Using reverse genetics techniques, we generated recombinant influenza A/WSN/33 (WSN) viruses carrying the neuraminidase (NA) of influenza B virus. Chimeric viruses expressing the full-length influenza B/Yamagata/16/88 virus NA grew to titers similar to that of wild-type influenza WSN virus. Recombinant viruses in which the cytoplasmic tail or the cytoplasmic tail and the transmembrane domain of the type B NA were replaced with those of the type A NA were impaired in tissue culture. This finding correlates with reduced NA content in virions. We also generated a recombinant influenza A virus expressing a chimeric hemagglutinin (HA) protein in which the ectodomain is derived from type B/Yamagata/16/88 virus HA, whereas both the cytoplasmic and the transmembrane domains are derived from type A/WSN virus HA. This A/B chimeric HA virus did not grow efficiently in MDCK cells. However, after serial passage we obtained a virus population that grew to titers as high as wild-type influenza A virus in MDCK cells. One amino acid change in position 545 (H545Y) was found to be responsible for the enhanced growth characteristics of the passaged virus. Taken together, we show here that the absence of reassortment between influenza viruses belonging to different A and B types is not due to spike glycoprotein incompatibility at the level of the full-length NA or of the HA ectodomain.     

Mutations at palmitylation sites of the influenza virus hemagglutinin affect virus formation.

The carboxy terminus of the hemagglutinin (HA) of influenza A viruses contains three cysteine residues which are highly conserved among HA subtypes. It has previously been shown for the H2, H3, and H7 subtypes of HA that these cysteine residues are modified by the covalent attachment of palmitic acid. In order to study the role of the acylated cysteines in the formation of infectious influenza viruses, we introduced mutations into the HA of influenza A/WSN/33 virus (H1 subtype) by reverse-genetics techniques. We found that the cysteine at position 563 of the cytoplasmic tail is required for infectious-particle formation. The cysteine at position 560 can be changed to alanine or tyrosine to yield virus strains that are attenuated in cell cultures. The change from cysteine at position 553 to serine or alanine does not significantly alter the phenotype of the virus. The requirement for a cysteine at position 563 suggests a functional role for palmitylation of the cytoplasmic tail. This interpretation is further supported by experiments in which two or more of the cysteine residues were mutated, eliminating potential palmitylation sites. None of these double or triple mutations resulted in infectious virus. Selection of revertants of the attenuated cysteine-to-tyrosine mutant (mutation at position 560) always resulted in reversion to cysteine rather than to other amino acids. Although our data indicate a biological role for the conserved cysteine residues in the cytoplasmic tail of the HA of influenza viruses, we cannot exclude the possibility that structural constraints in the cytoplasmic tail of the HA--rather than altered palmitylation--are the determining factors for infectious-particle formation.     

Palmitoylation of the Influenza A Virus M2 Protein Is Not Required for Virus Replication In Vitro but Contributes to Virus Virulence

The influenza A virus M2 protein has important roles during virus entry and in the assembly of infectious virus particles. The cytoplasmic tail of the protein can be palmitoylated at a cysteine residue, but this residue is not conserved in a number of human influenza A virus isolates. Recombinant viruses encoding M2 proteins with a serine substituted for the cysteine at position 50 were generated in the A/WSN/33 (H1N1) and A/Udorn/72 (H3N2) genetic backgrounds. The recombinant viruses were not attenuated for replication in MDCK cells, Calu-3 cells, or in primary differentiated murine trachea epithelial cell cultures, indicating there was no significant contribution of M2 palmitoylation to virus replication in vitro. The A/WSN/33 M2C50S virus displayed a slightly reduced virulence after infection of mice, suggesting that there may be novel functions for M2 palmitoylation during in vivo infection.Influenza A virus is a member of the Orthomyxoviridae and contains a segmented, negative-sense RNA genome that codes for 10 or 11 proteins, depending upon the virus strain (11). The integral membrane protein M2 is the viral ion channel protein that is required during virus entry (29) and for the production of infectious virus particles (4, 10, 12, 13). The sequences responsible for the latter map to the cytoplasmic tail of the protein and overlap with a number of sites for posttranslational modification, which include palmitoylation and phosphorylation (7, 26, 31). Palmitoylation occurs on the cysteine present at amino acid 50 and is not required for ion channel activity of the M2 protein from A/Udorn/72 (H3N2) (7). Palmitoylation of M2 appeared to be dispensable for the production of infectious virus particles using a reassortant virus consisting of seven segments from an H3N8 subtype virus (A/Equine/Miami/63) and the M segment from an H1N1 subtype virus (A/Puerto Rico/8/34) (2). No studies examining the role of M2 palmitoylation in the context of a naturally occurring influenza A virus strain have been published to date.The significance of palmitoylation of the influenza A virus hemagglutinin (HA) protein can vary among virus strains. Palmitoylation of HA from an H7 and an H1 but not an H3 subtype is required for efficient membrane fusion (5, 24, 32), whereas palmitoylation of HA from an H3 but not an H1 subtype is required for virus assembly (5). An analysis of 3,532 sequences of influenza isolates from humans revealed that the M2 residue C50 is conserved in a strain-specific manner. A total of 2,602 of 2,610 H3N2 sequences code for a cysteine at this position; the cysteine, however, is conserved in only 330 of 1,051 H1N1 sequences (data not shown). A serine residue is substituted for cysteine in the majority of the H1N1 viruses that do not have a cytoplasmic palmitoylation site; the newly emerged 2009 H1N1 influenza A viruses, however, do have a cysteine at this position (3). The sequence alignment data are consistent with a strain-specific selective pressure to maintain the palmitoylation site on the M2 protein. Interestingly, other M2 cytoplasmic tail sequences display differential effects on infectious virus production, depending on the strain used (12).To investigate the role of M2 palmitoylation in influenza A virus replication, we substituted a serine for the cysteine residue at position 50 (C50S) of the M2 protein in two influenza A virus strains, A/Udorn/72 (H3N2) (rUdorn) and A/WSN/33 (H1N1) (rWSN). The resultant viruses were tested for their ability to replicate in tissue culture cells, and the mouse-adapted virus was tested for virulence in a mouse model of infection. Neither mutant virus showed any defect in virus replication in tissue culture cells, in differentiated murine primary trachea epithelial cells (mTEC), or in the lungs of infected mice. The viruses lacking a palmitoylation site, however, did have a modest reduction in virulence, suggesting that M2 palmitoylation is dispensable for in vitro replication but contributes to virus virulence in vivo.     

Modulation of glycosylation and transport of viral membrane glycoproteins by a sodium ionophore

Analysis of viral glycoprotein expression on surfaces of monensin- treated cells using a fluorescence-activated cell sorter (FACS) demonstrated that the sodium ionophore completely inhibited the appearance of the vesicular stomatitis virus (VSV) G protein on (Madin- Darby canine kidney) MDCK cell surfaces. In contrast, the expression of the influenza virus hemagglutinin (HA) glycoprotein on the surfaces of MDCK cells was observed to occur at high levels, and the time course of its appearance was not altered by the ionophore. Viral protein synthesis was not inhibited by monensin in either VSV- or influenza virus-infected cells. However, the electrophoretic mobilities of viral glycoproteins were altered, and analysis of pronase-derived glycopeptides by gel filtration indicated that the addition of sialic acid residues to the VSV G protein was impaired in monensin-treated cells. Reduced incorporation of fucose and galactose into influenza virus HA was observed in the presence of the ionophore, but the incompletely processed HA protein was cleaved, transported to the cell surface, and incorporated into budding virus particles. In contrast to the differential effects of monensin on VSV and influenza virus replication previously observed in monolayer cultures of MDCK cells, yields of both viruses were found to be significantly reduced by high concentrations of monensin in suspension cultures, indicating that cellular architecture may play a role in determining the sensitivity of virus replication to the drug. Nigericin, an ionophore that facilitates transport of potassium ions across membranes, blocked the replication of both influenza virus and VSV in MDCK cell monolayers, indicating that the ion specificity of ionophores influences their effect on the replication of enveloped viruses.     

Phenotypic and Genetic Characterization of Avian Influenza H5N2 Viruses with Intra- and Inter-Duck Variations in Taiwan

Background

Human infections with avian influenza viruses (AIVs) have frequently raised global concerns of emerging, interspecies-transmissible viruses with pandemic potential. Waterfowl, the predominant reservoir of influenza viruses in nature, harbor precursors of different genetic lineages that have contributed to novel pandemic influenza viruses in the past.

Methods

Two duck influenza H5N2 viruses, DV518 and DV413, isolated through virological surveillance at a live-poultry market in Taiwan, showed phylogenetic relatedness but exhibited different replication capabilities in mammalian Madin-Darby Canine Kidney (MDCK) cells. This study characterizes the replication properties of the two duck H5N2 viruses and the determinants involved.

Results

The DV518 virus replicated more efficiently than DV413 in both MDCK and chicken DF1 cells. Interestingly, the infection of MDCK cells by DV518 formed heterogeneous plaques with great differences in size [large (L) and small (S)], and the two viral strains (p518-L and p518-S) obtained from plaque purification exhibited distinguishable replication kinetics in MDCK cells. Nonetheless, both plaque-purified DV518 strains still maintained their growth advantages over the plaque-purified p413 strain. Moreover, three amino acid substitutions in PA (P224S), PB2 (E72D), and M1 (A128T) were identified in intra-duck variations (p518-L vs p518-S), whereas other changes in HA (N170D), NA (I56T), and NP (Y289H) were present in inter-duck variations (DV518 vs DV413). Both p518-L and p518-S strains had the N170D substitution in HA, which might be related to their greater binding to MDCK cells. Additionally, polymerase activity assays on 293T cells demonstrated the role of vRNP in modulating the replication capability of the duck p518-L viruses in mammalian cells.

Conclusion

These results demonstrate that intra-host phenotypic variation occurs even within an individual duck. In view of recent human infections by low pathogenic AIVs, this study suggests possible determinants involved in the stepwise selection of virus variants from the duck influenza virus population which may facilitate inter-species transmission.     

用8质粒病毒拯救系统产生H9N2/WSN重组A型流行性感冒病毒

把禽流行性感冒(流感)病毒A／Chicken／Shanghai／F／98(H9N2)的血凝素(HA)和神经氨酸酶(NA)基因cDNA克隆至polⅠ-pol Ⅱ双向转录和表达载体pHW2000,用这两种质粒与8质粒病毒拯救系统中流感病毒A／WSN／33(H1N1)6个内部基因cDNA的质粒组合(6 2重排),共转染COS-1细胞,产生了能在鸡胚中高滴度增殖的H9N2／、WSN重组病毒。用A／WSN／33的8个基因cDNA质粒作对照,也产生了转染子病毒。经过EID50测定和MDCK感染实验,新基因型H9N2／WSN病毒感染鸡胚的能力强(EID50为10^-11／0．2m1),而且对鸡胚的毒力弱,在不加胰酶的情况下不使MDCK细胞产牛病变。经电镜观察,两个转染子病毒的形态与野生型流感病毒相似。反向遗传操作技术的建立,为对禽流感病毒基因功能和疫苗构建等方面的研究提供了新的手段。     

Direct sequencing of the HA gene of influenza (H3N2) virus in original clinical samples reveals sequence identity with mammalian cell-grown virus.

When influenza (H3N2) viruses from infected individuals are grown in embryonated chicken eggs, viruses are isolated which differ antigenically and structurally from viruses grown in mammalian Madin-Darby canine kidney (MDCK) cell culture [G.C. Schild, J.S. Oxford, J.C. de Jong, and R.G. Webster, Nature (London) 303:706-709, 1983]. To determine which of these viruses is most representative of virus replicating in the infected individual, a region of the HA gene of virus present in original clinical samples was amplified by using the polymerase chain reaction and sequenced directly. Comparison of 170 amino acid residues of HA1 flanking and containing the receptor-binding site and antigenic sites indicated that over this region, the HA of virus replicating in the infected individual was identical to that of virus after growth in MDCK cells and was distinct from the HA of viruses grown in eggs. Therefore, cultivation of human influenza H3N2 virus in mammalian MDCK cells results in a virus similar to the predominant population of virus found in the infected individual.     

Differential nucleocytoplasmic shuttling of the nucleoprotein of influenza a viruses and association with host tropism

The nucleoprotein (NP) of influenza A virus plays a crucial role in virus replication, infectivity, and host adaptation. As a major component of the viral ribonucleoprotein complexes (vRNP), NP initiates vRNP shuttling between the nucleus and cytoplasm in the host cell. However, the characteristics of the nucleocytoplasmic shuttling of NP from H1N1 influenza A virus still remain unclear. In the present study, the subcellular localization and the related key residues of the H1N1 influenza virus NP were identified and evaluated. The NP of influenza virus A/WSN/33 (H1N1; WSN) displayed a more obvious nuclear accumulation than A/Anhui/1/2013 (H7N9; AH) and A/chicken/Shandong/lx1023/2007 (H9N2; SD). NP residue K4, located in NLS1, and residue F253, located in NES3, from WSN NP are not conserved in H7N9 and H9N2, which instead encode Q4 and I253, respectively. Crucially, these residues are involved in the regulation of NP nucleocytoplasmic shuttling through interactions with CRM1 and importin‐α. Moreover, residues at position 253 also play important roles in the replication of the virus, resulting in an increase in vRNP polymerase activity and an alteration of the cell tropism and pathogenicity in mice. The present data revealed a pivotal role of the Q4 and I253 residues of NP from H7N9 in enhancing the cytoplasmic accumulation of NP and vRNP activity compared to the K4 and F253 residues in WSN‐NP. In addition, an F253I substitution in the NP of WSN altered the survival ratio of infected mice and the growth curve in infected avian‐origin cells (DF‐1). The current data indicate that the F253I mutation results in attenuated pathogenicity of the virus in mice and altered cell tropism. The present study demonstrated the dissimilarity in subcellular NP transport processes between H1N1 virus WSN and other influenza A virus strains, as well as uncovered the mechanism responsible for this difference.     

Sialidase activity of influenza A virus in an endocytic pathway enhances viral replication

N2 neuraminidase (NA) genes of the 1957 and 1968 pandemic influenza virus strains possessed avian-like low-pH stability of sialidase activity, unlike most epidemic strains. We generated four reverse-genetics viruses from a genetic background of A/WSN/33 (H1N1) that included parental N2 NAs of 1968 pandemic (H3N2) and epidemic (H2N2) strains or their counterpart N2 NAs in which the low-pH stability of the sialidase activity was changed by substitutions of one or two amino acid residues. We found that the transfectant viruses bearing low-pH-stable sialidase (WSN/Stable-NAs) showed 25- to 80-times-greater ability to replicate in Madin-Darby canine kidney (MDCK) cells than did the transfectant viruses bearing low-pH-unstable sialidase (WSN/Unstable-NAs). Enzymatic activities of WSN/Stable-NAs were detected in endosomes of MDCK cells after 90 min of virus internalization by in situ fluorescent detection with 5-bromo-4-chloro-indole-3-yl-alpha-N-acetylneuraminic acid and Fast Red Violet LB. Inhibition of sialidase activity of WSN/Stable-NAs on the endocytic pathway by pretreatment with 4-guanidino-2,4-dideoxy-N-acetylneuraminic acid (zanamivir) resulted in a significant decrease in progeny viruses. In contrast, the enzymatic activities of WSN/Unstable-NAs, the replication of which had no effect on pretreatment with zanamivir, were undetectable in cells under the same conditions. Hemadsorption assays of transfectant-virus-infected cells revealed that the low-pH stability of the sialidase had no effect on the process of removal of sialic acid from hemagglutinin in the Golgi regions. Moreover, high titers of viruses were recovered from the lungs of mice infected with WSN/Stable-NAs on day 3 after intranasal inoculation, but WSN/Unstable-NAs were cleared from the lungs of the mice. These results indicate that sialidase activity in late endosome/lysosome traffic enhances influenza A virus replication.     

Improvement of influenza A/Fujian/411/02 (H3N2) virus growth in embryonated chicken eggs by balancing the hemagglutinin and neuraminidase activities, using reverse genetics

The H3N2 influenza A/Fujian/411/02-like virus strains that circulated during the 2003-2004 influenza season caused influenza epidemics. Most of the A/Fujian/411/02 virus lineages did not replicate well in embryonated chicken eggs and had to be isolated originally by cell culture. The molecular basis for the poor replication of A/Fujian/411/02 virus was examined in this study by the reverse genetics technology. Two antigenically related strains that replicated well in embryonated chicken eggs, A/Sendai-H/F4962/02 and A/Wyoming/03/03, were compared with the prototype A/Fujian/411/02 virus. A/Sendai differed from A/Fujian by three amino acids in the neuraminidase (NA), whereas A/Wyoming differed from A/Fujian by five amino acids in the hemagglutinin (HA). The HA and NA segments of these three viruses were reassorted with cold-adapted A/Ann Arbor/6/60, the master donor virus for the live attenuated type A influenza vaccines (FluMist). The HA and NA residues differed between these three H3N2 viruses evaluated for their impact on virus replication in MDCK cells and in embryonated chicken eggs. It was determined that replication of A/Fujian/411/02 in eggs could be improved by either changing minimum of two HA residues (G186V and V226I) to increase the HA receptor-binding ability or by changing a minimum of two NA residues (E119Q and Q136K) to lower the NA enzymatic activity. Alternatively, recombinant A/Fujian/411/02 virus could be adapted to grow in eggs by two amino acid substitutions in the HA molecule (H183L and V226A), which also resulted in the increased HA receptor-binding activity. Thus, the balance between the HA and NA activities is critical for influenza virus replication in a different host system. The HA or NA changes that increased A/Fujian/411/02 virus replication in embryonated chicken eggs were found to have no significant impact on antigenicity of these recombinant viruses. This study demonstrated that the reverse genetics technology could be used to improve the manufacture of the influenza vaccines.     

The influenza virus hemagglutinin cytoplasmic tail is not essential for virus assembly or infectivity.

The influenza A virus hemagglutinin (HA) glycoprotein contains a cytoplasmic tail which consists of 10-11 amino acids, of which five residues re conserved in all subtypes of influenza A virus. As the cytoplasmic tail is not needed for intracellular transport to the plasma membrane, it has become virtually dogma that the role of the cytoplasmic tail is in forming protein-protein interactions necessary for creating an infectious budding virus. To investigate the role of the HA cytoplasmic tail in virus replication, reverse genetics was used to obtain an influenza virus that lacked an HA cytoplasmic tail. The rescued virus contained the HA of subtype A/Udorn/72 in a helper virus (subtype A/WSN/33) background. Biochemical analysis indicated that only the introduced tail- HA was incorporated into virions and these particles lacked a detectable fragment of the helper virus HA. The tail- HA rescued virus assembled and replicated almost as efficiently as virions containing wild-type HA, suggesting that the cytoplasmic tail is not essential for the virus assembly process. Nonetheless, a revertant virus was isolated, suggesting that possession of a cytoplasmic tail does confer an advantage.     

Modifications to the Hemagglutinin Cleavage Site Control the Virulence of a Neurotropic H1N1 Influenza Virus

A key determinant of influenza virus pathogenesis is mutation in the proteolytic cleavage site of the hemagglutinin (HA). Typically, low-pathogenicity forms of influenza virus are cleaved by trypsin-like proteases, whereas highly pathogenic forms are cleaved by different proteases (e.g., furin). Influenza virus A/WSN/33 (WSN) is a well-studied H1N1 strain that is trypsin independent in vitro and has the ability to replicate in mouse brain. Previous studies have indicated that mutations in the neuraminidase (NA) gene allow the recruitment of an alternate protease (plasminogen/plasmin) for HA activation. In this study we have identified an additional mutation in the P2 position of the WSN HA cleavage site (S328Y) that appears to control virus spread in a plasmin-dependent manner. We reconstructed recombinant WSN viruses containing tyrosine (Y), phenylalanine (F), or serine (S) in the P2 position of the cleavage site. The Y328 and F328 viruses allowed plaque formation in the absence of trypsin, whereas the S328 virus was unable to form plaques under these conditions. In mice, Y328 and F328 viruses were able to efficiently spread following intracranial inoculation; in contrast, the S328 virus showed only limited infection of mouse brain. Following intranasal inoculation, all viruses could replicate efficiently, but with Y328 and F328 viruses showing a limited growth defect. We also show that wild-type HA (Y328) was more efficiently cleaved by plasmin than S328 HA. Our studies form the foundation for a more complete understanding of the molecular determinants of influenza virus pathogenesis and the role of the plasminogen/plasmin system in activating HA.For all viruses, the infectious cycle begins with the penetration of the host cell (27). Enveloped viruses penetrate cells via a membrane fusion event mediated by a spike protein present in the virus envelope, with fusion triggered by conformational changes in the spike protein following exposure to low pH and/or receptor (45). In the case of influenza virus, the viral spike protein hemagglutinin (HA) mediates both receptor binding and membrane fusion (46). Many viral fusion proteins are activated following cleavage by host cell proteases (9, 20, 21), and this has been most extensively documented for influenza viruses, where cleavage is directly related to exposure of the fusion peptide and fusion activation (38). For proteases, a general nomenclature for the cleavage site positions of the substrate has been designated, with cleavage occurring between P1 and P1′ and with the position numbers increasing in the N-terminal direction relative to the cleaved peptide bond (P2, P3, P4, etc.). Low-pathogenicity influenza virus strains contain an HA cleavage site with a single arginine residue at the P1 position and are thus described as having monobasic cleavage sites. These viruses can utilize trypsin (or other trypsin-like serine proteases) for activation, with the tissue distribution of the activating protease typically restricting infection to the respiratory and/or intestinal organs. The presence of a polybasic cleavage site is critical for the systemic spread and increased virulence associated with highly pathogenic avian influenza (HPAI) viruses (33). In the case of HPAI viruses such as H5N1 and H7N7, it is well established that mutations in the region of the HA cleavage site lead to an insertion of several arginine or lysine residues in addition to the P1 arginine (specifically in the P2 to P6 cleavage site positions) that can be recognized by furin—an intracellular serine protease found in many cell types—allowing a widening of the cell tropism of the virus (18).Influenza virus is currently of major biomedical interest, both due to annual morbidity and the threat of new pandemic viruses. Influenza viruses exist as many different subtypes (H1 to H16), with H1 and H3 viruses currently infecting humans (10, 30). Normally, H1 viruses are considered to have low pathogenicity and have a monobasic cleavage site. However, two H1 isolates A/WSN/33 (WSN) and A/NWS/33 (NWS) have been selected to propagate in mouse brain and are thus considered to be highly pathogenic, neurovirulent viruses in mice (39, 40). The A/WSN/33 virus in particular has been used extensively for studies on influenza virus replication and pathogenesis, in part because this virus forms plaques in the absence of trypsin and serves as a model of highly pathogenic influenza virus. The HA of the A/WSN/33 virus was originally shown to be cleaved by plasmin, following activation of serum plasminogen in MDBK cells (22). The virulence properties of A/WSN/33 were subsequently linked to the neuraminidase (NA) gene (37) and the absence of a glycosylation site at position 130 of NA (25). In addition, the presence of a C-terminal lysine on NA was shown to be critical for the virulence properties of A/WSN/33, with the viral NA binding and sequestering of plasminogen on the cell surface, leading to increased cleavage of HA (15, 16). It has also been suggested that the HA of A/WSN/33 can be cleaved by an endosomal serine protease in MDBK cells (7).Based on the recent pandemic status of novel H1N1 viruses and the known importance of the HA cleavage site in viral pathogenicity (30), we assessed the presence of cleavage site changes, in addition to the conventional monobasic/polybasic cleavage sites, in the context of the virulence properties of H1 influenza viruses. We characterize the role of the bulky hydrophobic residues tyrosine and phenylalanine, found in the P2 cleavage positions of only A/WSN/33 and A/NWS/33 HA, respectively, and show that these residues are major virulence determinants for these viruses, allowing efficient use of plasmin for spread in vitro and in vivo.     

携带TAP标签的重组甲型流感病毒的构建与鉴定

【目的】将TAP标签构建到WSN病毒基因组上,得到含有TAP标签的重组流感病毒,以便进行后续的病毒追踪。【方法】利用反向遗传学技术,对甲型流感病毒A/WSN/33(H1N1)的PA片段进行改造来插入TAP(tandemaffinitypurification)标签序列。通过病毒拯救得到表达外源标签TAP的重组流感病毒WSNPA-TAP,并对拯救出的重组病毒进行生物学鉴定。【结果】成功拯救出重组流感病毒并命名为WSN PA-TAP。重组病毒基因组测序表明重组病毒的序列正确,利用RNA银染技术观察到重组病毒的全基因组片段。重组流感病毒WSN PA-TAP在MDCK细胞上测定生长曲线,发现该重组病毒的复制能力比野生型WSN弱;Westernblotting检测到PA-TAP融合蛋白的表达,其分子质量为96 kDa。【结论】成功拯救出能够表达外源标签TAP的重组流感病毒WSN PA-TAP,为筛选与甲型流感病毒聚合酶有关的宿主蛋白的研究提供了新思路,同时也为以甲型流感病毒为载体携带外源基因的探索提供了重要依据。     

Impact of host cell line adaptation on quasispecies composition and glycosylation of influenza A virus hemagglutinin

The genome of influenza A viruses is constantly changing (genetic drift) resulting in small, gradual changes in viral proteins. Alterations within antibody recognition sites of the viral membrane glycoproteins hemagglutinin (HA) and neuraminidase (NA) result in an antigenetic drift, which requires the seasonal update of human influenza virus vaccines. Generally, virus adaptation is necessary to obtain sufficiently high virus yields in cell culture-derived vaccine manufacturing. In this study detailed HA N-glycosylation pattern analysis was combined with in-depth pyrosequencing analysis of the virus genomic RNA. Forward and backward adaptation from Madin-Darby Canine Kidney (MDCK) cells to African green monkey kidney (Vero) cells was investigated for two closely related influenza A virus PR/8/34 (H1N1) strains: from the National Institute for Biological Standards and Control (NIBSC) or the Robert Koch Institute (RKI). Furthermore, stability of HA N-glycosylation patterns over ten consecutive passages and different harvest time points is demonstrated. Adaptation to Vero cells finally allowed efficient influenza A virus replication in Vero cells. In contrast, during back-adaptation the virus replicated well from the very beginning. HA N-glycosylation patterns were cell line dependent and stabilized fast within one (NIBSC-derived virus) or two (RKI-derived virus) successive passages during adaptation processes. However, during adaptation new virus variants were detected. These variants carried "rescue" mutations on the genomic level within the HA stem region, which result in amino acid substitutions. These substitutions finally allowed sufficient virus replication in the new host system. According to adaptation pressure the composition of the virus populations varied. In Vero cells a selection for "rescue" variants was characteristic. After back-adaptation to MDCK cells some variants persisted at indifferent frequencies, others slowly diminished and even dropped below the detection limit.     

Inhibition of influenza A virus (H1N1) fusion by benzenesulfonamide derivatives targeting viral hemagglutinin

Hemagglutinin (HA) of the influenza virus plays a crucial role in the early stage of the viral life cycle by binding to sialic acid on the surface of host epithelial cells and mediating fusion between virus envelope and endosome membrane for the release of viral genomes into the cytoplasm. To initiate virus fusion, endosome pH is lowered by acidification causing an irreversible conformational change of HA, which in turn results in a fusogenic HA. In this study, we describe characterization of an HA inhibitor of influenza H1N1 viruses, RO5464466. One-cycle time course study in MDCK cells showed that this compound acted at an early step of influenza virus replication. Results from HA-mediated hemolysis of chicken red blood cells and trypsin sensitivity assay of isolated HA clearly showed that RO5464466 targeted HA. In cell-based assays involving multiple rounds of virus infection and replication, RO5464466 inhibited an established influenza infection. The overall production of progeny viruses, as a result of the compound's inhibitory effect on fusion, was dramatically reduced by 8 log units when compared with a negative control. Furthermore, RO5487624, a close analogue of RO5464466, with pharmacokinetic properties suitable for in vivo efficacy studies displayed a protective effect on mice that were lethally challenged with influenza H1N1 virus. These results might benefit further characterization and development of novel anti-influenza agents by targeting viral hemagglutinin.     

Lipid raft and influenza virus--viral glycoproteins on a raft

Lipid molecules of the plasma membrane are not distributed homogeneously, but form a lateral organization resulting from preferential packaging of sphingolipid and cholesterol into lipid microdomain rafts, in which specific membrane proteins become incorporated. Evidence has accumulated that a variety of viruses including influenza virus use the raft during some steps of their replication cycles. Influenza virus glycoproteins, hemagglutinin (HA) and neuraminidase, associate intrinsically with the rafts. The HA protein is distributed in clusters at the plasma membrane and concentrated in the small area by interacting with the raft. A mutant influenza virus, whose HA protein lacks the ability to associate with the raft, contains reduced amounts of the HA proteins and exhibits a decreased virus to cell fusion activity, resulting in greatly reduced infectivity. Thus, the raft may play an important role in virus production by acting as a concentrating devise or an efficient carrier to transport the HA protein to the site of virus budding.