Characterization of a temperature-sensitive influenza B virus mutant defective in neuraminidase.

ts5, a temperature-sensitive mutant of influenza B virus, belongs to one of seven recombination groups. When the mutant infected MDCK cells at the nonpermissive temperature (37.5 degrees C), infectious virus was produced at very low levels compared with the yield at the permissive temperature (32 degrees C) and hemagglutinating and enzymatic activities were undetectable. However, viral protein synthesis and transport of hemagglutinin (HA) and neuraminidase (NA) to the cell surface were not affected. The NA was found as a monomer within cells even at 32 degrees C, in contrast to wild-type virus NA, existing mostly as an oligomer, but the mutant had oligomeric NA, like the wild-type virus. Its enzymatic activity was more thermolabile than that of wild-type virus. Despite the low yield, large aggregates of progeny virus particles were found to accumulate on the cell surface at the nonpermissive temperature, and these aggregates were broken by treatment with bacterial neuraminidase, with the concomitant appearance of hemagglutinating activity, suggesting that NA prevents the aggregation of progeny virus by removal of neuraminic acid from HA and cell receptor, allowing its release from the cells. Further treatment with trypsin resulted in the recovery of infectivity. When bacterial NA was added to the culture early in infection, many hemagglutinable infectious virus was produced. We also suggest that the removal of neuraminic acid from HA by NA is essential for the subsequent cleavage of HA by cellular protease. Nucleotide sequence analysis of RNA segment 6 revealed that ts5 encoded five amino acid changes in the NA molecule but not in NB.     

Interdependence of hemagglutinin glycosylation and neuraminidase as regulators of influenza virus growth: a study by reverse genetics

The hemagglutinin (HA) of fowl plague virus A/FPV/Rostock/34 (H7N1) carries two N-linked oligosaccharides attached to Asn123 and Asn149 in close vicinity to the receptor-binding pocket. In previous studies in which HA mutants lacking either one (mutants G1 and G2) or both (mutant G1,2) glycosylation sites had been expressed from a simian virus 40 vector, we showed that these glycans regulate receptor binding affinity (M. Ohuchi, R. Ohuchi, A. Feldmann, and H. D. Klenk, J. Virol. 71:8377-8384, 1997). We have now investigated the effect of these mutations on virus growth using recombinant viruses generated by an RNA polymerase I-based reverse genetics system. Two reassortants of influenza virus strain A/WSN/33 were used as helper viruses to obtain two series of HA mutant viruses differing only in the neuraminidase (NA). Studies using N1 NA viruses revealed that loss of the oligosaccharide from Asn149 (mutant G2) or loss of both oligosaccharides (mutant G1,2) has a pronounced effect on virus growth in MDCK cells. Growth of virus lacking both oligosaccharides from infected cells was retarded, and virus yields in the medium were decreased about 20-fold. Likewise, there was a reduction in plaque size that was distinct with G1,2 and less pronounced with G2. These effects could be attributed to a highly impaired release of mutant progeny viruses from host cells. In contrast, with recombinant viruses containing N2 NA, these restrictions were much less apparent. N1 recombinants showed lower neuraminidase activity than N2 recombinants, indicating that N2 NA is able to partly overrule the high-affinity binding of mutant HA to the receptor. These results demonstrate that N-glycans flanking the receptor-binding site of the HA molecule are potent regulators of influenza virus growth, with the glycan at Asn149 being dominant and that at Asn123 being less effective. In addition, we show here that HA and NA activities need to be highly balanced in order to allow productive influenza virus infection.     

Studies on the adaptation of influenza virus replicated at low temperature. III. Biochemical studies.

Five strains of influenza viruses A(H3N2) replicated at low temperature passaged in cotton rats were reisolated. The properties of these strains replicated at low temperature were compared before and after passage in susceptible animals to check the stability of some its markers. At the same time original viruses replicated at 37 degrees C--which are different in epidemiological potency--were compared. The following parameters being tested: NA activity, HA titers, heat inactivation NA and Ha, Michaelis constants and optimum pH. We observed some differences between strains both replicated at low temperature after passage in the susceptible animal organism and original viruses from 37 degrees C. Viruses replicated at low temperature from original epidemiostrain are really cold adapted and remained stable after passage in the animals when the others derived from no epidemic strain are not stable.     

流感病毒血凝素基因、神经氨酸酶基因在小鼠中的免疫效果观察

流感病毒血凝素基因,神经氨酸酶基因免疫BALB／c小鼠,获得特异阳性抗体反应,抗体滴度与基因免疫量呈正相关性,各实验组免疫小鼠抗同型流感病毒攻击存活率为100％,血凝素基因免疫小鼠抗异型流感病毒攻击存活率为100％,神经氨酸酶基因免疫小鼠抗异型流感病毒攻击存活率为75％,血凝素基因与神经氨酸酶基因联合免疫小鼠抗异型流感病毒攻击存活率为100％。     

Novel function of plasminogen-binding activity of the NA determines the pathogenicity of influenza A virus

Because cleavage of the hemagglutinin (HA) molecule by proteases is a prerequisite for infectivity of influenza A viruses, this molecule is a major determinant of viral pathogenicity. Although well documented in the pathogenicity of avian influenza viruses, the role of HA cleavage in the pathogenicity of mammalian viruses is not well understood. Therefore, we studied a mouse-adapted human isolate A/WSN/33 (WSN), a neurovirulent influenza virus strain that causes systemic infection when inoculated intranasally into mice. We found a novel mechanism of HA cleavage for WSN virus: the neuraminidase (NA) of WSN virus binds and sequesters plasminogen on the cell surface, leading to enhanced cleavage of the HA. The structural basis of this novel function of the NA molecule appears to be the presence of a carboxyl-terminal lysine and the absence of an oligosaccharide side chain at position 146. To obtain direct evidence that the plasminogen-binding activity of the NA enhances the pathogenicity of WSN virus, we generated mutant viruses that are deficient in plasminogen-binding activity by reverse genetics. The mutant viruses showed attenuated growth in mice and failed to grow at all in the brains of these animals. Therefore, we concluded that the novel function of plasminogen-binding activity of the NA determines the pathogenicity of WSN virus in mice.     

A Mutant Influenza Virus That Uses an N1 Neuraminidase as the Receptor-Binding Protein

In the vast majority of influenza A viruses characterized to date, hemagglutinin (HA) is the receptor-binding and fusion protein, whereas neuraminidase (NA) is a receptor-cleaving protein that facilitates viral release but is expendable for entry. However, the NAs of some recent human H3N2 isolates have acquired receptor-binding activity via the mutation D151G, although these isolates also appear to retain the ability to bind receptors via HA. We report here the laboratory generation of a mutation (G147R) that enables an N1 NA to completely co-opt the receptor-binding function normally performed by HA. Viruses with this mutant NA grow to high titers even in the presence of extensive mutations to conserved residues in HA''s receptor-binding pocket. When the receptor-binding NA is paired with this binding-deficient HA, viral infectivity and red blood cell agglutination are blocked by NA inhibitors. Furthermore, virus-like particles expressing only the receptor-binding NA agglutinate red blood cells in an NA-dependent manner. Although the G147R NA receptor-binding mutant virus that we characterize is a laboratory creation, this same mutation is found in several natural clusters of H1N1 and H5N1 viruses. Our results demonstrate that, at least in tissue culture, influenza virus receptor-binding activity can be entirely shifted from HA to NA.     

Expression of Functional Recombinant Hemagglutinin and Neuraminidase Proteins from the Novel H7N9 Influenza Virus Using the Baculovirus Expression System

The baculovirus expression system is a powerful tool for expression of recombinant proteins. Here we use it to produce correctly folded and glycosylated versions of the influenza A virus surface glycoproteins - the hemagglutinin (HA) and the neuraminidase (NA). As an example, we chose the HA and NA proteins expressed by the novel H7N9 virus that recently emerged in China. However the protocol can be easily adapted for HA and NA proteins expressed by any other influenza A and B virus strains. Recombinant HA (rHA) and NA (rNA) proteins are important reagents for immunological assays such as ELISPOT and ELISA, and are also in wide use for vaccine standardization, antibody discovery, isolation and characterization. Furthermore, recombinant NA molecules can be used to screen for small molecule inhibitors and are useful for characterization of the enzymatic function of the NA, as well as its sensitivity to antivirals. Recombinant HA proteins are also being tested as experimental vaccines in animal models, and a vaccine based on recombinant HA was recently licensed by the FDA for use in humans. The method we describe here to produce these molecules is straight forward and can facilitate research in influenza laboratories, since it allows for production of large amounts of proteins fast and at a low cost. Although here we focus on influenza virus surface glycoproteins, this method can also be used to produce other viral and cellular surface proteins.     

Global host immune response: pathogenesis and transcriptional profiling of type A influenza viruses expressing the hemagglutinin and neuraminidase genes from the 1918 pandemic virus

To understand more fully the molecular events associated with highly virulent or attenuated influenza virus infections, we have studied the effects of expression of the 1918 hemagglutinin (HA) and neuraminidase (NA) genes during viral infection in mice under biosafety level 3 (agricultural) conditions. Using histopathology and cDNA microarrays, we examined the consequences of expression of the HA and NA genes of the 1918 pandemic virus in a recombinant influenza A/WSN/33 virus compared to parental A/WSN/33 virus and to an attenuated virus expressing the HA and NA genes from A/New Caledonia/20/99. The 1918 HA/NA:WSN and WSN recombinant viruses were highly lethal for mice and displayed severe lung pathology in comparison to the nonlethal New Caledonia HA/NA:WSN recombinant virus. Expression microarray analysis performed on lung tissues isolated from the infected animals showed activation of many genes involved in the inflammatory response, including cytokine, apoptosis, and lymphocyte genes that were common to all three infection groups. However, consistent with the histopathology studies, the WSN and 1918 HA/NA:WSN recombinant viruses showed increased up-regulation of genes associated with activated T cells and macrophages, as well as genes involved in apoptosis, tissue injury, and oxidative damage that were not observed in the New Caledonia HA/NA:WSN recombinant virus-infected mice. These studies document clear differences in gene expression profiles that were correlated with pulmonary disease pathology induced by virulent and attenuated influenza virus infections.     

The low-pH stability discovered in neuraminidase of 1918 pandemic influenza A virus enhances virus replication

The "Spanish" pandemic influenza A virus, which killed more than 20 million worldwide in 1918-19, is one of the serious pathogens in recorded history. Characterization of the 1918 pandemic virus reconstructed by reverse genetics showed that PB1, hemagglutinin (HA), and neuraminidase (NA) genes contributed to the viral replication and virulence of the 1918 pandemic influenza virus. However, the function of the NA gene has remained unknown. Here we show that the avian-like low-pH stability of sialidase activity discovered in the 1918 pandemic virus NA contributes to the viral replication efficiency. We found that deletion of Thr at position 435 or deletion of Gly at position 455 in the 1918 pandemic virus NA was related to the low-pH stability of the sialidase activity in the 1918 pandemic virus NA by comparison with the sequences of other human N1 NAs and sialidase activity of chimeric constructs. Both amino acids were located in or near the amino acid resides that were important for stabilization of the native tetramer structure in a low-pH condition like the N2 NAs of pandemic viruses that emerged in 1957 and 1968. Two reverse-genetic viruses were generated from a genetic background of A/WSN/33 (H1N1) that included low-pH-unstable N1 NA from A/USSR/92/77 (H1N1) and its counterpart N1 NA in which sialidase activity was converted to a low-pH-stable property by a deletion and substitutions of two amino acid residues at position 435 and 455 related to the low-pH stability of the sialidase activity in 1918 NA. The mutant virus that included "Spanish Flu"-like low-pH-stable NA showed remarkable replication in comparison with the mutant virus that included low-pH-unstable N1 NA. Our results suggest that the avian-like low-pH stability of sialidase activity in the 1918 pandemic virus NA contributes to the viral replication efficiency.     

Pathogenicity of influenza viruses with genes from the 1918 pandemic virus: functional roles of alveolar macrophages and neutrophils in limiting virus replication and mortality in mice

The Spanish influenza pandemic of 1918 to 1919 swept the globe and resulted in the deaths of at least 20 million people. The basis of the pulmonary damage and high lethality caused by the 1918 H1N1 influenza virus remains largely unknown. Recombinant influenza viruses bearing the 1918 influenza virus hemagglutinin (HA) and neuraminidase (NA) glycoproteins were rescued in the genetic background of the human A/Texas/36/91 (H1N1) (1918 HA/NA:Tx/91) virus. Pathogenesis experiments revealed that the 1918 HA/NA:Tx/91 virus was lethal for BALB/c mice without the prior adaptation that is usually required for human influenza A H1N1 viruses. The increased mortality of 1918 HA/NA:Tx/91-infected mice was accompanied by (i) increased (>200-fold) viral replication, (ii) greater influx of neutrophils into the lung, (iii) increased numbers of alveolar macrophages (AMs), and (iv) increased protein expression of cytokines and chemokines in lung tissues compared with the levels seen for control Tx/91 virus-infected mice. Because pathological changes in AMs and neutrophil migration correlated with lung inflammation, we assessed the role of these cells in the pathogenesis associated with 1918 HA/NA:Tx/91 virus infection. Neutrophil and/or AM depletion initiated 3 or 5 days after infection did not have a significant effect on the disease outcome following a lethal 1918 HA/NA:Tx/91 virus infection. By contrast, depletion of these cells before a sublethal infection with 1918 HA/NA:Tx/91 virus resulted in uncontrolled virus growth and mortality in mice. In addition, neutrophil and/or AM depletion was associated with decreased expression of cytokines and chemokines. These results indicate that a human influenza H1N1 virus possessing the 1918 HA and NA glycoproteins can induce severe lung inflammation consisting of AMs and neutrophils, which play a role in controlling the replication and spread of 1918 HA/NA:Tx/91 virus after intranasal infection of mice.     

Specific residues of the influenza A virus hemagglutinin viral RNA are important for efficient packaging into budding virions

A final step in the influenza virus replication cycle is the assembly of the viral structural proteins and the packaging of the eight segments of viral RNA (vRNA) into a fully infectious virion. The process by which the RNA genome is packaged efficiently remains poorly understood. In an approach to analyze how vRNA is packaged, we rescued a seven-segmented virus lacking the hemagglutinin (HA) vRNA (deltaHA virus). This virus could be passaged in cells constitutively expressing HA protein, but it was attenuated in comparison to wild-type A/WSN/33 virus. Supplementing the deltaHA virus with an artificial segment containing green fluorescent protein (GFP) or red fluorescent protein (RFP) with HA packaging regions (45 3' and 80 5' nucleotides) partially restored the growth of this virus to wild-type levels. The absence of the HA vRNA in the deltaHA virus resulted in a 40 to 60% reduction in the packaging of the PA, NP, NA, M, and NS vRNAs, as measured by quantitative PCR (qPCR), and the packaging of these vRNAs was partially restored in the presence of GFP/RFP packaging constructs. To further define nucleotides of the HA coding sequence which are important for vRNA packaging, synonymous mutations were introduced into the full-length HA cDNA of influenza A/WSN/33 and A/Puerto Rico/8/34 viruses, and mutant viruses were rescued. qPCR analysis of vRNAs packaged in these mutant viruses identified a key region of the open reading frame (nucleotides 1659 to 1671) that is critical for the efficient packaging of an influenza virus H1 HA segment.     

Class II MHC-restricted T cell determinants processed from either endosomes or the cytosol show similar requirements for host protein transport but different kinetics of presentation.

We have studied the role of APC protein transport in presentation of class II MHC-restricted T cell determinants of influenza virus glycoproteins that have distinct Ag processing requirements. Two I-Ed-restricted epitopes were analyzed: hemagglutinin (HA) 111-119, which is processed by the exogenous/endocytic pathway, and neuraminidase (NA) 79-93, which has a requirement for cytosolic processing. NA 79-93 is presented from infectious but not non-replicative virus under ordinary conditions. This requirement for viral biosynthesis could be bypassed by using a soluble inhibitor of NA,2,3-dehydro-2-deoxy-N-acetyl neuraminic acid (DDAN), to facilitate cytosolic introduction of virus. APC exposed to UV virus/DDAN present HA and NA determinants derived directly from proteins of the input virus particles. This allows presentation of both endocytically and cytosolically processed epitopes in the same experiment using noninfectious virus. The inhibitor brefeldin A (BFA) was used to interrupt host protein transport at various times relative to virus/DDAN addition. We observed that BFA added simultaneously with virus blocked recognition of NA 79-93 but not HA 111-119. This distinction was found to be based upon different expression kinetics of the HA and NA determinants. Expression of NA 79-93 required 6 to 9 h, whereas HA 111-119 was presented by 1 h after Ag addition. When APC were incubated with BFA at intervals before virus addition, presentation of HA 111-119 was also blocked as a function of time. Data indicate that about 5 h of BFA treatment is needed to deplete host protein pools required for presentation of I-Ed-restricted T cell determinants processed from either endosomes or the cytosol.     

Human-like receptor specificity does not affect the neuraminidase-inhibitor susceptibility of H5N1 influenza viruses

If highly pathogenic H5N1 influenza viruses acquire affinity for human rather than avian respiratory epithelium, will their susceptibility to neuraminidase (NA) inhibitors (the likely first line of defense against an influenza pandemic) change as well? Adequate pandemic preparedness requires that this question be answered. We generated and tested 31 recombinants of A/Vietnam/1203/04 (H5N1) influenza virus carrying single, double, or triple mutations located within or near the receptor binding site in the hemagglutinin (HA) glycoprotein that alter H5 HA binding affinity or specificity. To gain insight into how combinations of HA and NA mutations can affect the sensitivity of H5N1 virus to NA inhibitors, we also rescued viruses carrying the HA changes together with the H274Y NA substitution, which was reported to confer resistance to the NA inhibitor oseltamivir. Twenty viruses were genetically stable. The triple N158S/Q226L/N248D HA mutation (which eliminates a glycosylation site at position 158) caused a switch from avian to human receptor specificity. In cultures of differentiated human airway epithelial (NHBE) cells, which provide an ex vivo model that recapitulates the receptors in the human respiratory tract, none of the HA-mutant recombinants showed reduced susceptibility to antiviral drugs (oseltamivir or zanamivir). This finding was consistent with the results of NA enzyme inhibition assay, which appears to predict influenza virus susceptibility in vivo. Therefore, acquisition of human-like receptor specificity does not affect susceptibility to NA inhibitors. Sequence analysis of the NA gene alone, rather than analysis of both the NA and HA genes, and phenotypic assays in NHBE cells are likely to adequately identify drug-resistant H5N1 variants isolated from humans during an outbreak.     

Characterization of mutants of influenza A virus selected with the neuraminidase inhibitor 4-guanidino-Neu5Ac2en.

The development of viral resistance to the neuraminidase (NA) inhibitor, 4-guanidino-Neu5Ac2en, of influenza viruses was studied by serial passage of A/Turkey/Minnesota/833/80 (H4N2) in Madin-Darby canine kidney cells in the presence of increasing concentrations of inhibitor. Resistant mutants selected after eight passages, had a 10,000-fold reduction in sensitivity to the inhibitor in plaque assays, but their affinity (1/Kd) to the inhibitor was similar to that of the parental virus. Electron microscopic analysis revealed aggregation of the mutant virus at the cell surface in the presence of the inhibitor. Sequence analysis established that a substitution had occurred in the NA (Arg-249 to Lys) and in the HA2 subunit of the hemagglutinin (Gly-75 to Glu), in the vicinity of the proposed second sialic acid binding site. The change of residue 249 appears to be a chance mutation, for we were unable to reisolate this mutant, whereas subsequent experiments indicate changes in the hemagglutinin. After 13 passages of the parental virus, mutants that were resistant to the high concentrations of inhibitor tested were obtained. These viruses retained their drug-resistant phenotype even after five passages without the inhibitor. Electron microscopic analysis revealed no aggregation of virus on the surface of infected cells in the presence of the inhibitor. Sequence analysis of the NA gene from these drug-resistant mutants revealed an additional substitution of Glu to Ala at the conserved amino acid residue 119. This substitution is responsible for reducing the affinity of the inhibitor to the NA. Our findings suggest that the emergence of mutants resistant to 4-guanidine-Neu5Ac2en is a multistep process requiring prolonged exposure to the inhibitor.     

Influenza A viruses possessing type B hemagglutinin and neuraminidase: potential as vaccine components

A licensed live attenuated influenza vaccine is available as a trivalent mixture of types A (H1N1 and H3N2) and B vaccine viruses. Thus, interference among these viruses could restrict their replication, affecting vaccine efficacy. One approach to overcoming this potential problem is to use a chimeric virus possessing type B hemagglutinin (HA) and neuraminidase (NA) in a type A vaccine virus background. We previously generated a type A virus possessing a chimeric HA in which the entire ectodomain of the type A HA molecule was replaced with that of the type B HA, and showed that this virus protected mice from challenge by a wild-type B virus. In the study described here, we generated type A/B chimeric viruses carrying not only the chimeric (A/B) HA, but also the full-length type B NA instead of the type A NA, resulting in (A/B) HA/NA chimeric viruses possessing type B HA and NA ectodomains in the background of a type A virus. These (A/B) HA/NA chimeric viruses were attenuated in both cell culture and mice as compared with the wild-type A virus. Our findings may allow an effective live influenza vaccine to be produced from a single master strain, providing a model for the design of future live influenza vaccines.     

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.     

共表达禽流感病毒HA和NA基因重组禽痘病毒的遗传稳定性

将表达禽流感病毒H5HA及N1NA基因的重组禽痘病毒rFPV HA NA连续传代至 2 5代 ,取第 5、15及 2 5代重组病毒作为受检代次 ,进行外源基因的PCR扩增与测序 ,同时比较这 3个代次重组禽痘病毒的免疫效力。结果对各代次重组病毒DNA的模板进行PCR扩增 ,均能够获得HA和NA两个目的片段 ;经测序证明两个外源基因在细胞传代过程中没有发生氨基酸水平的改变。动物试验结果表明 ,该重组病毒的毒力在细胞传代过程中没有发生变化 ,接种试验鸡后在鸡体内能够检测到外源基因的存在 ,各免疫组在免疫 2周后的抗体效价平均为 6 5log2 ,完全抵抗了高致病力禽流感病毒的攻击。以上结果表明此重组病毒具有较好的遗传稳定性 ,经过 2 5代的传代后 ,所插入的外源基因及其表达产物的免疫原性未见变化 ,重组病毒的免疫效力相当稳定。     

Adjustment of receptor-binding and neuraminidase substrate specificties in avian–human reassortant influenza viruses

Balanced action of hemagglutinin (HA) and neuraminidase (NA) is an important condition of influenza virus efficient replication, but a role of HA and NA specificities at oligosaccharide level in maintaining such a balance remains poorly studied. Avian virus HA binds exclusively and NA digests efficiently α2–3-sialylated carbohydrate chains, while human virus HA interacts with α2–6 chains and low-active NA cleaves both α2–3- and α2–6-sialosides. Reassortment between viruses leading to appearance of avian virus HA and human virus NA on the virion surface often resulted in decreasing the replicative potential of the formed variants because of disturbance of a functional balance between “alien” HA and NA. A restoration of the reassortant productivity happened due to the appearance of amino acid substitutions in HA and, sometimes, NA. Here, a role of NA and HA oligosaccharide specificities in a restoration of HA–NA functional balance in high-yield passage variants was studied. Postreassortment changes in HA receptor-binding and NA substrate specificities for three reassortant/passage variant virus pairs towards 3′SiaLac, 3′SiaLacNAc, SiaLe^c, SiaLe^a, SiaLe^x, 6′SiaLac, and 6′SiaLacNAc were determined. Selection of the high-yield variants of the human-avian reassortants led either to twofold decrease in the affinity of HA for most α2–3-sialosides and the appearance of affinity for α2–6-sialosides (H3N2 reassortant), or to decreasing the HA affinity for SiaLe^c and SiaLe^a (H3N1 reassortant), or to enhancing the ability of NA to discriminate between α2–3/2–6 substrates (H4N1 reassortant). Thus, all postreassortment changes in oligosaccharide specificities of “alien” HA and NA were directed towards their adjustment to each other, but by different manner.     

Influenza Virus Neuraminidases with Reduced Enzymatic Activity That Avidly Bind Sialic Acid Receptors

Influenza virus neuraminidase (NA) cleaves off sialic acid from cellular receptors of hemagglutinin (HA) to enable progeny escape from infected cells. However, NA variants (D151G) of recent human H3N2 viruses have also been reported to bind receptors on red blood cells, but the nature of these receptors and the effect of the mutation on NA activity were not established. Here, we compare the functional and structural properties of a human H3N2 NA from A/Tanzania/205/2010 and its D151G mutant, which supports HA-independent receptor binding. While the wild-type NA efficiently cleaves sialic acid from both α2-6- and α2-3-linked glycans, the mutant exhibits much reduced enzymatic activity toward both types of sialosides. Conversely, while wild-type NA shows no detectable binding to sialosides, the D151G NA exhibits avid binding with broad specificity toward α2-3 sialosides. D151G NA binds the 3′ sialyllactosamine (3′-SLN) and 6′-SLN sialosides with equilibrium dissociation constant (K_D) values of 30.0 μM and 645 μM, respectively, which correspond to much higher affinities than the corresponding affinities (low mM) of HA to these glycans. Crystal structures of wild-type and mutant NAs reveal the structural basis for glycan binding in the active site by exclusively impairing the glycosidic bond hydrolysis step. The general significance of D151 among influenza virus NAs was further explored by introducing the D151G mutation into three N1 NAs and one N2 NA, which all exhibited reduced enzymatic activity and preferential binding to α2-3 sialosides. Since the enzymatic and binding activities of NAs are not routinely assessed, the potential for NA receptor binding to contribute to influenza virus biology may be underappreciated.     

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.