A novel peptide inhibits the influenza virus replication by preventing the viral attachment to the host cells

Avian influenza viruses (AIV), the causative agent of avian flu or bird flu, cause widespread morbidity and mortality in poultry. The symptoms of the disease range from mild flu like symptoms to death. These viruses possess two important surface glycoproteins, namely hemagglutinin (HA) and neuraminidase (NA) against which neutralizing antibodies are produced. Due to the highly mutative nature of the genes which encode these proteins, the viruses often confer resistance to the current anti-viral drugs making the prevention and treatment of infection challenging. In our laboratory, we have recently identified a novel anti-viral peptide (P1) against the AIV H9N2 from a phage displayed peptide library. This peptide inhibits the replication of the virus in ovo and in vitro by its binding to the HA glycoprotein. In the current study, we demonstrate that the peptide inhibits the virus replication by preventing the attachment to the host cell but it does not have any effect on the viral fusion. The reduction in the viral nucleoprotein (NP) expression inside the host cell has also been observed during the peptide (P1) treatment. This novel peptide may have the potential to be developed as a therapeutic agent for the treatment and control of avian influenza virus H9N2 infections.     

Study of specific oligosaccharide structures related with swine flu (H1N1) and avian flu, and tamiflu as their remedy

The infection of pandemic influenza viruses such as swine flu (H1N1) and avian flu viruses to the host cells is related to the following two factors: First, the surface protein such as HA (hemagglutinin) and NA (neuraminidase) of the influenza virus. Second, the specific structure of the oligosaccharide [sialic acid(alpha2-6) galactose(beta1-4)glucose or sialic acid(alpha2-3)galactose(beta1-4)glucose] on the host cell. After recognizing the specific structure of the oligosaccharide on the surface of host cells by the surface protein of the influenza virus, the influenza virus can secrete sialidase and cleave the sialic acid attached on the final position of the specific structure of the oligosaccharide on the surface of host cells. Tamiflu (oseltamivir), known as a remedy of swine flu, has a saccharide analog structure, especially the sialic acid analog. Tamiflu can inhibit the invasion of influenza viruses (swine flu and avian flu viruses) into the host cells by competition with sialic acid on the terminal position of the specific oligosaccharide on the surface of the host cell. Because of the emergence of Tamiflu resistance, the development of new potent anti-influenza inhibitors is needed. The inhibitors with positive-charge groups have potential as antiviral therapeutics, and the strain specificity must also be resolved.     

Biophysical Measurement of the Balance of Influenza A Hemagglutinin and Neuraminidase Activities

The interaction of influenza A viruses with the cell surface is controlled by the surface glycoproteins hemagglutinin (HA) and neuraminidase (NA). These two glycoproteins have opposing activities: HA is responsible for binding the host receptor (sialic acid) to allow infection, and NA is responsible for cleaving the receptor to facilitate virus release. Several studies have demonstrated that compatible levels of HA and NA activity are required for a virus to replicate efficiently. This is consequently of great interest for determining virus transmissibility. The concurrent role of these two proteins in receptor binding has never been directly measured. We demonstrate a novel biophysical approach based on bio-layer interferometry to measure the balance of the activities of these two proteins in real time. This technique measures virus binding to and release from a surface coated with either the human-like receptor analog α2,6-linked sialic acid or the avian-like receptor analog α2,3-linked sialic acid in both the presence and absence of NA inhibitors. Bio-layer interferometry measurements were also carried out to determine the effect of altering HA receptor affinity and NA stalk length on receptor binding.     

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.     

NA Proteins of Influenza A Viruses H1N1/2009, H5N1, and H9N2 Show Differential Effects on Infection Initiation,Virus Release,and Cell-Cell Fusion

Two surface glycoproteins of influenza virus, haemagglutinin (HA) and neuraminidase (NA), play opposite roles in terms of their interaction with host sialic acid receptors. HA attaches to sialic acid on host cell surface receptors to initiate virus infection while NA removes these sialic acids to facilitate release of progeny virions. This functional opposition requires a balance. To explore what might happen when NA of an influenza virus was replaced by one from another isolate or subtype, in this study, we generated three recombinant influenza A viruses in the background of A/PR/8/34 (PR8) (H1N1) and with NA genes obtained respectively from the 2009 pandemic H1N1 virus, a highly pathogenic avian H5N1 virus, and a lowly pathogenic avian H9N2 virus. These recombinant viruses, rPR8-H1N1NA, rPR8-H5N1NA, and rPR8-H9N2NA, were shown to have similar growth kinetics in cells and pathogenicity in mice. However, much more rPR8-H5N1NA and PR8-wt virions were released from chicken erythrocytes than virions of rPR8-H1N1NA and rPR8-H9N2NA after 1 h. In addition, in MDCK cells, rPR8-H5N1NA and rPR8-H9N2NA infected a higher percentage of cells, and induced cell-cell fusion faster and more extensively than PR8-wt and rPR8-H1N1NA did in the early phase of infection. In conclusion, NA replacement in this study did not affect virus replication kinetics but had different effects on infection initiation, virus release and fusion of infected cells. These phenomena might be partially due to NA proteins’ different specificity to α2-3/2-6-sialylated carbohydrate chains, but the exact mechanism remains to be explored.     

Adaptation of influenza A viruses to cells expressing low levels of sialic acid leads to loss of neuraminidase activity

Influenza A viruses possess two virion surface proteins, hemagglutinin (HA) and neuraminidase (NA). The HA binds to sialyloligosaccharide viral receptors, while the NA removes sialic acids from the host cell and viral sialyloligosaccarides. Alterations of the HA occur during adaptation of influenza viruses to new host species, as in the 1957 and 1968 influenza pandemics. To gain a better understanding of the contributions of the HA and possibly the NA to this process, we generated cell lines expressing reduced levels of the influenza virus receptor determinant, sialic acid, by selecting Madin-Darby canine kidney cells resistant to a lectin specific for sialic acid linked to galactose by alpha(2-3) or alpha(2-6) linkages. One of these cell lines had less than 1/10 as much N-acetylneuraminic acid as its parent cell line. When serially passaged in this cell line, human H3N2 viruses lost sialidase activity due to a large internal deletion in the NA gene, without alteration of the HA gene. These findings indicate that NA mutations can contribute to the adaptation of influenza A virus to new host environments and hence may play a role in the transmission of virus across species.     

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.     

In vitro enhancement of human natural cell-mediated cytotoxicity by purified influenza virus glycoproteins.

The role of the glycoproteins of influenza virus, hemagglutinin (HA), and neuraminidase (NA) in the in vitro stimulation of natural cell-mediated cytotoxicity (NCMC) or natural killer activity of human peripheral blood lymphocytes was evaluated with radiolabeled K562 cells as target cells in an overnight chromium release assay. Three different approaches were used. (i) Purified viral proteins were obtained by extraction with Nonidet P-40, separation on a sucrose gradient, and further purification by affinity chromatography. Ficoll-Hypaque-purified peripheral blood lymphocytes exposed to HA or NA individually or to a mixture of both significantly increased NCMC (32 to 50%). (ii) Treatment of HA and NA with their respective homologous antisera or F(ab')2 antibody abrogated the stimulation of NCMC by these glycoproteins. (iii) Virions treated with proteolytic enzymes resulted in viral cores lacking either HA or NA or both activities. Compared to whole virions, viral cores devoid of HA activity only induced a 50% increase in NCMC, whereas viral cores lacking HA activity and with traces of NA activity stimulated only 10% of the NCMC. These results suggest that influenza virus-induced cell-mediated cytotoxicity is largely due to its glycoproteins.     

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.     

流感病毒神经氨酸酶抑制剂药物耐药现状及机制研究进展

流感病毒可引起急性呼吸道传染病,严重危害人们的身体健康。神经氨酸酶抑制剂(NAI)是以神经氨酸酶为靶点的药物,可以有效抑制甲型和乙型流感病毒复制,是目前流感预防和治疗的一线用药。然而由于NA或者HA的突变,导致病毒对该药耐药。不同亚型流感病毒对神经氨酸酶抑制剂的耐药情况也不同,不同的检测方法也对判断病毒是否耐药有影响。     

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.     

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.     

Influenza virus hemagglutinin and neuraminidase cytoplasmic tails control particle shape.

The cytoplasmic tails of the influenza virus glycoproteins hemagglutinin (HA) and neuraminidase (NA) are highly conserved in sequence for all virus subtypes and it is believed that assembly of this enveloped virus depends on interactions of these domains with cytoplasmic viral components. However, it is possible to rescue altered influenza viruses lacking either the HA or NA cytoplasmic tails. We have obtained an influenza virus that lacks both the cytoplasmic tail of HA and NA. Particle production is reduced approximately 10-fold but these particles, although having a fairly normal protein composition, are greatly elongated and of extended irregular shape. We propose a model in which the interactions of the cytoplasmic tails of HA and NA with an internal viral component are so important for spherical virion shape that there is dual redundancy in the interactions.     

Influenza virus assembly and lipid raft microdomains: a role for the cytoplasmic tails of the spike glycoproteins

Influenza viruses encoding hemagglutinin (HA) and neuraminidase (NA) glycoproteins with deletions in one or both cytoplasmic tails (HAt- or NAt-) have a reduced association with detergent-insoluble glycolipids (DIGs). Mutations which eliminated various combinations of the three palmitoylation sites in HA exhibited reduced amounts of DIG-associated HA in virus-infected cells. The influenza virus matrix (M(1)) protein was also found to be associated with DIGs, but this association was decreased in cells infected with HAt- or NAt- virus. Regardless of the amount of DIG-associated protein, the HA and NA glycoproteins were targeted primarily to the apical surface of virus-infected, polarized cells. The uncoupling of DIG association and apical transport was augmented by the observation that the influenza A virus M(2) protein as well as the influenza C virus HA-esterase-fusion glycoprotein were not associated with DIGs but were apically targeted. The reduced DIG association of HAt- and NAt- is an intrinsic property of the glycoproteins, as similar reductions in DIG association were observed when the proteins were expressed from cDNA. Examination of purified virions indicated reduced amounts of DIG-associated lipids in the envelope of HAt- and NAt- viruses. The data indicate that deletion of both the HA and NA cytoplasmic tails results in reduced DIG association and changes in both virus polypeptide and lipid composition.     

Modulation of murine macrophage responses stimulated with influenza glycoproteins.

Previously it was reported that influenza virus stimulated, nonspecific resistance was largely due to its glycoproteins, hemagglutinin (HA) and neuraminidase (NA). The enhancement of natural killer cell activity was the intrinsic property of NA and HA. In the present study, the stimulatory effect of these glycoproteins on the murine peritoneal macrophages was studied. Electrophoretically purified glycoproteins, NA and HA, of influenza virus A/USSR/90/77 (H1N1) were administered intraperitoneally to C3H/HeN mice, with or without stearyl tyrosine (ST). Macrophages were isolated and were restimulated with phorbol myristate acetate. H2O2 secretion was determined by horseradish peroxidase dependent oxidation of phenol red assay. HA enhanced H2O2 secretion only in the presence of ST (60 nmol.mg-1.h-1), whereas NA alone stimulated H2O2 secretion (83 nmol.mg-1.h-1), by 6-fold over control (13 nmol.mg-1.h-1), and this stimulation was further increased (136 nmol.mg-1.h-1) in the presence of ST. Interleukin 1 (IL-1) activity was determined by using D10.G4.1 cells. There was a little stimulation of IL-1 activity (less than 1 U/mL) of macrophages isolated from HA-primed of HA+ST-primed mice restimulated with HA. On the other hand, IL-1 activity of macrophages isolated from NA-primed mice restimulated with NA significantly increased (102 U/mL) over control (less than 1 U/mL), and an additional 2-fold increase (231 U/mL) resulted when macrophages from NA+ST-primed mice were used. Tumor necrosis factor (TNF) activity was examined by using L929 cells. Negligible TNF activity was observed in macrophages isolated from either HA-primed or HA+ST-primed mice restimulated with HA.(ABSTRACT TRUNCATED AT 250 WORDS)     

Syncytium formation and HIV-1 replication are both accentuated by purified influenza and virus-associated neuraminidase.

The degree of sialylation has been shown previously to modulate the process of human immunodeficiency virus type-1 (HIV-1) infection by affecting the interaction between the virus and CD4-expressing target cells. In the present study, we investigated whether HIV-1 replication cycle was affected by neuraminidase (NA) derived from the human influenza (flu) virus. We first demonstrate that the level of HIV-1-mediated syncytium formation was greatly enhanced in the presence of purified flu NA. Pretreatment of established monocytic and lymphocytic cell lines as well as primary mononuclear cells with purified flu NA augmented also the process of virus infection. A comparable up-regulating effect was observed when using several strains of UV-inactivated whole flu virus, thereby suggesting that virus-anchored NA enzymes positively modulate the HIV-1 life cycle. Furthermore, flu NA-mediated positive effect on HIV-1 biology was abrogated with zanamivir, a specific flu NA inhibitor. Our results provide a new model allowing the investigation of the potential benefit of using NA inhibitors in the treatment of HIV-1-infected patients suffering from coinfection with NA-bearing pathogens.     

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.     

In silico analysis of genes nucleoprotein, neuraminidase and hemagglutinin: a comparative study on different strains of influenza A (Bird flu) virus sub-type H5N1

The avian influenza (bird flu) is an infectious disease of birds, ranging from a mild to a severe form of illness. Influenza viruses pose significant challenges to both human and animal health. The proteins, nucleoprotein (NP), neuraminidase (NA) and hemagglutinin (HA) of influenza A virus (Bird flu virus) sub-type A/Hatay/2004/(H5N1) from chicken were selected for this study. Our in silico analysis predicted that HA of influenza A virus is highly sensitive to mutations and hence it is significant for its pathogenic nature. None of the mutations was detected as an important change except in NA where K332R was at a PKC phosphorylation site. Analysis of the sequence comparison showed that the maximum number of mutations were observed in HA. These mutations are significant as they are involved in change in polarity or hydrophobicity as well as in propensity of each amino acid residue to stabilize the secondary structure. The program MAPMUTATION can be used to monitor the mutations, and predict the trend of mutations.     

Purified glycoproteins of influenza virus stimulate cell-mediated cytotoxicity in vivo

Previously, we reported that purified surface influenza viral glycoproteins can induce cell-mediated cytotoxicity (CMC) in vitro. Both neuraminidase (NA) and hemagglutinin (HA) were equally good stimulators, on an equimolar basis. In order to broaden the scope of these observations, we examined whether these glycoproteins stimulate natural killer (NK) activity in vivo. Biologically active preparations of glycoproteins NA and HA were purified from virus A/USSR/90/77 (H1N1) and recombinant virus A/USSR/92/77 (H1) x A/Prague/1/56 (N7), respectively. The studies were carried out using the optimal doses of NA and HA. In a 4-hour NK assay, using NK-sensitive YAC-1 cells as targets, both viral glycoproteins stimulated the NK activity of splenocytes of BALB/c and C3H mice. This stimulation was independent of the route of administration (intravenous or intraperitoneal) of the antigen. The observed NK activity was viral antigen-specific and could be modulated to levels comparable to those observed with the standard stimulator, polyinosinic acid-polycytidylic acid, by the use of an appropriate synthetic adjuvant, stearyl tyrosinate. Direct and indirect evidences suggest that the enhanced CMC is due to NK cells. These observations imply that enhancement of NK activity is the intrinsic property of influenza NA and HA.     

Interaction of influenza virus proteins with planar lipid bilayers: A model for virion assembly

Changes in conductance of oxidized cholesterol planar lipid bilayers were measured following the incorporation of isolated surface glycoproteins; hemagglutinin and neuraminidase (HA+NA) or matrix protein (M-protein) of influenza virus. The conductance dependence of the lipid bilayers on the HA+NA or M-protein concentrations indicates different mechanisms of interaction of these viral proteins with the lipid bilayer. Adsorption of M-protein molecules on one side of the lipid bilayer affects the character of the HA+NA interaction with the opposite side. Planar lipid bilayers can be a useful model for investigation of the assembly of influenza virions and other enveloped viruses.