Architecture of a nascent viral fusion pore

Enveloped viruses use specialized protein machinery to fuse the viral membrane with that of the host cell during cell invasion. In influenza virus, hundreds of copies of the haemagglutinin (HA) fusion glycoprotein project from the virus surface. Despite intensive study of HA and its fusion activity, the protein's modus operandi in manipulating viral and target membranes to catalyse their fusion is poorly understood. Here, the three‐dimensional architecture of influenza virus–liposome complexes at pH 5.5 was investigated by electron cryo‐tomography. Tomographic reconstructions show that early stages of membrane remodeling take place in a target membrane‐centric manner, progressing from punctate dimples, to the formation of a pinched liposomal funnel that may impinge on the apparently unperturbed viral envelope. The results suggest that the M1 matrix layer serves as an endoskeleton for the virus and a foundation for HA during membrane fusion. Fluorescence spectroscopy monitoring fusion between liposomes and virions shows that leakage of liposome contents takes place more rapidly than lipid mixing at pH 5.5. The relation of ‘leaky’ fusion to the observed prefusion structures is discussed.     

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.     

Fusion of enveloped viruses with cells and liposomes. Activity and inactivation.

The fusion of viruses with cells and liposomes is reviewed with focus on the analysis of the final extents and kinetics of fusion. Influenza virus and Sendai virus exhibit 100% of fusion capacity with cells at pH 5 and pH 7.5, respectively. On the other hand, there may be in certain cases, a limit on the number of virions that can fuse with a single cell, that is significantly below the limit on binding. It still remains to be resolved whether this limit reflects a limited number of possible fusion sites, or a saturation limit on the amount of viral glycoproteins that can be incorporated in the cellular membrane, like the case of virus fusion with pure phospholipid vesicles, in which the fusion products were shown to consist of a single virus and several liposomes. Both viruses demonstrate incomplete fusion activity towards liposomes of a variety of compositions. In the case of Sendai virus, fusion inactive virions bind essentially irreversibly to liposomes. Yet, preliminary results revealed that such bound, unfused virions can be released by sucrose gradient centrifugation. The separated unfused virions subsequently fuse when incubated with a "fresh" batch of liposomes. We conclude, therefore, that the fraction of initially bound unfused virions does not consist of dective particles, but rather of particles bound to liposomes via "inactive" sites. Details of the low pH inactivation of fusion capacity of influenza virus towards cells and liposomes are presented. This inactivation is caused by protonation and exposure of the hydrophobic segment of HA2, and affects primarily the fusion rate constants. Some degree of inactivation also occurs when virions are bound to cellular membranes.     

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.     

The membrane fusion step of vaccinia virus entry is cooperatively mediated by multiple viral proteins and host cell components

For many viruses, one or two proteins allow cell attachment and entry, which occurs through the plasma membrane or following endocytosis at low pH. In contrast, vaccinia virus (VACV) enters cells by both neutral and low pH routes; four proteins mediate cell attachment and twelve that are associated in a membrane complex and conserved in all poxviruses are dedicated to entry. The aim of the present study was to determine the roles of cellular and viral proteins in initial stages of entry, specifically fusion of the membranes of the mature virion and cell. For analysis of the role of cellular components, we used well characterized inhibitors and measured binding of a recombinant VACV virion containing Gaussia luciferase fused to a core protein; viral and cellular membrane lipid mixing with a self-quenching fluorescent probe in the virion membrane; and core entry with a recombinant VACV expressing firefly luciferase and electron microscopy. We determined that inhibitors of tyrosine protein kinases, dynamin GTPase and actin dynamics had little effect on binding of virions to cells but impaired membrane fusion, whereas partial cholesterol depletion and inhibitors of endosomal acidification and membrane blebbing had a severe effect at the later stage of core entry. To determine the role of viral proteins, virions lacking individual membrane components were purified from cells infected with members of a panel of ten conditional-lethal inducible mutants. Each of the entry protein-deficient virions had severely reduced infectivity and except for A28, L1 and L5 greatly impaired membrane fusion. In addition, a potent neutralizing L1 monoclonal antibody blocked entry at a post-membrane lipid-mixing step. Taken together, these results suggested a 2-step entry model and implicated an unprecedented number of viral proteins and cellular components involved in signaling and actin rearrangement for initiation of virus-cell membrane fusion during poxvirus entry.     

Role of lipids in virus replication

Viruses intricately interact with and modulate cellular membranes at several stages of their replication, but much less is known about the role of viral lipids compared to proteins and nucleic acids. All animal viruses have to cross membranes for cell entry and exit, which occurs by membrane fusion (in enveloped viruses), by transient local disruption of membrane integrity, or by cell lysis. Furthermore, many viruses interact with cellular membrane compartments during their replication and often induce cytoplasmic membrane structures, in which genome replication and assembly occurs. Recent studies revealed details of membrane interaction, membrane bending, fission, and fusion for a number of viruses and unraveled the lipid composition of raft-dependent and -independent viruses. Alterations of membrane lipid composition can block viral release and entry, and certain lipids act as fusion inhibitors, suggesting a potential as antiviral drugs. Here, we review viral interactions with cellular membranes important for virus entry, cytoplasmic genome replication, and virus egress.     

Tamiflu-resistant but HA-mediated cell-to-cell transmission through apical membranes of cell-associated influenza viruses

The infection of viruses to a neighboring cell is considered to be beneficial in terms of evasion from host anti-virus defense systems. There are two pathways for viral infection to "right next door": one is the virus transmission through cell-cell fusion by forming syncytium without production of progeny virions, and the other is mediated by virions without virus diffusion, generally designated cell-to-cell transmission. Influenza viruses are believed to be transmitted as cell-free virus from infected cells to uninfected cells. Here, we demonstrated that influenza virus can utilize cell-to-cell transmission pathway through apical membranes, by handover of virions on the surface of an infected cell to adjacent host cells. Live cell imaging techniques showed that a recombinant influenza virus, in which the neuraminidase gene was replaced with the green fluorescence protein gene, spreads from an infected cell to adjacent cells forming infected cell clusters. This type of virus spreading requires HA activation by protease treatment. The cell-to-cell transmission was also blocked by amantadine, which inhibits the acidification of endosomes required for uncoating of influenza virus particles in endosomes, indicating that functional hemagglutinin and endosome acidification by M2 ion channel were essential for the cell-to-cell influenza virus transmission. Furthermore, in the cell-to-cell transmission of influenza virus, progeny virions could remain associated with the surface of infected cell even after budding, for the progeny virions to be passed on to adjacent uninfected cells. The evidence that cell-to-cell transmission occurs in influenza virus lead to the caution that local infection proceeds even when treated with neuraminidase inhibitors.     

Target Membrane Cholesterol Modulates Single Influenza Virus Membrane Fusion Efficiency but Not Rate

Host lipid composition influences many stages of the influenza A virus (IAV) entry process, including initial binding of IAV to sialylated glycans, fusion between the viral envelope and the host membrane, and the formation of a fusion pore through which the viral genome is transferred into a target cell. In particular, target membrane cholesterol has been shown to preferentially associate with virus receptors and alter physical properties of the membrane like fluidity and curvature. These properties affect both IAV binding and fusion, which makes it difficult to isolate the role of cholesterol in IAV fusion from receptor binding effects. Here, we develop a fusion assay that uses synthetic DNA-lipid conjugates as surrogate viral receptors to tether virions to target vesicles. To avoid the possibly perturbative effect of adding a self-quenched concentration of dye-labeled lipids to the viral membrane, we tether virions to lipid-labeled target vesicles and use fluorescence microscopy to detect individual, pH-triggered IAV membrane fusion events. Through this approach, we find that cholesterol in the target membrane enhances the efficiency of single-particle IAV lipid mixing, whereas the rate of lipid mixing is independent of cholesterol composition. We also find that the single-particle kinetics of influenza lipid mixing to target membranes with different cholesterol compositions is independent of receptor binding, suggesting that cholesterol-mediated spatial clustering of viral receptors within the target membrane does not significantly affect IAV hemifusion. These results are consistent with the hypothesis that target membrane cholesterol increases lipid mixing efficiency by altering host membrane curvature.     

A unique phosphatidylinositol bearing a novel branched-chain fatty acid from Rhodococcus equi binds to influenza virus hemagglutinin and inhibits the infection of cells

From the aquatic bacterium Rhodococcus equi strain S(420), we isolated a substance that strongly binds to influenza viruses. Structural analyses revealed that it is a unique type of phosphatidylinositol (PtdIns) bearing a branched-chain fatty acid (14-methyloctadecanoic acid). In a TLC/virus-binding immunostaining assay, this PtdIns bound to all subtypes of hemagglutinin (HA) of influenza A viruses tested, isolated from humans, ducks and swine, and also to human influenza B viruses. Furthermore, the PtdIns significantly prevented the infection of MDCK cells by influenza viruses, and also inhibited the virus-mediated hemagglutination and low pH-induced hemolysis of human erythrocytes, which represents the fusogenic activities of the viral HA. We also used purified hemagglutinin instead of virions to examine the interaction between viral HA and PtdIns, showing that the PtdIns binds to hemagglutinin. These findings indicate that the inhibitory mechanism of PtdIns on the influenza virus infection may be through its binding to viral HA spikes and host cell endosomal/lysosomal membranes, which are mediated by the function of viral HA.     

Stoichiometry of envelope glycoprotein trimers in the entry of human immunodeficiency virus type 1

The human immunodeficiency virus type 1 (HIV-1) envelope glycoproteins (Envs) function as a trimer, mediating virus entry by promoting the fusion of the viral and target cell membranes. HIV-1 Env trimers induce membrane fusion through a pH-independent pathway driven by the interaction between an Env trimer and its cellular receptors, CD4 and CCR5/CXCR4. We studied viruses with mixed heterotrimers of wild-type and dominant-negative Envs to determine the number (T) of Env trimers required for HIV-1 entry. To our surprise, we found that a single Env trimer is capable of supporting HIV-1 entry; i.e., T = 1. A similar approach was applied to investigate the entry stoichiometry of envelope glycoproteins from amphotropic murine leukemia virus (A-MLV), avian sarcoma/leukosis virus type A (ASLV-A), and influenza A virus. When pseudotyped on HIV-1 virions, the A-MLV and ASLV-A Envs also exhibit a T = 1 entry stoichiometry. In contrast, eight to nine influenza A virus hemagglutinin trimers function cooperatively to achieve membrane fusion and virus entry, using a pH-dependent pathway. The different entry requirements for cooperativity among Env trimers for retroviruses and influenza A virus may influence viral strategies for replication and evasion of the immune system.     

Mini-plasmin found in the epithelial cells of bronchioles triggers infection by broad-spectrum influenza A viruses and Sendai virus.

Extracellular cleavage of virus envelope fusion glycoproteins by host cellular proteases is a prerequisite for the infectivity of mammalian and nonpathogenic avian influenza viruses, and Sendai virus. Here we report a protease present in the airway that, like tryptase Clara, can process influenza A virus haemagglutinin and Sendai virus envelope fusion glycoprotein. This protease was extracted from the membrane fraction of rat lungs, purified and then identified as a mini-plasmin. Mini-plasmin was distributed predominantly in the epithelial cells of the upward divisions of bronchioles and potentiated the replication of broad-spectrum influenza A viruses and Sendai virus, even that of the plasmin-insensitive influenza A virus strain. In comparison with plasmin, its increased hydrophobicity, leading to its higher local concentrations on membranes, and decreased molecular mass may enable mini-plasmin to gain ready access to the cleavage sites of various haemagglutinins and fusion glycoproteins after expression of these viral proteins on the cell surface. These findings suggest that mini-plasmin in the airway may play a pivotal role in the spread of viruses and their pathogenicity.     

Molecular mechanisms of virus spread and virion components as tools of virulence. A review

Despite of differences in replication strategy among virus families, some basic principles have remained similar. Analogous mechanisms govern virus entry into cells and the use of enzymes which direct the replication of the virus genome. The function of many cell surface receptors (such as glycosoaminoglycans, glycoproteins, proteins) which interact with viral capsid proteins or envelope glycoproteins has recently been elucidated. The list of cellular receptors (Table I) is still far from being final. The capsid components, similarly as the envelope glycoproteins, may form specific pocket like sites, which interact with the cell surface receptors. Neutralizing antibodies usually react with antigenic domains adjacent to the receptor binding site(s) and hamper the close contact inevitable for virion attachment. In the case of more complex viruses, such as herpes simplex virus, different viral glycoproteins interact with several cellular receptors. At progressed phase of adsorption the virions are engulfed into endocytic vesicles and the virion fusion domain(s) become(s) activated. The outer capsid components of reoviruses which participate in adsorption and fusion may get activated already in the lumen of digestive tract, i.e. before their engulfment by resorptive epithelium cells. Activation of the hydrophobic fusion domain(s) is a further important step allowing to pass through the lipid bilayer when penetrating the cell membrane in order to reach the cytosol. Activation of the virion fusion domain is accomplished by a conformation change, which occurs at acid pH (influenza virus hemagglutinin, sigma 1 protein of the reovirus particle) and/or after protease treatment. The herpes simplex virus fusion factors (gD and gH) undergo conformation changes by a pH-independent mechanism triggered due to interaction with the cell surface receptor(s) and mediated by mutual interactions with the viral envelope glycoproteins. The virion capsid or envelope components participating in the entry and membrane fusion are not the only tools of virulence. The correct function of virus coded proteins, which participate in replication of the viral genome, and/or in the supply of necessary nucleotides, may be very essential. In the case of enteroviruses, which RNA interacts with ribosomes directly, the correct configuration of the non-coding viral RNA sequence is crucial for initiation of translation occurring in the absence of the classical "cap" structure.     

Cellular proteinases trigger the infectivity of the influenza A and Sendai viruses.

It has been proposed that the pathogenicity of the influenza and Sendai virus is primarily determined by host cellular proteases that activate viral infectivity. We isolated trypsin-type serine proteases from rat lungs, candidates for the processing proteases of viral envelope glycoproteins, such as tryptase Clara localized in the Clara cells of the bronchial epithelium and mini-plasmin. These enzymes specifically cleave the precursor of fusion glycoprotein HA of influenza virus at Arg325, and the F0 of Sendai virus at Arg116 in the consensus cleavage motif, Gln(Glu)-X-Arg, resulting in the induction of infectivity of these viruses. Proteolytic activation of viruses by these enzymes occurs extracellularly, probably on the surface and/or in the lumen of the respiratory tract. On the other hand, we isolated two compounds from human bronchial lavage, which inhibit the activity of tryptase Clara. One was a mucus protease inhibitor and the other was a pulmonary surfactant. These compounds inhibited multiple cycles of virus replication in vitro and in vivo, but did not themselves affect the hemagglutination and the infectivity of the virus. Administration of these compounds in the airway may be useful for preventing and treating infection with influenza virus and Sendai virus.     

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

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

Rapid membrane fusion of individual virus particles with supported lipid bilayers

Many enveloped viruses employ low-pH-triggered membrane fusion during cell penetration. Solution-based in vitro assays in which viruses fuse with liposomes have provided much of our current biochemical understanding of low-pH-triggered viral membrane fusion. Here, we extend this in vitro approach by introducing a fluorescence assay using single particle tracking to observe lipid mixing between individual virus particles (influenza or Sindbis) and supported lipid bilayers. Our single-particle experiments reproduce many of the observations of the solution assays. The single-particle approach naturally separates the processes of membrane binding and membrane fusion and therefore allows measurement of details that are not available in the bulk assays. We find that the dynamics of lipid mixing during individual Sindbis fusion events is faster than 30 ms. Although neither virus binds membranes at neutral pH, under acidic conditions, the delay between membrane binding and lipid mixing is less than half a second for nearly all virus-membrane combinations. The delay between binding and lipid mixing lengthened only for Sindbis virus at the lowest pH in a cholesterol-dependent manner, highlighting the complex interaction between lipids, virus proteins, and buffer conditions in membrane fusion.     

Autocatalytic activation of influenza hemagglutinin

Enveloped viruses contain surface proteins that mediate fusion between the viral and target cell membranes following an activating stimulus. Acidic pH induces the influenza virus fusion protein hemagglutinin (HA) via irreversible refolding of a trimeric conformational state leading to exposure of hydrophobic fusion peptides on each trimer subunit. Herein, we show that cells expressing fowl plague virus HA demonstrate discrete switching behavior with respect to the HA conformational change. Partially activated states do not exist at the scale of the cell, activation of HA leads to aggregation of cell surface trimers, and newly synthesized HA refold spontaneously in the presence of previously activated HA. These observations imply a feedback mechanism involving self-catalyzed refolding of HA and thus suggest a mechanism similar to the autocatalytic refolding and aggregation of prions.     

Effect of membrane curvature-modifying lipids on membrane fusion by tick-borne encephalitis virus

Enveloped viruses enter cells by fusion of their own membrane with a cellular membrane. Incorporation of inverted-cone-shaped lipids such as lysophosphatidylcholine (LPC) into the outer leaflet of target membranes has been shown previously to impair fusion mediated by class I viral fusion proteins, e.g., the influenza virus hemagglutinin. It has been suggested that these results provide evidence for the stalk-pore model of fusion, which involves a hemifusion intermediate (stalk) with highly bent outer membrane leaflets. Here, we investigated the effect of inverted-cone-shaped LPCs and the cone-shaped oleic acid (OA) on the membrane fusion activity of a virus with a class II fusion protein, the flavivirus tick-borne encephalitis virus (TBEV). This study included an analysis of lipid mixing, as well as of the steps preceding or accompanying fusion, i.e., binding to the target membrane and lipid-induced conformational changes in the fusion protein E. We show that the presence of LPC in the outer leaflet of target liposomes strongly inhibited TBEV-mediated fusion, whereas OA caused a very slight enhancement, consistent with a fusion mechanism involving a lipid stalk. However, LPC also impaired the low-pH-induced binding of a soluble form of the E protein to liposomes and its conversion into a trimeric postfusion structure that requires membrane binding at low pH. Because inhibition is already observed before the lipid-mixing step, it cannot be determined whether impairment of stalk formation is a contributing factor in the inhibition of fusion by LPC. These data emphasize, however, the importance of the composition of the target membrane in its interactions with the fusion peptide that are crucial for the initiation of fusion.     

Structural changes in Influenza virus at low pH characterized by cryo-electron tomography

Influenza virus enters host cells by endocytosis. The low pH of endosomes triggers conformational changes in hemagglutinin (HA) that mediate fusion of the viral and endosomal membranes. We have used cryo-electron tomography to visualize influenza A virus at pH 4.9, a condition known to induce fusogenicity. After 30 min, when all virions are in the postfusion state, dramatic changes in morphology are apparent: elongated particles are no longer observed, larger particles representing fused virions appear, the HA spikes become conspicuously disorganized, a layer of M1 matrix protein is no longer resolved on most virions, and the ribonucleoprotein complexes (RNPs) coagulate on the interior surface of the virion. To probe for intermediate states, preparations were imaged after 5 min at pH 4.9. These virions could be classified according to their glycoprotein arrays (organized or disorganized) and whether or not they have a resolved M1 layer. Employing subtomogram averaging, we found, in addition to the neutral-pH state of HA, two intermediate conformations that appear to reflect an outwards movement of the fusion peptide and rearrangement of the HA1 subunits, respectively. These changes are reversible. The tomograms also document pH-induced changes affecting the M1 layer that appear to render the envelope more pliable and hence conducive to fusion. However, it appears desirable for productive infection that fusion should proceed before the RNPs become coagulated with matrix protein, as eventually happens at low pH.     

Effect of the cytoplasmic domain of the simian immunodeficiency virus envelope protein on incorporation of heterologous envelope proteins and sensitivity to neutralization

In addition to the viral envelope (Env) proteins, host cell-derived proteins have been reported to be present in human immunodeficiency virus and simian immunodeficiency virus (SIV) envelopes, and it has been postulated that they may play a role in infection. We investigated whether the incorporation of host cell proteins is affected by the structure and level of incorporation of viral Env proteins. To compare the cellular components incorporated into SIV particles and how this is influenced by the structure of the cytoplasmic domain, we compared SIV virions with full-length and truncated Env proteins. The levels of HLA-I and HLA-II molecules were found to be significantly (15- to 25-fold) higher in virions with full-length Env than in those with a truncated Env. Virions with a truncated Env were also found to be less susceptible to neutralization by specific antibodies against HLA-I or HLA-II proteins. We also compared the level of incorporation into SIV virions of a coexpressed heterologous viral glycoprotein, the influenza virus hemagglutinin (HA) protein. We found that SIV infection of cells expressing influenza virus HA resulted in the production of phenotypically mixed SIV virions containing influenza virus HA as well as SIV envelope proteins. The HA proteins were more effectively incorporated into virions with full-length Env than in virions with truncated Env. The phenotypically mixed particles with full-length Env, containing higher levels of HA, were sensitive to neutralization with anti-HA antibody, whereas virions with truncated Env proteins and containing lower levels of HA were more resistant to neutralization by anti-HA antibody. In contrast, SIV virions with truncated Env proteins were found to be highly sensitive to neutralization by antisera to SIV, whereas virions with full-length Env proteins were relatively resistant to neutralization. These results indicate that the cytoplasmic domain of SIV Env affects the incorporation of cellular as well as heterologous viral membrane proteins into the SIV envelope and may be an important determinant of the sensitivity of the virus to neutralizing antibodies.     

Cellular proteins in influenza virus particles

Virions are thought to contain all the essential proteins that govern virus egress from the host cell and initiation of replication in the target cell. It has been known for some time that influenza virions contain nine viral proteins; however, analyses of other enveloped viruses have revealed that proteins from the host cell can also be detected in virions. To address whether the same is true for influenza virus, we used two complementary mass spectrometry approaches to perform a comprehensive proteomic analysis of purified influenza virus particles. In addition to the aforementioned nine virus-encoded proteins, we detected the presence of 36 host-encoded proteins. These include both cytoplasmic and membrane-bound proteins that can be grouped into several functional categories, such as cytoskeletal proteins, annexins, glycolytic enzymes, and tetraspanins. Interestingly, a significant number of these have also been reported to be present in virions of other virus families. Protease treatment of virions combined with immunoblot analysis was used to verify the presence of the cellular protein and also to determine whether it is located in the core of the influenza virus particle. Immunogold labeling confirmed the presence of membrane-bound host proteins on the influenza virus envelope. The identification of cellular constituents of influenza virions has important implications for understanding the interactions of influenza virus with its host and brings us a step closer to defining the cellular requirements for influenza virus replication. While not all of the host proteins are necessarily incorporated specifically, those that are and are found to have an essential role represent novel targets for antiviral drugs and for attenuation of viruses for vaccine purposes.