For influenza virus, we developed an efficient, noncytotoxic, plasmid-based virus-like particle (VLP) system to reflect authentic virus particles. This system was characterized biochemically by analysis of VLP protein composition, morphologically by electron microscopy, and functionally with a VLP infectivity assay. The VLP system was used to address the identity of the minimal set of viral proteins required for budding. Combinations of viral proteins were expressed in cells, and the polypeptide composition of the particles released into the culture media was analyzed. Contrary to previous findings in which matrix (M1) protein was considered to be the driving force of budding because M1 was found to be released copiously into the culture medium when M1 was expressed by using the vaccinia virus T7 RNA polymerase-driven overexpression system, in our noncytotoxic VLP system M1 was not released efficiently into the culture medium. Additionally, hemagglutinin (HA), when treated with exogenous neuraminidase (NA) or coexpressed with viral NA, could be released from cells independently of M1. Incorporation of M1 into VLPs required HA expression, although when M1 was omitted from VLPs, particles with morphologies similar to those of wild-type VLPs or viruses were observed. Furthermore, when HA and NA cytoplasmic tail mutants were included in the VLPs, M1 failed to be efficiently incorporated into VLPs, consistent with a model in which the glycoproteins control virus budding by sorting to lipid raft microdomains and recruiting the internal viral core components. VLP formation also occurred independently of the function of Vps4 in the multivesicular body pathway, as dominant-negative Vps4 proteins failed to inhibit influenza VLP budding.Viruses that derive their lipid envelope by budding from the plasma membrane undergo a complex, multistep assembly process involving the proper organization of viral proteins at the cell membrane and formation of the viral particle by pinching off from the cell surface (53). For influenza virus, an enveloped, negative-stranded, segmented-RNA virus, assembly requires the coalescence of hemagglutinin (HA), neuraminidase (NA), and the M2 ion channel protein (viral membrane proteins) and matrix protein (M1) and ribonucleoprotein (RNP) complexes (soluble viral components) (54). Influenza viruses bud from discrete lipid raft microdomains (52), and the concentration of HA and NA into lipid raft microdomains is both an intrinsic property of the surface glycoproteins (32) and a requirement for efficient virus replication (2, 59). Proper virion assembly is thought to involve a series of protein-protein interactions between the glycoproteins and the internal soluble components. The prevailing model of influenza virus assembly suggests that M1 underlies the lipid bilayer and coordinates assembly between the surface glycoproteins through interactions with their cytoplasmic tails, concomitantly forming a bridge to the internal RNP complexes making up the viral core (3,54).Studies concerning the roles of ...

The assembly and budding of influenza A virus is a complex, multistep process involving the proper organization of viral proteins at the plasma membrane, incorporation of a segmented genome, and formation of viral particles by pinching off from the cell surface (reviewed in reference 51). The segmented genome of influenza virus is made up of eight singlestranded, negative-sense RNA segments that are wrapped around the outside of the nucleoprotein (NP) subunits to form viral ribonucleoprotein (vRNP) complexes. The matrix protein (M1) is the major internal structural protein and underlies the viral envelope, and it is thought to form contacts between the vRNPs and the cytoplasmic tails of the viral integral membrane proteins. The spike glycoproteins hemagglutinin (HA) and neuraminidase (NA) are the predominant viral surface proteins and are the major antigenic determinants of influenza virus (reviewed in reference 27). The M2 protein is translated from a spliced mRNA derived from genome segment 7 (28); the protein is abundantly expressed on the cell surface, and small amounts of M2 are incorporated into virions (ϳ15 tetramers per virion) (19,29,54,62,63). The M2 protein has a proton-selective ion channel activity (13,36,43). Although only a low level of expression of M2 is necessary for virus replication (56), the ion channel activity is required for acidification of the viral particle within the late endosome during virus entry. The influx of H ϩ ions disrupts interactions between the vRNPs and M1, allowing the RNPs to be released into the cytoplasm devoid of M1 protein after fusion of the virus and cellular membrane has occurred (reviewed in references 17, 26, and 53). During virus assembly, there is evidence that each of the viral surface integral membrane proteins participates in forming a complete viral particle. Data obtained using mutant HA and NA protein-containing viruses generated by reverse genetics indicated a role for the HA and NA cytoplasmic tails in controlling virus morphology (25), virus assembly (66), and genome packaging (65). The accumulation of HA and NA into lipid microdomains on the plasma membrane is an intrinsic property of each protein that is required to facilitate efficient virus budding and replication (4,18,30,49,55). Furthermore, biochemical evidence supports a model in which HA and NA recruit M1 to lipid microdomains (1,15,66), presumably through specific features of the cytoplasmic domains of HA (e.g., palmitoylation sites [12]) and NA (e.g., a critical proline residue [5,35]). The importance of HA and NA in virus assembly was also observed in a biologically relevant influenza virus-like particle (influenza VLP) system (11).Whereas the M2 ion channel activity is essential for influenza virus replication primarily during virus entry, structural elements of the M2 protein appear to be important at other stages of the virus replicative cycle. Several studies have now shown that, although posttranslational modifications such as phosphorylation (20,57) and palmitoylation (8,20) of the ...

Many enveloped viruses complete their replication cycle by forming vesicles that bud from the plasma membrane. Some viruses encode "late" (L) domain motifs that are able to hijack host proteins involved in the vacuolar protein sorting (VPS) pathway, a cellular budding process that gives rise to multivesicular bodies and that is topologically equivalent to virus budding. Although many enveloped viruses share this mechanism, examples of viruses that require additional viral factors and viruses that appear to be independent of the VPS pathway have been identified. Alternative mechanisms for virus budding could involve other topologically similar process such as cell abscission, which occurs following cytokinesis, or virus budding could proceed spontaneously as a result of lipid microdomain accumulation of viral proteins. Further examination of novel virus-host protein interactions and characterization of other enveloped viruses for which budding requirements are currently unknown will lead to a better understanding of the cellular processes involved in virus assembly and budding.

JC virus (JCV) is a common human polyomavirus that infects greater than 70% of the general population worldwide. JCV is also the causative agent of progressive multifocal leukoencephalopathy (PML), a fatal demyelinating disease of the CNS. Currently, little is known about the mechanisms that restrict JCV tropism to a few human cell types and tissues. In vivo, JCV can be detected in oligodendrocytes and astrocytes in the CNS of patients with PML. The virus can also be detected in kidney, tonsil, and B lymphocytes of patients both with and without PML. In vitro, JCV can only be propagated in cultures of human fetal glial cells or in cell lines derived from this tissue. In contrast, the closely related monkey polyomavirus, SV40, has a broad tropism for primate cells, including those cells that are also susceptible to infection by JCV. We hypothesized that one potential block to infection is at the level of virus entry. To examine this, we constructed a JCV-SV40 chimeric viral genome that contains the regulatory region and the early genes of SV40 and the late structural genes of JCV. The hybrid virus (JCSV) induced SV40-like cytopathic effect in human glial cells and hemagglutinated human type O red blood cells similar to JCV. More importantly, the hybrid virus maintained the host range of JCV, suggesting that interactions between the virus capsid and host cell receptors contribute to JCV tropism.

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