Retroviruses acquire a lipid envelope during budding from the membrane of their hosts. Therefore, the composition of this envelope can provide important information about the budding process and its location. Here, we present mass spectrometry analysis of the lipid content of human immunodeficiency virus type 1 (HIV-1) and murine leukemia virus (MLV). The results of this comprehensive survey found that the overall lipid content of these viruses mostly matched that of the plasma membrane, which was considerably different from the total lipid content of the cells. However, several lipids are enriched in comparison to the composition of the plasma membrane: (i) cholesterol, ceramide, and GM3; and (ii) phosphoinositides, phosphorylated derivatives of phosphatidylinositol. Interestingly, microvesicles, which are similar in size to viruses and are also released from the cell periphery, lack phosphoinositides, suggesting a different budding mechanism/ location for these particles than for retroviruses. One phosphoinositide, phosphatidylinositol 4,5-bisphosphate [PI(4,5)P 2 ], has been implicated in membrane binding by HIV Gag. Consistent with this observation, we found that PI(4,5)P 2 was enriched in HIV-1 and that depleting this molecule in cells reduced HIV-1 budding. Analysis of mutant virions mapped the enrichment of PI(4,5)P 2 to the matrix domain of HIV Gag. Overall, these results suggest that HIV-1 and other retroviruses bud from cholesterol-rich regions of the plasma membrane and exploit matrix/PI(4,5)P 2 interactions for particle release from cells.Retroviruses rely on their host for many essential parts of the viral replication cycle. Biochemical and antibody-based analyses of the replication cycle and proteins found in the virions have revealed many details of the molecular interactions between human immunodeficiency virus (HIV) and its host (20). In contrast, the role of lipids has been less well studied. With the increasing recognition that lipids play an important role in cellular signaling, it is no coincidence that lipid factors are slowly gaining prominence in our understanding of retroviral replication.Retroviruses, including HIV and murine leukemia virus (MLV), acquire their lipid coats by budding through host plasma membranes. Two important issues arise when considering the roles of lipids in retrovirus assembly and budding. First, the idea that HIV and other retroviruses bud from lipid rafts has gained widespread acceptance (39, 45). Lipid rafts are liquid ordered domains that exist within the liquid disordered phase of the bulk cell membrane. These dynamic lipid-protein assemblies are characterized by high levels of cholesterol, sphingolipids, saturated glycerophospholipids, and raft proteins. Because the half-lives for lipid rafts are extremely short (50), the assignment of HIV to lipid rafts is commonly established through the colocalization of HIV proteins with putative raft proteins and the preponderance of raft lipids, including cholesterol, sphingomyelin (SM), dihydrosphingomyelin (dhSM), ce...

2Viral infections spread based on the ability of viruses to overcome multiple barriers and move from cell to cell, tissue to tissue, and person to person and even across species. While there are fundamental differences between these types of transmissions, it has emerged that the ability of viruses to utilize and manipulate cell-cell contact contributes to the success of viral infections. Central to the excitement in the field of virus cell-to-cell transmission is the idea that cell-to-cell spread is more than the sum of the processes of virus release and entry. This implies that virus release and entry are efficiently coordinated to sites of cell-cell contact, resulting in a process that is distinct from its individual components. In this review, we will present support for this model, illustrate the ability of viruses to utilize and manipulate cell adhesion molecules, and discuss the mechanism and driving forces of directional spreading. An understanding of viral cell-to-cell spreading will enhance our ability to intervene in the efficient spreading of viral infections.

SUMMARY We screened a panel of mouse and human monoclonal antibodies (MAbs) against chikungunya virus and identified several with inhibitory activity against multiple alphaviruses. Passive transfer of broadly neutralizing MAbs protected mice against infection by chikungunya, Mayaro, and O’nyong’nyong alphaviruses. Using alanine-scanning mutagenesis, loss-of-function recombinant proteins and viruses, and multiple functional assays, we determined that broadly neutralizing MAbs block multiple steps in the viral lifecycle including entry and egress, and bind to a conserved epitope on the B domain of the E2 glycoprotein. A 16 Å resolution cryo-electron microscopy structure of a Fab fragment bound to CHIKV E2 B domain provided an explanation for its neutralizing activity. Binding to the B domain was associated with repositioning of the A domain of E2 that enabled cross-linking of neighboring spikes. Our results suggest that B domain antigenic determinants could be targeted for vaccine or antibody therapeutic development against multiple alphaviruses of global concern.

Applying 4D imaging, this study investigates the mechanism by which cell-cell contact enhances retrovirus spreading and demonstrates that viral budding is highly polarized towards sites of cell-cell contact.

Summary We evaluated the mechanism by which neutralizing human monoclonal antibodies inhibit chikungunya virus (CHIKV) infection. Potently neutralizing antibodies (NAbs) blocked infection at multiple steps of the virus life cycle, including entry and release. Cryo-electron microscopy structures of Fab fragments of two human NAbs and chikungunya virus-like particles showed a binding footprint that spanned independent domains on neighboring E2 subunits within one viral spike, suggesting a mechanism for inhibiting low pH-dependent membrane fusion. Detailed epitope mapping identified residue E2-W64 as a critical interaction residue. An escape mutation (E2-W64G) at this residue rendered CHIKV attenuated in mice. Consistent with this data, CHIKV-E2-W64G failed to emerge in vivo under the selection pressure of one of the NAbs, IM-CKV063. As our study suggests that antibodies engaging the residue E2-W64 can potently inhibit CHIKV at multiple stages of infection, antibody-based therapies or immunogens that target this region might have protective value.

Claudin-1 (CLDN1), a tight junction (TJ) protein, has recently been identified as an entry co-receptor for hepatitis C virus (HCV). Ectopic expression of CLDN1 rendered several non-hepatic cell lines permissive to HCV infection. However, little is known about the mechanism by which CLDN1 mediates HCV entry. It is believed that an additional entry receptor(s) is required because ectopic expression of CLDN1 in both HeLa and NIH3T3 cells failed to confer susceptibility to viral infection. Here we found that CLDN1 was co-immunoprecipitated with both HCV envelope proteins when expressed in 293T cells. Results from biomolecular fluorescence complementation assay showed that overexpressed CLDN1 also formed complexes with CD81 and low density lipoprotein receptor. Subsequent imaging analysis revealed that CLDN1 was highly enriched at sites of cell-cell contact in permissive cell lines, co-localizing with the TJ marker, ZO-1. However, in both HeLa and NIH3T3 cells the ectopically expressed CLDN1 appeared to reside predominantly in intracellular vesicles. The CLDN1-CD81 complex formed in HeLa cells was also exclusively distributed intracellularly, co-localizing with EEA1, an early endosomal marker. Correspondingly, transepithelial electric resistance, obtained from the naturally susceptible human liver cell line, Huh7, was much higher than that of the HeLa-CLDN1 cell line, suggesting that Huh7 is likely to form functional tight junctions. Finally, the disruption of TJ-enriched CLDN1 by tumor necrosis factor-␣ treatment markedly reduced the susceptibility of Huh7.5.1 cells to HCV infection. Our results suggest that the specific localization pattern of CLDN1 may be crucial in the regulation of HCV cellular tropism.Hepatitis C virus (HCV), 3 a major human pathogen, specifically infects hepatocytes. In nature, the virus may exist in several forms: enveloped lipoprotein-free virus, enveloped lipoprotein-associated virus, non-enveloped lipoprotein-free virus, and non-enveloped lipoprotein-associated virus (1). While the nature of these different forms remains elusive, they may infect cells via different means (1). Importantly, pseudoviral particles that consist of an HIV core and HCV E1 and E2 (i.e. HCVpp) have been found to strictly infect human hepatocytes and a few hepatoma-derived cell lines (2-4). HCVpp most likely resembles enveloped lipoprotein-free viruses whose entry is dependent upon two of the HCV envelope proteins, E1 and E2 (5-9). A plethora of evidence that has been accumulated from studies employing HCVpp has allowed for a proposed model of HCV entry: HCV attaches to hepatocytes via specific receptor(s) followed by clathrin-dependent internalization (endocytosis) (10, 11). Internalized virions then traffic to early endosomes where the virion envelope proteins undergo conformational changes and then fuse with the cellular membrane for viral entry (4, 12). In numerous attempts to identify an HCV entry receptor(s), both human tetraspanin CD81 and the human scavenger receptor, SR-BI, were isolated in screens based on...

The matrix protein (M1) of influenza A virus is generally viewed as a key orchestrator in the release of influenza virions from the plasma membrane during infection. In contrast to this model, recent studies have indicated that influenza virus requires expression of the envelope proteins for budding of intracellular M1 into virus particles. Here we explored the mechanisms that control M1 budding. Similarly to previous studies, we found that M1 by itself fails to form virus-like-particles (VLPs). We further demonstrated that M1, in the absence of other viral proteins, was preferentially targeted to the nucleus/perinuclear region rather than to the plasma membrane, where influenza virions bud. Remarkably, we showed that a 10-residue membrane targeting peptide from either the Fyn or Lck oncoprotein appended to M1 at the N terminus redirected M1 to the plasma membrane and allowed M1 particle budding without additional viral envelope proteins. To further identify a functional link between plasma membrane targeting and VLP formation, we took advantage of the fact that M1 can interact with M2, unless the cytoplasmic tail is absent. Notably, native M2 but not mutant M2 effectively targeted M1 to the plasma membrane and produced extracellular M1 VLPs. Our results suggest that influenza virus M1 may not possess an inherent membrane targeting signal. Thus, the lack of efficient plasma membrane targeting is responsible for the failure of M1 in budding. This study highlights the fact that interactions of M1 with viral envelope proteins are essential to direct M1 to the plasma membrane for influenza virus particle release.The late phase of the influenza A virus replication cycle is marked by the occurrence of assembly and budding at the plasma membrane of infected cells, which leads to the separation of virion and host cell membranes and ultimately results in the production of infectious virus particles. This critical step is a highly concerted process driven largely by protein-protein, protein-lipid, and protein-nucleic acid interactions (34, 40). It has been established for many years that four viral structural components, namely, the matrix protein (M1), hemagglutinin (HA), neuraminidase (NA), and M2, are actively involved in the assembly and budding process (34,35,40), although the identities of these inter-and intramolecular interactions and regulatory mechanisms for influenza A virus assembly and budding are unclear. It has also been suggested that interactions of M1 with various cytoplasmic tails (CTs) of HA, NA, and M2 are critical to drive the assembly and release of influenza A virions from the surface of infected cells (1,5,10,18,25,29,30,68). To date, these interactions have been largely speculative because direct interactions have been demonstrated only for M1 and M2 (5, 18, 29).Early investigations into the budding machinery of influenza A virus using vaccinia virus-and baculovirus-based expression systems indicated that M1 was the only viral protein absolutely required for the assembly of virus particles (14,15...

The proline-rich L domains of human immunodeficiency virus 1 (HIV-1) and other retroviruses interact with late endocytic proteins during virion assembly and budding. In contrast, the YPDL L domain of equine infectious anemia virus (EIAV) is apparently unique in its reported ability to interact both with the 2 subunit of the AP-2 adaptor protein complex and with ALG-2-interacting protein 1 (AIP1/Alix) protein factors involved in early and late endosome formation, respectively. To define further the mechanisms by which EIAV adapts vesicle trafficking machinery to facilitate virion production, we have examined the specificity of EIAV p9 binding to endocytic factors and the effects on virion production of alterations in early and late endocytic protein expression. The results of these studies demonstrated that (i) an ϳ300-residue region of AIP1/Alix-(409 -715) was sufficient for binding to the EIAV YPDL motif; (ii) overexpression of AIP1/Alix or AP-2 2 subunit specifically inhibited YPDL-mediated EIAV budding; (iii) virion budding from a replication-competent EIAV variant with its L domain replaced by the HIV PTAP sequence was inhibited by wild type or mutant 2 to a level similar to that observed when a dominant-negative mutant of Tsg101 was expressed; and (iv) overexpression or siRNA silencing of AIP1/Alix and AP-2 revealed additive suppression of YPDL-mediated EIAV budding. Taken together, these results indicated that both early and late endocytic proteins facilitate EIAV production mediated by either YPDL or PTAP L domains, suggesting a comprehensive involvement of endocytic factors in retroviral assembly and budding that can be accessed by distinct L domain specificities. Equine infectious anemia virus (EIAV)2 is a member of the lentivirus subfamily of retroviruses, which also includes human immunodeficiency virus 1 (HIV-1). The EIAV genome is the simplest among the lentivirus family and encodes three major structural proteins (Gag, Pol, and Env) along with three accessory gene products (Tat, Rev, S2). Like all retroviruses, EIAV Gag protein is synthesized as a polyprotein that upon virion maturation is processed by virus-encoded protease to yield four major structural proteins: matrix, capsid, nucleocapsid, and p9 protein. All four structural proteins of Gag polyprotein play critical and distinctive roles in retrovirus assembly and budding. Matrix proteins direct targeting of Gag polyproteins to cell membranes for viral assembly and budding (1, 2). Both capsid and nucleocapsid have been shown to mediate Gag-Gag interactions during viral assembly, with capsid responsible for the dimerization of homologous Gag polyproteins (3, 4) and nucleocapsid essential for multimerization of Gag molecules (5, 6). The EIAV p9 protein contains a YPDL late (L) domain that is critical for virion release during the late stage of virus budding. Proline-rich L domains such as PTAP and PPPY with similar functions have also been identified from HIV-1, Rous sarcoma virus, and a variety of other enveloped viruses (7).L domains appear to...