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.