Enveloped viruses utilize glycoproteins on their surface to mediate entry into the target host cell. Conserved features in many viral glycoproteins suggest that they function in a similar manner to accomplish the common goal of viral entry into the host. Entry involves both recognition of specific receptors on the host surface and fusion of the viral and host cell membranes. The viral glycoproteins mediate both of these events. For many viruses, viral entry takes place after endocytosis where the acidic environment of the endosome triggers conformational changes in the viral glycoprotein(s) that result in membrane fusion. Fusion appears to require the concerted action of several oligomeric glycoproteins, thus the conformational changes are likely to also be synchronous and this synchrony is easily obtained with a trigger such as a low pH environment. Retroviruses, including Rous sarcoma virus (RSV), and a number of other virus families enter cells in a pH-independent manner. Thus, the cue for the proposed glycoprotein conformational changes for these pH-independent viruses is unclear. Indeed, the relevant conformational changes that lead to fusion are ill- defined for viruses utilizing a neutral pH entry pathway. Although the glycoproteins of many pH-dependent and independent viruses are similar, the study of neutral pH entry has been slowed by our lack of knowledge about, or ability to trigger, a fusogenic conformation for the viral glycoproteins. The overall goal of this project is to analyze viral entry in a model retroviral system. Because of the availability of a cloned host receptor (Tva) that is easily manipulated and a well characterized viral envelope protein, RSV represents an ideal system to study pH-independent retroviral entry. This analysis will focus on the molecular details of the interaction of the viral envelope protein and the receptor, the effects of this interaction on the Env protein, the regions in Env that mediate these effects, and finally the relationship of changes that occur in Env to membrane fusion.
The specific aims to be pursued are: 1) Produce mutations in defined regions of env and study their effects separately on a) receptor binding, b) receptor-induced conformational changes of envelope, c) membrane fusion, and d) infection. 2) Employ a genetic selection strategy to isolate viruses with alterations in envelope that allow escape from soluble receptor or that suppress of an envelope or receptor defect. 3) Determine the consequences of the Tva/EnvA interaction: cooperative effects on conformational changes, characterization of activated EnvA, and analysis of the effects of peptides to conserved regions of TM.
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