Human immunodeficiency virus (HIV) has been shown to be etiologic agent of acquired immune deficiency syndrome (AIDS). Recent studies have shown that HIV infection is dependent on the interaction of the virus envelope glycoprotein (gp 120/gp41) with the CD4 differentiation antigen on the cell surface of the helper/inducer subset of T-cells. Another property attributable to the HIV envelope glycoprotein is the ability to induce virus cypopathology mediated by the fusion of CD4 positive cells. This investigation proposes to define, at the molecular level, what structural features of the glypcoprotein are necessary for biological activities such as viral infectivity and cell fusion activity. We propose to use in vitro site directed mutagenesis techniques to modify the cleavage/activation site of the gp160 envelope precursor to generate HIV glycoprotein molecules that: a) are defective in cleavage from the precursor gp160 to gp120/gp41; b) are able to be cleaved into gp120 and gp41 only with the addition of exogenous proteases; or c) are able to be cleaved into gp120 and gp41 but lack fusogenic activity. Previous studies have demonstrated that vaccinia viruses can be utilized as vectors for efficient expression of foreign glycoprotein genes placed under vaccinia virus transcriptional regulatory signals. We propose to construct recombinant vaccinia viruses expressing these modified HIV glycoprotein genes to assay for the ability of these proteins to be transported to the cell surface, fusion activity, and differences in the transport from the endoplasmic reticulum to the plasma membrane. We also propose to use vaccinia recombinants expressing the wild type and mutant HIV glycoproteins to investigate the oligomeric structure of the HIV gp120/gp41 and determine whether proteolytic cleavage of gp160 to gp120/gp41 is necessary for proper oligomerization. These modified gp120/gp41 genes will be inserted into an infectious proviral clone of HIV that is replication competent and known to cause cytopathology when transfected into susceptible cells. The infectious proviral clones will be used to determine if: (a) cleavage of the gp160 precursor is necessary for virus infection; (b) loss of fusion activity correlates with the inability of virus to infect susceptible cells; and (c) the lack of fusion activity results in infectious virus of decreased cytopathic potential. In addition, we propose to further examine the mechanisms by which HIV enters the susceptible cells using ultrastructural and biochemical analyses. It is anticipated that the results from the proposed study will provide information concerning the structure-function relationships of the HIV glycoprotein that are necessary for virus infectivity and cytopathology.
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| Stephens, E B; Butfiloski, E J; Monck, E (1992) Analysis of the amino terminal presequence of the feline immunodeficiency virus glycoprotein: effect of deletions on the intracellular transport of gp95. Virology 190:569-78 |
| Britt, W J; Vugler, L; Stephens, E B (1988) Induction of complement-dependent and -independent neutralizing antibodies by recombinant-derived human cytomegalovirus gp55-116 (gB). J Virol 62:3309-18 |
| Stephens, E B; Compans, R W (1988) Assembly of animal viruses at cellular membranes. Annu Rev Microbiol 42:489-516 |
