The candidate's research focuses on the molecular mechanism of HIV-1 pathogenesis and will involve characterizing the role of HIV-1 Nef in downregulation of cell surface CD4. In order to circumvent problems that others have encountered due to the cytotoxic effects of constitutive Nef expression, nef will be stably transfected into CD4 positive (CD4+) cell lines in which Nef expression will be tightly regulated by an inducible tetracycline-responsive promotor. Cell surface delivery and endocytosis assays will be used to determine whether newly synthesized or cell surface CD4 is sequestered in Nef-expressing cells. CD4 will be examined for postranslational modification and phosphorylation status; proteins which co-immunoprecipitate with CD4 and/or Nef will be identified. During Phase II, based upon information obtained in Phase I, the candidate will far explore the mechanism of Nef-induced CD4 downregulation. Subcellular fractionation techniques, co-localization studies, and techniques to examine the biosynthesis of cell surface molecules will he used to study Nef-induced CD4 intracellular trafficking and localization. Primate studies have found that Nef expression is absolutely required for the development of AIDS; animals infected with SIV lacking nef do not become ill and have normal levels of circulating CD4+ lymphocytes. If Nef-induced modulation of cell surface CD4 expression is important for the pathogenesis of AIDS, pharmacologic manipulation of such modulation may slow or perhaps arrest the development of disease progression in HIV-l infected individuals.
| Bresnahan, P A; Yonemoto, W; Ferrell, S et al. (1998) A dileucine motif in HIV-1 Nef acts as an internalization signal for CD4 downregulation and binds the AP-1 clathrin adaptor. Curr Biol 8:1235-8 |
