HIV type I (HIV-1) can cause G2 cell cycle arrest and death of CD4 ؉ T lymphocytes in vitro and inexorable depletion of these cells in vivo. However, the molecular mechanism of viral cytopathicity has not been satisfactorily elucidated. Previously, we showed that HIV-1 kills T cells by a necrotic form of cell death that requires high level expression of an integrated provirus but not the env or nef genes. To determine which viral protein(s) are required for cell death, we systematically mutated, alone and in combination, the ORFs of the NL4-3 strain of HIV-1. We found that the elimination of the viral functions encoded by gag-pol and vpu, tat, and rev did not mitigate cytopathicity. However, elimination of the vif and vpr accessory genes together, but not individually, renders the virus incapable of causing cell death and G 2 cell cycle blockade. We thus identify vif and vpr as necessary for T cell cytopathic effects induced by HIV-1. These findings may provide an important insight into the molecular mechanism of viral pathogenesis in AIDS.AIDS ͉ apoptosis ͉ lymphocytes ͉ necrosis ͉ NL4-3

Id proteins act as negative regulators of bHLH transcription factors by forming transcriptionally inactive protein complexes. The proposed function of these proteins includes promotion of cell growth and cell cycle progression, induction of apoptosis, and inhibition of cellular differentiation. We investigated the role of the ubiquitin-mediated proteolytic pathway in the degradation of the Id3 protein. We found Id3 to be a short-lived protein and estimated the half-life to be approximately 20 min in 293 cells. Using specific inhibitors of the 26S proteasome and mutant fibroblast cells with a temperature-sensitive defect in the essential E1 ubiquitin-activating enzyme, we show that Id3 and the related Id1 and Id2 proteins are degraded through the ubiquitin-proteasome pathway. We found the Id4 protein to be much less sensitive to inhibitors of the 26S proteasome, but its degradation was dependent on the E1 enzyme. In addition, we observed that coexpression of the bHLH protein E47 with Id3 significantly reduced the rate of degradation of Id3, suggesting that Id3 is less susceptible to degradation by the 26S proteasome when complexed to a bHLH protein. -Bounpheng, M. A., Dimas, J. J., Dodds, S. G., Christy, B. A. Degradation of Id proteins by the ubiquitin-proteasome pathway.

An important unresolved issue of AIDS pathogenesis is the mechanism of human immunodeficiency virus (HIV)-induced CD4(+) T-lymphocyte destruction. We show here that HIV type 1 (HIV-1) exerts a profound cytopathic effect upon peripheral blood CD4(+) T lymphocytes that resembles necrosis rather than apoptosis. Necrotic cytopathology was found with both laboratory-adapted strains and primary isolates of HIV-1. We carefully investigated the role of env, which has been previously implicated in HIV cytopathicity. HIV-1 stocks with equivalent infectivity were prepared from constructs with either an intact or mutated env coding region and pseudotyped with the glycoprotein of vesicular stomatitis virus (VSV-G) so that the HIV envelope was not rate-limiting for infection. Infected Jurkat T cells died whether or not env was intact; however, the expression of env accelerated death significantly. The accelerated death was blocked by protease inhibitors, indicating that it was due to reinfection by newly produced virus in env(+) cultures. Accordingly, we found no disparity in kinetics in CD4(lo) Jurkat cells. In highly infected peripheral blood T cells, profound necrosis occurred equivalently with both env(+) and env(-) stocks of HIV-1. We also found that HIV-1 cytopathicity was undiminished by the absence of nef. However, viral stocks made by complementation or packaging of HIV-1 genomes with the natural protein-coding sequences replaced by the green fluorescent protein were highly infectious but not cytopathic. Thus, env can accelerate cell death chiefly as an entry function, but one or more viral functions other than env or nef is essential for necrosis of CD4(+) T cells induced by HIV-1.