3rd Conference on Retroviruses and Opportunistic Infections Washington, DC - January 28-February 1, 1996

Conf Retroviruses Opportunistic Infect 1996 Jan 28-Feb 1; 3rd:61 (abstract no. 46)

Fujita K, Maldarelli F, Silver J
Laboratory of Molecular Microbiology, NIAID, NIH, Bethesda MD 20892, USA.

Interaction between viral envelope (Env) and receptor is complex because it must mediate viral entry but not interfere with viral release. HIV has several mechanisms for downregulating its receptor, CD4, in infected cells. To study this process, we established CD4 (+) HeLa cell clones stably expressing HIV-1 Rev, Vpu, and Env and analyzed them for the expression of surface CD4 by FACS. Individual clones were found to contain two populations of cells differing by about ten-fold in level of surface CD4. The level of Env was comparable in high and low CD4 cells. Both Env and Vpu were required for downregulation of CD4 as mutants in either gene failed to downregulate CD4. When cells were treated with cycloheximide to block new protein synthesis, intracellular CD4 decreased very slowly in cells with low surface CD4 but rapidly in cells with high CD4. This suggests that transport of intracellular CD4 to the cell surface was blocked in cells with low surface CD4 and that the rate of degradation of intracellular CD4 was decreased in these cells in the presence of cycloheximide. However, Vpu and Env have been reported to accelerate the degradation of CD4. We propose that Vpu-dependent accelerated destruction of intracellular CD4 involves a labile cofactor whose synthesis is also blocked by cycloheximide. When high and low CD4-expressing cells were separated by FACS, each subpopulation gave rise to a mixture of high and low CD4-expressing cells after several days in culture, indicating that CD4 levels varied over time in individual cells or their progeny. Dual FACS analysis for CD4 and Vpu showed that CD4 downregulation was only seen in cells with the highest Vpu levels. Since Vpu is capable of multimerization, many of the observations in our system are explained by a semiquantitative model in which CD4 interacts with multimerized Vpu. When the concentration of Vpu exceeds a threshold required for multimerization, a Vpu-CD4 complex forms whose transport to the cell surface is blocked, resulting in decreased surface CD4.

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