1st National Conference Human Retroviruses and Related Infections Washington, DC - December 12-16, 1993

Natl Conf Hum Retrovir Relat Infect 1993 Dec 12-16;1: (abstract no. 14)

Raja NU, Vincent MJ, Jabbar MA
Research Institute, Cleveland Clinic Foundation, Cleveland, OH 44195

The HIV-1 proteins, gp160, Vpu and Nef, are involved in the down modulation of CD4 in HIV-1 infected cells. Previous experiments have demonstrated that gp160 traps CD4 in the endoplasmic reticulum, and the vpu protein induces rapid degradation of CD4 in the ER. In the present study, we have investigated the sequence requirement for vpu dependent proteolysis in the cell. To this end, we generated chimeric envelope glycoproteins having the ectodomain of HIV-1 gp160, the anchor domain of CD4, and 38, 25, 24, 18, 7, 4, and 1 aa of the CD4 cytoplasmic domain. Using the vaccinia virus-T7 RNA polymerase expression system, we analyzed the expression of chimeric proteins in the presence and absence of Vpu. In singly transfected cells, the chimeric envelope glycoproteins having 38, 24, 18 aa, and less of the CD4 cytoplasmic domain were endoproteolytically cleaved and biologically active in the fusion of HeLa CD4+ cells. However, one of the chimeras having 25 aa of the CD4 cytoplasmic tail was retained in the ER using the transmembrane ER retention signal and was defective in membrane fusion. Furthermore, biochemical analyses of the coexpressing cells revealed that the vpu protein induced degradation of the envelope glycoproteins having 38, 25, and 24 aa of the CD4+ cells expressing Vpu. However, the HIV-1 gp160 and envelope glycoprotein having the membrane proximal 18 aa or less of the CD4 cytoplasmic tail were stable and fusion-competent in cells expressing Vpu. Further analyses have revealed that the anchor domain of CD4 appear to provide initial `interaction motif' through which the vpu protein could recognize sequences or structural determinants in the cytoplasmic domain of CD4. We will discuss the mechanism by which Vpu induces the proteolysis of CD4 and chimeric proteins in the endoplasmic reticulum.

Keywords: Animals, Antigens, CD4, Base Sequence, Endoplasmic Reticulum, Gene Products, env, Gene Products, vpu, Glycoproteins, HIV-1, Hela Cells, Humans, Membrane Fusion, Protein Precursors, Recombinant Fusion Proteins, genetics, immunology

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1993-12-12
14

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