Faculty Bibliography

Human immunodeficiency virus type 1 Vpr is a virion-associated, regulatory protein that is required for efficient viral replication in monocytes/macrophages. The protein is believed to act in conjunction with the Gag matrix protein to allow import of the viral preintegration complex in nondividing cells. In cells, Vpr localizes to the nucleus. Recently, we showed that Vpr prevents the activation of p34cdc2-cyclin B. This results in arrest of Vpr-expressing cells in the G2/M phase of the cell cycle. Here, we use a panel of expression vectors encoding Vpr molecules mutated in the amino-terminal alpha-helical region, the central hydrophobic region, or the carboxy-terminal basic region to define the functional domains of the protein. The results showed cell cycle arrest was largely controlled by the carboxy-terminal basic domain of the protein. In contrast, the amino-terminal alpha-helical region of Vpr was required for nuclear localization and packaging into virions. The carboxy terminus appeared to be unnecessary for nuclear localization. In the alpha-helical region, mutation of Ala-30 to Pro resulted in a protein that localized to the cytoplasm. Surprisingly, fusion of Vpr to luciferase resulted in a molecule that failed to localize to the nucleus. In addition, we show that simian immunodeficiency virus Vpr, but not Vpx, induces G2 arrest. We speculate that Vpr has two sites for interaction with cellular factors: one in the alpha-helical region that specifies nuclear localization and one in the carboxy-terminal domain that is required for Cdc2 inhibition

Human immunodeficiency virus type 1 Nef down-regulates surface expression of murine and human CD4 but not human CD8. We recently reported that the cytoplasmic domain of CD4 is required for its down-regulation by Nef. Using a chimeric molecule composed of the extracellular and transmembrane domains of human CD8 fused to the cytoplasmic domain of human CD4, we show here that the cytoplasmic domain of CD4 is sufficient for down-regulation by Nef. Since the cytoplasmic domain of CD4 is also the site of its association with p56lck, we used a series of CD4 mutants to determine whether the regions of the cytoplasmic domain of CD4 required for down-regulation by Nef are the same as those required for p56lck binding. Our results indicate that the portion of the cytoplasmic domain required for the down-regulation of CD4 by Nef overlaps with the binding site of p56lck, but the cysteine residues which are essential for the association of CD4 with p56lck are not required. This observation raised the possibility that Nef competes with p56lck for binding to CD4. However, under conditions which are considerably milder than those permissive for coimmunoprecipitation of CD4 and p56lck, we found no evidence for an association between Nef and CD4. While a decrease in total CD4 was observed in lysates of cells expressing Nef, the levels of p56lck were not significantly affected. Pulse-chase experiments further revealed a decrease in the half-life of CD4 in Nef-expressing cells. These results show that the decrease in surface CD4 expression induced by Nef is mediated at least in part by a decrease in the half-life of CD4 protein. These results also indicate that a large portion of p56lck is free of CD4 in T cells expressing Nef, which could have a significant effect on T-cell function

CD4 is crucial for antigen-driven helper T cell signaling and is used as receptor by the human immunodeficiency virus (HIV). The HIV early protein Nef causes a loss of CD4 from cell surfaces through a previously undefined posttranscriptional mechanism. Here, we demonstrate that Nef acts by inducing CD4 endocytosis, resulting in its degradation in lysosomes. CD4 down-regulation is strongly enhanced by the association of Nef with cell membranes through myristoylation. The study of chimeric molecules reveals that 20 membrane-proximal residues of the CD4 cytoplasmic domain are sufficient to confer Nef sensitivity. Within this region, a dileucine motif, reminiscent of an endocytosis and lysosomal targeting signal found in the CD3 gamma and delta chains, is crucial for CD4 response to Nef

The product of the vpr open reading frame of human immunodeficiency virus type 1 (HIV-1) is a 15-kDa, arginine-rich protein that is present in virions in molar quantities equivalent to that of Gag. We report here the results of our investigations into the mechanism by which Vpr is incorporated into virions during assembly in infected cells. For these studies we used an expression vector encoding a Vpr molecule fused at its amino terminus to a nine-amino-acid peptide from influenza virus hemagglutinin. The tagged Vpr expression vector and a vpr mutant HIV-1 provirus were used to cotransfect COS cells, and the resulting virions were tested for the presence of the tagged protein on immunoblots probed with monoclonal antibody against the hemagglutinin peptide. The COS-produced virions were found to contain readily detectable amounts of tagged Vpr and smaller amounts of a putative tagged Vpr dimer. Infectivity of the particles was not altered by incorporation of tagged Vpr. Our results using this system in combination with mutant HIV-1 proviruses suggested that incorporation of Vpr into virions requires the carboxy-terminal Gag protein of HIV-1 (p6) but not gp160, Pol, or genomic viral RNA. In addition, analysis of mutated, tagged Vpr molecules suggested that amino acids near the carboxy terminus (amino acids 84 to 94) are required for incorporation of Vpr into HIV-1 virions. The single cysteine residue near the carboxy terminus was required for production of a stable protein. Arginine residues tested were not important for incorporation or stability of tagged Vpr. These results suggested a novel strategy for blocking HIV-1 replication

Two functions have been attributed to the product of the human immunodeficiency virus type 1 vpu open reading frame: it increases virion release from infected cells and induces rapid degradation of CD4 shortly after its synthesis. In the absence of Vpu, newly synthesized gp160 and CD4 associate in the endoplasmic reticulum (ER), forming a complex whose further maturation is blocked and which is eventually degraded. In studies using NL4-3-based expression vectors, it has been previously shown that Vpu induces the release of gp160 from the complex that it forms with CD4 in the ER. This release, which appears to be due to the rapid degradation of CD4 induced by Vpu, allows gp160 to transit to the Golgi, where it matures further. We investigated which regions of CD4 are important for its susceptibility to Vpu-induced degradation by transfecting HeLa cells with isogenic vpu-positive and vpu-negative proviruses and vectors expressing various truncated or mutated CD4 molecules. The results suggested that the cytoplasmic domain of CD4 contains a determinant lying within amino acids 418 to 425 that is critical for susceptibility to Vpu-induced degradation. Neither the phosphorylation sites in the cytoplasmic domain nor the Lck interaction region was required for the effect. Vpu-induced degradation was specific for CD4, since CD8, even when retained in the ER, was not degraded. In addition, under conditions of high-level Vpu expression, CD4 degradation could be observed in the absence of gp160 or other means of retaining CD4 in the ER