Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UMLS:C0021051 (immunodeficiency)
 71,517 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The small membrane protein Vpu of human immunodeficiency virus type 1 stimulates rapid degradation of CD4 molecules that are retained in the endoplasmic reticulum. To analyze the domain(s) of CD4 involved in Vpu-stimulated degradation, we examined degradation of hybrid proteins made between the vesicular stomatitis virus glycoprotein (VSV G) and CD4. Vpu expression stimulated rapid degradation of a hybrid consisting of the extracellular domain of VSV G linked to the transmembrane and cytoplasmic domains of CD4. Analysis of additional hybrids showed that both the cytoplasmic and transmembrane domains of CD4 were required for this Vpu-stimulated degradation. This conclusion is in apparent conflict with a recent study showing that the cytoplasmic domain of CD4 alone is sufficient to cause Vpu-stimulated degradation of a CD8-CD4 hybrid protein. The apparent conflict may be explained by the presence of related sequences or structures in the transmembrane domains of CD4 and CD8 that are involved in binding Vpu directly or that interact with the Vpu-stimulated degradation system.
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PMID:Stimulation of heterologous protein degradation by the Vpu protein of HIV-1 requires the transmembrane and cytoplasmic domains of CD4. 809 84

A processing protease for the human immunodeficiency virus type I (HIV-I) envelope glycoprotein gp160 precursor has been purified to homogeneity from the post-nuclear membrane fraction of a human T4+ lymphocyte clone. Most of the processing activity was found to be present in the fractions of endoplasmic reticulum and Golgi apparatus of the cells. The purified enzyme has a monomeric structure with a molecular mass of 26 +/- 3 kDa, as judged by gel-permeation liquid chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing and nonreducing conditions. The purified enzyme converted gp160 to gp120 and gp41, showing a pH optimum of 6.5-7.0. Direct amino acid sequencing of the amino terminus of the product gp41 revealed that the cleavage site of gp160 was between Arg511 and Ala512. The enzyme activity was inhibited by trypsin-type protease inhibitors, but was not affected by CaCl2, MgCl2 or chelating agents. The properties of the purified enzyme are clearly distinct from those of processing proteases reported previously. Although the significance of the enzyme in vivo is not currently certain, judging from its cleavage specificity and subcellular localization, this endopeptidase appears to be a processing enzyme for the human immunodeficiency virus type I gp160 precursor protein in human T cells.
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PMID:Processing protease for gp160 human immunodeficiency virus type I envelope glycoprotein precursor in human T4+ lymphocytes. Purification and characterization. 809 9

The human immunodeficiency virus type 1 (HIV-1) encoded Vpu is a small integral membrane phosphoprotein that functions in the enhancement of viral particle release and has more recently been shown to cause degradation of CD4 at the endoplasmic reticulum. We have demonstrated earlier that Vpu is phosphorylated by the ubiquitous casein kinase-2 (CK-2) in HIV-1 infected cells. The phosphoacceptor sites targeted by CK-2 in Vpu, however, have not been demonstrated and it was unclear whether Vpu was phosphorylated at one or more of its four serine residues. In this study we characterized the CK-2 phosphoacceptor sites in Vpu using recombinant CK-2 for in vitro phosphorylation of recombinant Vpu protein as well as synthetic peptides of Vpu. Phosphorylation of both Ser52 and Ser56 was demonstrated by in vitro phosphorylation using three 54-residue peptides comprising the entire hydrophilic part of Vpu and containing single serine to asparagine transitions in either position 52 or 56. The Km values of CK-2 to these peptides were established, revealing a preferential phosphorylation of Ser56. The Km values are: Ser56 = 31 microM; Ser 52 = 156 microM; wild type = 27 microM. In addition, we studied phosphorylation of Vpu by endogenous CK-2 following in vitro translation in rabbit reticulocyte lysate of wild-type Vpu or a mutant, Vpum2/6, carrying serine to asparagine changes at amino acid positions 52 and 56. The in vivo phosphorylation of Vpu was studied in transiently transfected human embryonic kidney (293) cells. In this system, the mutant Vpum2/6 was not phosphorylated, indicating that the seryl residues of Vpu at amino acid positions 52 and 56, but not those at positions 23 and 61, are phosphorylated by CK-2. The two CK-2 phosphorylation sites are conserved in all known Vpu sequences and represent the consensus Ser52GlyAsn(Glu/Asp)Ser(Glu/Asp)Gly(Glu/Asp)59. Prediction of the secondary structure revealed a conserved alpha-helix-turn-alpha-helix motif for the hydrophilic C-terminal part of Vpu. A structural model for Vpu is proposed in which the membrane anchor precedes a region comprising two amphipathic alpha-helices of opposed polarity, joined by a strongly acidic turn that protrudes into the cytoplasm and contains the CK-2 phosphorylation sites. Possible functional and structural homologies of Vpu to the membrane channel-forming M2 protein of influenza A viruses are discussed.
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PMID:The human immunodeficiency virus type 1 encoded Vpu protein is phosphorylated by casein kinase-2 (CK-2) at positions Ser52 and Ser56 within a predicted alpha-helix-turn-alpha-helix-motif. 810 1

The human immunodeficiency virus type 1 (HIV-1)-specific Vpu is an 81-amino-acid amphipathic integral membrane protein with at least two different biological functions: (i) enhancement of virus particle release from the plasma membrane of HIV-1-infected cells and (ii) degradation of the virus receptor CD4 in the endoplasmic reticulum (ER). We have previously found that Vpu is phosphorylated in infected cells at two seryl residues in positions 52 and 56 by the ubiquitous casein kinase 2. To study the role of Vpu phosphorylation on its biological activity, a mutant of the vpu gene lacking both phosphoacceptor sites was introduced into the infectious molecular clone of HIV-1, pNL4-3, as well as subgenomic Vpu expression vectors. This mutation did not affect the expression level or the stability of Vpu but had a significant effect on its biological activity in infected T cells as well as transfected HeLa cells. Despite the presence of comparable amounts of wild-type and nonphosphorylated Vpu, decay of CD4 was observed only in the presence of phosphorylated wild-type Vpu. Nonphosphorylated Vpu was unable to induce degradation of CD4 even if the proteins were artificially retained in the ER. In contrast, Vpu-mediated enhancement of virus secretion was only partially dependent on Vpu phosphorylation. Enhancement of particle release by wild-type Vpu was efficiently blocked when Vpu was artificially retained in the ER, suggesting that the two biological functions of Vpu are independent, occur at different sites within a cell, and exhibit different sensitivity to phosphorylation.
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PMID:Differential activities of the human immunodeficiency virus type 1-encoded Vpu protein are regulated by phosphorylation and occur in different cellular compartments. 813 11

The human immunodeficiency virus type-1 (HIV-1) encoded Vpu protein facilitates the release of budding virions from the surface of infected cells and delays the rate of syncytium formation of the virus. Furthermore, Vpu induces rapid degradation of nascent CD4 molecules that are retained in the endoplasmic reticulum by the formation of gp160-CD4 complexes. Currently, little is known of the precise mechanism(s) by which Vpu function. Whether or not these different events are related remain unclear. In this report, we describe our recent structure/function studies on vpu suggesting that the protein may have independent biological activities during the HIV-1 infection.
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PMID:Mutational analysis of the HIV-1 Vpu protein. 815 84

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.
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PMID:Vpu-induced degradation of CD4: requirement for specific amino acid residues in the cytoplasmic domain of CD4. 823 Apr 46

CD4 is an integral membrane glycoprotein which functions as the human immunodeficiency virus receptor for infection of human host cells. We have recently demonstrated that Vpu, a human immunodeficiency virus type 1-encoded integral membrane phosphoprotein, induces rapid degradation of CD4 in the endoplasmic reticulum. Using an in vitro model system, we demonstrated that Vpu targets specific sequences in the cytoplasmic domain of CD4 to promote its degradation. In this report, we have further delineated regions within CD4 which are required for susceptibility to Vpu. Transfer of the CD4 cytoplasmic region into a heterologous protein, CD8, rendered the chimeric protein sensitive to Vpu-dependent degradation. In contrast, substitution of the CD8 transmembrane domain with the analogous region from CD4 did not confer sensitivity to Vpu. Finally, mutant forms of the CD4 protein containing the extracellular region alone or the extracellular and transmembrane regions linked to a heterologous cytoplasmic domain were not targeted by Vpu. Thus, sequences present in the cytoplasmic domain of CD4 are necessary and sufficient to confer sensitivity to Vpu.
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PMID:Sequences present in the cytoplasmic domain of CD4 are necessary and sufficient to confer sensitivity to the human immunodeficiency virus type 1 Vpu protein. 828 53

Morphogenesis of retroviruses involves assembly of the structural Gag and Gag-Pol polyproteins with subsequent budding of the virus particle from the plasma membrane and proteolytic cleavage by the viral proteinase. The matrix (MA) domain, representing the N-terminal segment of Gag, plays a critical role in this process. We constructed an in-frame deletion in the MA coding region (lacking codons 16 to 99) of the human immunodeficiency virus (HIV) type 1 gag gene. Following transient transfection of the complete proviral DNA carrying the deletion, the mutant polyprotein was synthesized and proteolytically processed like the wild-type polyprotein. However, release of virus particles was reduced approximately 10-fold. The extracellular particles that were released did not contain viral glycoproteins and were noninfectious. Electron micrographs revealed budding of virus particles into the endoplasmic reticulum (ER) of transfected cells and large numbers of particles within the ER. These particles were all immature and morphologically indistinguishable from intracisternal A-type particles, a class of murine endogenous retrovirus elements. Budding structures at the plasma membrane were rarely seen and only a few extracellular particles were observed, but in contrast to those in the ER, these particles had the morphology of mature particles, similar to that of wild-type HIV, except for the lack of surface projections.
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PMID:A large deletion in the matrix domain of the human immunodeficiency virus gag gene redirects virus particle assembly from the plasma membrane to the endoplasmic reticulum. 833 36

The human immunodeficiency virus type 1 (HIV-1) Vpu protein is a transmembrane phosphoprotein which induces rapid degradation of CD4 in the endoplasmic reticulum (ER). To identify sequences in CD4 for Vpu-induced degradation, we generated four chimeric envelope glycoproteins having the ectodomain of HIV-1 gp160, the anchor domain of CD4, and 38, 25, 24, and 18 amino acids (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, and 18 aa 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 cytoplasmic tail and degradation occurred in the ER. Consequently, the fusion-competent glycoproteins did not induce the formation of syncytia in HeLa CD4+ cells expressing Vpu. However, the HIV-1 gp160 and chimeric envelope glycoprotein having the membrane-proximal 18 aa of the CD4 cytoplasmic tail were stable and fusion competent in cells expressing Vpu. In addition, we examined the stability of CD4 molecules in the presence of Vpu. Coexpression analyses revealed that the Vpu protein induced degradation of CD4 whereas mutant CD4 having the membrane-proximal 18 aa of the cytoplasmic domain was relatively stable in the presence of Vpu. Taken together, these studies have elucidated that the Vpu protein requires sequences or sequence determinants in the cytoplasmic domain of CD4 to induce degradation of the glycoproteins in the cell.
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PMID:Human immunodeficiency virus type 1 Vpu protein induces degradation of chimeric envelope glycoproteins bearing the cytoplasmic and anchor domains of CD4: role of the cytoplasmic domain in Vpu-induced degradation in the endoplasmic reticulum. 835 Apr 11

A single-chain antibody, derived from a human monoclonal antibody that recognizes the CD4 binding region of the human immunodeficiency virus type 1 (HIV-1) envelope protein, has been designed for intracellular expression in eukaryotic cells. The single-chain antibody is composed of an immunoglobulin heavy-chain leader sequence and heavy- and light-chain variable regions that are joined by an interchain linker. The antibody is stably expressed and retained in the endoplasmic reticulum and is not toxic to the cells. The antibody binds to the envelope protein within the cell and inhibits processing of the envelope precursor and syncytia formation. The infectivity of the HIV-1 particles produced by cells that express the single-chain antibody is substantially reduced. These studies illustrate the feasibility of designing antibodies that bind and inactivate molecules intracellularly. Antibodies that act on target molecules within cells should provide a useful tool for research as well as for control of infectious and other diseases.
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PMID:Design, intracellular expression, and activity of a human anti-human immunodeficiency virus type 1 gp120 single-chain antibody. 835 37


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