Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: 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 human immunodeficiency virus type 1 (HIV-1) and HIV-2 Vpr and Vpx proteins are packaged into virions through virus type-specific interactions with the Gag polyprotein precursor. To examine whether HIV-1 Vpr (Vpr1) and HIV-2 Vpx (Vpx2) could be used to target foreign proteins to the HIV particle, their open reading frames were fused in frame with genes encoding the bacterial staphylococcal nuclease (SN), an enzymatically inactive mutant of SN (SN*), and chloramphenicol acetyltransferase (CAT). Transient expression in a T7-based vaccinia virus system demonstrated the synthesis of appropriately sized Vpr1-SN/SN* and Vpx2-SN/SN* fusion proteins which, when coexpressed with their cognate p55Gag protein, were efficiently incorporated into virus-like particles. Packaging of the fusion proteins was dependent on virus type-specific determinants, as previously seen with wild-type Vpr and Vpx proteins. Particle-associated Vpr1-SN and Vpx2-SN fusion proteins were enzymatically active, as determined by in vitro digestion of lambda phage DNA. To determine whether functional Vpr1 and Vpx2 fusion proteins could be targeted to HIV particles, the gene fusions were cloned into an HIV-2 long terminal repeat/Rev response element-regulated expression vector and cotransfected with wild-type HIV-1 and HIV-2 proviruses. Western blot (immunoblot) analysis of sucrose gradient-purified virions revealed that both Vpr1 and Vpx2 fusion proteins were efficiently packaged regardless of whether SN, SN*, or CAT was used as the C-terminal fusion partner. Moreover, the fusion proteins remained enzymatically active and were packaged in the presence of wild-type Vpr and Vpx proteins. Interestingly, virions also contained smaller proteins that reacted with antibodies specific for the accessory proteins as well as SN and CAT fusion partners. Since similar proteins were absent from Gag-derived virus-like particles and from virions propagated in the presence of an HIV protease inhibitor, they must represent cleavage products produced by the viral protease. Taken together, these results demonstrate that Vpr and Vpx can be used to target functional proteins, including potentially deleterious enzymes, to the human or simian immunodeficiency virus particle. These properties may be exploitable for studies of HIV particle assembly and maturation and for the development of novel antiviral strategies.
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PMID:Targeting foreign proteins to human immunodeficiency virus particles via fusion with Vpr and Vpx. 774 85

Morphogenesis of retroviruses involves ordered assembly of the structural Gag- and Gag-Pol polyproteins, with subsequent budding from the plasma membrane and proteolytic cleavage by the viral proteinase (PR). Two cleavage sites exist between the capsid (CA) and nucleocapsid (NC) domains of the human immunodeficiency virus (HIV) type 1 Gag polyprotein which are separated by a 14-amino-acid spacer peptide of unknown function. To analyze the role of the two cleavage sites and the spacer peptide, both sites were individually mutated and a deletion mutation that precisely removes the spacer peptide was constructed. Following transfection of proviral DNA carrying the point mutations, mutant polyproteins were synthesized and assembled like wild-type polyprotein, and release of particles was not significantly altered. Both mutations abolished cleavage at the respective site and reduced or abolished viral infectivity. Deletion of the spacer peptide severely affected ordered assembly and reduced particle release. The extracellular particles that were released exhibited normal density but were heterogeneous in size. Electron micrographs revealed large electron-dense plaques underneath the plasma membrane of transfected cells which appeared like confluent ribonucleoprotein complexes arrested early in the budding process. Extracellular particles exhibited very aberrant and heterogeneous morphology and were incapable of inducing viral spread. These particles may correspond to membrane vesicles sequestered by the rigid structures underneath the cell membrane and not released by a regular budding process.
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PMID:The spacer peptide between human immunodeficiency virus capsid and nucleocapsid proteins is essential for ordered assembly and viral infectivity. 774 87

The human immunodeficiency virus type 1 (HIV-1) Pr55gag precursors were previously shown to assemble and bud efficiently as noninfectious virus-like particles (VLPs) when expressed in baculovirus-infected insect cells. In this study, we examined the abilities of foreign antigens to be incorporated on the outer surface of HIV-1 Gag particles. We have used a dual recombinant baculovirus, expressing the HIV-1 Gag gene and gD gene under the control of the P10 and polyhedrin promoters, respectively, to obtain hybrid VLPs. Transmission electron microscopy of insect cells infected with the dual recombinant revealed very large aggregates of particles budding from the cell membrane. The release of VLPs into the culture medium was clearly different for a recombinant baculovirus producing solely HIV-1 Gag, for which particles were uniformly distributed all around the cell surface. Biochemical analysis of hybrid particles indicated that glycoprotein gD was packaged into HIV-1 Gag VLPs. Moreover, the carboxy-terminal p6 region of Gag polyprotein and the glycoprotein gD intracytoplasmic domain were not required for gD incorporation. The experiments described here clearly demonstrate that glycoprotein gD can be packaged with HIV-1 Gag particles and released from insect cells.
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PMID:Incorporation of pseudorabies virus gD into human immunodeficiency virus type 1 Gag particles produced in baculovirus-infected cells. 776 63

Productive, spreading infection of peripheral blood lymphocytes (PBL) with human immunodeficiency virus type 1 (HIV-1) requires the viral protein Vif. To study the requirement for vif in this system, we infected PBL with a phenotypically complemented HIV-1 clone mutated in vif. Progeny virus was produced which was noninfectious in PBL but replicated in SupT1 cells. Analysis of metabolically labeled proteins of sedimentable extracellular particles made in PBL by radioimmunoprecipitation with either serum from a patient with AIDS or a monoclonal antibody reactive with HIV-1 Gag proteins revealed that vif-negative but not wild-type particles carry higher levels of p55, p41, and p38 Gag-specific proteins compared with those of p24. Similar results were obtained with sucrose-purified virions. Our data indicate that vif plays a role in Gag protein processing or in incorporation of processed Gag products into mature virions. The presence of unprocessed precursor Gag polyprotein (Pr55gag) and other Gag processing intermediates in PBL-derived vif-negative extracellular particles may contribute to the reduced infectivity of this virus.
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PMID:Aberrant Gag protein composition of a human immunodeficiency virus type 1 vif mutant produced in primary lymphocytes. 776 28

Substitution of glycine with glutamic acid at position 48 of the human immunodeficiency virus protease resulted in an enzyme with reduced activity on one of the protease processing sites in the viral Pol polyprotein precursor. Cleavage at this site was restored by a second-site substitution in the substrate replacing an aspartic acid with either glycine or asparagine. These results suggest that the glutamic acid side chain in the mutant protease has an unfavorable charge-charge interaction with this position in the substrate. Cleavage of a processing site in the viral Gag polyprotein precursor with the mutant enzyme was enhanced, and this enhancement was dependent on the presence of an arginine residue in the substrate, again suggesting a charge-charge interaction. The potential for such interactions was confirmed using molecular modeling. The effect of the position 48 substitution was attributed to a 10-fold increase in Km for the processing site in Pol. These results indicate that the addition of a side chain at position 48 can alter the specificity of the HIV-1 protease to substrate in a sequence specific manner and that compensatory changes can be made in the substrate.
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PMID:A side chain at position 48 of the human immunodeficiency virus type-1 protease flap provides an additional specificity determinant. 788 51

Simian immunodeficiency virus (SIV) and human immunodeficiency virus type 2 (HIV-2) encode the accessory viral protein X (Vpx) known to be incorporated into virions in amounts comparable to those of the Gag proteins. The localization of Vpx within SIVmac-infected HUT-78 cells and SIVmac virions was studied by immunoelectron microscopy. Vpx appeared to be associated with extracellular virions as well as budding viral particles at the surface of infected cells. Immunolabelling of purified viral cores suggested that Vpx was a component of the amorphous material surrounding the core structure. Furthermore, a detergent insoluble fraction containing SIV core proteins was devoid of Vpx. To investigate the protein requirement for packaging of Vpx, BHK-21 cells were co-infected with vaccinia virus recombinants encoding Vpx and other SIV proteins able to assemble into virus-like particles. Analysis by immunoprecipitation of the extracellular particulate material as well as immunoelectron microscopy demonstrated that co-expression of Vpx with the Pr56gag polyprotein was sufficient for the formation of pseudo-virions containing Vpx. Virus-like particles that appeared upon expression of p16gag did not contain Vpx. The results suggest that Vpx is packaged into viral particles through its binding to the Gag polyprotein. The precise positioning of Vpx within the space separating the viral envelope from the core structure is postulated to result from the reorganization of viral proteins that occurs upon Gag polyprotein cleavage and budding.
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PMID:Localization of viral protein X in simian immunodeficiency virus macaque strain and analysis of its packaging requirements. 796 5

A series of deletions was introduced into the CA domain of the human immunodeficiency virus type 1 Gag polyprotein to examine its role in virus particle and core formation. The mutations resulted in two phenotypes, indicating the existence of two functionally distinct regions within the CA domain. Deletions within a conserved stretch of 20 amino acids referred to as the major homology region (MHR) and deletions C terminal to this region blocked virus replication and significantly reduced the ability to form viral particles. Deletions N terminal to the MHR also prevented virus replication, but the mutants retained the ability to assemble and release viral particles with the same efficiency as the wild-type virus. The mutant particles contained circular rather than cone-shaped cores, and while they were of a density similar to that of wild-type particles, they were more heterogeneous in size. These results indicate that CA domain sequences N terminal to the MHR are essential for the morphogenesis of the mature cone-shaped core.
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PMID:Functional domains of the capsid protein of human immunodeficiency virus type 1. 796 9

Little is known about host factors necessary for retroviral virion assembly or uncoating. We have previously shown that the principal structural protein of the human immunodeficiency virus HIV-1, the Gag polyprotein, binds the cyclophilin peptidyl-prolyl isomerases; cyclophilins catalyse a rate-limiting step in protein folding and protect cells from heat shock. Here we demonstrate that cyclophilin A is specifically incorporated into HIV-1 virions but not into virions of other primate immunodeficiency viruses. A proline-rich region conserved in all HIV-1 Gag polyproteins is required for cyclophilin A binding and incorporation. Disruption of a single proline blocks the Gag-cyclophilin interaction in vitro, prevents cyclophilin A incorporation into virions, and inhibits HIV-1 replication. Our results indicate that the interaction of Gag with cyclophilin A is necessary for the formation of infectious HIV-1 virions.
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PMID:Specific incorporation of cyclophilin A into HIV-1 virions. 752 24

Cyclophilins are a family of proteins that bind the immunosuppressant cyclosporin A, possess peptidyl-prolyl cis-trans isomerase activity, and assist in the folding of proteins. Human cyclophilins A and B are host cell proteins that bind specifically to the HIV-1 Gag polyprotein p55gag in vitro. Here we report that viral particles formed by p55gag, in contrast to particles formed by the Gag polyproteins of other retroviruses, contain significant amounts of cyclophilin A. Sequences in the capsid domain of p55gag are both required and sufficient for the virion-association of cyclophilin A. The association of cyclophilin A with HIV-1 virions was inhibited in a dose-dependent manner by cyclosporin A as well as by SDZ NIM811 ([Melle-4]cyclosporin), a non-immunosuppressive analogue of cyclosporin A. Drug-induced reductions in virion-associated cyclophilin A levels were accompanied by reductions in virion infectivity, indicating that the association is functionally relevant. Moreover, SDZ NIM811 inhibited the replication of HIV-1 but was inactive against SIVMAC, a primate immunodeficiency virus closely related to HIV-1, which does not incorporate cyclophilin A.
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PMID:Functional association of cyclophilin A with HIV-1 virions. 752 24

The role of the matrix protein (MA) of human immunodeficiency virus type 1 in intracellular transport, assembly, and extracellular release of Gag polyprotein precursor (Pr55gag) was investigated by deletion mutagenesis of the MA domain of recombinant Gag precursor expressed in baculovirus-infected cells. In addition, three carboxy-terminally truncated forms of the Gag precursor, representing mainly the MA, were constructed. One corresponded to an MA with a deletion of its last 12 residues (amb120), while the others corresponded to the entire MA with an additional sequence from the N-terminal portion of the CA (amb143 and och180). Deletions within the MA central region (residues 41 to 78) appeared to be detrimental to Gag particle assembly and budding from the plasma membrane. A slightly narrower domain, between amino acids 41 and 68, was found to be critical for soluble Gag secretion. Mutations which totally or partially deleted one or the other of the two polybasic signals altered the transport of N-myristylated Gag precursor to the plasma membrane. In coexpression with wild-type Gag precursor, a discrete trans-dominant negative effect on wild-type Gag particle assembly and release was observed with deletion mutants located in the central MA region (residues 41 to 78). A more significant negative effect was obtained with the two recombinant proteins of amb120 and och180, which redirected the Gag particle assembly pathway from the plasma membrane compartment to intracellular vesicles (amb120) and to the nuclear compartment (och180).
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PMID:Human immunodeficiency virus type 1 MA deletion mutants expressed in baculovirus-infected cells: cis and trans effects on the Gag precursor assembly pathway. 798 31


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