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Query: UMLS:C0021051 (immunodeficiency)
 71,517 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

To develop a test for diagnosis of human immunodeficiency virus-1 (HIV-1) exposure sensitivity, a part of the gag gene was cloned and expressed in Escherichia coli, using expression vectors containing a trp promoter. The immunoreactivity of recombinant protein was determined using HIV-1 specific antibodies in a Western blot analysis. The recombinant plasmid, pYHCgag3, gag gene was fused to the trpE' gene linked to the hydroxylamine (HA) cleavage recognition sequence which was induced to overexpress a core antigen (gag a.a. 121-398 from plasmid BH10) as fusion protein in the form of insoluble inclusion body. Recombinant gag was purified by a simple single step purification procedure. After partial purification of inclusion bodies and subject to the HA-cleavage treatment, gag protein was further purified to homogeneity using DEAE-Sepharose chromatography. The purified core antigen offered reliable results with high sensitivity and specificity for identification of HIV-1 antibodies when tested in the enzyme-linked immunosorbent assay (ELISA). These results suggest that mass production of recombinant core antigen will provide a valuable resource to HIV-1 serodiagnostics for the screening of large groups of blood donors to prevent HIV-1 infection.
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PMID:Overexpression and simple purification of human immunodeficiency virus-1 gag epitope derived from a recombinant antigen in E. coli and its use in ELISA. 776 47

The human immunodeficiency virus type 1 internal structural protein precursor, p55, and its corresponding matrix proteolytic fragment, p17, are phosphorylated at Ser111 by protein kinase C. COS-7 cells transfected with plasmids encoding either the wild-type or Ser111-->Ala mutated human immunodeficiency virus type 1 gag gene matrix domain proteins were treated with phorbol 12-myristate 13-acetate (PMA), and the phosphorylation of the expressed p17 proteins was examined by radioimmunoprecipitation, SDS-polyacrylamide gel electrophoresis, and autoradiography. PMA treatment of transfected cells resulted in a 4-5-fold increase in wild-type p17 (but not mutated p17) phosphorylation; however, mutated p17 exhibited a low basal level of phosphorylation that was not affected by PMA, suggesting that additional sites were phosphorylated. PMA treatment of cells expressing wild-type p17 produced a dramatic shift in the localization of p17 from the cytosol to the membrane fraction within 8-15 min, followed by a slow quantitative dissociation of p17 back into the cytosol by 90 min. The cytosol-to-membrane translocation was dependent on N-myristoylated p17 since cells expressing p17 with a Gly2-->Ala mutation did not localize to the membrane. PMA also failed to induce the translocation of fully N-myristoylated Ser111-->Ala p17, suggesting that p17 phosphorylation at Ser111 was responsible for membrane association. This conclusion was confirmed by the finding of phosphorylated wild-type p17 in the membrane fraction only after PMA treatment. These results suggest that a "myristoyl-protein switch" regulates the reversible membrane targeting of p17 by protein kinase C-mediated phosphorylation. This signal may provide a mechanism for the cellular regulation of virus development through modulation of gag protein-related developmental steps such as capsid targeting, assembly, encapsidation, budding, and maturation.
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PMID:Regulation of HIV-1 gag protein subcellular targeting by protein kinase C. 787 52

The human immunodeficiency virus type 2 gag precursor protein, pr41, self assembles as virus-like particles (VLP) when the gag gene is expressed in insect cells. To map the functional domains for HIV-2 gag VLP formation, a series of deletion mutants was constructed by removing sequentially the C-terminal region of HIV-2 gag precursor protein and expressing the truncated gag genes in SF9 insect cells by means of recombinant baculoviruses. We found that deletion of up to 143 amino acids at the C-terminus of HIV-2 gag, leaving 376 amino acids at the N-terminus of the protein, did not prevent VLP formation. However, an additional four amino acids deletion from the C-terminus, which represents 372 amino acids at the N-terminus, made gag protein fail to form VLP. There is a proline-rich region at amino acid positions 372 and 377 of HIV-2 gag. To analyze the role of these proline residues, we generated five mutants in which proline was changed sequentially into leucine. Our results showed that replacement of one or two prolines did not stop gag VLP formation, whereas replacement of all three prolines by leucine residues completely abolished VLP assembly. Our data demonstrate that the C-terminal p12 region of HIV-2 gag precursor protein and the zinc finger domain are dispensable for gag VLP assembly, but the presence of at least one of the three proline residues located between amino acid positions 372 and 377 of HIV-2NIH-Z is required.
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PMID:Mapping of functional domains for HIV-2 gag assembly into virus-like particles. 797 51

Inbred strains of mice differ markedly in their relative susceptibility to the development of lymphoproliferation and immunodeficiency, a syndrome termed mouse AIDS (MAIDS), after infection with the LP-BM5 mixture of murine leukemia viruses (MuLV). The etiologic virus in this mixture is replication defective (BM5def) and encodes only a variant gag protein. Genetic determinants of resistance and susceptibility to induction of MAIDS reside both within and outside the MHC. In strains with C57BL background genes, the MHC haplotypes associated with resistance to disease include d and a, whereas haplotypes b, s, and q are associated with sensitivity. Previous studies showed that MHC class I genes (H-2Dd, H-2Ld) mapping in the D end of H-2 and other genes mapping proximal to the D end determine resistance to MAIDS. This paper examines the nature of these non-D end MHC genes using assays of MHC recombinant and transgenic mice. We demonstrate that expression of E alpha d confers significant resistance to MAIDS, even in mice that do not express H-2Dd/H-2Ld. Unexpectedly, we found that E alpha polymorphisms can significantly influence resistance, with H-2b mice bearing E alpha d as a transgene having greater resistance to MAIDS than mice bearing an E alpha k transgene. E alpha d-mediated resistance to MAIDS was associated with decreased levels of the BM5def genome in splenic DNA, suggesting that E alpha genes exert their effect by enhancing antiviral activity.
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PMID:Influence of H-2 class II antigens on the development of murine AIDS. 814 77

In order to monitor the antibody response to feline immunodeficiency virus (FIV) in cats, following experimental and natural infection, enzyme-linked immunosorbent assays (ELISAs) were developed using recombinant env and gag proteins and p24-specific monoclonal antibodies. It was shown that in experimentally infected cats an env protein-specific antibody response was directly followed by a gag protein-specific response. Furthermore, an ELISA for the detection of env protein-specific serum antibodies proved more sensitive in identifying experimentally and naturally infected cats than ELISAs demonstrating gag protein-specific antibodies. It was concluded that, like in HIV infection of humans, the detection of env protein-specific serum antibodies in addition to gag protein-specific antibodies is not only an important tool in the diagnosis of the infection but also in studies concerning the pathogenesis of the disease.
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PMID:gag- and env-specific serum antibodies in cats after natural and experimental infection with feline immunodeficiency virus. 820 21

Packaging of retroviral genomic RNA during virion assembly is thought to be mediated by specific interactions between the gag polyprotein and RNA sequences (often termed the psi or E region) near the 5' end of the genome. For many retroviruses, including human immunodeficiency virus type 1 (HIV-1), the portions of the gag protein and the RNA that are required for this interaction remain poorly defined. We have used an RNA gel mobility shift assay to measure the in vitro binding of purified glutathione S-transferase-HIV-1 gag fusion proteins to RNA riboprobes. Both the complete gag polyprotein and the nucleocapsid (NC) protein alone were found to bind specifically to an HIV-1 riboprobe. Either Cys-His box of NC could be removed without eliminating specific binding to the psi riboprobe, but portions of gag containing only the MA and CA proteins without NC did not bind to RNA. There were at least two binding sites in HIV-1 genomic RNA that bound to the gag polyprotein: one entirely 5' to gag and one entirely within gag. The HIV-1 NC protein bound to riboprobes containing other retroviral psi sequences almost as well as to the HIV-1 psi riboprobe.
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PMID:Specific binding of human immunodeficiency virus type 1 gag polyprotein and nucleocapsid protein to viral RNAs detected by RNA mobility shift assays. 823 Apr 41

The human immunodeficiency virus type 1 (HIV-1) nucleocapsid protein contains two copies of a sequence motif, the cysteine-histidine box, that is conserved among retroviruses. To identify the functionally relevant positions of a cysteine-histidine box, each amino acid in the proximal copy of the motif was individually substituted by site-directed mutagenesis. Mutations at 5 of 14 positions abolished virus replication and reduced the viral RNA content of mutant particles to between 10 and 20% of parental levels. Mutations at other positions had either no or only a minor effect on virus replication and virion RNA content. In vitro binding of RNA to bacterially expressed mutant Pr55gag polyprotein correlated well with the effects of the mutations on particle-associated viral RNA levels. The two different copies of the motif in the HIV-1 nucleocapsid protein are not functionally equivalent, since the conversion of the proximal motif to an exact copy of the distal motif results in a defect in virus replication and a reduction in the viral RNA content of mutant particles. The simultaneous substitution of functionally relevant positions in both motifs led to a significant decline in gag protein export, indicating that the nucleocapsid domain of the gag precursor is also required for efficient assembly or release of the virion.
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PMID:Mapping of functionally important residues of a cysteine-histidine box in the human immunodeficiency virus type 1 nucleocapsid protein. 837 56

An effective HIV vaccine should be capable of eliciting virus-specific cytotoxic T lymphocytes (CTL). We have characterized the cellular and molecular features of a simian immunodeficiency virus of macaques (SIVmac) gag-specific CTL response in rhesus monkeys. We have shown that SIVmac-infected rhesus monkeys expressing the major histocompatibility complex (MHC) class I molecule Mamu-A*01 develop a SIVmac gag-specific CTL response which recognizes a 9 amino acid fragment of the gag protein in association with Mamu-A*01. Moreover, this peptide/MHC class I recognition is mediated by T cell receptors (TCR) employing a predominant V beta gene family and J beta gene. Using this understanding of a SIVmac-specific CTL response, we have shown that SIVmac-specific CTL can be elicited through three novel approaches to vaccination: a recombinant viral vector, a recombinant bacterial vector and a peptide vaccine. These studies illustrate the utility of the SIV/macaque model in AIDS vaccine research.
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PMID:Simian immunodeficiency virus-specific cytotoxic T lymphocytes in rhesus monkeys: characterization and vaccine induction. 839 61

To determine the interaction between the gag precursor and the viral protease and to confirm the role of gag precursor in formation of human immunodeficiency virus type 1 particles, the gag and protease encoding regions of a proviral genome with mutations at the site between p17 and p24 or p24 and p15 were expressed by recombinant baculoviruses under the transcriptional control of the strong polyhedrin promoter. Western blot analyses of the expressed products of p17-p24 mutated viruses revealed that both 41- and 55-kDa proteins were synthesized. However, free p24, p17, and the other smaller cleavage products (p9, p6) could not be detected in infected insect cells. The second recombinant virus (p24-p15) synthesized not only a 55k-Da protein, but also a number of smaller products including a 40k-Da protein, p24, and p17. Examination of the insect cells infected by either of these two recombinant viruses by electron microscopy failed to detect any gag particle formation, although some irregular membrane protrusions and profound distortions of the cell surface were clearly visible in the cells infected with recombinant mutant p17-p24 virus, but not with recombinant p24-p15 mutants. To investigate the morphogenic capability of the gag-pol fusion protein, a mutant gag-pol gene containing an inactive protease as well as a modified gag-pol gene lacking the frameshifting activity were expressed in insect cells. While the inactive protease mutant was capable of forming immature particles that were secreted, the frameshifting mutant synthesized only an aberrant form of gag particles with a large radius of curvature in lieu of spherical particles. However, when this mutant was expressed in insect cells in the presence of a truncated gag protein with M(r) of 46 kDa (lacking only the p6 domain), normal immature particles containing both antigens were formed.
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PMID:Morphogenic capabilities of human immunodeficiency virus type 1 gag and gag-pol proteins in insect cells. 843 69

The role of the RNA secondary structure in the 5' packaging signal region of human immunodeficiency virus type 1 (HIV-1) in initiating translation of gag mRNA has been investigated both in vitro and in the presence of cellular cofactors in vivo. Heat denaturation of the structure and mutagenic deletion both lead to an increase in levels of translated products, indicating that the structure is a significant inhibitor of translation. The proximity of the gag AUG to the packaging signal structure suggested that it might function as an internal ribosome entry site. However, in both a cell-free system and eukaryotic cells, translation will initiate at a novel upstream initiation codon introduced within the 5' noncoding region. This codon is utilized exclusively, resulting in gag protein products with an extra 11 amino acids at the amino terminus, which, when expressed in T lymphocytes, are confined intracellularly, probably because of the lack of an N-terminal glycine myristoylation signal. Deletion of the secondary structure abolishes gag production even in the presence of tat and rev in trans. Using dicistronic constructs containing the HIV-1 5' leader cloned between two heterologous open reading frames, we were unable to detect any significant expression of the second open reading frame that would have been supportive of an internal ribosome entry site mechanism. Using mutant proviruses either lacking the entire packaging signal structure region or containing the introduced upstream initiation codon in long-term replication studies, we were unable to detect reverse transcriptase activity in culture supernatants. The 5' packaging signal structure of HIV-1 does not serve as an internal ribosome entry site. The translation of gag is consistent with ribosomal scanning. However, the packaging signal structure causes significant translational inhibition.
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PMID:The human immunodeficiency virus type 1 5' packaging signal structure affects translation but does not function as an internal ribosome entry site structure. 855 34


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