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Query: UMLS:C0019693 (HIV)
 170,526 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The development of an effective HIV vaccine is both a pressing and a formidable problem. The most encouraging results to date have been achieved using live-attenuated immunodeficiency viruses. However, the frequency of pathogenic breakthroughs has been a deterrent to their development. We suggest that expression libraries generated from viral DNA can produce the immunologic advantages of live vaccines without risk of reversion to pathogenic viruses. The plasmid libraries could be deconvoluted into useful components or administered as complex mixtures. To explore this approach, we designed and tested several of these genetic live vaccines (GLVs) for HIV. We constructed libraries by cloning overlapping fragments of the proviral genome into mammalian expression plasmids, then used them to immunize mice. We found that inserting library fragments into a vector downstream of a secretory gene sequence led to augmented antibody responses, and insertion downstream of a ubiquitin sequence enhanced cytotoxic lymphocyte responses. Also, fragmentation of gag into subgenes broadened T-cell epitope recognition. We have fragmented the genome by sequence-directed and random methods to create libraries with different features. We propose that the characteristics of GLVs support their further investigation as an approach to protection against HIV and other viral pathogens.
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PMID:Genetic live vaccines mimic the antigenicity but not pathogenicity of live viruses. 1043 51

Beta-catenin, IkappaBalpha, and HIV Vpu are recruited to the ubiquitin-proteasome degradation pathway by betaTRCP, one of the components of the ubiquitin ligase complex. betaTRCP2, a related gene of betaTRCP, was cloned and characterized. Three isoforms, betaTRCP2A, betaTRCP2B, and betaTRCP2C, were identified. All of these betaTRCP2 isoforms consist of an F-box and seven WD repeats. Human betaTRCP2A shows 86% total amino acid identity with human betaTRCP. betaTRCP2 mRNA of 4.5 kb in size was detected almost ubiquitously. Sequence analyses on betaTRCP2 genomic clones revealed that the betaTRCP2 gene consists of at least 14 exons. Exons 1 and 4-14 are shared among all betaTRCP2 isoforms. betaTRCP2A of 508 amino acids lacks exons 2 and 3, betaTRCP2B of 529 amino acids contains exon 3, and betaTRCP2C of 542 amino acids contains exon 2. These results indicate that three betaTRCP2 isoforms are transcribed due to alternative splicing. The betaTRCP2 gene has been mapped to human chromosome 5q35.1 by fluorescence in situ hybridization.
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PMID:Molecular cloning and genomic structure of the betaTRCP2 gene on chromosome 5q35.1. 1069 85

Expression of the human immunodeficiency virus type 1 (HIV-1) Env glycoprotein is stringently regulated in infected cells. The majority of the glycoprotein does not reach the cell surface but rather is retained in the endoplasmic reticulum or a cis-Golgi compartment and subsequently degraded. We here report that Env of various HIV-1 isolates is ubiquitinated at the extracellular domain of gp41 and that Env expression could be increased by lactacystin, a specific proteasome inhibitor, suggesting that the ubiquitin/proteasome system is involved in control of expression and degradation.
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PMID:Ubiquitination of the human immunodeficiency virus type 1 env glycoprotein. 1079 17

Human immunodeficiency virus type-1 (HIV-1) integrase catalyzes the irreversible insertion of the viral genome into host chromosomal DNA. We have developed a mammalian expression system for the synthesis of authentic HIV-1 integrase in the absence of other viral proteins. Integrase, which bears a N-terminal phenylalanine, was found to be a short-lived protein in human embryo kidney 293T cells. The degradation of integrase could be suppressed by proteasome inhibitors. N-terminal phenylalanine is recognized as a degradation signal by a ubiquitin-proteasome proteolytic system known as the N-end rule pathway. The replacement of N-terminal phenylalanine with methionine, valine, or glycine, which are stabilizing residues in the N-end rule, resulted in metabolically stabilized integrase proteins (half-life of N-terminal Met-integrase was at least 3 h). Conversely, the substitution of N-terminal phenylalanine with other destabilizing residues retained the metabolic instability of integrase. These findings indicate that the HIV-1 integrase is a physiological substrate of the N-end rule. We discuss a possible functional similarity to the better understood turnover of the bacteriophage Mu transposase and functions of integrase instability to the maintenance and integrity of the host cell genome.
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PMID:Degradation of HIV-1 integrase by the N-end rule pathway. 1089 19

The DNA repair protein HHR23A is a highly conserved protein that functions in nucleotide excision repair. HHR23A contains two ubiquitin associated domains (UBA) that are conserved in a number of proteins with diverse functions involved in ubiquitination, UV excision repair, and signaling pathways via protein kinases. The cellular binding partners of UBA domains remain unclear; however, we previously found that the HHR23A UBA(2) domain interacts specifically with the HIV-1 Vpr protein. Analysis of the low resolution solution structure of HHR23A UBA(2) revealed a hydrophobic loop region of the UBA(2) domain that we predicted was the interface for protein/protein interactions. Here we present results of in vitro binding studies that demonstrate the requirement of this hydrophobic loop region for interaction with human immunodeficiency virus (HIV-1) Vpr. A single point mutation of the Pro at residue 333 to a Glu totally abolishes the binding of HIV-1 Vpr to UBA(2). High resolution NMR structures of the binding deficient UBA(2) mutant P333E as well as of the wild-type UBA(2) domain were determined to compare the effect of this mutation on the structure. Small but significant differences are observed only locally at the site of the mutation. The biochemical and structural analysis confirms the function of the HHR23A UBA(2) GFP-loop as the protein/protein interacting domain.
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PMID:Biochemical and structural analysis of the interaction between the UBA(2) domain of the DNA repair protein HHR23A and HIV-1 Vpr. 1108 58

Retrovirus assembly and maturation involve folding and transport of viral proteins to the virus assembly site followed by subsequent proteolytic cleavage of the Gag polyprotein within the nascent virion. We report that inhibiting proteasomes severely decreases the budding, maturation, and infectivity of HIV. Although processing of the Env glycoproteins is not changed, proteasome inhibitors inhibit processing of Gag polyprotein by the viral protease without affecting the activity of the HIV-1 viral protease itself, as demonstrated by in vitro processing of HIV-1 Gag polyprotein Pr55. Furthermore, this effect occurs independently of the virus release function of the HIV-1 accessory protein Vpu and is not limited to HIV-1, as proteasome inhibitors also reduce virus release and Gag processing of HIV-2. Electron microscopy analysis revealed ultrastructural changes in budding virions similar to mutants in the late assembly domain of p6(gag), a C-terminal domain of Pr55 required for efficient virus maturation and release. Proteasome inhibition reduced the level of free ubiquitin in HIV-1-infected cells and prevented monoubiquitination of p6(gag). Consistent with this, viruses with mutations in PR or p6(gag) were resistant to detrimental effects mediated by proteasome inhibitors. These results indicate the requirement for an active proteasome/ubiquitin system in release and maturation of infectious HIV particles and provide a potential pharmaceutical strategy for interfering with retrovirus replication.
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PMID:Proteasome inhibition interferes with gag polyprotein processing, release, and maturation of HIV-1 and HIV-2. 1108 48

Retroviral Gag polyproteins have specific regions, commonly referred to as late assembly (L) domains, which are required for the efficient separation of assembled virions from the host cell. The L domain of HIV-1 is in the C-terminal p6(gag) domain and contains an essential P(T/S)AP core motif that is widely conserved among lentiviruses. In contrast, the L domains of oncoretroviruses such as Rous sarcoma virus (RSV) have a more N-terminal location and a PPxY core motif. In the present study, we used chimeric Gag constructs to probe for L domain activity, and observed that the unrelated L domains of RSV and HIV-1 both induced the appearance of Gag-ubiquitin conjugates in virus-like particles (VLP). Furthermore, a single-amino acid substitution that abolished the activity of the RSV L domain in VLP release also abrogated its ability to induce Gag ubiquitination. Particularly robust Gag ubiquitination and enhancement of VLP release were observed in the presence of the candidate L domain of Ebola virus, which contains overlapping P(T/S)AP and PPxY motifs. The release defect of a minimal Gag construct could also be corrected through the attachment of a peptide that serves as a physiological docking site for the ubiquitin ligase Nedd4. Furthermore, VLP formation by a full-length Gag polyprotein was sensitive to lactacystin, which depletes the levels of free ubiquitin through inhibition of the proteasome. Our findings suggest that the engagement of the ubiquitin conjugation machinery by L domains plays a crucial role in the release of a diverse group of enveloped viruses.
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PMID:A role for ubiquitin ligase recruitment in retrovirus release. 1108 48

Our previous biochemical studies of HIV-1 and MuLV virions isolated and identified mature Gag products, HIV-1 p6(Gag) and MuLV p12(Gag), that were conjugated to a single ubiquitin. To study the importance of the monoubiquitination of Gag, a series of lysine to arginine mutants were constructed that eliminated ubiquitination at one or both of the lysines in HIV-1(NL4-3) p6(Gag) and both lysines in Moloney MuLV p12(Gag). HPLC and immunoblot analysis of the HIV-1 mutants demonstrated that either of the lysines in p6(Gag), K27 or K33, could be monoubiquitinated. However, infectivity assays showed that monoubiquitination of HIV-1 p6(Gag) or MuLV p12(Gag) is not required for viral replication in vitro. Pulse-chase radiolabeling of HIV-1-producing cells revealed that monoubiquitination of p6(Gag) does not affect the short-term release of virus from the cell, the maturation of Pr55(Gag), or the sensitivity of these processes to proteasome inhibitors. Experiments with protease-deficient HIV-1 showed that Pr55(Gag) can be monoubiquitinated, suggesting that p6(Gag) is first modified as a domain within Gag. Examination of the proteins inside an HIV-1 mutant found that free ubiquitin was incorporated into the virions in the absence of the lysines in p6(Gag), showing that the ubiquitin inside the virus is not initially brought in as a p6(Gag) conjugate. Although our results establish that monoubiquitination of p6(Gag) and p12(Gag) is not required for viral replication in vitro, this modification may be a by-product of interactions between Gag and cellular proteins during assembly and budding.
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PMID:Ubiquitination of HIV-1 and MuLV Gag. 1111 87

The diffusive properties of biomacromolecules within the aqueous phase of polyacrylamide gels are described. High quality NMR spectra can be obtained under such conditions. As compared to water, a fivefold reduction in the translational diffusion constant, but only a 1.6-fold decrease (1.4-fold increase) in amide-15N T2 (T1) are observed for human ubiquitin within a 10% acrylamide gel. Weak alignment of the solute macromolecules can be achieved within such gels by vertical or radial compression or by the embedding of magnetically oriented purple membrane fragments. The methods are applied to deriveresidual dipolar couplings for human HIV-1 Nef and ubiquitin.
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PMID:Solution NMR of proteins within polyacrylamide gels: diffusional properties and residual alignment by mechanical stress or embedding of oriented purple membranes. 1120 May 24

Ubiquitination appears to be involved in virus particle release from infected cells. Free ubiquitin (Ub), as well as Ub covalently bound to a small fraction of p6 Gag, is detected in mature HIV particles. Here we report that the p6 region in the Pr55(Gag) structural precursor polyprotein binds to Tsg101, a putative Ub regulator that is involved in trafficking of plasma membrane-associated proteins. Tsg101 was found to interact with Gag in (i) a yeast two-hybrid assay, (ii) in vitro coimmunoprecipitation by using purified Pr55(Gag) and rabbit reticulocyte lysate-synthesized Tsg101, and (iii) in vivo in the cytoplasm of COS cells transfected with gag. The PTAPP motif [or late (L) domain] within p6, which is required for release of mature virus from the plasma membrane, was the determinant for binding Pr55(Gag). The N-terminal region in Tsg101, which is homologous to the Ubc4 class of Ub-conjugating (E2) enzymes, was the determinant of interaction with p6. Mutation of Tyr-110 in Tsg101, present in place of the active-site Cys that binds Ub in E2 enzymes, and other residues unique to Tsg101, impaired p6 interaction, indicating that features that distinguish Tsg101 from active E2 enzymes were important for binding the viral protein. The results link L-domain function in HIV to the Ub machinery and a specific component of the cellular trafficking apparatus.
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PMID:Tsg101, a homologue of ubiquitin-conjugating (E2) enzymes, binds the L domain in HIV type 1 Pr55(Gag). 1142 3


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