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)

Lentiviruses infect both dividing and nondividing cells. In this study we characterized a lentiviral vector system consisting of a packaging vector (pHP) and a transducing vector (pTV) derived from a recombinant human immunodeficiency virus type 1 (HIV-1). In pHP, the long terminal repeats (LTRs), the 5' untranslated leader and portions of the env and nef genes were deleted. The leader sequence of pHP was substituted with a modified Rous sarcoma virus (RSV) 59 bp leader containing a mutated RSV gag AUG and a functional 5' splice site. The pHP construct was found to direct Gag-Pol synthesis as efficiently as wild-type HIV-1. The pTV construct contains sequences required for RNA packaging, reverse transcription and integration, but lacks viral genes. Co-transfection of pHP, pTV and a vesicular stomatitis virus G (VSV-G) envelope plasmid produced vectors at titers of 10(5)-10(6) transducing units per milliliter in 48 h. Replication-competent virus (RCV) was not detected when deletions were made in the env gene in pHP. The ability of this vector system to transduce dividing and nondividing cell in vitro and in vivo was also demonstrated. Compared with a Moloney murine leukemia virus (MLV) vector, the HP/TV vectors transduced human muscle-, kidney-, liver-derived cell lines and CD34+ primary hematopoietic progenitor cells more efficiently. Although the levels of the pTV transgene expression were high soon after transduction, the expression tended to decrease with time due either to the loss of proviral DNA or to the inactivation of promoter activity, which was found to be cell type-dependent. Analyses of extrachromosomal DNA showed that the unintegrated proviral DNA of lentiviral vectors survived much longer than that of the retroviral vectors. We demonstrate that the HP/TV vector is capable of high efficiency transduction and that long-term expression of lentiviral vectors is dependent on target cell type, the internal promoter and the transgene itself in the transducing vector.
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PMID:Efficacy and safety analyses of a recombinant human immunodeficiency virus type 1 derived vector system. 1050 94

The retrovirus matrix (MA) sequence of the Gag polyprotein has been shown to contain functions required for membrane targeting and binding during particle assembly and budding. Additional functions for MA have been proposed based on the existence of MA mutants in Rous sarcoma virus (RSV), murine leukemia virus, human immunodeficiency virus type 1, and human T-cell leukemia virus type 1 that lack infectivity even though they release particles of normal composition. Here we describe an RSV MA mutant with a surprising and previously unreported phenotype. In the mutant known as Myr1E, the small membrane-binding domain of the Src oncoprotein has been added as an N-terminal extension of Gag. While Myr1E is not infectious, full infectivity can be reestablished by a single amino acid substitution in the Src sequence (G2E), which eliminates the addition of myristic acid and the membrane-binding capacity of this foreign sequence. The presence of myristic acid at the N terminus of the Myr1E Gag protein does not explain its replication defect, because other myristylated derivatives of RSV Gag are fully infectious (e.g., Myr2 [C. R. Erdie and J. W. Wills, J. Virol. 64:5204-5208, 1990]). Biochemical analyses of Myr1E particles reveal that they contain wild-type levels of the Gag cleavage products, Env glycoproteins, and reverse transcriptase activity when measured on an exogenous template. Genomic RNA incorporation appears to be mildly reduced compared to the wild-type level. Unexpectedly, RNA isolated from Myr1E particles is monomeric when analyzed on nondenaturing Northern blots. Importantly, the insertional mutation does not lie within previously identified dimer linkage sites. In spite of the dimerization defect, the genomic RNA from Myr1E particles serves efficiently as a template for reverse transcription as measured by an endogenous reverse transcriptase assay. In marked contrast, after infection of avian cells, the products of reverse transcription are nearly undetectable. These findings might be explained either by the loss of a normal function of MA needed in the formation or stabilization of RNA dimers or by the interference in such events by the mutant MA molecules. It is possible that Myr1E viruses package a single copy of viral RNA.
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PMID:RNA dimerization defect in a Rous sarcoma virus matrix mutant. 1059 Jan 3

After the polyprotein precursor of retroviral envelope proteins is proteolytically cleaved, the surface (SU) and transmembrane (TM) subunits remain associated with each other by noncovalent interactions or by disulfide bonds. Disulfide linkages confer a relatively stable association between the SU and TM envelope protein subunits of Rous sarcoma virus and murine leukemia virus. In contrast, the noncovalent association between SU and TM of human immunodeficiency virus leads to significant shedding of SU from the surface of infected cells. The SU and TM proteins of bovine leukemia virus (BLV) initially were reported to be disulfide linked but later were concluded not to be, since TM is often lost during purification of SU protein. Here, we show that SU and TM of BLV do, indeed, associate through disulfide bonds, whether the envelope proteins are overexpressed in transfected cells, are produced in virus-infected cells, or are present in newly produced virions.
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PMID:The SU and TM envelope protein subunits of bovine leukemia virus are linked by disulfide bonds, both in cells and in virions. 1068 14

Retroviral proteases form a unique subclass of the family of aspartic proteases. These homodimeric enzymes from a number of viral sources have by now been extensively characterized, both structurally and biochemically. The importance of such knowledge to the development of new drugs against AIDS has been, to a large extent, the driving force behind this progress. High-resolution structures are now available for enzymes from human immunodeficiency virus types 1 and 2, simian immunodeficiency virus, feline immunodeficiency virus, Rous sarcoma virus, and equine infectious anemia virus. In this review, structural and biochemical data for retroviral proteases are compared in order to analyze the similarities and differences between the enzymes from different sources and to enhance our understanding of their properties.
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PMID:Structural and biochemical studies of retroviral proteases. 1070 46

The p12 region of the Moloney murine leukemia virus (M-MuLV) Gag protein contains a PPPY motif important for efficient virion assembly and release. To probe the function of the PPPY motif, a series of insertions of homologous and heterologous motifs from other retroviruses were introduced at various positions in a mutant gag gene lacking the PPPY motif. The assembly defects of the PPPY deletion mutant could be rescued by insertion of a wild-type PPPY motif and flanking sequences at several ectopic positions in the Gag protein. The late assembly domain (L-domain) of Rous sarcoma virus (RSV) or human immunodeficiency virus type 1 (HIV-1) could also fully or partially restore M-MuLV assembly when introduced into matrix, p12, or nucleocapsid domains of the mutant M-MuLV Gag protein lacking the PPPY motif. Strikingly, mutant viruses carrying the RSV or the HIV-1 L-domain at the original location of the deleted PPPY motif were replication competent in rodent cells. These data suggest that the PPPY motif of M-MuLV acts in a partially position-independent manner and is functionally interchangeable with L-domains of other retroviruses. Electron microscopy studies revealed that deletion of the entire p12 region resulted in the formation of tube-like rather than spherical particles. Remarkably, the PPPY deletion mutant formed chain structures composed of multiple viral particles linked on the cell surface. Many of the mutants with heterologous L-domains released virions with wild-type morphology.
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PMID:Infectivity of Moloney murine leukemia virus defective in late assembly events is restored by late assembly domains of other retroviruses. 1090 79

Rous sarcoma virus (RSV), a simple retrovirus, needs to export unspliced viral RNA from the nucleus to the cytoplasm, circumventing the host cell restriction on cytoplasmic expression of intron-containing RNA. The cytoplasmic accumulation of full-length viral RNA is promoted by two cis-acting direct repeat (DR) elements that flank the src gene; at least one copy of the DR sequence is necessary for viral replication. We show here that the DR mediates export of a reporter construct from the nucleus, suggesting it is a constitutive transport element (CTE). In contrast, human immunodeficiency virus type 1 (HIV-1) and other complex retroviruses encode accessory proteins, Rev or Rex, which promote export of incompletely spliced viral transcripts. This RNA export pathway is CRM1 dependent and can be blocked by the cytotoxic agent leptomycin B. We show here that DR-mediated export is CRM1 independent, suggesting that RSV uses a different export pathway from that of HIV-1 and other complex retroviruses. The simian retroviruses have a CTE which interacts with the cellular Tap export protein. However, we were unable to detect binding of the RSV DR RNA to Tap, suggesting it may use a different export pathway from that of the simian retroviruses. These data suggest that the RSV DR element uses a novel nucleocytoplasmic export pathway.
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PMID:Rous sarcoma virus DR posttranscriptional elements use a novel RNA export pathway. 1100 Feb 20

Human T-cell leukemia virus type-1 (HTLV-1) is associated with a number of human diseases. Based on the therapeutic success of human immunodeficiency virus type 1 (HIV-1) PR inhibitors, the proteinase (PR) of HTLV-1 is a potential target for chemotherapy. To facilitate the design of potent inhibitors, the subsite specificity of HTLV-1 PR was characterized and compared to that of HIV-1 PR. Two sets of substrates were used that contained single amino-acid substitutions in peptides representing naturally occurring cleavage sites in HIV-1 and HTLV-1. The original HIV-1 matrix/capsid cleavage site substrate and most of its substituted peptides were not hydrolyzed by the HTLV-1 enzyme, except for those with hydrophobic residues at the P4 and P2 positions. On the other hand, most of the peptides representing the HTLV-1 capsid/nucleocapsid cleavage site were substrates of both enzymes. A large difference in the specificity of HTLV-1 and HIV-1 proteinases was demonstrated by kinetic measurements, particularly with regard to the S4 and S2 subsites, whereas the S1 subsite appeared to be more conserved. A molecular model of the HTLV-1 PR in complex with this substrate was built, based on the crystal structure of the S9 mutant of Rous sarcoma virus PR, in order to understand the molecular basis of the enzyme specificity. Based on the kinetics of shortened analogs of the HTLV-1 substrate and on analysis of the modeled complex of HTLV-1 PR with substrate, the substrate binding site of the HTLV-1 PR appeared to be more extended than that of HIV-1 PR. Kinetic results also suggested that the cleavage site between the capsid and nucleocapsid protein of HTLV-1 is evolutionarily optimized for rapid hydrolysis.
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PMID:Comparison of the substrate specificity of the human T-cell leukemia virus and human immunodeficiency virus proteinases. 1101 83

The 5' leader of Rous sarcoma virus (RSV) genomic RNA and of retroviruses in general is long and contains stable secondary structures that are critical in the early and late steps of virus replication such as RNA dimerization and packaging and in the process of reverse transcription. The initiation of RSV Gag translation has been reported to be 5' cap dependent and controlled by three short open reading frames located in the 380-nucleotide leader upstream of the Gag start codon. Translation of RSV Gag would thus differ from that prevailing in other retroviruses such as murine leukemia virus, reticuloendotheliosis virus type A, and simian immunodeficiency virus, in which an internal ribosome entry segment (IRES) in the 5' end of the genomic RNA directs efficient Gag expression despite stable 5' secondary structures. This prompted us to investigate whether RSV Gag translation might be controlled by an IRES-dependent mechanism. The results show that the 5' leaders of RSV and v-Src RNA exhibit IRES properties, since these viral elements can promote efficient translation of monocistronic RNAs in conditions inhibiting 5' cap-dependent translation. When inserted between two cistrons in a canonical bicistronic construct, both the RSV and v-Src leaders promote expression of the 3' cistron. A genetic analysis of the RSV leader allowed the identification of two nonoverlapping 5' and 3' leader domains with IRES activity. In addition, the v-Src leader was found to contain unique 3' sequences promoting an efficient reinitiation of translation. Taken together, these data lead us to propose a new model for RSV translation.
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PMID:Rous sarcoma virus translation revisited: characterization of an internal ribosome entry segment in the 5' leader of the genomic RNA. 1109 Jan 56

Purified retrovirus Gag proteins or Gag protein fragments are able to assemble into virus-like particles (VLPs) in vitro in the presence of RNA. We have examined the role of nucleic acid and of the NC domain in assembly of VLPs from a Rous sarcoma virus (RSV) Gag protein and have characterized these VLPs using transmission electron microscopy (TEM), scanning TEM (STEM), and cryoelectron microscopy (cryo-EM). RNAs of diverse sizes, single-stranded DNA oligonucleotides as small as 22 nucleotides, double-stranded DNA, and heparin all promoted efficient assembly. The percentages of nucleic acid by mass, in the VLPs varied from 5 to 8%. The mean mass of VLPs, as determined by STEM, was 6.5 x 10(7) Da for both RNA-containing and DNA oligonucleotide-containing particles, corresponding to a stoichiometry of about 1,200 protein molecules per VLP, slightly lower than the 1,500 Gag molecules estimated previously for infectious RSV. By cryo-EM, the VLPs showed the characteristic morphology of immature retroviruses, with discernible regions of high density corresponding to the two domains of the CA protein. In spherically averaged density distributions, the mean radial distance to the density corresponding to the C-terminal domain of CA was 33 nm, considerably smaller than that of equivalent human immunodeficiency virus type 1 particles. Deletions of the distal portion of NC, including the second Zn-binding motif, had little effect on assembly, but deletions including the charged residues between the two Zn-binding motifs abrogated assembly. Mutation of the cysteine and histidine residues in the first Zn-binding motif to alanine did not affect assembly, but mutation of the basic residues between the two Zn-binding motifs, or of the basic residues in the N-terminal portion of NC, abrogated assembly. Together, these findings establish VLPs as a good model for immature virions and establish a foundation for dissection of the interactions that lead to assembly.
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PMID:Characterization of Rous sarcoma virus Gag particles assembled in vitro. 1122 98

The N-terminal domain of the retroviral capsid (CA) protein is one of the least conserved regions encoded in the genome. Surprisingly, the three-dimensional structures of the CA from different genera exhibit alpha-helical structural features that are highly conserved. The N-terminal residues of the human immunodeficiency virus type 1 (HIV-1) and Rous sarcoma virus (RSV) capsid proteins form a beta-hairpin. To determine if this feature is conserved in the retroviral family, we cloned, expressed, purified, and solved the structure of a N-terminal 134 amino acid fragment (CA(134)) from the human T-cell leukemia virus type 1 (HTLV-I) using high resolution nuclear magnetic resonance (NMR) spectroscopy. The CA(134) fragment contains an N-terminal beta-hairpin and a central coiled-coil-like structure composed of six alpha-helices. The N-terminal Pro1 residue contacts Asp54 in the helical cluster through a salt bridge. Thus, the beta-hairpin is conserved and the helical cluster is structurally similar to other retroviral CA domains. However, although the same Asp residue defines the orientation of the hairpin in both the HTLV-1 and HIV-1 CA proteins, the HTLV-I hairpin is oriented away, rather than towards, the helical core. Significant differences were also detected in the spatial orientation and helical content of the long centrally located loop connecting the helices in the core. It has been proposed that the salt bridge allows the formation of a CA-CA interface that is important for the assembly of the conical cores that are characteristic of HIV-1. As HTLV-I forms spherical cores, the salt-bridge feature is apparently not conserved for this function although its role in determining the orientation of the beta-hairpin may be critical, along with the central loop. Comparison of three-dimensional structures is expected to elucidate the relationships between the retroviral capsid protein structure and its function.
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PMID:Structural analysis of the N-terminal domain of the human T-cell leukemia virus capsid protein. 1124 88


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