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

Reverse transcriptase (RT) was first discovered as an essential catalyst in the biological cycle of retroviruses. However, in the past years evidence has accumulated showing that RTs are involved in a surprisingly large number of RNA-mediated transpositional events that include both viral and nonviral genetic entities. Although it is probable that some RT-bearing genetic elements like the different types of AIDS viruses and the mammalian LINE family have arisen in recent geological times, the possibility that reverse transcription first took place in the early Archean is supported by (1) the hypothesis that RNA preceded DNA as cellular genetic material; (2) the existence of homologous regions of the subunit tau of the E. coli DNA polymerase III with the simian immunodeficiency virus RT, the hepatitis B virus RT, and the beta' subunit of the E. coli RNA polymerase (McHenry et al. 1988); (3) the presence of several conserved motifs, including a 14-amino-acid segment that consists of an Asp-Asp pair flanked by hydrophobic amino acids, which are found in all RTs and in most cellular and viral RNA polymerases. However, whether extant RTs descend from the primitive polymerase involved in the RNA-to-DNA transition remains unproven. Substrate specificity of the AMV and HIV-1 RTs can be modified in the presence of Mn2+, a cation which allows them to add ribonucleotides to an oligo (dG) primer in a template-dependent reaction. This change in specificity is comparable to that observed under similar conditions in other nucleic acid polymerases. This experimentally induced change in RT substrate specificity may explain previous observations on the misincorporation of ribonucleotides by the Maloney murine sarcoma virus RT in the minus and plus DNA of this retrovirus (Chen and Temin 1980). Our results also suggest that HIV-infected macrophages and T-cell cells may contain mixed polynucleotides containing both ribo- and deoxyribonucleotides. The evolutionary significance of these changes in substrate specificities of nucleic acid polymerases is also discussed.
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PMID:On the early emergence of reverse transcription: theoretical basis and experimental evidence. 128 61

The activity of the avian myeloblastosis virus (AMV) or the human immunodeficiency virus type 1 (HIV-1) protease on peptide substrates which represent cleavage sites found in the gag and gag-pol polyproteins of Rous sarcoma virus (RSV) and HIV-1 has been analyzed. Each protease efficiently processed cleavage site substrates found in their cognate polyprotein precursors. Additionally, in some instances heterologous activity was detected. The catalytic efficiency of the RSV protease on cognate substrates varied by as much as 30-fold. The least efficiently processed substrate, p2-p10, represents the cleavage site between the RSV p2 and p10 proteins. This peptide was inhibitory to the AMV as well as the HIV-1 and HIV-2 protease cleavage of other substrate peptides with Ki values in the 5-20 microM range. Molecular modeling of the RSV protease with the p2-p10 peptide docked in the substrate binding pocket and analysis of a series of single-amino acid-substituted p2-p10 peptide analogues suggested that this peptide is inhibitory because of the potential of a serine residue in the P1' position to interact with one of the catalytic aspartic acid residues. To open the binding pocket and allow rotational freedom for the serine in P1', there is a further requirement for either a glycine or a polar residue in P2' and/or a large amino acid residue in P3'. The amino acid residues in P1-P4 provide interactions for tight binding of the peptide in the substrate binding pocket.
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PMID:Mechanism of inhibition of the retroviral protease by a Rous sarcoma virus peptide substrate representing the cleavage site between the gag p2 and p10 proteins. 133 Oct 99

Recently we reported (D. B. Evans, W. G. Tarpley, and S. K. Sharma, 1991, Protein Expression Purif. 2, 205-213) the cloning, expression, and characterization of recombinant chimeric proteins with an N-terminal metal-binding peptide (mbp), His-Asp-His-Asp-His, and a renin cleavage site. Using these chimerics as examples, we describe here the use of genetically engineered alternating histidines in the purification of these chimerics by immobilized metal affinity chromatography (IMAC). In these chimerics, an alternate histidine-containing peptide was fused to the N-termini of HIV reverse transcriptase (HIV RT) and beta-galactosidase. These chimerics were retarded on immobilized nickel very strongly and could be completely eluted only by the use of 100 mM imidazole, whereas the wildtype HIV RT and Escherichia coli contaminating proteins were eluted between 10 and 35 mM imidazole. When the DNA coding for the mbp was removed, the resulting chimerics were recovered from the IMAC column at 35 mM imidazole. The strong and specific interaction between the chimeric protein and the immobilized metal ion was also abolished when the mbp was specifically cleaved by human renin. It is concluded from these studies that tailoring recombinant proteins with three or more alternate histidines should result in the isolation of such chimeric proteins from crude mixtures in a single step. Since IMAC is amendable to scale up, the tailored specificity engineered into the protein of interest via an mbp should allow one to achieve large-scale isolation of recombinant proteins from bacterial and nonbacterial hosts in a highly predictable manner.
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PMID:On the engineering of rDNA proteins for purification by immobilized metal affinity chromatography: applications to alternating histidine-containing chimeric proteins from recombinant Escherichia coli. 138 56

The human immunodeficiency virus type 1 (HIV-1) integrase enzyme exhibits significant amino acid sequence conservation with integrase proteins of other retroviruses. We introduced specific amino acid substitutions at a number of the conserved residue positions of recombinant HIV-1 integrase. Some of these substitutions resulted in proteins which were not able to be purified in the same manner as the wild-type enzyme, and these were not studied further. The remaining mutant enzymes were assessed for their abilities to perform functions characteristic of the integrase protein. These included specific removal of the terminal dinucleotides from oligonucleotide substrates representative of the viral U5-long terminal repeat, nonspecific cleavage of oligonucleotide substrates, and mediation of the strand transfer (integration) reaction. Substitution at position 43, within the protein's zinc finger motif region, resulted in an enzyme with reduced specificity for cleavage of the terminal dinucleotide. In addition, a double substitution of aspartic acid and glutamine for valine and glutamic acid, respectively, at positions 151 and 152 within the D,D(35)E motif region rendered the integrase protein inactive for all of its functions. The introduction of this double substitution into an infectious HIV-1 provirus yielded a mutant virus that was incapable of productively infecting human T-lymphoid cells in culture.
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PMID:Requirement of active human immunodeficiency virus type 1 integrase enzyme for productive infection of human T-lymphoid cells. 143 23

The protease from simian immunodeficiency virus (SIV) was chemically synthesized by automated solid-phase technology as an NH2-terminally extended derivative, capped with biotin. Biotin-linker-(SIV protease (1-99)): the linker segment, Gly-Gly-Asp-Arg-Gly-Phe-Ala-Ala, corresponds to the amino acid sequence preceding that of the protease in the SIV gag/pol precursor polyprotein. Accordingly, the Ala-Pro bond joining the octapeptide linker to the protease constitutes a site naturally cleaved by the protease during viral maturation. This strategy for synthesis was designed to facilitate purification of the biotinylated protein derivative from a complex mixture of reaction products by avidin/agarose-affinity chromatography and to provide the means for autocatalytic removal of the biotin-linker segment. As anticipated, folding of the full-length construct leads to activation of the enzyme and excision of the desired 99-residue SIV protease (overall yield, approximately). The specificity of the synthetic SIV protease toward a number of well characterized protein substrates was the same as observed for the nearly identical enzyme from human immunodeficiency virus type 2 (HIV-2 protease) and distinct from that of the more disparate HIV-1 protease. The same functional ordering with respect to the human retroviral proteases was reflected in Ki values observed with a number of protease inhibitors. Thus, the folded synthetic SIV protease shows patterns of specificity and susceptibility to inhibition that are in accord with what would be expected based upon its degree of structural similarity to proteases from HIV-1 and HIV-2.
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PMID:Chemical synthesis of a biotinylated derivative of the simian immunodeficiency virus protease. Purification by avidin affinity chromatography and autocatalytic activation. 158 12

1. Certain metal ions have been identified as inhibitors (IC50 1-20 microM) of the aspartic proteinase of Human Immunodeficiency Virus Type 1 (HIV-PR). 2. By contrast most simple metal ions do not inhibit this enzyme. 3. Those that did inhibit have in common a high charge/size ratio or "hard" acidic nature, preferring to combine covalently with oxygen donor ligands. 4. Some evidence from independent X-ray crystal structure determinations suggests that the metalloinhibitors identified here may bind in the active site of the enzyme via coordination to the carboxylate side chains of the essential active site residues Asp 25 and 125. 5. Although the measured inhibition is only microM, very few enzyme-inhibitor interactions can be taking place and so more complex metalloinhibitors with ligands that can also bind to peptide side chains of the enzyme might be significantly more potent inhibitors of HIV-PR and of viral replication.
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PMID:Inhibition of HIV-1 proteinase by metal ions. 161 81

Substitution of the conserved Asp-443 residue of HIV-1 reverse transcriptase by asparagine specifically suppressed the ribonuclease H activity of the enzyme without affecting the reverse transcriptase activity, suggesting involvement of this ionizable residue at the ribonuclease H active site. An analogous asparagine substitution of the Asp-498 residue yielded an unstable enzyme that was difficult to enzymatically characterize. However, the instability caused by the Asn-498 mutation was relieved by the introduction of a second distal Asn-443 substitution, yielding an enzyme with wild type reverse transcriptase activity, but lacking ribonuclease H activity.
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PMID:Site-directed mutagenesis of the conserved Asp-443 and Asp-498 carboxy-terminal residues of HIV-1 reverse transcriptase. 169 2

Monoclonal antibody SF8/5E11, which recognizes the transmembrane protein (TMP) of simian immunodeficiency virus of macaque monkeys (SIVmac), displayed strict strain specificity. It reacted with cloned and uncloned SIVmac251 but not with cloned SIVmac142 and SIVmac239 on immunoblots. This monoclonal antibody neutralized infection by cloned, cell-free SIVmac251 and inhibited formation of syncytia by cloned SIVmac251-infected cells; these activities were specific to cloned SIVmac251 and did not occur with the other viruses. Site-specific mutagenesis was used to show that TMP amino acids 106 to 110 (Asp-Trp-Asn-Asn-Asp) determined the strain specificity of the monoclonal antibody. This strain-specific neutralizing determinant is located within a variable region of SIVmac and human immunodeficiency virus type 2 (HIV-2) which includes conserved, clustered sites for N-linked glycosylation. The determinant corresponds exactly to a variable, weak neutralizing epitope in HIV-1 TMP which also includes conserved, clustered sites for N-linked glycosylation. Thus, the location of at least one neutralizing epitope appears to be common to both SIVmac and HIV-1. Our results suggest a role for this determinant in the viral entry process. Genetic variation was observed in this neutralizing determinant following infection of a rhesus monkey with molecularly cloned SIVmac239; variant forms of the strain-specific, neutralizing determinant accumulated during persistent infection in vivo. Selective pressure from the host immune response in vivo may result in sequence variation in this neutralizing determinant.
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PMID:Strain-specific neutralizing determinant in the transmembrane protein of simian immunodeficiency virus. 170 94

Three human immunodeficiency virus type 1 (HIV-1) mutants were constructed with mutations in their protease genes: AH2-pSVL, with an in-phase deletion; BH27-pSVL, with an out-of-phase deletion creating a stop codon immediately after the deletion site; and CA-pSVL, with a point mutation creating an Asp-to-Ala substitution at the putative protease active site. The wild-type, HXB2-pSVL, and the mutated viral genomes were used to transfect COS-M6 cells and to produce virions. Immunoblotting assays with a monoclonal antibody (MAb) specific for p24 showed that all three mutant contained a gag precursor, Pr56gag, with AH2 and CA expressing an extra band of about 160 kDa. Similar assays with a MAb specific for HIV-1 reverse transcriptase (RT) also revealed a 160-kDa protein from AH2 and CA virions and two mature p66 and p51 RT subunits from HXB2 virions. In addition, HXB2, AH2, and CA but not BH27 virions exhibited RT activity. The same protein in the 160-kDa band seemed to possess both p24 and RT components, since the MAb against p24 was able to immunoadsorb RT antigen and enzymatic activity. These results indicate that the HIV-1 gag-pol fusion protein produced in mammalian cells expressed significant RT activity.
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PMID:Identification and characterization of human immunodeficiency virus type 1 gag-pol fusion protein in transfected mammalian cells. 170 86

The C-terminal catalytic domain (residues 704-1047) of the human ras GTPase-activating protein (GAP) has been engineered so as to incorporate the tripeptide, Glu-Glu-Phe, at its C terminus. This motif is recognized by the commercially available YL1/2 monoclonal antibody to alpha-tubulin and has previously been used for the immunoaffinity purification of HIV enzymes engineered to contain this epitope (Stammers, D. K., Tisdale, M., Court, S., Parmar, V., Bradley, C., and Ross, C. K. (1991) FEBS Lett. 283, 298-302). The engineered GAP catalytic domain (GAP-344) was obtained in high yield and purity from Escherichia coli extracts by means of a single affinity column of immobilized YL1/2, eluted under mild conditions with the dipeptide, Asp-Phe. The protein had similar activity to that previously described for full-length GAP, suggesting that the addition of the epitope did not grossly affect the activity. R903K and L902I mutants of GAP-344 were constructed, and the immunoaffinity purification procedure allowed their rapid characterization. The R903K mutant had less than 3% the activity of the normal protein, whereas the L902I substitution had less than 0.5% of normal activity, suggesting an important role for Leu-902 and Arg-903, residues absolutely conserved among GAP-related proteins. This work exemplifies the general utility of the C-terminal Glu-Glu-Phe motif for the rapid purification of proteins whose function is not altered by C-terminal modification.
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PMID:Use of the Glu-Glu-Phe C-terminal epitope for rapid purification of the catalytic domain of normal and mutant ras GTPase-activating proteins. 171 77


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