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)

COS-7 cells transfected with human immunodeficiency virus type 1 (HIV-1) proviral DNA produce virus in which three tRNA species are most abundant in the viral tRNA population. These tRNAs have been identified through RNA sequencing techniques as tRNA(3Lys) the primer tRNA in HIV-1, and members of the tRNA(1,2Lys) isoacceptor family. These RNAs represent 60% of the low-molecular-weight RNA isolated from virus particles, while they represent only 6% of the low-molecular-weight RNA isolated from the COS cell cytoplasm. Thus, tRNA(Lys) is selectively incorporated into HIV-1 particles. We have measured the ratio of tRNA(3Lys) molecules to copies of genomic RNA in viral RNA samples and have calculated that HIV-1 contains approximately eight molecules of tRNA(3Lys) per two copies of genomic RNA. We have also obtained evidence that the Pr160gag-pol precursor is involved in primer tRNA(3Lys) incorporation into virus. First, selective tRNA(Lys) incorporation and wild-type amounts of tRNA(3Lys) were maintained in a protease-negative virus unable to process Pr55gag and Pr160gag-pol precursors, indicating that precursor processing was not required for primer tRNA incorporation. Second, viral particles containing only unprocessed Pr55gag protein did not selectively incorporate tRNA(Lys), while virions containing both unprocessed Pr55gag and Pr160gag-pol proteins demonstrated select tRNA(3Lys) packaging. Third, studies with a proviral mutant containing a deletion of most of the reverse transcriptase sequences and approximately one-third of the integrase sequence in the Pr160gag-pol precursor resulted in the loss of selective tRNA incorporation and an eightfold decrease in the amount of tRNA(3Lys) per two copies of genomic RNA. We have also confirmed herein finding of a previous study which indicated that the primer binding site is not required for the selective incorporation of tRNA(Lys).
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PMID:Role of Pr160gag-pol in mediating the selective incorporation of tRNA(Lys) into human immunodeficiency virus type 1 particles. 751 Nov 67

Mutation in the human immunodeficiency virus type 1 reverse transcriptase (RT) at codon 215 has been shown to play a significant role in resistance to zidovudine (AZT). Substitution of threonine with tyrosine or phenylalanine alone confers decreased susceptibility to the inhibitor. In this study we constructed a panel of 10 viruses with different amino acids at this codon, including 7 novel mutants, and assessed their susceptibilities to AZT. The majority of the new mutants were AZT sensitive, whereas the Thr-215-->Trp mutant was partially resistant (threefold less susceptible). A combination of the Thr-215-->Trp with the other AZT resistance mutations Lys-70-->Arg and Met-41-->Leu gave additive resistance. The Thr-215-->Phe virus was less AZT resistant than the Thr-215-->Tyr mutant, both on its own and when each was combined with the Met-41-->Leu mutant. These observations confirm the general hypothesis that increased bulk of the amino acid side chains at this position confers decreased AZT sensitivity. A leucine-to-valine substitution at codon 74 has previously been found to confer dideoxynucleoside resistance. We constructed mutants with five novel amino acid substitutions (Ala, Gly, Glu, Met, and Asp) at codon 74. Of these, only one (that with the Met substitution) retained enough RT activity to yield viable virus. It thus appears that there are severe structure-function constraints on the amino acid side chains at this position in the enzyme. The activities of the Leu-74-->Ala and Leu-74-->Met RT enzymes expressed in Escherichia coli appeared to have reduced susceptibility to ddGTP compared with the wild-type enzyme. The mutants described in this work may prove useful for correlation with structural studies of the human immunodeficiency virus type 1 RT.
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PMID:Mutagenic study of codons 74 and 215 of the human immunodeficiency virus type 1 reverse transcriptase, which are significant in nucleoside analog resistance. 751 65

We generated variants of the human immunodeficiency virus type 1 (HIV-1) that are resistant to 2',3'-dideoxycytidine (ddC) and 2',3'-didehydro-3'-deoxythymidine (d4T) by in vitro selection in MT-4 cells. Portions of flanking protease and integrase sequences as well as the complete reverse transcriptase (RT) open-reading frame of these viruses were cloned and sequenced, using polymerase chain reaction (PCR)-based methods. Mutations were observed at amino acid position 65 (Lys-->Arg; AAA-->AGA) when ddC was employed in the selection procedure and at site 50 (Ile-->Thr; ATT-->ACT) when d4T was used. We confirmed the ability of these mutations to confer diminished sensitivity for these compounds by site-directed mutagenesis, in which these mutations were inserted into the pol gene of infectious recombinant HXB2-D DNA. Viruses that contained the site 65 mutation possessed approximately 5-10 fold resistance against ddC when compared with wild-type HXB2-D. The site 50 mutation conferred approximately 30-fold resistance to d4T in these same assays. Similar results were obtained using primary cord blood lymphocytes in drug resistance assays, indicating that these mutations could confer drug resistance in more than one cell type and that the respective mutations could be expressed in cells of primary origin. No cross-resistance against 3'-azido-3'-deoxythymidine (AZT) was noted for either the site 65 or 50 mutations.
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PMID:Identification of novel mutations that confer drug resistance in the human immunodeficiency virus polymerase gene. 751 78

We developed an endogenous in vitro reverse transcription assay to study the properties of priming and template switching during human immunodeficiency virus (HIV) replication. Reactions were primed with HIV reverse transcriptase (RT) and either a deoxyoligonucleotide primer (dPR) or tRNA(Lys-3), the natural primer for reverse transcription. The RNA templates utilized were the actual HIV sequences involved in the first template switch, namely a primer binding sequence (PBS)/U5/R RNA donor template and a R/U3 RNA acceptor template. Reverse transcription reactions using the latter templates and dPR or tRNA(Lys-3) as primers yielded four major products: (-)-strong-stop DNA, a partial template-switched DNA, full template-switched DNA, and a pseudo-PBS-primed product. Use of dPR resulted in three times less template switching than was obtained with tRNA(Lys-3). When reactions were primed with either dPR or tRNA(Lys-3), increases in acceptor:donor template ratios resulted in augmented template switching. Increasing the concentration of RT resulted in increased priming from the PBS but had no effect on the efficiency of template switching. Decreasing the extent of R region overlap resulted in a drop in efficiency of template switching. Decreases in the R region on the donor template also caused a drop in initiation of transcription that was primed by tRNA(Lys-3) from the PBS. In contrast, a corresponding reduction of the R region on the acceptor template had no effect on priming. We conclude that a transcriptional complex of tRNA(Lys-3) and RT may be associated not only with the PBS but also with other cis RNA sequences and secondary structures in a manner essential for efficient priming and template switching.
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PMID:Comparison of deoxyoligonucleotide and tRNA(Lys-3) as primers in an endogenous human immunodeficiency virus-1 in vitro reverse transcription/template-switching reaction. 751 78

Previously we identified the highly conserved amino acids Glu-Leu-Asp-Lys-Trp-Ala (ELDKWA) on the ecto-domain of gp41 as the epitope of a neutralizing monoclonal antibody (2F5) directed against human immunodeficiency virus type 1. In the present study, the sequence defining the epitope was introduced into the loop of antigenic site B of the influenza virus hemagglutinin. The resulting chimeric virus was able to elicit ELDKWA-specific immunoglobulins G and A in antisera of mice. Moreover, the distantly related human immunodeficiency virus type 1 isolates MN, RF, and IIIB were neutralized by these antisera. These data suggest that this conserved B-cell epitope is a promising candidate for inclusion in a vaccine against AIDS. The results also show that influenza virus can be used to effectively present the antigenic structure of this B-cell epitope.
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PMID:Cross-neutralizing activity against divergent human immunodeficiency virus type 1 isolates induced by the gp41 sequence ELDKWAS. 751 84

The technique of in vitro selection was used to generate variants of the human immunodeficiency virus type 1 that are resistant to 2',3'-dideoxycytidine (ddC). Most of the pol regions of such viruses, including the complete reverse transcriptase open reading frame and portions of flanking protease and integrase genes, were cloned and sequenced, using PCR-based procedures. Mutations were variously detected at amino acid site 65 (Lys-->Arg; AAA-->AGA) and at a previously reported codon, site 184 (Met-->Val; ATG-->GTG). We introduced the site 65 mutation into the pol gene of infectious, cloned HxB2-D DNA by site-directed mutagenesis in order to confirm by viral replication assay the importance of this site in conferring resistance to ddC. The recombinant virus possessed greater than 10-fold resistance against this compound in comparison with parental HxB2-D. Cross-resistance of approximately 20- and 3-fold, respectively, was detectable against the (-) enantiomer of 2',3'-dideoxy-3'-thiacytidine and 2',3'-dideoxyinosine but not against 3'-azido-3'-deoxythymidine. Combinations of the site 65 and 184 mutations did not yield levels of resistance higher than those attained with the site 65 mutation alone. The presence of the site 65 mutation was confirmed by PCR analysis of peripheral blood mononuclear cells from patients on long-term ddC therapy in 4 of 11 cases tested. Viruses that possessed a ddC resistance phenotype were isolated from subjects whose viruses contained the site 65 mutation in each of four instances. Four of these clinical samples were also demonstrated to possess the Met-184-->Val mutation, and one of them possessed both the Lys-65-->Arg and Met-184-->Val substitutions. Direct cloning and sequencing revealed the site 65 mutation in viruses isolated from these individuals.
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PMID:Identification of a mutation at codon 65 in the IKKK motif of reverse transcriptase that encodes human immunodeficiency virus resistance to 2',3'-dideoxycytidine and 2',3'-dideoxy-3'-thiacytidine. 751 55

A human immunodeficiency virus type 1 variant resistant to zalcitabine (2',3'-dideoxycytidine [ddC]) was selected by sequential passage in the presence of increasing concentrations of ddC in peripheral blood mononuclear cell cultures. A mutation causing a lysine-to-arginine substitution was noted in reverse transcriptase (RT) codon 65 of this ddC-selected virus. A cloned mutant virus with this codon 65 mutation was constructed by using a novel PCR approach for site-directed mutagenesis. Characterization of this virus confirmed that the RT Lys-65-->Arg substitution was necessary and sufficient for a fourfold increase in the ddC 50% inhibitory concentration, as well as for resistance to didanosine (2',3'-dideoxyinosine [ddI]). Lys-65-->Arg and virus resistance to ddC and ddI also developed during therapy in isolates from one ddC-treated patient and two ddI-treated patients. Recombinant-expressed codon 65 mutant RT enzyme was resistant to ddCTP and ddATP in cell-free polymerase assays. Results of mutant enzyme studies are consistent with Lys-65-->Arg leading to changes in binding of the triphosphate forms of these nucleoside analogs to the RT. These data have implications for future studies of ddC resistance, particularly those aimed at defining its clinical relevance.
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PMID:Resistance to 2',3'-dideoxycytidine conferred by a mutation in codon 65 of the human immunodeficiency virus type 1 reverse transcriptase. 751 56

Determination of the three-dimensional structure of the human immunodeficiency virus type-1 (HIV-1) reverse transcriptase (RT) has indicated a totally different folding for the 51-kDa subunit (p51) than for the 66-kDa subunit (p66). The polymerase catalytic site is located on the p66 subunit. Moreover, the HIV-1-specific RT inhibitors, also designated as the non-nucleoside RT inhibitors (NNRTIs), select for amino acid mutations that afford resistance to these compounds and are clustered in the palm domain of the HIV-1 RT p66 subunit. This pocket is located in the vicinity of, but clearly distinct from, the polymerase active site. However, for the NNRTIs that belong to the class of the [2',5'-bis-O-(tert-butyldimethylsilyl)-3'-spiro-5''-(4''-amino-1'',2''- oxathiole- 2'',2''-dioxide)] (TSAO) derivatives, the resistance mutation is located at position Glu138. On the p66 subunit, this amino acid is distant from the binding site of the HIV-1-specific RT inhibitors. When the TSAO-specific resistance mutation Glu138-->Lys was introduced solely in the p51 subunit of the RT p66/p51 heterodimer, the enzyme proved completely resistant to TSAO-m3T but retained full sensitivity to TIBO R82150 and ddGTP. On the other hand, when the mutation was introduced only in the p66 subunit the enzyme remained equally sensitive to the inhibitory effects of TSAO-m3T, TIBO R82150, and ddGTP. Our data provide compelling evidence for a structural and functional role of the p51 subunit in the sensitivity and/or resistance of the enzyme to the NNRTIs.
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PMID:Resistance of HIV-1 reverse transcriptase against [2',5'-bis-O-(tert-butyldimethylsilyl)-3'-spiro-5''-(4''-amino-1'',2''- oxathiole-2'',2''-dioxide)] (TSAO) derivatives is determined by the mutation Glu138-->Lys on the p51 subunit. 752 83

"BcgI cassette" mutagenesis was used to prepare variants of p66 human immunodeficiency virus (HIV)-1 reverse transcriptase with amino acid substitutions between residues Glu224 and Trp229. Mutant polypeptides were reconstituted in vitro with wild type p51 to generate the "selectively mutated" heterodimer series p66(224A)/p51-p66(229A)/p51. Purified enzymes were characterized with respect to dimerization, DNA polymerase, RNase H, and tRNA(Lys-3) binding. The combined analyses indicate that while alteration of p66 residues Glu224-Leu228 has minimal consequences, the DNA polymerase activities of mutant p66(229A)/p51 are impaired. DNase I footprinting illustrates that this mutant does not form a stable replication complex with a model template-primer. In vivo studies indicate that the equivalent mutation eliminates viral infectivity, suggesting a contribution of Trp229 toward architecture of the p66 primer grip.
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PMID:Mutating the "primer grip" of p66 HIV-1 reverse transcriptase implicates tryptophan-229 in template-primer utilization. 752 8

The structural proteins of human immunodeficiency virus type 1, for example, Gag and Env, are encoded by unspliced and incompletely spliced viral transcripts. The expression of these mRNAs in the cytoplasm, along with their commensurate translation, is absolutely dependent on the virally encoded Rev trans activator. Previous studies have demonstrated that Rev binds directly to its substrate mRNAs via an arginine-rich element that also serves as its nuclear localization sequence. In an attempt to define the specific amino acid residues that are important for in vivo activity, we have constructed a series of missense mutations that scan across this region. Our data demonstrate that all eight arginine residues within this element can, individually, be substituted for either leucine or lysine with no apparent loss of function. Importantly, these findings suggest that no single amino acid within the arginine-rich domain of Rev is, by itself, essential for activity and that considerable functional redundancy is therefore likely to exist within this region. Interestingly, one mutant in which a tryptophan had been substituted for a serine failed to accumulate exclusively in the nucleus but still bound RNA in a manner that was indistinguishable from that of the wild-type protein. This observation indicates that features of the arginine-rich region that are additional to those required for RNA binding are important for Rev's correct accumulation in the nucleus.
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PMID:Scanning mutagenesis of the arginine-rich region of the human immunodeficiency virus type 1 Rev trans activator. 752 98


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