Biomedical subjects

M A Parniak

Publications and source records attributed to M A Parniak.

At least 55 records · Page 3Linked to original sources

PubMed1996

Studies of neutralizing monoclonal antibody to human immunodeficiency virus type 1 reverse transcriptase: antagonistic and synergistic effects in reactions performed in the presence of nucleoside and nonnucleoside inhibitors, respectively.

We have assessed interactions between the reverse transcriptase (RT) of human immunodeficiency virus type 1 (HIV-1) and a neutralizing monoclonal antibody (1E8) that hinders binding of deoxynucleoside triphosphate (dNTP) substrates. Steady-state reactions with homopolymeric template-primers revealed that 1E8 antagonized inhibition of RT activity mediated by 3'-azido-3'-deoxythymidine triphosphate and 2',3'-dideoxycytidine triphosphate. However, an additive or synergistic inhibition of RT polymerase activity was noted when 1E8 and the nonnucleoside RT inhibitors nevirapine and delavirdine were studied. Chain elongation and dNTP incorporation studies using an HIV-1 genome-derived heteropolymeric template and either oligodeoxynucleotide or tRNA3(Lys) as the primer yielded results consistent with the above observations. 1E8 also increased pausing at certain sites during synthesis of negative-strand, strong-stop DNA, whether or not ddNTP and nonnucleoside RT inhibitors were present.

Animals↗

PubMed1996

Human immunodeficiency virus Type 1 nucleocapsid protein (NCp7) directs specific initiation of minus-strand DNA synthesis primed by human tRNA(Lys3) in vitro: studies of viral RNA molecules mutated in regions that flank the primer binding site.

Retroviral reverse transcription starts near the 5' end of unspliced viral RNA at a sequence called the primer binding site (PBS), where the tRNA primer anneals to the RNA template for initiation of DNA synthesis. We have investigated the roles of NCp7 in annealing of primer tRNA(Lys3) to the PBS and in reverse transcriptase (RT) activity, using a cell-free reverse transcription reaction mixture consisting of various 5' viral RNA templates, natural primer tRNA(Lys3) or synthetic primer, human immunodeficiency virus type I (HIV-1) nucleocapsid protein (NCp7), and HIV-1 RT. In the presence of tRNA(Lys3), NCp7 was found to stimulate synthesis of minus-strand strong-stop DNA [(-)ssDNA], consistent with previous reports. However, specific DNA synthesis was observed only at a NCp7/RNA ratio similar to that predicted to be present in virions. Moreover, at these concentrations, NCp7 inhibited the synthesis of nonspecific reverse-transcribed DNA products, which are initiated because of self-priming by RNA templates. In contrast to results obtained with tRNA(Lys3) as primer, NCp7 inhibited the synthesis of (-)ssDNA products primed by an 18-nucleotide (nt) ribonucleotide (rPR), complementary to the PBS, even though rPR can initiate synthesis of such material in the absence of preannealing with NCp7. Primer placement band shift assays showed that NCp7 was necessary for efficient formation of the tRNA-RNA complex. In contrast, NCp7 was found to prevent formation of the rPR-RNA complex. Since NCp7 appears to exert opposite effects (annealing versus dissociation) on tRNA(Lys3) and rPR substrates, the non-PBS binding regions of the tRNA(Lys3) molecule may play a role in the annealing of tRNA to the template. We also investigated the roles of an A-rich loop upstream of the PBS, a 7-nt region immediately downstream of the PBS, and a 54-nt deletion further downstream of the PBS in interactions with tRNA(Lys3). We found that deletions in the 54-nt region that may prevent formation of the U5-leader stem prevented tRNA(Lys3) placement and priming, while deletions in the A-rich loop or the 7-nt sequence had relatively minor effects in this regard.

Binding Sites↗

PubMed1996

Differences in the inhibition of human immunodeficiency virus type 1 reverse transcriptase DNA polymerase activity by analogs of nevirapine and [2',5'-bis-O-(tert-butyldimethylsilyl)-3'-spiro-5"-(4"-amino-1", 2"-oxathiole-2",2"-dioxide] (TSAO).

We compared the inhibition of HIV-1 reverse transcriptase (RT) by 1-[2',5'-bis-O-(t-butyldimethylsilyl)-beta-D-ribofuranosyl]-3'- spiro-5"-(4"-amino-1", 2"-oxathiole-2",2"-dioxide)-3-ethylthymine (TSAOe3T) and the nonnucleoside RT inhibitor (NNRTI) 9-aminonevirapine (9-NH2N). Both compounds were equally effective against p51/p66 heterodimeric RT RNA-dependent DNA polymerase activity, although TSAOe3T was a much better inhibitor of the p51/p51 and p66/p66 RT homodimers. Inhibition by TSAOe3T and 9-NH2N combinations was essentially additive. TSAOe3T did not protect either free RT or the RT-template/ primer-deoxynucleoside triphosphate ternary complex from irreversible inactivation by the photolabel 9-azidonevirapine. Slight protection of the RT-template/primer binary complex was noted, but only at high TSAOe3T/photolabel ratios. Analysis of RT polymerization product profiles under both continuous- and single-processive cycle conditions showed that 9-NH2N prevented the formation of full-length product with a corresponding accumulation of smaller polymerization products. In contrast, all products formed in the absence of inhibitor, including full-length product, were noted in TSAOe3T-inhibited reactions, albeit at reduced levels. TSAOe3T thus inhibits HIV-1 RT by a different mechanism than NNRTI such as nevirapine. Our data suggest that TSAOe3T and 9-NH2N interact differently with HIV-1 RT, perhaps by binding to distinct sites on the enzyme.

Antiviral Agents↗

PubMed1995

Synergistic inhibition of HIV-1 reverse transcriptase DNA polymerase activity and virus replication in vitro by combinations of carboxanilide nonnucleoside compounds.

The carboxanilides UC84 and UC38 are nonnucleoside inhibitors of both the RNA-dependent and DNA-dependent DNA polymerase activities of HIV-1 reverse transcriptase (RT). We have previously shown that UC84 and UC38 bind to the same site as nevirapine but interact with different RT mechanistic forms, with UC84 preferentially binding to the RT-primer/template complex and UC38 binding only to the RT-primer/template-dNTP ternary complex [Fletcher, R. S., et al. (1995) Biochemistry 34, 4346-4353]. Here we demonstrate that combinations of UC84 and UC38 inhibit RT DNA polymerase activity in vitro in a synergistic manner. This synergy was noted primarily in reactions containing high concentrations of primer/template and Km levels of dNTP substrate and was independent of both primer/template identity and the molar ratio of UC84:UC38. Combination indices were in the range of 0.4-0.6, indicating substantial synergy in the inhibition of RT activity. More importantly, combinations of UC84 and UC38 also showed a high degree of synergy in inhibiting HIV-1 replication in both MT-4 and cord blood mononuclear cells. We believe this to be the first example of synergistic inhibition of HIV-1 RT by combinations of structurally related nonnucleoside inhibitors.

Antiviral Agents↗

PubMed1995

Carboxanilide derivative non-nucleoside inhibitors of HIV-1 reverse transcriptase interact with different mechanistic forms of the enzyme.

Researchers at the National Cancer Institute first recognized the anti-HIV potential of the carboxanilide compound oxathiin carboxanilide (UC84) [Bader, J. P., et al. (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 6740-6744]. We have compared the inhibitory effect of UC84 and a second-generation thiocarboxanilide derivative, UC38, on HIV-1 reverse transcriptase (RT) RNA-dependent DNA polymerase activity. UC38 was a much better inhibitor (IC50 = 0.8 microM) than UC84 (IC50 = 4.3 microM). Inhibition by UC84 was competitive with respect to primer/template (P/T), whereas that by UC38 was uncompetitive. Both compounds were mixed noncompetitive inhibitors with respect to deoxynucleoside triphosphate (dNTP). Both compounds protected RT from irreversible photoinactivation by an azido derivative of nevirapine, implying that UC84 and UC38 bind to the same region of RT as nevirapine. UC84 photoprotected both free RT and the RT-P/T binary complex, but did not protect the RT-P/T-dNTP ternary complex. In contrast, UC38 completely photoprotected the RT-P/T-dNTP ternary complex, but not free RT or the RT-P/T binary complex. UC84 and UC38 thus appear to bind to different mechanistic forms of RT in the polymerase reaction sequence.

Affinity Labels↗

PubMed1995

Mutated K65R recombinant reverse transcriptase of human immunodeficiency virus type 1 shows diminished chain termination in the presence of 2',3'-dideoxycytidine 5'-triphosphate and other drugs.

A lysine-to-arginine substitution at amino acid 65 (K65R) in human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) is associated with resistance to 2',3'-dideoxycytidine (ddC), 2',3'-dideoxyinosine (ddI), and the (-) enantiomer of 2',3'-dideoxy-3'-thiacytidine (3TC). To further characterize the molecular basis of such resistance, we expressed the pp6/p51 heterodimer of wild-type RT, K65R mutated RT, and a doubly mutated (K65R/M184V) RT in Escherichia coli and assessed the characteristics of nucleotide incorporation and chain termination in cell-free reverse transcription reactions in the presence and absence of various nucleoside triphosphate analogs. These reactions employed a HIV RNA template (HIV-PBS) that contained the primer binding sequence (PBS) and the U5 and R regions of HIV-1 genomic RNA and an oligodeoxynucleotide (dPR) complementary to the HIV-1 PBS as primer. The K65R and K65R/M184V RTs showed significantly decreased chain-termination effects during polymerization with the 5'-triphosphates of ddC, 3TC, 2',3'-dideoxyadenosine, and AZT (3'-azido-3'-deoxythymidine) in comparison with wild-type RT. Detailed analysis with ddCTP and wild-type RT revealed that chain termination occurred at all guanines in the RNA template. However, the frequency of dideoxynucleoside triphosphate (ddNTP)-induced chain termination was decreased at certain guanines but not others in reactions catalyzed by K65R RT. Both the K65R mutant RT and wild-type RT had similar processive activity. These results indicate that decreased chain termination of K65R RT in the presence of ddNTPs is consistent with data obtained in viral replication assays.

Arginine↗

PubMed1995

[Molecular basis and clinical significance of HIV-1 resistance to nucleoside compounds].

The prolonged use of anti-viral nucleosides (ZDV, ddI, ddC) in HIV-infected patients has given rise to the isolation of viral variants that display resistance against these compounds. Tissue culture selection experiments, involving increasing concentrations of anti-viral compounds, have likewise been shown to select for drug-resistant strains of HIV. Cloning, sequencing and site-directed mutagenesis have shown that a series of point mutations in the viral reverse transcriptase (RT) are responsible for this phenomenon. A different series of mutations in RT are responsible for resistance against non-nucleoside inhibitors of this enzyme. These mutations are due to the error-prone nature of RT during viral replication. Mutated forms of recombinant RT, that derive from drug-resistant viruses, have reduced affinity for relevant triphosphorylated nucleosides.

Acquired Immunodeficiency Syndrome↗

PubMed1994

The K65R mutant reverse transcriptase of HIV-1 cross-resistant to 2', 3'-dideoxycytidine, 2',3'-dideoxy-3'-thiacytidine, and 2',3'-dideoxyinosine shows reduced sensitivity to specific dideoxynucleoside triphosphate inhibitors in vitro.

The K65R mutation in human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) encodes cross-resistance to 2',3'-dideoxycytidine (ddC), 2',3'-dideoxy-3'-thiacytidine (3TC), and 2',3'-dideoxyinosine (ddI). We characterized the in vitro sensitivities of recombinant wild type (wt) and K65R mutant RT to dideoxynucleoside triphosphate (ddNTP) inhibitors, using a variety of primer-templates. With poly(rA)-oligo(dT), the K65R mutant showed slight increases in Ki for ddTTP and 3'-azido, 3'-deoxythymidine triphosphate (AZTTP) compared to wt RT, but neither wt nor K65R RT was inhibited by ddCTP or ddATP. With poly(rI)-oligo(dC), the K65R mutant showed a 2-fold increase in Km for dCTP and a 20-fold increase in Ki for ddCTP compared to wt, whereas ddATP, ddTTP, and AZTTP failed to inhibit either enzyme. With a heteropolymeric primer-template, the K65R mutant showed 10-fold reduced sensitivities to ddCTP, 3TCTP, and ddATP, and 4-fold reduced sensitivity to AZTTP, compared to wt. In contrast, both enzymes were equally inhibited by ddTTP and ddGTP. HIV-1 cross-resistance to ddC/3TC/ddI resulting from the K65R mutation may therefore involve selective alterations in substrate/inhibitor recognition. Additionally, competitive inhibition by ddNTPs noncomplementary to the template base appears to be unimportant in the mechanism of inhibition of HIV-1 RT by dideoxynucleoside analogs.

Amino Acid Sequence↗

PubMed1994

Comparison of deoxyoligonucleotide and tRNA(Lys-3) as primers in an endogenous human immunodeficiency virus-1 in vitro reverse transcription/template-switching reaction.

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.

DNA Primers↗

PubMed1994

Generation of multiple drug resistance by sequential in vitro passage of the human immunodeficiency virus type 1.

We have sequentially passaged both laboratory and clinical isolates of the human immunodeficiency virus type 1 (HIV-1) in MT-4 cells in the presence of increasing concentrations of different drugs to derive viral variants that are multiply resistant to various combinations of ddC, ddI, d4T and AZT. The EC50 values obtained for the viruses thus generated varied between 50-100 times above those of parental wild-type strains in the case of AZT, 20-30 times for d4T, but only 10-15 times for ddI and ddC. Cultivation of AZT-resistant viruses in the presence of increasing concentrations of ddI yielded viruses that were resistant to the latter compound, with no apparent decrease in susceptibility to AZT. Sometimes, viruses selected for resistance against ddI were cross-resistant as well against ddC, although most viruses selected for resistance to ddC were not cross-resistant to ddI. Combinations of two or three of these compounds inhibited replication of HIV variants that displayed resistance to the same drugs when tested individually. No emergence of drug resistance was demonstrable when combinations of drugs were employed simultaneously in these selection protocols or when single drugs were used in concert with interferon-2 alpha or high dilutions of virus-neutralizing antisera. Cloning and sequencing of some viruses resistant to each of AZT, ddI, and ddC revealed the simultaneous presence of mutations at sites 41, 74, 184 and 215 within the HIV pol gene open reading frame.

Antiviral Agents↗

PubMed1994

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.

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.

Amino Acid Sequence↗

PubMed1994

Effects of alterations of primer-binding site sequences on human immunodeficiency virus type 1 replication.

The human immunodeficiency virus type 1 genomic RNA primer-binding site (PBS) sequence comprises 18 nucleotides which are complementary to those at the 3' end of the replication initiation primer tRNA(3Lys). To investigate the role of the PBS in viral replication, we either deleted the original wild-type PBS (complementary to tRNA(3Lys) or replaced it with DNA sequences complementary to either tRNA(1,2Lys) or tRNA(Phe). Transfection of COS cells with such molecular constructs yielded similar levels of viral progeny that were indistinguishable with regard to viral proteins and tRNA content. Virus particles derived from PBS-deleted molecular clones were noninfectious for MT-4, Jurkat, and CEM-T4 cells. However, infectious viruses were derived from constructs in which the PBS had been altered to sequences complementary to either tRNA(1,2Lys) or tRNA(Phe), although mutated forms showed significant lags in replication efficiency in comparison with wild types. Molecular analysis of reverse-transcribed DNA in cells infected by the mutated viruses indicated that both tRNA(1,2Lys) and tRNA(Phe) could function as primers for reverse transcription during the early stages of infection. Sequencing of full-length proviral DNA, obtained 6 days after infection, revealed the mutated PBS, indicating that a complete cycle of reverse transcription had occurred. During subsequent rounds of infection, reversion of the mutated PBS to wild-type sequences was observed, accompanied by increased production of viral gene products. Reversion to wild-type PBS sequences was confirmed both by specific PCR analysis, using distinct primer pairs, and by direct sequencing of amplified segments. We also performed endogenous in vitro reverse transcription experiments in which synthesis of minus-strand strong-stop viral DNA was primed from a synthetic RNA template containing a PBS complementary to various tRNA isoacceptors. These results showed that tRNA(3Lys) was a much more efficient primer of such reactions than either tRNA(1,2Lys) or tRNA(Phe).

Animals↗

PubMed1994

Identification of novel mutations that confer drug resistance in the human immunodeficiency virus polymerase gene.

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.

Drug Resistance↗

PubMed1993

Non-enzymic glycation of collagen inhibits binding of oxidized low-density lipoprotein.

We have examined the effect of non-enzymic glycation of native soluble collagen, in solution or in gels, on binding of oxidized low-density lipoprotein (LDL). We found the following. (1) Glycation markedly inhibited binding of LDL. This is contrary to results previously reported; the difference may be attributable to the use of detergent- and heat-denatured collagen, covalently bound to agarose beads, in the earlier study. (2) With increased duration of glycation, collagen solution would not gel, and preformed gels dissolved. (3) [14C]Glucose bound to collagen gels dissociated slowly, even at pH 5, suggesting that it was not present as a Schiff's base; in addition, ketoamines, pentosidine and fluorescent advanced glycation products were not detectable in glycated collagen gels, although they accumulated in tendon collagen glycated under the same conditions. It is hypothesized that the difference in glycation effects between gel and tendon may be due to the strength of cross-linking before glycation: the increase in intermolecular distance in collagen fibrils which results from glycation disrupts the fibrils in gels, preventing binding of LDL and formation of glycation-dependent cross-links, whereas the extensive cross-linking in tendon maintains the intermolecular distances within a range which permits formation of glycation cross-links.

Animals↗

PubMed1993

Monoclonal antibody-mediated inhibition of HIV-1 reverse transcriptase polymerase activity. Interaction with a possible deoxynucleoside triphosphate binding domain.

A series of monoclonal antibodies against p51/p66 human immunodeficiency virus-1 (HIV-1) reverse transcriptase (RT) were prepared by immunizing mice with the native enzyme immobilized on nitrocellulose. One of these antibodies, designated 1E8, was found to inhibit both RNA-dependent and DNA-dependent polymerase activities of RT but had no effect on the RNase H activity of the enzyme. This inhibition was noncompetitive with respect to primer/template and competitive with respect to deoxynucleoside triphosphate (dNTP). The extent of 1E8 inhibition of RT polymerase activity decreased with increasing concentrations of dNTP in the incubation but was not affected by changes in primer/template concentration. 1E8 bound equally well in solution to both free RT and to the RT-primer/template complex. However, binding to the latter was significantly reduced by the addition of increasing concentrations of dNTP. The ability of dNTP to inhibit the interaction of 1E8 with the RT-primer/template complex was dependent on the identity of the homopolymeric primer/template used; only that dNTP complementary to the template was effective in this respect. 1E8 bound to the p51/p66 reverse transcriptase heterodimer in solution and reacted with both p51 and p66 subunits of reverse transcriptase on Western blots. The antibody is therefore presumed to recognize a linear surface epitope on the enzyme. 1E8 was found to specifically recognize a peptide with the sequence KKDSTKWRK. This sequence corresponds to residues 65-73 of HIV-1 reverse transcriptase, a region identified as highly antigenic by several computer algorithms. Two mutations within this sequence have been identified with resistance to 3'-azido,3'-deoxythymidine. We conclude that residues 65-73 of HIV-1 reverse transcriptase may be at or near the polymerase active site of the enzyme, and may form part of the deoxynucleoside triphosphate binding domain of the enzyme.

Amino Acid Sequence↗

PubMed1993

Generation and characterization of murine monoclonal antibodies reactive against N-terminal and other regions of HIV-1 reverse transcriptase.

We produced a series of monoclonal antibodies against the human immunodeficiency virus (HIV-1) reverse transcriptase by immunizing mice with either purified recombinant HIV-1 p66 protein or with recombinant vaccinia virus which expresses HIV-1 pol sequences. The antibodies generated were specific for the reverse transcriptase protein, and recognized only the p51 and p66 subunits of the enzyme in each of the HIV-1 viral lysates and lysates of HIV-1 infected cells. The antibodies did not cross-react with HIV-2 reverse transcriptase. Most important, several of the antibodies are unique, in that they are the first that can bind to sites close to the N-terminal. This latter region has been suggested to form part of the polymerase domain of the reverse transcriptase. None of the antibodies could neutralize either the RNA-dependent DNA polymerase or RNase H activities of either p66 or p51/66 proteins. The binding patterns of these various antibodies to p66 and p51/66 were dependent on each of three independent variables: the source of antigen amployed, the individual specificity of the antibody, and the method employed to detect reactivity. These monoclonal antibodies provide useful reagents for the study of reverse transcriptase native structure-function relationships.

Animals↗

PubMed1993

Expression, purification, and RNA-binding properties of HIV-1 p15gag nucleocapsid protein.

We have cloned and expressed HIV-1 gag p15 nucleocapsid protein (NCp15) in the form of a 41-kDa fusion polypeptide with glutathione-S-transferase (GST-NCp). The recombinant protein was rapidly degraded in bacterial lysates unless Zn2+ and Cd2+ were present in the extraction buffer. Inclusion of these metals stabilized the protein, allowing facile purification of GST-NCp by affinity chromatography. The native NCp15 was readily prepared from GST-NCp by proteolytic cleavage with thrombin. Both GST-NCp and the processed NCp15 were able to bind RNA containing sequences from the 5'-end of the HIV-1 genome. This binding was unaffected by the absence or the presence of Zn2+; however, the binding of RNA was absolutely dependent on the presence of K+. The GST-NCp fusion protein was nonselective in the binding of RNA, with all transcripts, including antisense and non-HIV RNA, binding with equal efficiency. In contrast, NCp15 was highly selective in binding of RNA. Sequences within nucleotides 1244-1412 of the HIV-1 proviral genome were found necessary for maximal binding of RNA to NCp15.

Base Sequence↗

PubMed1993

The same mutation that encodes low-level human immunodeficiency virus type 1 resistance to 2',3'-dideoxyinosine and 2',3'-dideoxycytidine confers high-level resistance to the (-) enantiomer of 2',3'-dideoxy-3'-thiacytidine.

Variants of human immunodeficiency virus type 1 that display 500- to 1,000-fold resistance to the (-) enantiomer of 2'-deoxy-3'-thiacytidine and approximately 4- to 8-fold resistance to 2',3'-dideoxycytidine and 2',3'-dideoxyinosine have been generated through in vitro selection with the former compound. The polymerase regions of several of these resistant viruses shared a codon alteration at site 184 (ATG-->GTG; methionine-->valine), a mutation previously associated with low-level resistance to 2',3'-dideoxycytidine. The biological relevance of this mutation for the (-) enantiomer of 2'-deoxy-3'-thiacytidine was confirmed by site-directed mutagenesis with the HXB2-D clone of human immunodeficiency virus type 1.

Antiviral Agents↗