UMLS:C0019693 - FACTA Search

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

A single amino acid substitution from methionine-184 to valine (M184V) of HIV-1 reverse transcriptase (RT) evokes the 1000-fold 3TC (Lamivudine) resistance by the HIV-1 virus observed in the clinic. The M184V mutant HIV-1 RT was studied to assess its catalytic efficiency during single nucleotide incorporation using a transient kinetic approach. The maximum rate of polymerization (k(pol)), binding affinity (K(d)), and incorporation efficiency (k(pol)/K(d)) were determined for incorporating dCTP and 3TC-TP by wild-type and 3TC-resistant HIV-1 RT. The 3TC-resistant HIV-1 RT showed a similar efficiency of incorporation compared with the wild-type enzyme during DNA-dependent DNA polymerization; however, the incorporation efficiency is reduced 3.5-fold during RNA-dependent polymerization. A dramatic 146- and 117-fold decrease in incorporation efficiency was observed for 3TC-MP incorporation by M184V RT for DNA- and RNA-dependent DNA polymerization, respectively, as compared with wild-type HIV-1 RT. While the k(pol) was slower and the K(d) was weaker for 3TC-TP incorporation by the M184V RT, the decrease in the efficiency of incorporation is primarily due to a substantially reduced binding affinity for the 3TC-TP to the enzyme.DNA (or RNA) complex poised for DNA elongation. The fidelity of M184V RT was also examined to evaluate mispair formation since this mutant has been suggested to exhibit a higher level of fidelity. The results of our studies indicate that there is a maximum 2.4-fold increase in fidelity for M184V RT as compared with wild-type HIV-1 RT. Both the wild-type and 3TC-resistant mutant RT showed higher fidelity using an RNA template as contrasted with the corresponding DNA template. This mechanistic information provides insight into our understanding of the molecular mechanism of 3TC-drug resistance and supports suggestions that increased RT fidelity and decreased fitness of the M184V HIV-1 virus may be factors contributing to the strong antiviral effect of AZT-3TC combination therapy.
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PMID:Mechanistic studies examining the efficiency and fidelity of DNA synthesis by the 3TC-resistant mutant (184V) of HIV-1 reverse transcriptase. 1041 20

We report a new approach for target quantification directly within DNA duplex. Our assay is based on the formation of a new biomolecular structure, the PD-loop. The approach takes advantage of a selective hybridization of a probe to double-stranded DNA (dsDNA), which is locally opened by a pair of bis-PNA oligomers. To optimize the technique, several experimental formats are tested with the use of PNA and oligonucleotide probes. The highest sensitivity is achieved when the hybridized probe is extended and multiply labeled with 125I-dCTP by DNA polymerase via strand displacement in the presence of single-strand binding (SSB) protein. In this case, the PNA-assisted probe hybridization combined with the method of multiphoton detection (MPD) allows to monitor sub-attomolar amounts of the HIV-1 target on the background of unrelated DNA at sub-nCi level of radioactivity. The developed robust methodology is highly discriminative to single mutations, thus being of practical use for DNA analysis.
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PMID:PNA openers as a tool for direct quantification of specific targets in duplex DNA. 1056 73

The initiation of (-) strand DNA synthesis by HIV-1 reverse transcriptase was examined using a transient kinetic approach and a physiologically relevant RNA 18-mer/RNA 36-mer primer-template substrate. HIV-1 reverse transcriptase (RT) was found to bind with reasonably high affinity to the RNA/RNA substrate (K(d) = 90 nM), although the affinity for DNA/RNA and DNA/DNA substrates is higher (K(d) approximately 5 nM). A pre-steady-state burst of deoxynucleotide incorporation (k(obsd) = 1.0 s(-)(1)) into the RNA duplex was observed followed by a slower steady-state release of the elongated primer-template product (k(ss) = 0.58 s(-)(1)). The observation of a burst provides evidence that the release of the product is most likely the rate-limiting step in the overall kinetic pathway for the enzymatic reaction during a single deoxynucleotide incorporation event. Furthermore, the release of this product was 5-fold faster than that for elongated DNA/RNA and DNA/DNA products. Single-turnover experiments showed that there is a hyperbolic dependence of the rate of deoxynucleotide incorporation on the concentration of dCTP and demonstrated that the maximum rate of dCTP incorporation (k(pol) = 1.4 s(-)(1)) is 33- and 12-fold slower than the values for DNA/RNA and DNA/DNA primer-template substrates, respectively, while the affinity of dCTP (K(d) = 780 microM) for the HIV-1 RT.RNA/RNA complex is 56- and 71-fold weaker than the affinities for HIV-1 RT.DNA/RNA and HIV-1 RT.DNA/DNA complexes, respectively. Consequently, the overall efficiency of dCTP incorporation (k(pol)/K(d)) into the RNA/RNA substrate is approximately 1800- and 800-fold less than that for DNA/RNA and DNA/DNA substrates, respectively. These findings provide evidence which suggests that the HIV-1 RT.RNA/RNA.dCTP ternary complex exists in a significantly different conformation compared to ternary complexes involving DNA/RNA and DNA/DNA substrates. A model summarizing these results is presented, and implications for the molecular mechanism of initiation of (-) strand DNA synthesis by RT are discussed.
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PMID:Initiation of minus-strand DNA synthesis by human immunodeficiency virus type 1 reverse transcriptase. 1062 65

Low levels of the CTP synthase inhibitor 3-deazauridine (3-DU) strongly potentiated the anti-HIV-1 activity of the 5'-triphosphates of the cytidine-based analogues [-]2'-deoxy-3'-thiacytidine (3TC; lamivudine) and 2',3'-dideoxycytidine (ddC). The potentiation was associated with a 3-DU-induced decrease in dCTP pool size; no changes were seen in cellular pool sizes of dATP, dGTP or dTTP.
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PMID:Potentiation of the anti-HIV activity of zalcitabine and lamivudine by a CTP synthase inhibitor, 3-deazauridine. 1077 21

A series of unnatural L-nucleosides such as 3TC, FTC and L-FMAU have been found to be potent antiviral agents. The mode of action of L-nucleosides has been found to be similar to that of D-nucleosides as antiviral agents, despite their unnatural stereochemistry, that is, nucleotide formation by kinases followed by interaction with the reverse transcriptase (RT) of HIV or DNA polymerase. To date, the mode of action of nucleoside inhibitors at the molecular level with respect to the active conformations of the 5'-triphosphates as well as the interaction with the RT is not known. Recently, the X-ray crystal structure of the RT-DNA-dTTP catalytic complex has been reported. Computer modeling has been performed for several pairs of D- and L-nucleoside inhibitors using the HIV-1 RT model and crystal coordinate data from a subset of the protein surrounding the deoxynucleoside triphosphate (dNTP) binding pocket region. Results from our modeling studies of D-/L-zidovudine, D-/L-3TC, D-/L-dideoxycytosine triphosphates, dTTP and dCTP show that their binding energies correlate with the reported 50% effective concentrations. Modeling results are also discussed with respect to favorable conformations of each inhibitor at the dNTP site in the polymerization process. Additionally, the clinically important M184V mutation, which confers resistance against 3TC and FTC, was studied with our modeling system. The binding energy patterns of nucleoside inhibitors at the M184V mutation site correlate with the reported antiviral data.
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PMID:Molecular modeling approach to understanding the mode of action of L-nucleosides as antiviral agents. 1112 Sep 56

Following intracellular activation of HIV nucleoside analogue reverse transcriptase inhibitors, their triphosphates (ddNTPs) compete with endogenous nucleoside triphosphates (dNTPs) for incorporation into proviral DNA. In this study we have examined the effect of combinations of two thymidine analogues, stavudine (d4T) and zidovudine (ZDV), and two cytidine analogues, lamivudine (3TC) and zalcitabine (ddC) on intracellular drug activation and on the relevant competing dNTP in uninfected and persistently HIV-infected cells. Endogenous triphosphates of deoxycytidine (dCTP) and deoxythymidine (dTTP) were measured using a template primer assay and the ratio of ddNTP:dNTP was calculated. Antiviral activity of two-drug combinations was also assayed by p24 ELISA. A significant reduction in d4T triphosphate (d4TTP) [0.11+/-0.09 pmol/10(6) cells to undetectable (<0.01); P=0.039] in the presence of equimolar concentrations of ZDV and d4T, resulted in a decrease in the d4TTP/dTTP ratio of 90%. ZDVTP/dTTP was not significantly altered in the presence of d4T. 3TC (10 microM) reduced total ddC phosphates by 57% and ddCTP/dCTP by 27%. 3TC phosphorylation was comparatively unaffected by ddC, up to a concentration of 10 microM ddC (>100 times the plasma concentration achieved following standard dosing). 3TC plus ddC resulted in greater p24 inhibition than 3TC or ddC alone (P<0.001). Combining one thymidine analogue (ZDV or d4T) with one cytidine analogue (3TC or ddC) resulted in greater inhibition of p24 inhibition than with any single agent. From a pharmacological viewpoint, the combination of ZDV plus d4T should be avoided, but in vitro the combination of 3TC plus ddC confers modest benefit over either drug alone. This in vitro study illustrates that decreases in ddNTP/dNTP are consistent with a reduction in antiviral effect.
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PMID:Correlation between intracellular pharmacological activation of nucleoside analogues and HIV suppression in vitro. 1122 92

The RNA genome of the lentivirus human immunodeficiency virus type 1 (HIV-1) is significantly richer in adenine nucleotides than the statistically equal distribution of the four different nucleotides that is expected. This compositional bias may be due to the guanine-to-adenine (G-->A) nucleotide hypermutation of the HIV genome, which has been explained by dCTP pool imbalances during reverse transcription. The adenine nucleotide bias together with the poor fidelity of HIV-1 reverse transcriptase markedly enhances the genetic variation of HIV and may be responsible for the rapid emergence of drug-resistant HIV-1 strains. We have now attempted to counteract the normal mutational pattern of HIV-1 in response to anti-HIV-1 drugs by altering the endogenous deoxynucleoside triphosphate pool ratios with antimetabolites in virus-infected cell cultures. We showed that administration of these antimetabolic compounds resulted in an altered drug resistance pattern due to the reversal of the predominant mutational flow of HIV (G-->A) to an adenine-to-guanine (A-->G) nucleotide pattern in the intact HIV-1-infected lymphocyte cultures. Forcing the virus to change its inherent nucleotide bias may lead to better control of viral drug resistance development.
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PMID:Exploitation of the low fidelity of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase and the nucleotide composition bias in the HIV-1 genome to alter the drug resistance development of HIV. 1139 May 79

Substrate properties of the earlier synthesized and characterized dCTP derivatives bearing in the exo-N-position of cytosine 2-(4-azido-2,3,5,6-tetrafluorobenzoylamino)ethyl (I), 2-(2-nitro-5-azidobenzoylamino)ethyl (II), 2-(4-azido-2,3,5,6-tetrafluorobenzylideneaminooxymethylcarbonylamino)ethyl (III), 4-(4-azido-2,3,5,6-tetrafluorobenzylideneaminooxy)butyloxy (IV), or 4-(4-azido-2,3,5,6-tetrafluorobenzylidenehydrazinocarbonyl)butyl- carbonylamino (V) groups were studied in the primer extension reaction catalyzed by rat DNA polymerase beta. Unlike the earlier results obtained with HIV reverse transcriptase, dCTP derivatives (I)-(III) were not recognized by rat DNA polymerase beta as dTTP analogues, and all the five nucleotides were utilized as dCTP analogues. When compared with dCTP, Km values for the synthesized dCTP derivatives were higher by a factor of 4-20; Vmax were 1-2.3 times higher for (I)-(III) and (V) but 20-fold lower for derivative (IV). Site-specific photomodifications of the primer-template-DNA polymerase beta complexes were carried out using photoreactive reagents PRI-PRV, obtained in situ by extension of 5'-32P-labeled primers with dCTP analogues (I)-(V), respectively, when exposed to UV irradiation at 303-313 nm. Reagents PRI and PRIV provided the maximum photocrosslinking of the 5'-32P-labeled primer to the DNA template (56%) and to the enzyme (20%), respectively. The lowest efficiency of photocrosslinking was observed for PRII (about 1%).
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PMID:[Reagents for modification of protein-nucleic acids complexes. II. Site-specific photomodification of DNA-polymerase beta complexes with primers elongated by the dCTP exo-N-substituted arylazido derivatives]. 1144 42

Misincorporation at a DNA-carcinogen adduct may contribute to formation of mutations if a polymerase proceeds past the lesion, compromising fidelity, as in the G:C to A:T mutations caused by O(6)-alkylguanine. Replication of primer/templates containing guanine (G), O(6)-methylguanine (O(6)-MeG), or O(6)-benzylguanine (O(6)-BzG) was assessed using T7 DNA polymerase exo(-) (T7(-)) and HIV-1 reverse transcriptase (RT). The steady-state parameters indicated that T7(-) and RT preferentially incorporated dTTP opposite O(6)-MeG and O(6)-BzG. The incorporation efficiencies (k(cat)/K(m)) were less for O(6)-BzG than O(6)-MeG for both dCTP and dTTP insertion. Pre-steady-state analysis indicated that the product formed during the burst phase, i.e., the burst amplitude, differed significantly between the unmodified 24-mer/36-G-mer and the O(6)-alkylG-containing substrates. Extension of the O(6)-BzG-containing duplexes was much more difficult for both polymerases as compared to O(6)-MeG, except when RT easily extended the O(6)-BzG:T base pair. The for binding of dCTP or dTTP to a RT*DNA complex containing O(6)-MeG was 8-fold greater than for dNTP binding to a complex containing unmodified DNA. The for a RT*DNA complex containing O(6)-BzG was 50-fold greater. In conclusion, the bulkier O(6)-BzG is a greater block to polymerization by T7(-) and RT than is O(6)-MeG, but some polymerization does occur with an O(6)-BzG substrate. Pre-steady-state analysis indicates that neither dCTP nor dTTP insertion is strongly preferred during polymerization of O(6)-BzG-containing DNA, unlike the case of O(6)-MeG. These results and others regarding polymerase stalling opposite O(6)-MeG and O(6)-BzG are discussed in the following paper in this issue [Woodside, A. M., and Guengerich, F. P. (2002) Biochemistry 41, 1039-1050].
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PMID:Effect of the O6 substituent on misincorporation kinetics catalyzed by DNA polymerases at O(6)-methylguanine and O(6)-benzylguanine. 1179 Jan 27

Oxidatively modified deoxynucleotide triphosphates (dN(oxo)TPs) present in nucleotide precursor pools may contribute to retroviral mutagenesis as a result of incorporation and ambiguous base pairing during reverse transcriptase mediated replication. We have examined the incorporation of 5-hydroxy-2'-deoxycytosine triphosphate (5-HO-dCTP) and 2'-deoxyinosine triphosphate (dITP) by HIV-1 reverse transcriptase (HIV-1 RT) on DNA and RNA templates of the same sequence in order to evaluate their mutagenic potential. Significant variations in insertion frequencies at homologous nucleotide positions were observed for each dN(oxo)TP, in general favoring the RNA template. A comparison of steady-state kinetics revealed a 10-fold preference for 5-HO-dCTP incorporation opposite G in RNA. Insertion frequencies for dITP were 2- to 20-fold greater on RNA for every base position examined. One exception to this general trend was observed for the insertion of 5-HO-dCTP by HIV-1 RT opposite A, which favored the DNA template by 4-fold. Deoxyinosine triphosphate was inserted opposite C with an 8-fold higher frequency compared to dGTP in RNA, while on DNA templates, the incorporation frequencies were equivalent. However, incorporation of dITP opposite other bases was characterized by relatively low frequencies. The RNA template bias observed for dN(oxo)TP incorporation is discussed in terms of recent efforts to utilize 5-OH-dCTP as an anti-HIV agent.
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PMID:Incorporation of oxidatively modified 2'-deoxynucleotide triphosphates by HIV-1 RT on RNA and DNA templates. 1201 86

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