Functional Analysis of Amino Acid Residues Constituting the dNTP Binding Pocket of HIV-1 Reverse Transcriptase

Harris, Dylan; Kaushik, Neerja; Pandey, Pradeep K.; Yadav, Prem N.; Pandey, Virendra N.

doi:10.1074/jbc.273.50.33624

Cited by 80 publications

(112 citation statements)

References 73 publications

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“…We analyzed the strand-displacement activity of RT in the presence of both 75 mM KCl and 35 mM KCl, because Fuentes et al (29) showed that 75 mM KCl (the concentration typically used in various RT assays) can inhibit strand displacement, presumably by stabilizing the nucleic acid duplex. They also showed that decreasing the concentration of KCl in the reaction increases the amount of strand displacement (14). As shown in Fig.…”

Section: Resultsmentioning

confidence: 81%

“…In analyzing mutations at Tyr-115, several groups have found that the only amino acid substitution that has little deleterious effect on the enzyme is Tyr-115-Phe (7)(8)(9)(10)(11)(12)(13)(14). Substitution of Tyr-115 with polar amino acids is extremely deleterious (9)(10)(11)(12)(13) whereas substitution with hydrophobic amino acids usually has an intermediate effect (7,8,11,12,14).…”

mentioning

confidence: 99%

“…Substitution of Tyr-115 with polar amino acids is extremely deleterious (9)(10)(11)(12)(13) whereas substitution with hydrophobic amino acids usually has an intermediate effect (7,8,11,12,14). Recently, the Tyr-115-Phe mutation, along with the mutations Lys-65-Arg, Leu-74-Val, and Met-184-Val, has been detected in HIV-1 RTs resistant to the carbocyclic nucleoside analog 1592U89 (abacavir) (15,16).…”

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confidence: 99%

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Analysis of mutations at positions 115 and 116 in the dNTP binding site of HIV-1 reverse transcriptase

Boyer

Sarafianos

Arnold³

et al. 2000

Proc. Natl. Acad. Sci. U.S.A.

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We have examined amino acid substitutions at residues 115 and 116 in the reverse transcriptase (RT) of HIV-1. A number of properties were examined, including polymerization and processivity on both DNA and RNA templates, strand displacement, ribonucleotide misincorporation, and resistance to nucleoside analogs. The RT variants Tyr-115-Phe and Phe-116 -Tyr are similar to wild-type HIV-1 RT in most, but not all, respects. In contrast, the RT variant Tyr-115-Val is significantly impaired in polymerase activity compared with wild-type RT; however, Tyr-115-Val is able to incorporate ribonucleotides as well as deoxyribonucleotides during polymerization and is resistant to a variety of nucleoside analogs.

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Section: Resultsmentioning

confidence: 81%

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confidence: 99%

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confidence: 99%

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Analysis of mutations at positions 115 and 116 in the dNTP binding site of HIV-1 reverse transcriptase

Boyer

Sarafianos

Arnold³

et al. 2000

Proc. Natl. Acad. Sci. U.S.A.

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“…It is possible that the more rigid Ile side chain in position 296 and its interactions with the hydrophobic cavity could affect the conformation and flexibility of loop ␤9-␣11, altering the interactions either with the incoming rNTP or with the template acceptor base. In different DNA polymerases and HIV RT, substitutions located both, near to, and remote from the active site can have significant effects on fidelity, and such effects appear to be the result of static and/or dynamic changes in the shape and the flexibility of the active site (28)(29)(30)(31).…”

Section: Incorporation Of Mutagenic Nucleotide Analogs By Fmdv 3d: Immentioning

confidence: 99%

Sequential structures provide insights into the fidelity of RNA replication

Ferrer-Orta

Arias²,

Pérez-Luque³

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

120

141

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RNA virus replication is an error-prone event caused by the low fidelity of viral RNA-dependent RNA polymerases. Replication fidelity can be decreased further by the use of mutagenic ribonucleoside analogs to a point where viral genetic information can no longer be maintained. For foot-and-mouth disease virus, the antiviral analogs ribavirin and 5-fluorouracil have been shown to be mutagenic, contributing to virus extinction through lethal mutagenesis. Here, we report the x-ray structure of four elongation complexes of foot-and-mouth disease virus polymerase 3D obtained in presence of natural substrates, ATP and UTP, or mutagenic nucleotides, ribavirin triphosphate and 5-fluorouridine triphosphate with different RNAs as template-primer molecules. The ability of these complexes to synthesize RNA in crystals allowed us to capture different successive replication events and to define the critical amino acids involved in (i) the recognition and positioning of the incoming nucleotide or analog; (ii) the positioning of the acceptor base of the template strand; and (iii) the positioning of the 3 -OH group of the primer nucleotide during RNA replication. The structures identify key interactions involved in viral RNA replication and provide insights into the molecular basis of the low fidelity of viral RNA polymerases.5-fluorouracil ͉ foot-and-mouth disease virus ͉ replication fidelity ͉ ribavirin ͉ RNA elongation

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“…Our observation that Gln 151 mutations result in AZT hypersensitivity was unexpected, since a methionine substitution at this site confers weak AZT resistance in clinical isolates, and the combination of four additional mutations with Q151M results in high level resistance to AZT and several other nucleoside analogs (46,47) (Table I). Analyses of purified RTs show that the Q151M and Q151N mutations confer resistance to nucleoside analogs as well as mild to moderate increases in the fidelity of nucleotide incorporation in cell-free assays (11,55,56). Substitutions of alanine or asparagine at the equivalent position of murine leukemia virus RT (Gln 190 ) also confer increased fidelity but render the enzyme hypersensitive to dideoxynucleoside triphosphates (57).…”

Section: Discussionmentioning

confidence: 99%

Purifying Selection Masks the Mutational Flexibility of HIV-1 Reverse Transcriptase

Smith

Anderson

Preston

2004

Journal of Biological Chemistry

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DNA and RNA polymerases share a core architecture composed of three structurally conserved motifs: A, B, and C. Although the amino acid sequences of these motifs are highly conserved between closely related organisms, variation across broader evolutionary distances suggests that only a few residues in each motif are indispensable for polymerase function. To test this, we constructed libraries of human immunodeficiency virus type-1 (HIV-1) containing random single amino acid replacements in motif B of reverse transcriptase (RT), and we used selection in culture to assess RT function. Despite the nearly absolute constancy of motif B in vivo, virus replicating in culture tolerated a range of conservative and nonconservative substitutions at 10 of the 11 amino acid positions examined. These included residues that are invariant across all retroviral subfamilies and highly conversed in diverse retroelements. Several mutants retained wild type infectivity, and serial passage experiments revealed replacements that were neutral or even beneficial to viral fitness. In addition, a number of the selected variants exhibited altered susceptibility to the nucleoside analog inhibitors AZT and 3TC. Taken together, these data indicate that HIV-1 tolerates a range of substitutions at conserved RT residues and that selection against slightly deleterious mutations (purifying selection) in vivo masks a large repertoire of viable phenotypic variants. This mutational flexibility likely contributes to HIV-1 evolution in response to changing selection pressures in infected individuals.

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Functional Analysis of Amino Acid Residues Constituting the dNTP Binding Pocket of HIV-1 Reverse Transcriptase

Cited by 80 publications

References 73 publications

Analysis of mutations at positions 115 and 116 in the dNTP binding site of HIV-1 reverse transcriptase

Analysis of mutations at positions 115 and 116 in the dNTP binding site of HIV-1 reverse transcriptase

Sequential structures provide insights into the fidelity of RNA replication

Purifying Selection Masks the Mutational Flexibility of HIV-1 Reverse Transcriptase

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