Molecular Architecture of the Mutagenic Active Site of Human Immunodeficiency Virus Type 1 Reverse Transcriptase:  Roles of the β8-αE Loop in Fidelity, Processivity, and Substrate Interactions

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It has previously been reported that mutations in the Gln 151 residue of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) greatly enhance RT fidelity. In this study, we employed pre-steady state kinetic assays to elucidate the mechanistic role of residue Gln 151 in highly error prone DNA synthesis by HIV-1 RT. Using our Q151N high fidelity mutant, which is structurally altered in its ability to interact with the 3-OH on the sugar moiety of the incoming deoxynucleotide triphosphate (dNTP), we examined how this change in RT-dNTP interaction affects HIV-1 RT fidelity. First, we found the binding affinity (K D ) of wild type and Q151N RT proteins to different template/primers to be similar. These results indicate that the Gln 151 residue is not involved in the formation of the binary complex (RT⅐template/primer) during DNA polymerization. We also found that by changing residue 151 from a Gln3 Asn, the maximum rate of dNTP incorporation (k pol ) for both correct and incorrect dNTPs was not affected. In contrast, the ability of the Q151N mutant to bind both correct and incorrect dNTPs (K d ) was diminished. The Q151N mutant was 120-fold less efficient at binding correct dNTP than wild type RT, and its decrease in binding was such that we were unable to measure the actual binding affinity of Q151N for incorrect dNTPs. Presumably, the fidelity increase observed during the steady state is explained by this defect in Q151N binding to incorrect dNTP. In wild type RT, residue Gln 151 is important for tight binding of incorrect dNTPs and may contribute to the low fidelity nature of HIV-1 RT. Since the Q151N mutation also alters RT binding to correct dNTPs, the wild type Gln 151 residue may play an important role in efficient binding of RT to correct dNTPs. Our findings suggest that residue Gln 151 is an important element for the execution of both highly error prone and efficient DNA synthesis by HIV-1 RT.It is becoming more apparent that many organisms employ multiple DNA polymerases to replicate their genomes. Some of these DNA polymerases are specifically involved in error prone DNA synthesis required for either spontaneous mutagenesis or bypassing DNA damage (1-3). Recent biochemical studies of DNA polymerases and and their bacterial UmuC/DinB homologs (4 -6) show that these are actually very low fidelity polymerases. It is possible that these organisms have evolved functionally diverse DNA polymerases for the different activities of genomic replication and mutagenesis. As demonstrated in a series of kinetic experiments with various DNA polymerases, it is clear that the ability to incorporate incorrect dNTPs 1 affects the efficiency of DNA synthesis (7). In other words, low fidelity and poor ability to discriminate between correct and incorrect dNTPs are detrimental to efficient DNA polymerization, which is likely essential for chromosomal DNA replication. The fact that efficient DNA synthesis and error prone DNA synthesis are kinetically at odds with one another may explain why possession of separat...

supporting

confidence: 83%

“…Recently, we reported that the Q151N mutant has 12.5 times higher fidelity than wild type HIV-1 RT based on the M13 lacZ␣ forward mutation assay (21). Data from single nucleotide steady state kinetic assays using the Q151N mutant also substantiated our findings (22).…”

supporting

confidence: 80%

Mechanistic Role of Residue Gln151 in Error Prone DNA Synthesis by Human Immunodeficiency Virus Type 1 (HIV-1) Reverse Transcriptase (RT)

Weiss¹,

Bambara²,

Kim³

2002

Self Cite

“…Hexahistidine-tagged HXB2 HIV-1 RT gene (23) was introduced into pET28a (Novagen) and overexpressed in BL21 Escherichia coli (Novagen). The RT protein was purified using Ni 2ϩ chelating chromatography as described previously (24,25). The concentration and purity of the protein was analyzed by 10% SDSpolyacrylamide gel using 1.5 g of bovine serum albumin (Sigma) as a control.…”

Section: Methodsmentioning

confidence: 99%

Effect of Ribonucleotides Embedded in a DNA Template on HIV-1 Reverse Transcription Kinetics and Fidelity

Daddacha

Noble²,

Nguyen

et al. 2013

Self Cite

Background: Under limiting dNTP concentrations, HIV-1 RT incorporates rNTPs during DNA synthesis. Results: HIV-1 RT utilizes dNTP less efficiently around rNMPs, and mismatch extension fidelity is significantly reduced. Conclusion: Presence of an rNMP in DNA template slows HIV-1 RT-mediated DNA synthesis and reduces fidelity. Significance: This study provides insight into how rNMP incorporation during proviral DNA synthesis can affect HIV-1 replication kinetics and fidelity.

“…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%

“…Two lines of evidence support this suggestion. First, biochemical analyses of mutant RTs and RDRPs have identified a number of amino acid replacements in motif B that severely compromise polymerase activity (7)(8)(9)(10)(11)(12)(13)(14)(15). Second, the primary sequence of motif B is strictly conserved in independent virus isolates; in human immunodeficiency virus type-1 (HIV-1), this region is nearly invariant (16).…”

mentioning

confidence: 99%

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

Smith

Anderson

Preston

2004

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.