Nucleotide sequence context influences HIV replication fidelity by modulating reverse transcriptase binding and product release

Division of Molecular Biology, Beckman Research Institute of the City of Hope Cancer Center, Duarte, CA, USA.
Bioscience trends (Impact Factor: 1.66). 08/2007; 1(1):52-61.
Source: PubMed


An RNA template/DNA primer (T/P) complex derived from the env gene of HIV-1 was used to examine the kinetic effects of specific basepair substitutions on dNTP incorporation and RNase H cleavage by HIV-1 reverse transcriptase (RT). Single basepair substitutions 2 or 6 nucleotides upstream from a defined polymerization site (denoted -2 and -6) were engineered by oligonucleotide synthesis to provide 7 T/P substrates for kinetic analysis. A -6 A/T substitution in the wild type sequence resulted in 14- and 7-fold increases in the apparent second order rate constants (k(2app)) for U/A and U/G basepair formation. The k(2app) for U/A formation was relatively unchanged for all other T/P basepair changes. The -6A/T substitution also uniquely lowered the RNase H cleavage rate by 3-fold. Combined kinetic and thermodynamic analyses indicated that these effects were due almost exclusively to increases in the KD (k(off)/k(on)) of initial binding of RT to the T/P and the rate of product release. The data suggest that certain sequence contexts may influence RT fidelity by modulating enzyme binding/dissociation rather than by altering dNTP binding affinity or the rate of the bond forming step.

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    • "The significance of our results lies in demonstration of viral access to multiple avenues of mutational escape from immune recognition. Infidelity and lack of proof-reading for reverse transcription results in 1 to 2 mutations/viral genome/round of replication [71]–[75]. While this level of mutation ensures that escape mutations occur at the level of the overall virus population, it would still leave the majority of viruses susceptible until selective expansion of the mutated virus allowed it to become predominant. "
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    ABSTRACT: Due to constitutive expression in cells targeted by human immunodeficiency virus (HIV), and immediate mode of viral restriction upon HIV entry into the host cell, APOBEC3G (A3G) and APOBEC3F (A3F) have been considered primarily as agents of innate immunity. Recent bioinformatic and mouse model studies hint at the possibility that mutation of the HIV genome by these enzymes may also affect adaptive immunity but whether this occurs in HIV-infected individuals has not been examined. We evaluated whether APOBEC-mediated mutations within common HIV CD8+ T cell epitopes can potentially enhance or diminish activation of HIV-specific CD8+ T cells from infected individuals. We compared ex vivo activation of CD8+ T lymphocytes from HIV-infected individuals by wild type HIV peptide epitopes and synthetic variants bearing simulated A3G/F-induced mutations by measuring interferon-γ (IFN-γ) production. We found that A3G/F-induced mutations consistently diminished HIV-specific CD8+ T cell responses against the common epitopes we tested. If this reflects a significant trend in vivo, then adaptation by HIV to enrich sequences that are favored for mutation by A3G/F (A3G/F hotspots) in portions of its genome that encode immunogenic CD8+ T cell epitopes would favor CTL escape. Indeed, we found the most frequently mutated A3G motif (CCC) is enriched up to 6-fold within viral genomic sequences encoding immunodominant CD8+ T cell epitopes in Gag, Pol and Nef. Within each gene, A3G/F hotspots are more abundant in sequences encoding epitopes that are commonly recognized due to their HLA restriction. Thus, in our system, mutations of the HIV genome, mimicking A3G/F activity, appeared to abrogate or severely reduce CTL recognition. We suggest that the physiological significance of this potential effect in facilitating CTL escape is echoed in the adaptation of the HIV genome to enrich A3G/F hotspots in sequences encoding CTL epitopes that are more immunogenic at the population level.
    PLoS ONE 04/2014; 9(4):e93428. DOI:10.1371/journal.pone.0093428 · 3.23 Impact Factor

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