Abstract
HIV-1 evolves rapidly, which is thought to result from one or more error-prone steps in the virus life cycle. Because HIV-1 reverse transcriptase (RT) does not possess 3'- to 5'-exonucleolytic proofreading activity and because RT has been shown to be error-prone in cell free systems, it should be an important contributor to the high rate of HIV-1 mutation. However, because RNA polymerase II (pol II) synthesizes viral RNA, it might also contribute significantly to HIV-1 mutagenesis. To assess the relative contributions of RT and RNA pol II to HIV-1 mutagenesis, a system was established to study the rate and nature of mutations in HIV-1 long terminal repeats (LTRs). Owing to the unique nature of replication at the ends of the viral genome, mutational analysis of retroviral LTRs provides an opportunity to evaluate the relative contribution of HIV-1 RT and RNA pol II to viral mutagenesis. Mutational analysis was performed on both LTRs of 215 proviruses, restricted to a single cycle of replication, employing single-stranded conformational polymorphism and DNA sequencing allowing direct identification of mutations in the absence of selection and within autologous viral sequences. A total of 21 independent mutations was identified. Ten mutations were observed in both LTRs, which could have been introduced by either RT or RNA pol II, whereas the other eleven mutations were only present in a single LTR and could only have been introduced by RT. This provides the first direct evidence that HIV-1 RT contributes significantly to HIV-1 mutagenesis and is likely to be the primary engine for HIV-1 mutagenesis. Moreover, mutations were observed at the U3-R border, but the nature of the mutations and their frequency differed from experiments performed using cell-free systems suggesting that other viral and/or cellular factors contribute to fidelity at the ends of the viral genome.
Highlights
HIV-11 genomes evolve rapidly [1,2,3] with genetic diversity having been extensively documented in sequential isolates taken from patients (4 – 6)
HIV-1 long terminal repeats (LTRs) Mutagenesis be introduced by reverse transcriptase (RT), whereas the same mutation found in both LTRs could be introduced by either RT or RNA polymerase II (pol II)
Cell-free studies using purified HIV-1 RT indicated that, when DNA synthesis reaches the end of the viral RNA template, non-templated additions of at least one nucleotide occur with a frequency approaching 50%, which would result in the introduction of one or more base pairs at the U3-R junctions of an integrated provirus (Fig. 1) [22, 23]
Summary
Plasmid Construction—pHIV-gpt and NL4-3 proviral DNA plasmids were obtained from the AIDS Research and Reference Reagent Program. pHIV-gpt is an HIV-1 vector based on the HXB2 strain (GenBankTM accession number K03455), which has deletions, mutations, and disruptions in the env, nef, vif, and vpr genes [24, 25]. PHIV-gpt is an HIV-1 vector based on the HXB2 strain (GenBankTM accession number K03455), which has deletions, mutations, and disruptions in the env, nef, vif, and vpr genes [24, 25]. The #69TIRevEnv packaging cell line [29] was grown in DMEM supplemented with 10% fetal bovine serum, 0.2 mg/ml G418 (Sigma-Aldrich Inc.), 0.1 mg/ml hygromycin Amplification of 3Ј-LTRs was performed using primers B9017ϩ (5Ј-ACCTTTAAGACCAATGACAAGG-3Ј) and B634Ϫ (5Ј-TGCTAGAGATTTTCCACACTGAC-3Ј) anticipated to generate a product of 702 bp. Amplification conditions for this PCR were: 95 °C for 30 s, 60 °C for 40 s, and 72 °C for 50 s, for 30 cycles. Date mutations detected by SSCP were confirmed by independent PCR amplification and DNA sequencing
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