Resolution of Specific Nucleotide Mismatches by Wild-Type and AZT-Resistant Reverse Transcriptases during HIV-1 Replication

Abstract

A key contributor to HIV-1 genetic variation is reverse transcriptase errors. Some mutations result because reverse transcriptase (RT) lacks 3' to 5' proofreading exonuclease and can extend mismatches. However, RT also excises terminal nucleotides to a limited extent, and this activity contributes to AZT resistance. Because HIV-1 mismatch resolution has been studied in vitro but only indirectly during replication, we developed a novel system to study mismatched base pair resolution during HIV-1 replication in cultured cells using vectors that force template switching at defined locations. These vectors generated mismatched reverse transcription intermediates, with proviral products diagnostic of mismatch resolution mechanisms. Outcomes for wild-type (WT) RT and an AZT-resistant (AZT(R)) RT containing a thymidine analog mutation set-D67N, K70R, D215F, and K219Q-were compared. AZT(R) RT did not excise terminal nucleotides more frequently than WT, and for the majority of tested mismatches, both WT and AZT(R) RTs extended mismatches in more than 90% of proviruses. However, striking enzyme-specific differences were observed for one mispair, with WT RT preferentially resolving dC-rC pairs either by excising the mismatched base or switching templates prematurely, while AZT(R) RT primarily misaligned the primer strand, causing deletions via dislocation mutagenesis. Overall, the results confirmed HIV-1 RT's high capacity for mismatch extension during virus replication and revealed dramatic differences in aberrant intermediate resolution repertoires between WT and AZT(R) RTs on one mismatched replication intermediate. Correlating mismatch extension frequencies observed here with reported viral mutation rates suggests a complex interplay of nucleotide discrimination and mismatch extension drives HIV-1 mutagenesis.

Keywords: forced copy-choice recombination; retroviral error mechanisms.

Figure 1

Schematic explanation of Retroviral Inside-Out (RIO) vector design, and steps of RIO vector reverse transcription that differ from those of HIV-1 and conventional HIV-1 based vectors. (A) Schematic of the conventional pWA18puro vector (Figure 1A top line; not to scale). The conventional HIV-based vector plasmid, pWA18puro contains native 5′ and 3′ LTRs (long terminal repeats consisting of U3, R, and U5), other essential cis-acting elements {primer binding site (pbs), packaging signal (Ψ), Rev response element (RRE), and polypurine tract (ppt)} and a selectable marker gene for puromycin resistance gene (Puro) driven by the SV40 virus promoter. How RIO vectors mimic recombination intermediates (Figure 1A; WA18 puro RNA vectors’ recombination). Heavy lines indicate nascent DNA strand; single-stranded wavy lines represent RNA; double-stranded wavy lines indicate host/non-viral DNA; portions shaded blue indicate the reverse-transcribed regions whose sequences contribute to recombination products; (D) indicates recombination donor and (A) indicates recombination acceptor sequences. (Figure 1A; RIO RNA) The curved dotted lines indicate how RIO vector design involved fusing segments that contribute to retroviral recombination products in reversed order (Figure 1A; RIO plasmid). The RIO RNA expression plasmid, which contains a Rous Sarcoma virus-derived promoter and SV40 polyadenylation signal, is indicated. (B) RIO vector reverse transcription. (Figure 1B: RIO vector initial minus strand DNA product) Minus strand DNA synthesis initiates from a tRNA annealed to the pbs on a RIO RNA transcript. However, whereas retroviral RNAs’ 5′ ends terminate at R, RIO vector RNAs contain additional upstream sequences. Thus, the 5′ ends of RIO RNAs resemble templates that arise after minus strand transfer for conventional vectors, and contain forced copy choice donor sequences (D) at their 5′ ends (Figure 1B: RIO vector forced template switch) After reaching RIO vectors’ 5′ end, the donor-terminated minus strand DNA switches to the acceptor site: allowing continued minus strand synthesis. (Figure 1B: RIO provirus) The double-stranded DNA that results after completion of subsequent RIO vector reverse transcription steps.

Figure 2

RIO vector forced copy choice recombination, mismatch generation and resolution (A) Formation of RIO RNA 5′ ends. Start site junctions in plasmid DNA are indicated below the schematic of RIO plasmids’ transcription start site. Two start site sequences were used in this study: one engineered to introduce an A at RIO RNA’s 5′ end, and the second with the native RSV 5′ G. Note that because this study’s focus is on template switch outcomes, the residue upstream of the transcription start site is designated +1, because it marks the position of the first residue incorporated after template switching. (B) Matched and mismatched replication intermediates that result during RIO vector reverse transcription. Each of the two 5′ end sequences represented in Figure 2A is paired with one of four accepter template sequences, to generate the two matched-end and six mismatched-end intermediates indicated in Figure 2B. (C) Outcomes of RIO vector reverse transcription. Anticipated possible proviral DNA outcomes, using the dC:rC mismatch as an example, are shown. For this mismatch, precise extension of the mispair would generate a Not I site that is not present in the donor template region. If template switching occurred before completion of the initial minus strand DNA product, or if the terminal base were excised before synthesis resumed upon template switching, then proviral products would contain a junctional Asc I site. Two additional possible mechanisms of mismatch resolution and their outcomes are presented.

Figure 3. Packaging and replication of HIV RIO vectors

(A) Vector RNA packaging. Conventional WA18puro and RIO vector RNAs transiently expressed with helper function plasmids in 293T cells (lanes 6–10) and packaged into virus particles (lanes 1–5) were detected by RNase protection assay (RPA). Probes protect portions of vector RNA (puro) and cellular 7SL RNA (7SL), respectively. Lane marked PS contains undigested probes; lane marked M contains molecular size standards of lengths indicated at the panel’s left. (B) Puromycin resistant colony forming titers. Titers were determined for conventional HIV vector WA18puro and terminal matched and mismatched RIO vectors, mobilized by either WT or AZT^R RT.The Y axis indicates cfu titers per ml after samples’ virion RNA content was normalized to values for WA18puro, with values based on infections using virus from three independent transfections.

Figure 4. Distribution of HIV RIO vector reverse transcription products revealed by diagnostic restriction enzyme digestion

Proviral DNA fragments were amplified from uncloned pools of RIO-transduced cells and digested with restriction enzymes diagnostic for mismatch extension or excision/premature jump. (A) Primary data example, showing a polyacrylamide gel of digested PCR fragments containing pooled dC-rA and dC:rC products generated by either WT or AZT^R RT, is shown. Enzymes used are indicated, with those used in odd-numbered lanes diagnostic of mismatch extension, and those in even-numbered lanes diagnostic of terminal base excision or premature template switching (B) Quantification of products, as determined by restriction analysis. Each RIO vector was designed so that precise extension and premature jump/excision led to the generation of one of two restriction sites. Data are from 3–5 independent digestion experiment repetitions. The percent (on Y axis) of total PCR product digestible by each diagnostic enzyme, as well as the % not cut by either enzyme, are represented in the graphs.

Figure 5

Mechanisms of mismatch resolution, as determined by high throughput sequencing. The graph depicts sequencing reads for products of two matched intermediate RIO vectors and two mismatched intermediate vectors generated by wild type and AZT^R RT. Reads were collated and the data indicate the percent of total high quality reads that were diagnostic of each mechanism of mismatch resolution coded at the bottom of the figure. See Materials and Methods for quality controls and other experimental details.