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. 2010 Oct;84(19):9864-78.
doi: 10.1128/JVI.00915-10. Epub 2010 Jul 21.

Nature, position, and frequency of mutations made in a single cycle of HIV-1 replication

Michael E Abram  1 , Andrea L Ferris, Wei Shao, W Gregory Alvord, Stephen H Hughes
Affiliations

Nature, position, and frequency of mutations made in a single cycle of HIV-1 replication

Michael E Abram et al. J Virol. 2010 Oct.

Abstract

There is considerable HIV-1 variation in patients. The extent of the variation is due to the high rate of viral replication, the high viral load, and the errors made during viral replication. Mutations can arise from errors made either by host DNA-dependent RNA polymerase II or by HIV-1 reverse transcriptase (RT), but the relative contributions of these two enzymes to the mutation rate are unknown. In addition, mutations in RT can affect its fidelity, but the effect of mutations in RT on the nature of the mutations that arise in vivo is poorly understood. We have developed an efficient system, based on existing technology, to analyze the mutations that arise in an HIV-1 vector in a single cycle of replication. A lacZalpha reporter gene is used to identify viral DNAs that contain mutations which are analyzed by DNA sequencing. The forward mutation rate in this system is 1.4 x 10(-5) mutations/bp/cycle, equivalent to the retroviral average. This rate is about 3-fold lower than previously reported for HIV-1 in vivo and is much lower than what has been reported for purified HIV-1 RT in vitro. Although the mutation rate was not affected by the orientation of lacZalpha, the sites favored for mutations (hot spots) in lacZalpha depended on which strand of lacZalpha was present in the viral RNA. The pattern of hot spots seen in lacZalpha in vivo did not match any of the published data obtained when purified RT was used to copy lacZalpha in vitro.

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Figures

FIG. 1.
FIG. 1.
Vector-based strategies used to measure HIV-1 replication fidelity. (A) Shuttle cassette. EM-zeor, EM-7 promoter zeocin resistance gene; lacZα-F and lacZα-R, α-complementing peptides of E. coli β-galactosidase in the forward and reverse orientations, respectively; ColE1 (oriE), E. coli bacterial origin of replication. Shuttle cassettes carrying lacZα as a mutational target in both orientations were cloned into the indicated vectors (see below), creating the substrates for transcription by RNA Pol II, RNA packaging, reverse transcription, and circularization of unintegrated viral lacZα DNA. (B and C) The 2-vector and 4-vector systems. The plasmids were transfected into 293T cells to produce pseudotyped HIV-1 viruses which were used to infect HOS cells. In panel B, on the left is shown the viral RNA-encoding vector plasmid for the 2-vector system. Two shaded boxes separated by an FspI site represent the HIV-1 long terminal repeats (LTRs). Retroviral genes are indicated in rectangular boxes as gag, pol, and env. On the right is shown the VSV-G expression plasmid, which contains a CMV promoter. In the 4-vector system, shown in panel C, the viral RNA-encoding vector plasmid does not contain the gag and pol genes, which are expressed from a second plasmid that contains a CMV promoter. The Rev protein is also expressed from a separate plasmid, but from a Rous sarcoma virus (RSV) promoter. In both the 2-vector and 4-vector systems, the viral vector plasmid has a nonviral sequence between the 3′ LTR and the 5′ LTR; this insert is not present in replicated viral DNAs. Both of the viral vector systems are limited to a single round of replication by an HIV-1 Env deletion. An integrase inactivating mutation, D116N, was used to increase the amount of replicated circular viral lacZα DNA present in HOS cells infected with these vectors. WRE, woodchuck regulatory element.
FIG. 2.
FIG. 2.
Class 1 mutations: single nucleotide substitutions detected in lacZα (4-vector system). The numbers, types, and locations of the independent substitution errors are shown for both the forward lacZα orientation (plus-strand nucleotide sequence) and the reverse lacZα orientation (negative-strand nucleotide sequence). Opposing directional arrows indicate the actual sequence context and direction of minus-strand DNA synthesis during reverse transcription. The total length of the lacZα target sequence was defined as 174 nt, representing codons 6 to 63 from GGA to the first TAA termination codon. Single nucleotide substitution errors are shown as letters above the original wild-type template sequence, limited to 11 per position, with additional errors indicated by +n. Runs of 3 or more identical nucleotides are underlined. Misalignment/slippage of the primer or template strand that could result in a substitution error is highlighted in orange or green, respectively. Mutational hot spots for which there are significant differences in the forward and reverse lacZα orientations are indicated by asterisks: *, P < 0.01; **, P < 0.001; ***, P < 0.0001 (see Materials and Methods). The mutation frequency is the number of lacZα DNAs with a mutation(s) divided by the total number of adjusted recovered clones.
FIG. 3.
FIG. 3.
Class 2 mutations: single nucleotide frameshifts detected in lacZα (4-vector system). Single nucleotide frameshift errors are indicated as triangles above the wild-type sequence: single nucleotide deletions (−1) are shown as upright triangles and single nucleotide additions (+1) as inverted triangles. Runs of 3 or more identical nucleotides are underlined. If a frameshift occurred within a run, its exact position within the run cannot be determined, and the deletions and additions are marked over the last nucleotide in the run in the direction of minus-strand DNA synthesis. Frameshift errors in nucleotide runs that could have arisen by a misalignment/slippage of the primer or template strand are highlighted in orange or green, respectively. Mutational hot spots for which there are significant differences in the forward and reverse lacZα orientations are indicated by asterisks: *, P < 0.01; **, P < 0.001; ***, P < 0.0001 (see Materials and Methods). The mutation frequency is the number of lacZα DNAs with a mutation(s) divided by the total number of adjusted recovered clones.
FIG. 4.
FIG. 4.
Class 3 mutations: multiple nucleotide substitutions detected in lacZα (4-vector system). Multiple mutations are shown as letters above the original wild-type template sequence, limited to 11 per position, with additional errors indicated by +n. Runs of 3 or more identical nucleotides are underlined. Mutational hot spots for which there are significant differences in the forward and reverse lacZα orientations are indicated by asterisks: *, P < 0.01; **, P < 0.001; ***, P < 0.0001 (see Materials and Methods). The mutation frequency is the number of lacZα DNAs with a mutation(s) divided by the total number of adjusted recovered clones.
FIG. 5.
FIG. 5.
Class 4 mutations: insertions, deletions, and/or deletions with insertions (indels) detected in lacZα (4-vector system). Indel mutations (deletions, deletions with insertions, and duplications) are shown as red lines extending over the positions of the mutations, with each lacZα DNA sequence numbered accordingly. The lines approximate the sizes of the indels, with the actual number of nucleotides inserted or deleted preceded by an inverted or upright triangle, respectively. Additional substitution errors within indels are shown as letters above the original wild-type template sequence. Runs of 3 or more identical nucleotides are underlined. The mutation frequency is the number of lacZα DNAs with a mutation(s) divided by the total number of adjusted recovered clones.
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