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A template-dependent dislocation mechanism potentiates K65R reverse transcriptase mutation development in subtype C variants of HIV-1.

Coutsinos D, Invernizzi CF, Moisi D, Oliveira M, Martinez-Cajas JL, Brenner BG, Wainberg MA - PLoS ONE (2011)

Bottom Line: However, the mechanism underlying this observation and the elevated rates of K65R development remained unknown.These findings confirm that the mechanism involved is template-specific and RT-independent.These findings provide additional mechanistic evidence for the facilitated development of the K65R mutation in subtype C HIV-1.

View Article: PubMed Central - PubMed

Affiliation: McGill University AIDS Center, Lady Davis Institute for Medical Research, Sir Mortimer B. Davis Jewish General Hospital, Montréal, Québec, Canada.

ABSTRACT
Numerous studies have suggested that the K65R reverse transcriptase (RT) mutation develops more readily in subtype C than subtype B HIV-1. We recently showed that this discrepancy lies partly in the subtype C template coding sequence that predisposes RT to pause at the site of K65R mutagenesis. However, the mechanism underlying this observation and the elevated rates of K65R development remained unknown. Here, we report that DNA synthesis performed with subtype C templates consistently produced more K65R-containing transcripts than subtype B templates, regardless of the subtype-origin of the RT enzymes employed. These findings confirm that the mechanism involved is template-specific and RT-independent. In addition, a pattern of DNA synthesis characteristic of site-specific primer/template slippage and dislocation was only observed with the subtype C sequence. Analysis of RNA secondary structure suggested that the latter was unlikely to impact on K65R development between subtypes and that Streisinger strand slippage during DNA synthesis at the homopolymeric nucleotide stretch of the subtype C K65 region might occur, resulting in misalignment of the primer and template. Consequently, slippage would lead to a deletion of the middle adenine of codon K65 and the production of a -1 frameshift mutation, which upon dislocation and realignment of the primer and template, would lead to development of the K65R mutation. These findings provide additional mechanistic evidence for the facilitated development of the K65R mutation in subtype C HIV-1.

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Sequence comparison of the RT region of the pol gene derived from subtype B and C HIV-1 spanning codons 62 to 68.(A) Outline of the reverse-transcription process in subtype B HIV-1 leading to either wild-type or K65R-containing transcripts. The K65R mutation results from an AAA-to-AGA mutation in subtype B HIV-1. (B) Outline of the reverse-transcription process in subtype C HIV-1 leading to either wild-type or K65R-containing transcripts. The K65R mutation results from an AAG-to-AGG mutation in subtype C HIV-1. The genomic sequences of the subtype B and C RT segments of pol spanning codons 62 to 68 are indicated. (−)ssDNA synthesis from the viral (+)ssRNA genome and (+)dsDNA synthesis from the (−)ssDNA intermediate are also depicted. Underlined are the homopolymeric nt stretches of both templates at their specific locations. In bold are the nt polymorphisms that exist between both subtypes in this region. Highlighted in a square is the base that, once mutated, gives rise to the K65R drug resistance mutation.
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pone-0020208-g001: Sequence comparison of the RT region of the pol gene derived from subtype B and C HIV-1 spanning codons 62 to 68.(A) Outline of the reverse-transcription process in subtype B HIV-1 leading to either wild-type or K65R-containing transcripts. The K65R mutation results from an AAA-to-AGA mutation in subtype B HIV-1. (B) Outline of the reverse-transcription process in subtype C HIV-1 leading to either wild-type or K65R-containing transcripts. The K65R mutation results from an AAG-to-AGG mutation in subtype C HIV-1. The genomic sequences of the subtype B and C RT segments of pol spanning codons 62 to 68 are indicated. (−)ssDNA synthesis from the viral (+)ssRNA genome and (+)dsDNA synthesis from the (−)ssDNA intermediate are also depicted. Underlined are the homopolymeric nt stretches of both templates at their specific locations. In bold are the nt polymorphisms that exist between both subtypes in this region. Highlighted in a square is the base that, once mutated, gives rise to the K65R drug resistance mutation.

Mentions: A single nucleotide (nt) mutation is required for both subtype B (AAA→AGA) (Figure 1A) and subtype C (AAG→AGG) (Figure 1B) viruses to develop the K65R mutation. Interestingly, subtype C viruses harbor a unique homopolymeric nucleic acid sequence at codons 64 and 65 of the RT segment of the pol gene which is also shared by subtypes F2, and H, and CRFs 07_BC, 08_BC and 31_BC. In addition, viruses from HIV-1 groups N and O as well as SIVcpz also share this particular subtype C sequence (Table 1). To investigate whether these polymorphisms in the viral templates might be involved in the preferential acquisition of K65R, we studied DNA synthesis from viral RNA and DNA templates derived from either subtype B or C HIV-1 [48]. The data showed that a strong pause site was present at the exact nt position responsible for K65R development during positive double-stranded DNA ((+)dsDNA) synthesis from the negative single-stranded DNA ((-)ssDNA) intermediate template. These findings were independent of the subtype-origin of the RT enzyme employed but were specific to the subtype-origin of the template. When two silent nt polymorphisms at codons 64 and 65 of the subtype C RT sequence were introduced into a wild-type NL4-3 virus of subtype B origin in cell culture, the resultant virus behaved like a subtype C virus in terms of K65R development [49]. Further investigation showed that the pausing reaction could not be alleviated by increasing individual nt concentrations and reaffirmed the template-specific and enzyme-independent nature of the findings [50]. Moreover, the pause site occurred at the end of a homopolymeric nt stretch; the synthesis of DNA from such regions is difficult for RT [51], [52], [53]. In addition, pause sites have been associated with strand transfer, recombination, misalignment and misincorporation events, all of which can lead to drug resistance [54], [55], [56], [57]. However, these data cannot confirm that the transcripts produced at the pause site contain K65R or some other mutational byproduct, or that the mutations produced may generate viable virus. Another recent report suggests that high-fidelity enzymes used in DNA amplification reactions as well as HIV-1 RT can also erroneously produce K65R [58]. These supplement our previous work but are not in contradiction with our findings. The current manuscript extends our previous results by providing an additional mechanistic basis for the hyper-presence of K65R in subtype C HIV-1.


A template-dependent dislocation mechanism potentiates K65R reverse transcriptase mutation development in subtype C variants of HIV-1.

Coutsinos D, Invernizzi CF, Moisi D, Oliveira M, Martinez-Cajas JL, Brenner BG, Wainberg MA - PLoS ONE (2011)

Sequence comparison of the RT region of the pol gene derived from subtype B and C HIV-1 spanning codons 62 to 68.(A) Outline of the reverse-transcription process in subtype B HIV-1 leading to either wild-type or K65R-containing transcripts. The K65R mutation results from an AAA-to-AGA mutation in subtype B HIV-1. (B) Outline of the reverse-transcription process in subtype C HIV-1 leading to either wild-type or K65R-containing transcripts. The K65R mutation results from an AAG-to-AGG mutation in subtype C HIV-1. The genomic sequences of the subtype B and C RT segments of pol spanning codons 62 to 68 are indicated. (−)ssDNA synthesis from the viral (+)ssRNA genome and (+)dsDNA synthesis from the (−)ssDNA intermediate are also depicted. Underlined are the homopolymeric nt stretches of both templates at their specific locations. In bold are the nt polymorphisms that exist between both subtypes in this region. Highlighted in a square is the base that, once mutated, gives rise to the K65R drug resistance mutation.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3105016&req=5

pone-0020208-g001: Sequence comparison of the RT region of the pol gene derived from subtype B and C HIV-1 spanning codons 62 to 68.(A) Outline of the reverse-transcription process in subtype B HIV-1 leading to either wild-type or K65R-containing transcripts. The K65R mutation results from an AAA-to-AGA mutation in subtype B HIV-1. (B) Outline of the reverse-transcription process in subtype C HIV-1 leading to either wild-type or K65R-containing transcripts. The K65R mutation results from an AAG-to-AGG mutation in subtype C HIV-1. The genomic sequences of the subtype B and C RT segments of pol spanning codons 62 to 68 are indicated. (−)ssDNA synthesis from the viral (+)ssRNA genome and (+)dsDNA synthesis from the (−)ssDNA intermediate are also depicted. Underlined are the homopolymeric nt stretches of both templates at their specific locations. In bold are the nt polymorphisms that exist between both subtypes in this region. Highlighted in a square is the base that, once mutated, gives rise to the K65R drug resistance mutation.
Mentions: A single nucleotide (nt) mutation is required for both subtype B (AAA→AGA) (Figure 1A) and subtype C (AAG→AGG) (Figure 1B) viruses to develop the K65R mutation. Interestingly, subtype C viruses harbor a unique homopolymeric nucleic acid sequence at codons 64 and 65 of the RT segment of the pol gene which is also shared by subtypes F2, and H, and CRFs 07_BC, 08_BC and 31_BC. In addition, viruses from HIV-1 groups N and O as well as SIVcpz also share this particular subtype C sequence (Table 1). To investigate whether these polymorphisms in the viral templates might be involved in the preferential acquisition of K65R, we studied DNA synthesis from viral RNA and DNA templates derived from either subtype B or C HIV-1 [48]. The data showed that a strong pause site was present at the exact nt position responsible for K65R development during positive double-stranded DNA ((+)dsDNA) synthesis from the negative single-stranded DNA ((-)ssDNA) intermediate template. These findings were independent of the subtype-origin of the RT enzyme employed but were specific to the subtype-origin of the template. When two silent nt polymorphisms at codons 64 and 65 of the subtype C RT sequence were introduced into a wild-type NL4-3 virus of subtype B origin in cell culture, the resultant virus behaved like a subtype C virus in terms of K65R development [49]. Further investigation showed that the pausing reaction could not be alleviated by increasing individual nt concentrations and reaffirmed the template-specific and enzyme-independent nature of the findings [50]. Moreover, the pause site occurred at the end of a homopolymeric nt stretch; the synthesis of DNA from such regions is difficult for RT [51], [52], [53]. In addition, pause sites have been associated with strand transfer, recombination, misalignment and misincorporation events, all of which can lead to drug resistance [54], [55], [56], [57]. However, these data cannot confirm that the transcripts produced at the pause site contain K65R or some other mutational byproduct, or that the mutations produced may generate viable virus. Another recent report suggests that high-fidelity enzymes used in DNA amplification reactions as well as HIV-1 RT can also erroneously produce K65R [58]. These supplement our previous work but are not in contradiction with our findings. The current manuscript extends our previous results by providing an additional mechanistic basis for the hyper-presence of K65R in subtype C HIV-1.

Bottom Line: However, the mechanism underlying this observation and the elevated rates of K65R development remained unknown.These findings confirm that the mechanism involved is template-specific and RT-independent.These findings provide additional mechanistic evidence for the facilitated development of the K65R mutation in subtype C HIV-1.

View Article: PubMed Central - PubMed

Affiliation: McGill University AIDS Center, Lady Davis Institute for Medical Research, Sir Mortimer B. Davis Jewish General Hospital, Montréal, Québec, Canada.

ABSTRACT
Numerous studies have suggested that the K65R reverse transcriptase (RT) mutation develops more readily in subtype C than subtype B HIV-1. We recently showed that this discrepancy lies partly in the subtype C template coding sequence that predisposes RT to pause at the site of K65R mutagenesis. However, the mechanism underlying this observation and the elevated rates of K65R development remained unknown. Here, we report that DNA synthesis performed with subtype C templates consistently produced more K65R-containing transcripts than subtype B templates, regardless of the subtype-origin of the RT enzymes employed. These findings confirm that the mechanism involved is template-specific and RT-independent. In addition, a pattern of DNA synthesis characteristic of site-specific primer/template slippage and dislocation was only observed with the subtype C sequence. Analysis of RNA secondary structure suggested that the latter was unlikely to impact on K65R development between subtypes and that Streisinger strand slippage during DNA synthesis at the homopolymeric nucleotide stretch of the subtype C K65 region might occur, resulting in misalignment of the primer and template. Consequently, slippage would lead to a deletion of the middle adenine of codon K65 and the production of a -1 frameshift mutation, which upon dislocation and realignment of the primer and template, would lead to development of the K65R mutation. These findings provide additional mechanistic evidence for the facilitated development of the K65R mutation in subtype C HIV-1.

Show MeSH
Related in: MedlinePlus