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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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Single incorrect nt incorporation and K65R production rates with subtype C RT on subtype B and C templates.(A) Lanes 1 through 10 depict (+)dsDNA synthesis from the (−)ssDNA intermediate with subtype C RT on the subtype B template. The full-length product contains a single dGTP incorporation at the P+1nt position. Lanes 11 through 20 depict (+)dsDNA synthesis from the (−)ssDNA intermediate with subtype C RT on the subtype C template. The full-length product contains two distinct dGTP incorporations at the P+1nt and P+2nt positions, indicative of dislocation with the subtype C template. (B) Graphical representation of transcript production with subtype C RT on both the subtype B and C templates. The same trend as when the subtype B-derived RT enzyme was employed was observed, further reinforcing the template-specific and enzyme independent notion of the mechanism. (C) Depiction of the primer and template systems used. The homopolymeric regions of both templates are underlined and the base responsible for the K65R mutation is indicated in bold.
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pone-0020208-g005: Single incorrect nt incorporation and K65R production rates with subtype C RT on subtype B and C templates.(A) Lanes 1 through 10 depict (+)dsDNA synthesis from the (−)ssDNA intermediate with subtype C RT on the subtype B template. The full-length product contains a single dGTP incorporation at the P+1nt position. Lanes 11 through 20 depict (+)dsDNA synthesis from the (−)ssDNA intermediate with subtype C RT on the subtype C template. The full-length product contains two distinct dGTP incorporations at the P+1nt and P+2nt positions, indicative of dislocation with the subtype C template. (B) Graphical representation of transcript production with subtype C RT on both the subtype B and C templates. The same trend as when the subtype B-derived RT enzyme was employed was observed, further reinforcing the template-specific and enzyme independent notion of the mechanism. (C) Depiction of the primer and template systems used. The homopolymeric regions of both templates are underlined and the base responsible for the K65R mutation is indicated in bold.

Mentions: To reaffirm that the findings were specific to the subtype origin of the template and not the RT, the same reactions were performed using CWT RT (Figure 5). Similar patterns of DNA synthesis were seen as with CWT RT on both the subtype B and C templates (Figure 5A) and more transcripts were again produced on the subtype C template after 120 min (37% vs. 22%, p<0.05) (Figure 5B). Again, dislocation occurred at the K65 site only when the subtype C template and primer were employed (Figure 5C).


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)

Single incorrect nt incorporation and K65R production rates with subtype C RT on subtype B and C templates.(A) Lanes 1 through 10 depict (+)dsDNA synthesis from the (−)ssDNA intermediate with subtype C RT on the subtype B template. The full-length product contains a single dGTP incorporation at the P+1nt position. Lanes 11 through 20 depict (+)dsDNA synthesis from the (−)ssDNA intermediate with subtype C RT on the subtype C template. The full-length product contains two distinct dGTP incorporations at the P+1nt and P+2nt positions, indicative of dislocation with the subtype C template. (B) Graphical representation of transcript production with subtype C RT on both the subtype B and C templates. The same trend as when the subtype B-derived RT enzyme was employed was observed, further reinforcing the template-specific and enzyme independent notion of the mechanism. (C) Depiction of the primer and template systems used. The homopolymeric regions of both templates are underlined and the base responsible for the K65R mutation is indicated in bold.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3105016&req=5

pone-0020208-g005: Single incorrect nt incorporation and K65R production rates with subtype C RT on subtype B and C templates.(A) Lanes 1 through 10 depict (+)dsDNA synthesis from the (−)ssDNA intermediate with subtype C RT on the subtype B template. The full-length product contains a single dGTP incorporation at the P+1nt position. Lanes 11 through 20 depict (+)dsDNA synthesis from the (−)ssDNA intermediate with subtype C RT on the subtype C template. The full-length product contains two distinct dGTP incorporations at the P+1nt and P+2nt positions, indicative of dislocation with the subtype C template. (B) Graphical representation of transcript production with subtype C RT on both the subtype B and C templates. The same trend as when the subtype B-derived RT enzyme was employed was observed, further reinforcing the template-specific and enzyme independent notion of the mechanism. (C) Depiction of the primer and template systems used. The homopolymeric regions of both templates are underlined and the base responsible for the K65R mutation is indicated in bold.
Mentions: To reaffirm that the findings were specific to the subtype origin of the template and not the RT, the same reactions were performed using CWT RT (Figure 5). Similar patterns of DNA synthesis were seen as with CWT RT on both the subtype B and C templates (Figure 5A) and more transcripts were again produced on the subtype C template after 120 min (37% vs. 22%, p<0.05) (Figure 5B). Again, dislocation occurred at the K65 site only when the subtype C template and primer were employed (Figure 5C).

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