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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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K65R production rates with subtype C RT on subtype B and C templates with primers containing the mutagenic nt.(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 is observed as a single band at the FL 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 is observed as two distinct bands at the FL and FL-1nt positions, which is indicative of dislocation on the subtype C template. The results are similar to those obtained when subtype B RT was used on the same templates, thus highlighting the template-specific and enzyme-independent nature of the mechanism. (B) Graphical representation of the amount of transcript production with subtype C RT on both subtype B and C templates. The values indicated with an asterisk have a p-value <0.05 when the amount of K65R-production between both subtypes at the given time-point is compared. More transcripts containing the mutagenic nt are produced with the subtype C template than with the subtype B template. The same trend is present when subtype B RT was used on the same templates. (C) Depiction of the primer and template systems used. The primers contain a G base at their 3′-end that becomes mismatched on the T on the template strand thus yielding K65R-containing transcripts. 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-g003: K65R production rates with subtype C RT on subtype B and C templates with primers containing the mutagenic nt.(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 is observed as a single band at the FL 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 is observed as two distinct bands at the FL and FL-1nt positions, which is indicative of dislocation on the subtype C template. The results are similar to those obtained when subtype B RT was used on the same templates, thus highlighting the template-specific and enzyme-independent nature of the mechanism. (B) Graphical representation of the amount of transcript production with subtype C RT on both subtype B and C templates. The values indicated with an asterisk have a p-value <0.05 when the amount of K65R-production between both subtypes at the given time-point is compared. More transcripts containing the mutagenic nt are produced with the subtype C template than with the subtype B template. The same trend is present when subtype B RT was used on the same templates. (C) Depiction of the primer and template systems used. The primers contain a G base at their 3′-end that becomes mismatched on the T on the template strand thus yielding K65R-containing transcripts. The homopolymeric regions of both templates are underlined and the base responsible for the K65R mutation is indicated in bold.

Mentions: To ensure the template-specific and enzyme-independent nature of both K65R production and primer/template dislocation on the subtype C template, similar experiments were performed using wild-type subtype C (CWT) RT. Similar patterns of DNA synthesis were observed as for the BWT RT reaction (Figure 3A) with the subtype C template again yielding more transcripts with the mutagenic nt than the subtype B template (95% vs. 70%, p<0.05) after 120 min (Figure 3B). A comparison of primer and template sequences shows that similar mechanisms of primer/template misalignment, slippage and dislocation are the likely cause of the elevated rates of K65R with the subtype C template (Figure 3C).


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)

K65R production rates with subtype C RT on subtype B and C templates with primers containing the mutagenic nt.(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 is observed as a single band at the FL 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 is observed as two distinct bands at the FL and FL-1nt positions, which is indicative of dislocation on the subtype C template. The results are similar to those obtained when subtype B RT was used on the same templates, thus highlighting the template-specific and enzyme-independent nature of the mechanism. (B) Graphical representation of the amount of transcript production with subtype C RT on both subtype B and C templates. The values indicated with an asterisk have a p-value <0.05 when the amount of K65R-production between both subtypes at the given time-point is compared. More transcripts containing the mutagenic nt are produced with the subtype C template than with the subtype B template. The same trend is present when subtype B RT was used on the same templates. (C) Depiction of the primer and template systems used. The primers contain a G base at their 3′-end that becomes mismatched on the T on the template strand thus yielding K65R-containing transcripts. 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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pone-0020208-g003: K65R production rates with subtype C RT on subtype B and C templates with primers containing the mutagenic nt.(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 is observed as a single band at the FL 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 is observed as two distinct bands at the FL and FL-1nt positions, which is indicative of dislocation on the subtype C template. The results are similar to those obtained when subtype B RT was used on the same templates, thus highlighting the template-specific and enzyme-independent nature of the mechanism. (B) Graphical representation of the amount of transcript production with subtype C RT on both subtype B and C templates. The values indicated with an asterisk have a p-value <0.05 when the amount of K65R-production between both subtypes at the given time-point is compared. More transcripts containing the mutagenic nt are produced with the subtype C template than with the subtype B template. The same trend is present when subtype B RT was used on the same templates. (C) Depiction of the primer and template systems used. The primers contain a G base at their 3′-end that becomes mismatched on the T on the template strand thus yielding K65R-containing transcripts. The homopolymeric regions of both templates are underlined and the base responsible for the K65R mutation is indicated in bold.
Mentions: To ensure the template-specific and enzyme-independent nature of both K65R production and primer/template dislocation on the subtype C template, similar experiments were performed using wild-type subtype C (CWT) RT. Similar patterns of DNA synthesis were observed as for the BWT RT reaction (Figure 3A) with the subtype C template again yielding more transcripts with the mutagenic nt than the subtype B template (95% vs. 70%, p<0.05) after 120 min (Figure 3B). A comparison of primer and template sequences shows that similar mechanisms of primer/template misalignment, slippage and dislocation are the likely cause of the elevated rates of K65R with the subtype C template (Figure 3C).

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