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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 B 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 B 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 B 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. (B) Graphical representation of the amount of transcripts containing the mutagenic nt produced with subtype B 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 with the mutagenic G at position 65 are produced with the subtype C template than with the subtype B template. (C) Depiction of the primer and template systems used. The primers contain a G base on their 3′-end that becomes mismatched on the T on the template strand thus yielding transcripts with the mutagenic nt. 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-g002: K65R production rates with subtype B 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 B 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 B 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. (B) Graphical representation of the amount of transcripts containing the mutagenic nt produced with subtype B 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 with the mutagenic G at position 65 are produced with the subtype C template than with the subtype B template. (C) Depiction of the primer and template systems used. The primers contain a G base on their 3′-end that becomes mismatched on the T on the template strand thus yielding transcripts with the mutagenic nt. The homopolymeric regions of both templates are underlined and the base responsible for the K65R mutation is indicated in bold.

Mentions: The production of DNA transcripts that contained the mutagenic G at the central position of codon 65 was evaluated using the subtype B-1MM2 and C-1MM2 primers on their respective subtype-matched (−)ssDNA templates. (+)dsDNA synthesis was evaluated simultaneously on both subtype B and C templates using wild-type subtype B (BWT) RT (Figure 2A). With the subtype B template, no pausing was seen and a distinct, 13 nt DNA product band containing K65R was observed at the full-length (FL) position. In contrast, use of the subtype C template led to two distinct DNA product bands at the FL and full-length -1 nt (FL-1nt) positions of 13 and 12 nt respectively, suggesting that a probable primer/template misalignment had occurred. Quantification results showed that larger amounts of DNA transcripts containing the mutagenic G nt were produced from the subtype C than subtype B template (68% vs. 40%, p<0.05) after 120 min (Figure 2B). The subtype B and C 1MM2 primers used contained a G at their 3′-end that should mismatch opposite a T (Figure 2C). Since the nt immediately downstream of the T on the subtype C template is a C, the template probably folded onto itself at that position to allow for the 3′-G of the primer to bind to the C of the template strand. Thus, misalignment would result in dislocation to produce either transcripts with a -1 nt frameshift mutation (FL-1nt band) or, upon subsequent primer/template realignment, transcripts containing the K65R mutation (FL band). It is important to note that the full-length products (FL) in the context of the subtype C primer/template sequences could have also been the result of either a realigned dislocation or of direct mispair extension. However, the later scenario would be less likely to occur as is observed on the subtype B sequence where dislocation is not present and in the assessment of the pre- and post-realignment products presented later.


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 B 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 B 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 B 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. (B) Graphical representation of the amount of transcripts containing the mutagenic nt produced with subtype B 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 with the mutagenic G at position 65 are produced with the subtype C template than with the subtype B template. (C) Depiction of the primer and template systems used. The primers contain a G base on their 3′-end that becomes mismatched on the T on the template strand thus yielding transcripts with the mutagenic nt. 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-g002: K65R production rates with subtype B 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 B 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 B 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. (B) Graphical representation of the amount of transcripts containing the mutagenic nt produced with subtype B 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 with the mutagenic G at position 65 are produced with the subtype C template than with the subtype B template. (C) Depiction of the primer and template systems used. The primers contain a G base on their 3′-end that becomes mismatched on the T on the template strand thus yielding transcripts with the mutagenic nt. The homopolymeric regions of both templates are underlined and the base responsible for the K65R mutation is indicated in bold.
Mentions: The production of DNA transcripts that contained the mutagenic G at the central position of codon 65 was evaluated using the subtype B-1MM2 and C-1MM2 primers on their respective subtype-matched (−)ssDNA templates. (+)dsDNA synthesis was evaluated simultaneously on both subtype B and C templates using wild-type subtype B (BWT) RT (Figure 2A). With the subtype B template, no pausing was seen and a distinct, 13 nt DNA product band containing K65R was observed at the full-length (FL) position. In contrast, use of the subtype C template led to two distinct DNA product bands at the FL and full-length -1 nt (FL-1nt) positions of 13 and 12 nt respectively, suggesting that a probable primer/template misalignment had occurred. Quantification results showed that larger amounts of DNA transcripts containing the mutagenic G nt were produced from the subtype C than subtype B template (68% vs. 40%, p<0.05) after 120 min (Figure 2B). The subtype B and C 1MM2 primers used contained a G at their 3′-end that should mismatch opposite a T (Figure 2C). Since the nt immediately downstream of the T on the subtype C template is a C, the template probably folded onto itself at that position to allow for the 3′-G of the primer to bind to the C of the template strand. Thus, misalignment would result in dislocation to produce either transcripts with a -1 nt frameshift mutation (FL-1nt band) or, upon subsequent primer/template realignment, transcripts containing the K65R mutation (FL band). It is important to note that the full-length products (FL) in the context of the subtype C primer/template sequences could have also been the result of either a realigned dislocation or of direct mispair extension. However, the later scenario would be less likely to occur as is observed on the subtype B sequence where dislocation is not present and in the assessment of the pre- and post-realignment products presented later.

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