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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 B RT on subtype B and C templates.(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 dGTP incorporation occurred at the P+1nt 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 containing 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 B RT on both the subtype B and C templates. The values indicated with an asterisk have a p-value <0.05 when the amount of transcripts containing the mutagenic nt produced between both subtypes at the given time-point is compared. More transcripts containing the mutagenic G nt at codon 65 are produced with the subtype C template than with the subtype B template. (C) Depiction of the primer and template systems used. dGTP was the only nt employed in the reaction to allow for the production of the mutagenic 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-g004: Single incorrect nt incorporation and K65R production rates with subtype B RT on subtype B and C templates.(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 dGTP incorporation occurred at the P+1nt 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 containing 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 B RT on both the subtype B and C templates. The values indicated with an asterisk have a p-value <0.05 when the amount of transcripts containing the mutagenic nt produced between both subtypes at the given time-point is compared. More transcripts containing the mutagenic G nt at codon 65 are produced with the subtype C template than with the subtype B template. (C) Depiction of the primer and template systems used. dGTP was the only nt employed in the reaction to allow for the production of the mutagenic transcripts. The homopolymeric regions of both templates are underlined and the base responsible for the K65R mutation is indicated in bold.

Mentions: To evaluate the production of DNA transcripts containing the mutagenic nt and to identify the exact location of dislocation on the subtype C template, (+)dsDNA synthesis was evaluated using the Subtype B-0MM1 and C-0MM1 primers on their respective, subtype-matched (−)ssDNA templates. As stated, the 0MM1 primers did not contain a mutagenic nt at the 3′ position and dGTP was the only nt provided to ensure production of K65R-containing transcripts. Only a single band was present at the primer +1 nt (P+1nt) for the subtype B template sequence whereas a double band was present at the P+1nt and P+2nt positions with the subtype C template (Figure 4A). This is indicative of primer/template slippage misalignment and confirms that the dislocation occurred at the K65 site. As anticipated, more transcripts containing the mutagenic G nt were produced from the subtype C than subtype B template (40% vs. 15%, p<0.05) after 120 min (Figure 4B). Although fewer DNA transcripts containing the mutagenic nt were present than in the primer-dependent reaction, this may reflect additional thermodynamic energy barriers necessary for the primer-independent reaction that requires misincorporation of a dGTP opposite a T on the template. When comparing the subtype B and C primers and templates in the primer-independent setting, dislocation occurred only with the subtype C template at the exact location of K65R-development (Figure 4C), probably because the dislocation allowed the primer and template to misalign, permitting dGTP to correctly bind opposite the C to yield the P+1nt product. The P+2nt product was obtained when the primer and template realigned and allowed for a second dGTP to be incorporated immediately following the first G on the subtype C but not B template. As described earlier, direct misincorporation, although less likely considering the template-specific conditions, could have also produced the band at position P+2nt. Indeed, the differences observed between the subtype B and C templates with regard to the development of transcripts containing the mutagenic nt are likely due to the added propensity for dislocation mutagenesis to occur on the subtype C template.


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 B RT on subtype B and C templates.(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 dGTP incorporation occurred at the P+1nt 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 containing 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 B RT on both the subtype B and C templates. The values indicated with an asterisk have a p-value <0.05 when the amount of transcripts containing the mutagenic nt produced between both subtypes at the given time-point is compared. More transcripts containing the mutagenic G nt at codon 65 are produced with the subtype C template than with the subtype B template. (C) Depiction of the primer and template systems used. dGTP was the only nt employed in the reaction to allow for the production of the mutagenic 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-g004: Single incorrect nt incorporation and K65R production rates with subtype B RT on subtype B and C templates.(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 dGTP incorporation occurred at the P+1nt 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 containing 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 B RT on both the subtype B and C templates. The values indicated with an asterisk have a p-value <0.05 when the amount of transcripts containing the mutagenic nt produced between both subtypes at the given time-point is compared. More transcripts containing the mutagenic G nt at codon 65 are produced with the subtype C template than with the subtype B template. (C) Depiction of the primer and template systems used. dGTP was the only nt employed in the reaction to allow for the production of the mutagenic transcripts. The homopolymeric regions of both templates are underlined and the base responsible for the K65R mutation is indicated in bold.
Mentions: To evaluate the production of DNA transcripts containing the mutagenic nt and to identify the exact location of dislocation on the subtype C template, (+)dsDNA synthesis was evaluated using the Subtype B-0MM1 and C-0MM1 primers on their respective, subtype-matched (−)ssDNA templates. As stated, the 0MM1 primers did not contain a mutagenic nt at the 3′ position and dGTP was the only nt provided to ensure production of K65R-containing transcripts. Only a single band was present at the primer +1 nt (P+1nt) for the subtype B template sequence whereas a double band was present at the P+1nt and P+2nt positions with the subtype C template (Figure 4A). This is indicative of primer/template slippage misalignment and confirms that the dislocation occurred at the K65 site. As anticipated, more transcripts containing the mutagenic G nt were produced from the subtype C than subtype B template (40% vs. 15%, p<0.05) after 120 min (Figure 4B). Although fewer DNA transcripts containing the mutagenic nt were present than in the primer-dependent reaction, this may reflect additional thermodynamic energy barriers necessary for the primer-independent reaction that requires misincorporation of a dGTP opposite a T on the template. When comparing the subtype B and C primers and templates in the primer-independent setting, dislocation occurred only with the subtype C template at the exact location of K65R-development (Figure 4C), probably because the dislocation allowed the primer and template to misalign, permitting dGTP to correctly bind opposite the C to yield the P+1nt product. The P+2nt product was obtained when the primer and template realigned and allowed for a second dGTP to be incorporated immediately following the first G on the subtype C but not B template. As described earlier, direct misincorporation, although less likely considering the template-specific conditions, could have also produced the band at position P+2nt. Indeed, the differences observed between the subtype B and C templates with regard to the development of transcripts containing the mutagenic nt are likely due to the added propensity for dislocation mutagenesis to occur on the subtype C template.

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