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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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Multiple incorrect nt incorporations at the K65 position 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 contains a single dGTP, dCTP or dTTP incorporation opposite the T in the template strand 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. Dislocation is observed with the subtype C template as two distinct nt incorporations at the P+1nt and P+2nt positions. (B) Graphical representation of DNA synthesis with subtype B RT on the subtype B and C templates. The values indicated with an asterisk have a p-value <0.05 when the amount of transcript-production between both subtypes at the given time-point is compared. (C) Depiction of the primer, template and nt employed in the system. The homopolymeric regions of both templates are underlined and the base responsible for the K65R generation is bolded.
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pone-0020208-g006: Multiple incorrect nt incorporations at the K65 position 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 contains a single dGTP, dCTP or dTTP incorporation opposite the T in the template strand 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. Dislocation is observed with the subtype C template as two distinct nt incorporations at the P+1nt and P+2nt positions. (B) Graphical representation of DNA synthesis with subtype B RT on the subtype B and C templates. The values indicated with an asterisk have a p-value <0.05 when the amount of transcript-production between both subtypes at the given time-point is compared. (C) Depiction of the primer, template and nt employed in the system. The homopolymeric regions of both templates are underlined and the base responsible for the K65R generation is bolded.

Mentions: To evaluate the mutagenic potential of the two other nt that might be incorrectly incorporated at the K65 site, we conducted RT reactions in the presence of dCTP, dTTP and dGTP. When BWT RT was employed with either the subtype B and C primer/template systems, the characteristic double-banding pattern of dislocation was only seen with the subtype C template (Figure 6A). More transcripts were again produced with the subtype C than subtype B template after 120 min (35% vs. 22%, p<0.05) (Figure 6B). In the presence of dCTP, dTTP or dGTP, it is only dGTP incorporation on a dislocated subtype C primer/template that might simultaneously produce a band at the P+1nt position and, upon realignment, a band at the P+2nt position, which would have allowed a second dGTP to be correctly incorporated (Figure 6C). We speculated that addition of dCTP and dTTP to the reaction would cause the P+1nt band produced from the C template to diminish significantly if dislocation at this position was not specific to dGTP. In this context, dCTP or dTTP could occupy the P+1nt position and allow for correct dGTP binding to occur at the P+2nt position, thereby eliminating the majority of P+1nt product. However, this did not occur, showing that dGTP is preferentially incorporated over other nt at the K65 site on the subtype C template via primer/template slippage and dislocation mutagenesis.


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)

Multiple incorrect nt incorporations at the K65 position 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 contains a single dGTP, dCTP or dTTP incorporation opposite the T in the template strand 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. Dislocation is observed with the subtype C template as two distinct nt incorporations at the P+1nt and P+2nt positions. (B) Graphical representation of DNA synthesis with subtype B RT on the subtype B and C templates. The values indicated with an asterisk have a p-value <0.05 when the amount of transcript-production between both subtypes at the given time-point is compared. (C) Depiction of the primer, template and nt employed in the system. The homopolymeric regions of both templates are underlined and the base responsible for the K65R generation is bolded.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3105016&req=5

pone-0020208-g006: Multiple incorrect nt incorporations at the K65 position 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 contains a single dGTP, dCTP or dTTP incorporation opposite the T in the template strand 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. Dislocation is observed with the subtype C template as two distinct nt incorporations at the P+1nt and P+2nt positions. (B) Graphical representation of DNA synthesis with subtype B RT on the subtype B and C templates. The values indicated with an asterisk have a p-value <0.05 when the amount of transcript-production between both subtypes at the given time-point is compared. (C) Depiction of the primer, template and nt employed in the system. The homopolymeric regions of both templates are underlined and the base responsible for the K65R generation is bolded.
Mentions: To evaluate the mutagenic potential of the two other nt that might be incorrectly incorporated at the K65 site, we conducted RT reactions in the presence of dCTP, dTTP and dGTP. When BWT RT was employed with either the subtype B and C primer/template systems, the characteristic double-banding pattern of dislocation was only seen with the subtype C template (Figure 6A). More transcripts were again produced with the subtype C than subtype B template after 120 min (35% vs. 22%, p<0.05) (Figure 6B). In the presence of dCTP, dTTP or dGTP, it is only dGTP incorporation on a dislocated subtype C primer/template that might simultaneously produce a band at the P+1nt position and, upon realignment, a band at the P+2nt position, which would have allowed a second dGTP to be correctly incorporated (Figure 6C). We speculated that addition of dCTP and dTTP to the reaction would cause the P+1nt band produced from the C template to diminish significantly if dislocation at this position was not specific to dGTP. In this context, dCTP or dTTP could occupy the P+1nt position and allow for correct dGTP binding to occur at the P+2nt position, thereby eliminating the majority of P+1nt product. However, this did not occur, showing that dGTP is preferentially incorporated over other nt at the K65 site on the subtype C template via primer/template slippage and dislocation mutagenesis.

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