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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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Related in: MedlinePlus

Multiple incorrect nt incorporations at the K65 position 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 is observed as a single dGTP, dCTP or dTTP incorporation occurred 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 C RT on the subtype C template. Dislocation is only present with the subtype C template. (B) Graphical representation of DNA synthesis with subtype C RT on the subtype B and C templates. The same trend is observed regardless of origin of the RT enzyme used. (C) Depiction of the primer, template and nt used in the reaction. The homopolymeric regions are underlined and the base responsible for the K65R mutation is indicated in bold.
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pone-0020208-g007: Multiple incorrect nt incorporations at the K65 position 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 is observed as a single dGTP, dCTP or dTTP incorporation occurred 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 C RT on the subtype C template. Dislocation is only present with the subtype C template. (B) Graphical representation of DNA synthesis with subtype C RT on the subtype B and C templates. The same trend is observed regardless of origin of the RT enzyme used. (C) Depiction of the primer, template and nt used in the reaction. The homopolymeric regions are underlined and the base responsible for the K65R mutation is indicated in bold.

Mentions: Similar levels of P+1nt and P+2nt products were obtained when CWT RT was used for the reaction in the presence of dCTP, dTTP and dGTP (Figure 7A) and the subtype C templates produced more transcripts than the subtype B transcripts after 120 min (30% vs. 18%, p<0.05) (Figure 7B). Thus, dGTP is preferentially and erroneously incorporated at the K65 site despite the presence of other mutagenic nt (Figure 7C).


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 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 is observed as a single dGTP, dCTP or dTTP incorporation occurred 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 C RT on the subtype C template. Dislocation is only present with the subtype C template. (B) Graphical representation of DNA synthesis with subtype C RT on the subtype B and C templates. The same trend is observed regardless of origin of the RT enzyme used. (C) Depiction of the primer, template and nt used in the reaction. The homopolymeric regions are underlined and the base responsible for the K65R mutation is indicated in bold.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3105016&req=5

pone-0020208-g007: Multiple incorrect nt incorporations at the K65 position 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 is observed as a single dGTP, dCTP or dTTP incorporation occurred 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 C RT on the subtype C template. Dislocation is only present with the subtype C template. (B) Graphical representation of DNA synthesis with subtype C RT on the subtype B and C templates. The same trend is observed regardless of origin of the RT enzyme used. (C) Depiction of the primer, template and nt used in the reaction. The homopolymeric regions are underlined and the base responsible for the K65R mutation is indicated in bold.
Mentions: Similar levels of P+1nt and P+2nt products were obtained when CWT RT was used for the reaction in the presence of dCTP, dTTP and dGTP (Figure 7A) and the subtype C templates produced more transcripts than the subtype B transcripts after 120 min (30% vs. 18%, p<0.05) (Figure 7B). Thus, dGTP is preferentially and erroneously incorporated at the K65 site despite the presence of other mutagenic nt (Figure 7C).

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