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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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K65K, K65T and K65I production as a result of DNA synthesis with primers containing the three nt alternatives and their impact on dislocation.(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 again as a distinct band at the FL and positions without any dislocation products. When A is present at the 3′-end of the primer strand, it correctly binds with the T of the template strand to yield wild-type K65K-containing transcripts. (B) 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. No dislocation products were observed. When C is present at the 3′-end of the primer strand, it incorrectly binds with the T of the template strand to yield K65T-containing transcripts. (C) 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 without dislocation. Lanes 11 through 20 depict (+)dsDNA synthesis from the (−)ssDNA intermediate with subtype B RT on the subtype C template. When T is present at the 3′-end of the primer strand, it incorrectly binds with the T of the template strand to yield K65I-containing transcripts. Dislocation products are only observed when G is included on the primer strand yielding K65R-containing transcripts.
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pone-0020208-g009: K65K, K65T and K65I production as a result of DNA synthesis with primers containing the three nt alternatives and their impact on dislocation.(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 again as a distinct band at the FL and positions without any dislocation products. When A is present at the 3′-end of the primer strand, it correctly binds with the T of the template strand to yield wild-type K65K-containing transcripts. (B) 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. No dislocation products were observed. When C is present at the 3′-end of the primer strand, it incorrectly binds with the T of the template strand to yield K65T-containing transcripts. (C) 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 without dislocation. Lanes 11 through 20 depict (+)dsDNA synthesis from the (−)ssDNA intermediate with subtype B RT on the subtype C template. When T is present at the 3′-end of the primer strand, it incorrectly binds with the T of the template strand to yield K65I-containing transcripts. Dislocation products are only observed when G is included on the primer strand yielding K65R-containing transcripts.

Mentions: To confirm that dislocation only occurs with dGTP, primers containing the three other bases at their 3′-end were used to determine whether dislocation would occur at the K65 site during DNA synthesis. We first evaluated wild-type K65K production by using an A base at the 3′-end of the primer with BWT RT on both the subtype B and C templates. The data show that the 13 nt FL product was present as a distinct single band when either the subtype B or C templates were used (Figure 9A). The absence of the double band in the context of the subtype C template indicates that dislocation does not occur at K65 when the primer contains an A at its 3′-end. Instead, complete primer extension was accomplished early (15 min) in the reaction because no incorrect incorporations or mismatches were necessary for the completion of DNA synthesis.


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)

K65K, K65T and K65I production as a result of DNA synthesis with primers containing the three nt alternatives and their impact on dislocation.(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 again as a distinct band at the FL and positions without any dislocation products. When A is present at the 3′-end of the primer strand, it correctly binds with the T of the template strand to yield wild-type K65K-containing transcripts. (B) 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. No dislocation products were observed. When C is present at the 3′-end of the primer strand, it incorrectly binds with the T of the template strand to yield K65T-containing transcripts. (C) 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 without dislocation. Lanes 11 through 20 depict (+)dsDNA synthesis from the (−)ssDNA intermediate with subtype B RT on the subtype C template. When T is present at the 3′-end of the primer strand, it incorrectly binds with the T of the template strand to yield K65I-containing transcripts. Dislocation products are only observed when G is included on the primer strand yielding K65R-containing transcripts.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3105016&req=5

pone-0020208-g009: K65K, K65T and K65I production as a result of DNA synthesis with primers containing the three nt alternatives and their impact on dislocation.(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 again as a distinct band at the FL and positions without any dislocation products. When A is present at the 3′-end of the primer strand, it correctly binds with the T of the template strand to yield wild-type K65K-containing transcripts. (B) 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. No dislocation products were observed. When C is present at the 3′-end of the primer strand, it incorrectly binds with the T of the template strand to yield K65T-containing transcripts. (C) 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 without dislocation. Lanes 11 through 20 depict (+)dsDNA synthesis from the (−)ssDNA intermediate with subtype B RT on the subtype C template. When T is present at the 3′-end of the primer strand, it incorrectly binds with the T of the template strand to yield K65I-containing transcripts. Dislocation products are only observed when G is included on the primer strand yielding K65R-containing transcripts.
Mentions: To confirm that dislocation only occurs with dGTP, primers containing the three other bases at their 3′-end were used to determine whether dislocation would occur at the K65 site during DNA synthesis. We first evaluated wild-type K65K production by using an A base at the 3′-end of the primer with BWT RT on both the subtype B and C templates. The data show that the 13 nt FL product was present as a distinct single band when either the subtype B or C templates were used (Figure 9A). The absence of the double band in the context of the subtype C template indicates that dislocation does not occur at K65 when the primer contains an A at its 3′-end. Instead, complete primer extension was accomplished early (15 min) in the reaction because no incorrect incorporations or mismatches were necessary for the completion of DNA synthesis.

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