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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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Evaluation of the dislocation products in the various subtype C primer/template conditions.(A) Graphical representation of K65R-containing transcript production with primers already harboring the mutagenic nt. Left: K65R production with subtype B RT on the subtype C template. Right: K65R production with subtype C RT on the subtype C template. The product of dislocation without realignment (FL-1nt product) is depicted as empty circles and the products of dislocation with realignment or misincorporation without dislocation (FL product) are depicted as empty triangles. When the mutagenic nt is already incorporated into the primer strand, the amount of FL and FL-1nt products is identical. (B) Schematic of the dislocation without realignment (FL-1nt) and the dislocation with realignment or misincorporation without dislocation (FL) products with the primer strand already containing the mutagenic nt. The sequence of the unextended primer is depicted in bold. (C) Graphical representation of K65R-containing transcript production with dGTP incorporation at the site of K65R development. Left: K65R production with subtype B RT on the subtype C template. Right: K65R production with subtype C RT on the subtype C template. The product of dislocation without realignment (P+1nt product) is depicted as empty circles and the products of dislocation with realignment or misincorporation without dislocation (P+2nt product) are depicted as empty triangles. During the process of incorporation of the mutagenic nt, the amount of P+1nt product reaches a maximum at 60 min whereas the amount of P+2nt product continues to rise steadily beyond 60 min. (D) Schematic of the dislocation without realignment (P+1nt) and the dislocation with realignment or misincorporation without dislocation (P+2nt) products during dGTP incorporation at the site of K65R development. The sequence of the unextended primer is depicted in bold.
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pone-0020208-g008: Evaluation of the dislocation products in the various subtype C primer/template conditions.(A) Graphical representation of K65R-containing transcript production with primers already harboring the mutagenic nt. Left: K65R production with subtype B RT on the subtype C template. Right: K65R production with subtype C RT on the subtype C template. The product of dislocation without realignment (FL-1nt product) is depicted as empty circles and the products of dislocation with realignment or misincorporation without dislocation (FL product) are depicted as empty triangles. When the mutagenic nt is already incorporated into the primer strand, the amount of FL and FL-1nt products is identical. (B) Schematic of the dislocation without realignment (FL-1nt) and the dislocation with realignment or misincorporation without dislocation (FL) products with the primer strand already containing the mutagenic nt. The sequence of the unextended primer is depicted in bold. (C) Graphical representation of K65R-containing transcript production with dGTP incorporation at the site of K65R development. Left: K65R production with subtype B RT on the subtype C template. Right: K65R production with subtype C RT on the subtype C template. The product of dislocation without realignment (P+1nt product) is depicted as empty circles and the products of dislocation with realignment or misincorporation without dislocation (P+2nt product) are depicted as empty triangles. During the process of incorporation of the mutagenic nt, the amount of P+1nt product reaches a maximum at 60 min whereas the amount of P+2nt product continues to rise steadily beyond 60 min. (D) Schematic of the dislocation without realignment (P+1nt) and the dislocation with realignment or misincorporation without dislocation (P+2nt) products during dGTP incorporation at the site of K65R development. The sequence of the unextended primer is depicted in bold.

Mentions: The levels of pre and post-realignment products following dislocation can shed light on the proportion of transcripts that contain the K65R mutation or that harbor a -1 frameshift mutation during primer-dependent transcription from the subtype C template. The data of Figure 8A show that the amount of transcripts that resulted from the primer and template that have not realigned (FL-1nt products) is equivalent to that which has realigned (FL products). With BWT RT, 36% of transcripts contained the FL product and 35% contained the FL-1nt product (Figure 8A, left panel). In the case of CWT RT, the FL and FL-1 products each represent 41% of the transcripts (Figure 8A, right panel). Dislocation and realignment will yield a 13 nt product that corresponds to the band at the FL position (Figure 8B). All of the transcripts that resulted from dislocation and subsequent realignment contained K65R. As discussed earlier, misincorporation of the 3′-G of the primer sequence opposite the T in the absence of dislocation might also generate, although with lower probability, the 13 nt FL product. In contrast, dislocation that is not followed by realignment will yield a 12 nt product that corresponds to the band at the FL-1 position (Figure 8B). These transcripts contain a -1 frameshift mutation with a deletion of the A at the middle position of codon 65.


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)

Evaluation of the dislocation products in the various subtype C primer/template conditions.(A) Graphical representation of K65R-containing transcript production with primers already harboring the mutagenic nt. Left: K65R production with subtype B RT on the subtype C template. Right: K65R production with subtype C RT on the subtype C template. The product of dislocation without realignment (FL-1nt product) is depicted as empty circles and the products of dislocation with realignment or misincorporation without dislocation (FL product) are depicted as empty triangles. When the mutagenic nt is already incorporated into the primer strand, the amount of FL and FL-1nt products is identical. (B) Schematic of the dislocation without realignment (FL-1nt) and the dislocation with realignment or misincorporation without dislocation (FL) products with the primer strand already containing the mutagenic nt. The sequence of the unextended primer is depicted in bold. (C) Graphical representation of K65R-containing transcript production with dGTP incorporation at the site of K65R development. Left: K65R production with subtype B RT on the subtype C template. Right: K65R production with subtype C RT on the subtype C template. The product of dislocation without realignment (P+1nt product) is depicted as empty circles and the products of dislocation with realignment or misincorporation without dislocation (P+2nt product) are depicted as empty triangles. During the process of incorporation of the mutagenic nt, the amount of P+1nt product reaches a maximum at 60 min whereas the amount of P+2nt product continues to rise steadily beyond 60 min. (D) Schematic of the dislocation without realignment (P+1nt) and the dislocation with realignment or misincorporation without dislocation (P+2nt) products during dGTP incorporation at the site of K65R development. The sequence of the unextended primer is depicted in bold.
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Related In: Results  -  Collection

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

pone-0020208-g008: Evaluation of the dislocation products in the various subtype C primer/template conditions.(A) Graphical representation of K65R-containing transcript production with primers already harboring the mutagenic nt. Left: K65R production with subtype B RT on the subtype C template. Right: K65R production with subtype C RT on the subtype C template. The product of dislocation without realignment (FL-1nt product) is depicted as empty circles and the products of dislocation with realignment or misincorporation without dislocation (FL product) are depicted as empty triangles. When the mutagenic nt is already incorporated into the primer strand, the amount of FL and FL-1nt products is identical. (B) Schematic of the dislocation without realignment (FL-1nt) and the dislocation with realignment or misincorporation without dislocation (FL) products with the primer strand already containing the mutagenic nt. The sequence of the unextended primer is depicted in bold. (C) Graphical representation of K65R-containing transcript production with dGTP incorporation at the site of K65R development. Left: K65R production with subtype B RT on the subtype C template. Right: K65R production with subtype C RT on the subtype C template. The product of dislocation without realignment (P+1nt product) is depicted as empty circles and the products of dislocation with realignment or misincorporation without dislocation (P+2nt product) are depicted as empty triangles. During the process of incorporation of the mutagenic nt, the amount of P+1nt product reaches a maximum at 60 min whereas the amount of P+2nt product continues to rise steadily beyond 60 min. (D) Schematic of the dislocation without realignment (P+1nt) and the dislocation with realignment or misincorporation without dislocation (P+2nt) products during dGTP incorporation at the site of K65R development. The sequence of the unextended primer is depicted in bold.
Mentions: The levels of pre and post-realignment products following dislocation can shed light on the proportion of transcripts that contain the K65R mutation or that harbor a -1 frameshift mutation during primer-dependent transcription from the subtype C template. The data of Figure 8A show that the amount of transcripts that resulted from the primer and template that have not realigned (FL-1nt products) is equivalent to that which has realigned (FL products). With BWT RT, 36% of transcripts contained the FL product and 35% contained the FL-1nt product (Figure 8A, left panel). In the case of CWT RT, the FL and FL-1 products each represent 41% of the transcripts (Figure 8A, right panel). Dislocation and realignment will yield a 13 nt product that corresponds to the band at the FL position (Figure 8B). All of the transcripts that resulted from dislocation and subsequent realignment contained K65R. As discussed earlier, misincorporation of the 3′-G of the primer sequence opposite the T in the absence of dislocation might also generate, although with lower probability, the 13 nt FL product. In contrast, dislocation that is not followed by realignment will yield a 12 nt product that corresponds to the band at the FL-1 position (Figure 8B). These transcripts contain a -1 frameshift mutation with a deletion of the A at the middle position of codon 65.

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