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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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Schematic of dislocation mutagenesis and the development of the K65R mutation in subtype C HIV-1.Step 1: DNA synthesis approaches the end of a homopolymeric nt stretch that ends precisely at the location of K65R development. Step 2: At the end of the sequence, the RT enzyme exhibits characteristic pausing of DNA synthesis. The template-strand folds onto itself and exposes a C in the folded-over template strand. Step 3: A dGTP nt correctly binds opposite the C base of the misaligned template strand as DNA synthesis continues. Step 4: The primer and template strands realign and the same C base becomes re-exposed on the now correctly aligned template strand. Step 5: A second dGTP becomes incorporated opposite the re-exposed C on the correctly aligned primer/template strands and DNA synthesis continues normally. This series of events yields the AAG-to-AGG change that is responsible for the more facilitated appearance of the K65R mutation in subtype C HIV-1.
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pone-0020208-g010: Schematic of dislocation mutagenesis and the development of the K65R mutation in subtype C HIV-1.Step 1: DNA synthesis approaches the end of a homopolymeric nt stretch that ends precisely at the location of K65R development. Step 2: At the end of the sequence, the RT enzyme exhibits characteristic pausing of DNA synthesis. The template-strand folds onto itself and exposes a C in the folded-over template strand. Step 3: A dGTP nt correctly binds opposite the C base of the misaligned template strand as DNA synthesis continues. Step 4: The primer and template strands realign and the same C base becomes re-exposed on the now correctly aligned template strand. Step 5: A second dGTP becomes incorporated opposite the re-exposed C on the correctly aligned primer/template strands and DNA synthesis continues normally. This series of events yields the AAG-to-AGG change that is responsible for the more facilitated appearance of the K65R mutation in subtype C HIV-1.

Mentions: We have now explored the mechanisms responsible for these observations as described in Figure 10. Initially, RT synthesizes (+)dsDNA from a subtype C (−)ssDNA intermediate template. As the enzyme approaches the K65 site, it encounters a homopolymeric stretch of 6 T nt followed by a single C immediately adjacent to the site of K65R development. The two nt polymorphisms that are present in subtype C but not B are responsible for this positioning of the homopolymeric stretch in subtype C HIV-1, resulting in pausing and slippage of the primer and template strands. During slippage, the template folds over itself, resulting in misalignment of the template and primer, concealing the final T of the homopolymeric stretch and prematurely uncovering the following C. Hydrogen bonding provided by the incoming nt might then enhance the stability of the misaligned primer and template, resulting in a G being correctly introduced into the newly synthesized primer strand opposite the exposed C on the misaligned template. These events will produce DNA transcripts containing the -1 frameshift mutation that involves deletion of the middle A of codon K65. These findings are consistent with the one-base frameshift mutations that result from a well-described and characterized Streisinger strand slippage mechanism [66], [67].


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)

Schematic of dislocation mutagenesis and the development of the K65R mutation in subtype C HIV-1.Step 1: DNA synthesis approaches the end of a homopolymeric nt stretch that ends precisely at the location of K65R development. Step 2: At the end of the sequence, the RT enzyme exhibits characteristic pausing of DNA synthesis. The template-strand folds onto itself and exposes a C in the folded-over template strand. Step 3: A dGTP nt correctly binds opposite the C base of the misaligned template strand as DNA synthesis continues. Step 4: The primer and template strands realign and the same C base becomes re-exposed on the now correctly aligned template strand. Step 5: A second dGTP becomes incorporated opposite the re-exposed C on the correctly aligned primer/template strands and DNA synthesis continues normally. This series of events yields the AAG-to-AGG change that is responsible for the more facilitated appearance of the K65R mutation in subtype C HIV-1.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3105016&req=5

pone-0020208-g010: Schematic of dislocation mutagenesis and the development of the K65R mutation in subtype C HIV-1.Step 1: DNA synthesis approaches the end of a homopolymeric nt stretch that ends precisely at the location of K65R development. Step 2: At the end of the sequence, the RT enzyme exhibits characteristic pausing of DNA synthesis. The template-strand folds onto itself and exposes a C in the folded-over template strand. Step 3: A dGTP nt correctly binds opposite the C base of the misaligned template strand as DNA synthesis continues. Step 4: The primer and template strands realign and the same C base becomes re-exposed on the now correctly aligned template strand. Step 5: A second dGTP becomes incorporated opposite the re-exposed C on the correctly aligned primer/template strands and DNA synthesis continues normally. This series of events yields the AAG-to-AGG change that is responsible for the more facilitated appearance of the K65R mutation in subtype C HIV-1.
Mentions: We have now explored the mechanisms responsible for these observations as described in Figure 10. Initially, RT synthesizes (+)dsDNA from a subtype C (−)ssDNA intermediate template. As the enzyme approaches the K65 site, it encounters a homopolymeric stretch of 6 T nt followed by a single C immediately adjacent to the site of K65R development. The two nt polymorphisms that are present in subtype C but not B are responsible for this positioning of the homopolymeric stretch in subtype C HIV-1, resulting in pausing and slippage of the primer and template strands. During slippage, the template folds over itself, resulting in misalignment of the template and primer, concealing the final T of the homopolymeric stretch and prematurely uncovering the following C. Hydrogen bonding provided by the incoming nt might then enhance the stability of the misaligned primer and template, resulting in a G being correctly introduced into the newly synthesized primer strand opposite the exposed C on the misaligned template. These events will produce DNA transcripts containing the -1 frameshift mutation that involves deletion of the middle A of codon K65. These findings are consistent with the one-base frameshift mutations that result from a well-described and characterized Streisinger strand slippage mechanism [66], [67].

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