A Template-Dependent Dislocation Mechanism Potentiates K65R Reverse Transcriptase Mutation Development in Subtype C Variants of HIV-1
A Template-Dependent Dislocation Mechanism Potentiates K65R Reverse Transcriptase Mutation Development in Subtype C Variants of HIV-1
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DOI：
10.1371/journal.pone.0020208
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发表时间：
2011-05-31
期刊：
影响因子：
3.7
通讯作者：
Wainberg, Mark A.
中科院分区：
文献类型：
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作者：
Coutsinos, Dimitrios;Invernizzi, Cedric F.;Wainberg, Mark A.
大量研究表明，K65R逆转录酶(RT)突变在C亚型中比在B亚型HIV-1中更容易发生。我们最近发现，这种差异部分存在于C亚型模板编码序列中，该序列使RT在K65R突变位点暂停。然而，这种观察的机制和K65R发育率升高的机制仍不清楚。在这里，我们报告了使用C亚型模板进行的DNA合成始终比B亚型模板产生更多含有K65R的转录本，而不考虑所使用的RT酶的亚型来源。这些发现证实了所涉及的机制是模板特定的和RT无关的。此外，仅在C亚型序列中观察到了DNA合成的特点，即位点特异的引物/模板滑移和错位。对RNA二级结构的分析表明，后者不太可能影响K65R亚型之间的发育，并且在CK65亚型区域的DNA合成过程中，可能会发生Streisinger链滑移，导致引物和模板的错位。因此，滑移会导致K65密码子中间腺嘌呤的缺失，并产生a-1移码突变，当引物和模板错位和重排时，将导致K65R突变的发生。这些发现为促进C亚型HIV-1中K65R突变的发展提供了额外的机制证据。
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.