Retroviral reverse transcriptases (RTs) have the ability to carry out strand displacement DNA synthesis in the absence of accessory proteins. Although studies with RTs and other DNA polymerases suggest that fingers subdomain residues participate in strand displacement, molecular determinants of this activity are still unknown. A mutant human immunodeficiency virus type 2 (HIV-2) RT (M41L/D67N/K70R/S215Y) with low strand displacement activity was identified after screening a panel of purified enzymes, including several antiretroviral drug-resistant HIV-1 and HIV-2 RTs. In HIV-1, resistance to zidovudine and other thymidine analogues is conferred by different combinations of M41L, D67N, K70R, L210W, T215F/Y, and K219E/Q (designated as thymidine analogue resistance-associated mutations (TAMs)). However, those changes are rarely selected in HIV-2. We show that the strand displacement activity of HIV-2ROD mutants M41L/S215Y and D67N/K70R was only slightly reduced compared to the wild-type RT. In contrast, mutants D67N/K70R/S215Y and M41L/D67N/K70R/S215Y were the most defective RTs in reactions carried out with nicked and gapped substrates. Moreover, these enzymes showed the lowest nucleotide incorporation rates in assays carried out with strand displacement substrates. Unlike in HIV-2, substitutions M41L/T215Y and D67N/K70R/T215Y/K219Q had no effect on the strand displacement activity of HIV-1BH10 RT. The strand displacement efficiencies of HIV-2ROD RTs were consistent with the lower replication capacity of HIV-2 strains bearing the four major TAMs in their RT. Our results highlight the role of the fingers subdomain in strand displacement. These findings might be important for the development of strand-displacement defective RTs.

中文翻译：

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由于积累的HIV-2逆转录酶中的胸苷类似抗性突变，导致缺陷的链置换DNA合成。

逆转录病毒逆转录酶（RTs）具有在不存在辅助蛋白的情况下进行链置换DNA合成的能力。尽管对RT和其他DNA聚合酶的研究表明手指亚结构域残基参与了链置换，但这种活性的分子决定因素仍然未知。在筛选一组纯化的酶（包括几种抗逆转录病毒药物耐药的HIV-1和HIV-2）后，鉴定了一种具有低链置换活性的突变型2型人类免疫缺陷病毒（HIV-2）RT（M41L / D67N / K70R / S215Y） RTs。在HIV-1中，M41L，D67N，K70R，L210W，T215F / Y和K219E / Q（称为胸苷类似物耐药相关突变（TAMs））的不同组合赋予了对齐多夫定和其他胸苷类似物的耐药性。但是，在HIV-2中很少选择这些变化。我们显示，与野生型RT相比，HIV-2ROD突变体M41L / S215Y和D67N / K70R的链置换活性仅略有降低。相反，突变体D67N / K70R / S215Y和M41L / D67N / K70R / S215Y是在带有缺口和缺口的底物上进行的反应中最有缺陷的RT。此外，在用链置换底物进行的测定中，这些酶显示出最低的核苷酸掺入率。与HIV-2不同，取代M41L / T215Y和D67N / K70R / T215Y / K219Q对HIV-1BH10 RT的链置换活性没有影响。HIV-2ROD RTs的链置换效率与在其RT中带有四个主要TAM的HIV-2菌株复制能力较低相符。我们的结果强调了手指亚域在链置换中的作用。