Defective Strand-Displacement DNA Synthesis Due to Accumulation of Thymidine Analogue Resistance Mutations in HIV-2 Reverse Transcriptase

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-2_ROD 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-1_BH10 RT. The strand displacement efficiencies of HIV-2_ROD 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.