Substitution of alanine for tyrosine-64 in the fingers subdomain of M-MuLV reverse transcriptase impairs strand displacement synthesis and blocks viral replication in vivo

A distinctive property of reverse transcriptase is the ability to carry out strand displacement synthesis in the absence of accessory proteins such as helicases or single-strand DNA binding proteins. Structure–function studies indicate that the fingers subdomain in HIV-1 reverse transcriptase contacts the template strand downstream of the primer terminus and is involved in strand displacement synthesis. Based on structural comparisons to the HIV-1 enzyme, we made single amino acid substitutions at the Tyr-64 and Leu-99 positions in the fingers subdomain of the M-MuLV reverse transcriptase to ask whether this subdomain has a similar role in displacement synthesis. In vitro assays comparing non-displacement versus displacement synthesis revealed that substitution of alanine at Tyr-64 generated a reverse transcriptase that was impaired in its capacity to carry out DNA and RNA displacement synthesis without affecting polymerase processivity or RNase H activity. However, substitution of Tyr-64 with phenylalanine and a variety of substitutions at position Leu-99 had no specific effect on displacement synthesis. The Y64A substitution prevented viral replication in vivo, and Y64A virus generated reduced levels of reverse transcription intermediates at all steps beyond the synthesis of minus strong stop DNA. The role of the fingers subdomain and in particular the possible contributions of the Tyr-64 residue in displacement synthesis are discussed.