Zinc finger function of HIV-1 nucleocapsid protein is required for removal of 5′-terminal genomic RNA fragments: A paradigm for RNA removal reactions in HIV-1 reverse transcription

► Mechanism for removal of HIV-1 5′-viral RNA fragments annealed to (−) strong-stop DNA. ► Removal most efficient with HIV-1 NC protein and functional RNase H. ► HIV-1 NC alone is sufficient, but must have helix destabilizing activity. ► Small RNA in duplex displaced by longer 3′ viral RNA in a strand exchange reaction. ► Common mechanism for RNA removal reactions in reverse transcription. During (−) strong-stop DNA [(−) SSDNA] synthesis, RNase H cleavage of genomic viral RNA generates small 5′-terminal RNA fragments (14–18 nt) that remain annealed to the DNA. Unless these fragments are removed, the minus-strand transfer reaction, required for (−) SSDNA elongation, cannot occur. Here, we describe the mechanism of 5′-terminal RNA removal and the roles of HIV-1 nucleocapsid protein (NC) and RNase H cleavage in this process. Using an NC-dependent system that models minus-strand transfer, we show that the presence of short terminal fragments pre-annealed to (−) SSDNA has no impact on strand transfer, implying efficient fragment removal. Moreover, in reactions with an RNase H− reverse transcriptase mutant, NC alone is able to facilitate fragment removal, albeit less efficiently than in the presence of both RNase H activity and NC. Results obtained from novel electrophoretic gel mobility shift and Förster Resonance Energy Transfer assays, which each directly measure RNA fragment release from a duplex in the absence of DNA synthesis, demonstrate for the first time that the architectural integrity of NC's zinc finger (ZF) domains is absolutely required for this reaction. This suggests that NC's helix destabilizing activity (associated with the ZFs) facilitates strand exchange through the displacement of these short terminal RNAs by the longer 3′ acceptor RNA, which forms a more stable duplex with (−) SSDNA. Taken together with previously published results, we conclude that NC-mediated fragment removal is linked mechanistically with selection of the correct primer for plus-strand DNA synthesis and tRNA removal step prior to plus-strand transfer. Thus, HIV-1 has evolved a single mechanism for these RNA removal reactions that are critical for successful reverse transcription.