Acceptor RNA Cleavage Profile Supports an Invasion Mechanism for HIV-1 Minus Strand Transfer

Abstract

We previously proposed that HIV-1 minus strand transfer occurs by an acceptor invasion-initiated multistep mechanism. During synthesis of minus strong stop DNA, reverse transcriptase (RT) transiently pauses at the base of TAR before continuing synthesis. Pausing promotes RT-RNase H cleavage of the donor RNA, exposing regions of the cDNA. The acceptor RNA then invades at these locations to interact with the minus strong stop DNA. Whereas primer extension continues on the donor RNA, the cDNA-acceptor hybrid expands by branch migration until transfer of the primer terminus is completed. We present results here showing that the interaction of the acceptor RNA and the cDNA can be determined by examining the time-dependent cleavage of the acceptor RNA by RNase H. Our approach utilizes a combination of RT-RNase H and Escherichia coli RNase H to allow assessment of acceptor-cDNA interactions at high sensitivity. Results show an initial interaction of the acceptor RNA with cDNA at the base of TAR. We observe a time-dependent shift in RNase H susceptibility along the length of the acceptor toward the 5 end, suggesting hybrid propagation from the initial invasion point. Control experiments validate that the RNase H cleavage profile represents the formation and expansion of the acceptor-DNA interaction and that the process is promoted by the nucleocapsid. Observations with this new approach lend additional support to the proposed multistep transfer mechanism.During retrovirus reverse transcription, minus strand DNA synthesis initiates near the 5Ј end of the RNA genome. In HIV-1, 1 synthesis to the genome 5Ј end results in a DNA fragment about 200 nt long called minus strand strong stop DNA (ϪsssDNA). Direct repeat (R) sequences at either ends of the genomic RNA facilitate transfer of the strong stop DNA from the 5Ј end to the 3Ј end of either the same or the copackaged RNA (Ref. 1 and references therein). The 5Ј and 3Ј repeat regions thereby serve as the donor and acceptor templates, respectively, in this transfer process. The transfer process is obligatory to the completion of reverse transcription and synthesis of double-stranded viral DNA.The HIV-1 genomic RNA contains 97-nucleotide-long repeat regions, the 5Ј-terminal 56 nts of which form the TAR hairpin.Deletion and mutational analysis demonstrate that strong stop transfer in HIV-1 does not require all of the R-region homology (2-7). Studies in murine leukemia virus have suggested that homology-independent factors, such as template structure and sequence, are also involved in facilitating minus strand transfer (8).The RT-RNase H activity is essential at various steps of reverse transcription, including minus strand transfer (Ref. 9 and references therein). During minus strand synthesis, RTRNase H cleaves the donor RNA within the RNA-DNA hybrid regions. This enables the ϪsssDNA to transfer to the acceptor. Viruses defective in RT-RNase H activity are unable to complete reverse transcription and show accumulation of ϪsssDNA in the cytoplasm (10 -13). Addition of RNase H ...

Insights into the Multiple Roles of Pausing in HIV-1 Reverse Transcriptase-promoted Strand Transfers

“…These cleavages increased until 15 min, and were then chased away, suggesting that the acceptor first interacted with the cDNA in the region upstream of the hairpin. Addition of E. coli RNase H to acceptor cleavage reactions increases the sensitivity of detection of the acceptor RNA and nascent DNA hybrid (56). With E. coli RNase H, most cleavages were concentrated in the 3Ј-end homology region (30 ϳ 50 nt before the hairpin) at 1 min, later shifting in the 5Ј direction, and then chased away at 5 min.…”

Dissection of a Circumscribed Recombination Hot Spot in HIV-1 after a Single Infectious Cycle

“…However, mounting evidence indicates that preferential sites for recombination do not necessarily correlate with stalling of reverse transcription. A particularly well documented case is the occurrence of preferential transfer in structured regions of the RNA template (20,23,(27)(28)(29)(30)(31)(32)(33). In some of these cases the hairpin structures were suggested to induce stalling of reverse transcription at their base favoring the annealing of the acceptor RNA on the nascent DNA (20).…”

Proximity and Branch Migration Mechanisms in HIV-1 Minus Strand Strong Stop DNA Transfer

“…The cDNA 3Ј terminus would then transfer to the acceptor RNA (5,6). In the second, transfer would occur through an acceptor-mediated invasion-driven pathway (6,(13)(14)(15)(16). In this case, the cleavage of the RNA segment remaining at the donor 5Ј end is not required (6).…”

“…Although the RT continues extension of the cDNA, the acceptor-cDNA hybrid would propagate, displacing donor RNA oligomers remaining from RT RNase H cleavage. Eventually, the 3Ј end of the cDNA would switch from donor to acceptor in a region favorable for transfer completion (14,15). The acceptor invasion is proposed to be triggered by RT pausing at one or two strong pause sites on the donor, leading to a clustering of cuts (18 -22), generating a gap for acceptor invasion (14).…”