Journal of virology 2017 09 20() pii 10.1128/JVI.01377-17
Previous studies in our laboratory showed that the RNA debranching enzyme (DBR1) is not required for early steps in HIV cDNA formation, but is necessary for synthesis of intermediate and late cDNA products. To further characterize this effect, we evaluated the topology of the 5′ end of the HIV-1 RNA genome during early infection with and without inhibition of DBR1 synthesis. Cells were transfected with DBR1 shRNA followed 48 hours later by infection with an HIV-1 derived vector containing an RNase H deficient reverse transcriptase. RNA was isolated at several times post-infection and treated with various RNA modifying enzymes prior to rapid amplification of 5′ ends (5′ RACE) for HIV-1 RNA and quantitative RT-PCR. In infected cells, DBR1 knockdown inhibited detection of free HIV-1 RNA 5′ ends at all time points. The difference in detection of free HIV-1 RNA 5′ ends in infected DBR1 knockdown vs. control cells was eliminated by in vitro incubation of infected cell RNAs with yeast or human DBR1 enzyme prior to 5′ RACE and qRT-PCR. This was dependent on the 2′ -5′ phosphatase activity of DBR1, since it did not occur when we used the catalytically inactive DBR1 N85A mutant. Finally, HIV-1 RNA from infected DBR1 knockdown cells was resistant to RNase R that degrades linear RNAs but not RNAs in circular or lariat-like conformations. These results provide evidence for formation of a lariat-like structure involving the 5′ end of HIV-1 RNA during an early step in infection, and the involvement of DBR1 in resolving it.IMPORTANCE Our findings support a new view of the early steps in HIV genome replication. We show that the HIV genomic RNA is rapidly de-capped and forms a lariat-like structure after entering a cell. The lariat-like structure is subsequently resolved by the cellular enzyme, DBR1, leaving a 5′ phosphate. This pathway is similar to the formation and resolution of pre-mRNA intron lariats and therefore suggests that similar mechanisms may be used by HIV. Our work therefore opens a new area of investigation in HIV replication and may ultimately uncover new targets for inhibiting HIV replication and for preventing the development of AIDS.