Conformational in the End of the HIV-1 Genome Dependent on the Debranching Enzyme During Early Stages of Infection

29 Previous studies in our laboratory showed that the RNA debranching enzyme (DBR1) is not 31 required for early steps in HIV cDNA formation, but is necessary for synthesis of intermediate 32 and late cDNA products. To further characterize this effect, we evaluated the topology of the 5’ 33 end of the HIV-1 RNA genome during early infection with and without inhibition of DBR1 34 synthesis. Cells were transfected with DBR1 shRNA followed 48 hours later by infection with 35 an HIV-1 derived vector containing an RNase H deficient reverse transcriptase. RNA was 36 isolated at several times post-infection and treated with various RNA modifying enzymes prior 37 to rapid amplification of 5’ ends (5’ RACE) for HIV-1 RNA and quantitative RT-PCR. In infected 38 cells, DBR1 knockdown inhibited detection of free HIV-1 RNA 5’ ends at all time points. The 39 difference in detection of free HIV-1 RNA 5’ ends in infected DBR1 knockdown vs. control cells 40 was eliminated by in vitro incubation of infected cell RNAs with yeast or human DBR1 enzyme 41 prior to 5’ RACE and qRT-PCR. This was dependent on the 2'-5' phosphatase activity of 42 DBR1, since it did not occur when we used the catalytically inactive DBR1 N85A mutant. 43 Finally, HIV-1 RNA from infected DBR1 knockdown cells was resistant to RNase R that 44 degrades linear RNAs but not RNAs in circular or lariat-like conformations. These results 45 provide evidence for formation of a lariat-like structure involving the 5’ end of HIV-1 RNA 46 during an early step in infection, and the involvement of DBR1 in resolving it. 47 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 52 cell. The lariat-like structure is subsequently resolved by the cellular enzyme, DBR1, leaving a 53 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. RNA. Features of these experiments included comparison of cells with and without DBR1 knockdown, and use of a viral vector preparation containing RT with the E478Q mutation that allowed analysis of the genomic RNA without the complication of RT-mediated degradation of the template RNA. Our studies employed 5’ RACE to detect free (uncapped and phosphorylated) 5’ ends of genomic RNAs, and quantitative RT-PCR of the products allowed improved analysis. In addition, in vitro digestion of RNAs from infected cells with different RNA modifying enzymes prior to the 5’ RACE allowed us to distinguish capped linear viral RNA, viral RNA in a circular or lariat-like configuration, and uncapped linear viral RNA. Input viral vector RNA was capped and linear, as expected, but in infected cells the viral RNA was converted rapidly (beginning by 2 hours post-infection) into other forms. In control cells, the temporal order of these forms was: [1] capped linear, [2] lariat-like, [3] uncapped linear, and the timing was consistent with resolution of the lariat-like form concomitant with minus-strand transfer during cDNA synthesis. In DBR1 knockdown cells, there was an accumulation of lariat-like structures and a decrease in uncapped linear viral RNA, consistent with a requirement for DBR1 to resolve the lariats into linear viral RNA. In support of this, in vitro incubation of RNA from infected DBR1 knockdown cells with purified DBR1 enzyme increased the amount of ligatable 5’ viral RNA ends. Moreover, this was dependent on the catalytic activity of DBR1. The accumulation of lariat-like viral RNAs in infected DBR1 knockdown cells was also consistent with the persistence of the 5' end of viral RNA in a form that was resistant to RNase R. The results reported here are consistent with formation and resolution of a lariat- like structure involving the 5' end of HIV-1 RNA during reverse transcription in infected cells, as previously proposed for yeast Ty1 retrotransposition. Previous studies the Ty1 RNA in wild type and dbr1 mutant yeast strains was differentially accessible by 5’ RACE (5). In their study, they pretreated the RNAs with phosphatases and pyrophosphatases prior to annealing the RNA oligonucleotide to enable amplification of only capped RNAs; they compared the results with reactions where there was no pretreatment, which would detect all uncapped RNAs. They found that in dbr1 mutants, there was little or no detection of free 5’ ends regardless of the treatments, while in wild-type controls, 5’ ends were detectable in both 327 the capped and uncapped forms (5) . However, the results were not reproduced by Coombes and Boeke (10), who used the same 5’ RACE strategy and a semi-quantitative RT-PCR 329 analysis as well as sequencing of the amplified product. The latter investigators reported that there was no difference in the sequenced amplified products and that no obvious difference in the amplification of 5’ ends between wild-type and dbr1 mutant samples. These differing results prompted development of our strategy for analysis of the 5’ ends of HIV-1 RNA in infected cells, which employed 5’ RACE and quantitative RT-PCR and which allowed identification of differences that may not have been evident in standard or semi-quantitative RT-PCR. Moreover, in vitro digestion of the infected cell RNAs with different RNA-modifying enzymes prior to 5’ RACE allowed us to distinguish capped, lariat-like and linear uncapped 337 RNAs; the results with the different enzymes were internally consistent.