Functional Study Of Reverse Transcriptases

The human immunodeficiency virus(HIV) is the causal agent of the acquired immune deficiency syndrome(AIDS) that affects millions of lives in the world. HIV is a retrovirus that contains diploid viral RNA genomes in a single virion. To complete each viral replication cycle, HIV needs to convert its RNA genomes into a doublestranded proviral DNA genome using its unique reverse transcriptase(RT). Two precise strand-transfers(disassociation and realignment) of nascent DNA fragments at the ends of the templates are required to complete the synthesis of the double-stranded proviral DNA genome with both long terminal repeats(LTR)Strand-transfers have been studied in three systems. First, viral genome sequences that switch between two distinct templates or misalign at different locations of the same template are studied in a single-cycle infection system. However, only strand-transfers in the same direction on the RNA templates will result in viral genomes that can be integrated into the host cell chromosomes. Thus, proviral genomes that are prematurely terminated or generated through strand-transfers but without two LTRs are not detected. Second, sizes of nascent cDNA fragments generated with a pair of short donor and acceptor RNA templates are used to study strand-transfers that occur only after cDNA fragments reach the 5’ end of donor templates. Third, strand-transfers are characterized by analyzing individual viral sequences(for deletions, insertions and recombination) generated by PCR from HIV RNA genomes. However, whether illegitimate strand-transfers result in nascent DNA fragments without two LTRs or PCR primer binding sites have not been studied since they would not have been detected by above methods.RT plays a critical role in retrovirus replication and is the only enzyme that allows studies of viral RNA genomes and host mRNAs. However, the frequency and requirements for strand-transfers, RT pausing as well as addition mutations at the end of the RNA template are still not well defined. To better understand mechanisms of strand-transfers, we analyzed a large number of nascent c DNA sequences by directly sequencing nascent reverse transcripts generated by reverse transcriptases from HIV-1, HIV-2 and murine leukemia virus(MLV). Among 1067 nascent reverse transcripts, 72% of them matched to the template sequences, although they randomly stopped across the RNA templates(~400bp). However, the rest 28% of them contained mismatched 3’ sequences due to illegitimate strand-transfers. The majority(81%) of the illegitimate strand-transfers were disassociated from RNA templates and realigned onto opposite complementary DNA strands. Up to three strand-transfers were detected in a single sequence although the majority of them(93%) contained one strand-transfer. Since the majority of illegitimate strand-transfer fragments were generated from templates at two opposite orientations, they resulted in defective viral genomes and could not been detected by previous methods. Further analysis showed that mutations at pause/disassociation sites of new reverse transcripts resulted significantly higher strand-transfer rates. We also found significantly higher illegitimate strand-transfer rates for HIV-2 RT(38.2%) and MLV RT(44.6%) than for HIV-1 RT(5.1%).Thus, the majority of illegitimate strand-transfers would result in defective viral genomes. This indicates that complete HIV genome replication through two precise strand-transfers is rare and explain why only less than 0.1% of HIV virions are infectious. A significantly higher illegitimate strand-transfer rate for HIV-2 RT than HIV-1 RT might account for lower viral loads and slower disease progression in individuals infected with HIV-2. We also studied the potential random-priming mechanisms of RT. The results showed that reverse transcriptase could often initiate reverse transcription through random-priming without primer. This random-priming had no preference for base composition or RNA structure. Finally, we found that the in vitro error rate of reverse transcriptase was much higher than those previously reported.The new discoveries about the processivity, strand-transfer,random primiung, and high error rate of different RTs made in this study will have significant implications for better understanding of biochemical characteristics of RTs as well as viral replication, biology and pathogenesis. Exploration of this unique intrinsic RT characteristic by increasing the illegitimate strand-transfer rate can serves as a novel therapeutic target for HIV infection.