578-T. Pausing during Initiation of HIV-1 Reverse Transcription Represents an Important Determinant for Viral Replication Fitness

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Session 76 Poster Session
HIV Resistance and Fitness
Session Time: 4:30-6:30 pm
Room 4E-F

578-T.

Pausing during Initiation of HIV-1 Reverse Transcription Represents an Important Determinant for Viral Replication Fitness
X. Wei*, C. Liang, M. Gštte, and M. A. Wainberg
Lady Davis Inst., Jewish Gen. Hosp., Montreal, QC, Canada

Background: The human immunodeficiency virus type-1 (HIV-1) recruits tRNALys3 as primer to initiate synthesis of the first DNA strand. The tRNA binds with its18 terminal nucleotides to a complementary primer binding site (PBS) near the 5' end of viral RNA. Previous studies have indicated that interactions between the anticodon loop of tRNALys3 and an 'A-rich loop' upstream of the PBS (169 AAAA 172) play important roles during the initiation process. We have recently shown that a deletion of the A-rich loop caused diminished viral replication fitness. Long-term culture of this virus resulted in the emergence of breakthrough variants with mutations in the vicinity of the A-rich loop. In this study, we have analyzed the underlying mechanisms involved in altered replication capacities of deletion-containing viruses and revertants.
Methods: We have analyzed and compared the emergence of breakthrough viruses in the context of wild-type HIV-1 and the M184V mutant, known to confer high-level resistance to lamivudine. The mutant variant is associated with diminished replication fitness and may thus compromise the emergence of revertants. Cell-free, biochemical studies were also conducted to provide mechanistic explanations.
Results: Long-term culture of viruses with the deletion in a wild-type background, yielded a variety of revertants with both or either G->A substitutions upstream of the former A-rich loop, and T-> A or C->A substitutions downstream of this region. In contrast, viruses containing the deletion together with the M184V mutation in the reverse transcriptase (RT) gene did not recover over protracted periods of time. Analyses of tRNA-primed DNA synthesis in cell-free assays showed diminished rates of the initiation of DNA synthesis when the RNA template contained the above A-rich loop deletion. Clearance from pausing at position +3 was identified as a sensitive step and the mutant RT enzyme with the M184V substitution was not able to efficiently bypass this obstacle. RNA templates that contained mutations formed in breakthrough viruses were able to facilitate the efficiency of initiation.
Conclusions: The initiation of tRNA-prime DNA synthesis is an important rate-limiting step in the process of reverse transcription. Our results show that RT, which contains the 184V mutation, may recognize the initiation complex for synthesis of viral DNA less well than wild-type enzyme.