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101 The M184V Mutation in Reverse Transcriptase Can Delay Reversion of Attenuated Variants of Simian Immunodeficiency Virus
J. B. Whitney* and M. A. Wainberg
McGill AIDS Ctr., Lady Davis Inst., Montreal, Quebec, Canada
Background: The development of an effective HIV vaccine has presented a formidable challenge. Toward this end a significant number of SIV-based models have already been developed, most employing deletion of viral accessory genes. A caveat of live-attenuated approaches is viral reversion and increased replicative fitness. In contrast to these approaches, our lab has developed a set of novel attenuated SIVmac239 candidates containing deletions within the 5 region of the leader sequence. These constructs displayed marked attenuation in several cell lines as well as monkey PBMCs. Over extended passage several deletion mutants exhibited increased replicative capacity in conjunction with the appearance of compensatory mutations. Therefore, in the context of vaccine development, we sought to determine the utility of additional mutations within the viral RT in delaying viral reversion.
Methods: We have already constructed and characterized a series of mutants containing deletions within a 97nt expanse of leader sequence downstream of the PBS. Those variants displaying moderate reversion kinetics (SD2+398-418, SD5+380-397, and SD6 +371-379) were selected as candidates for the insertion of the M184V substitution in reverse transcriptase. Replication capacities and reversion characteristics of these variants were assessed in CEMx174 cells and rhesus PMBC as determined by RT assay and p27 ELISA, respectively. Specific mutations were determined by cloning and sequencing
Results: The reversion of several SIV mutants can be further delayed by the addition of the M184V mutation. Serial passage of 2 mutants (SD5-M184V and SD6-M184V) resulted in the loss of the M184V mutation with a commensurate increase in reversion rates. Interestingly, 1 mutant in particular, SD2-M184V, retained the M184V mutation and also exhibits a different mutational outcome compared with SD2 controls. In addition, the major impediments to viral replication, i.e., dimerzation, have been determined.
Conclusions: The abolition of select regions of the leader sequence that comprise the DIS stem loop in conjunction with the M184V mutation can synergistically diminish viral fitness, delay reversion kinetics, and affect the mutational outcome.
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