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89 RNA Packaging and Gag Processing in SIV Require Multiple Interactions between Gag and Leader Sequences
Y. Guan*, C. Liang, and M. A. Wainberg
McGill Univ. AIDS Ctr., Lady Davis Inst.-Jewish Gen. Hosp., Montreal, QC, Canada
Background: Studies on the leader sequences of HIV-1 and SIV showed that the sequences between the primer binding site (PBS) and the major splice donor site are important for viral replication due to their multiple roles in RNA packaging, Gag processing, reverse transcription, etc. Deletions of selected sequences in this region resulted in severely attenuated viruses that retained all viral proteins. Therefore, this might represent a novel concept of live viral attenuation vaccine research. We have performed mechanistic studies with SIV to pursue this hypothesis.
Methods: Using the SIVmac239 clone, we generated a series of SIV constructs containing deletions in leader sequences downstream of the PBS. Replication capacity was investigated in CEMx174 cells and Monkey PBMCs. RNA packaging was assessed by quantitative RT-PCR. Gag processing was evaluated by radio-labeling and immunoprecipitation. A “forced evolution” strategy was used to investigate the mechanisms whereby these deletions impair viral replication. The relationships between the RNA structure of the leader sequences and their functions were analyzed.
Results: Deletions in sequences downstream of the PBS severely impaired replication of SIV, due to effects on RNA packaging and Gag processing. Several mutants restored replication capacity to levels similar to those of wild-type virus after forced evolution due to compensatory mutations located in the leader sequences and/or in the Gag region. RNA structural analysis and functional studies demonstrated that an intact U5-leader stem RNA structure was important for RNA packaging. Compensatory mutations in both leader sequences and the Gag region were required to rescue a deletion in the leader sequence, due to their roles in both RNA packaging and Gag processing. A series of these live-attenuated SIV constructs, ranging in replication ability from low to moderate degrees of attenuation, were generated.
Conclusions: Both RNA packaging and Gag processing require multiple interactions between Gag and the leader sequences that must exist within a context of proper structure. Deletions in leader sequences might be a novel way to generate a live-attenuated vaccine for AIDS.
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