Single Genome Analysis of HIV-1 Rev and RRE Variation in Natural Infections and in Vivo Characterization of NL4-3 Rev Phosphorylation

HIV-1 Rev and RRE sequences from eight patients were obtained by single genome amplification (SGA) of viral RNA extracted from plasma collected at early (0-6 months) and later times (18 mo-15 yrs) post-seroconversion. A maximum likelihood-based analysis was conducted to determine Rev and RRE population diversity and evolution. The activities of selected Rev (10 early; 12 late) and RRE (6 early; 9 late) variants were measured as cognate pairs and individual components in dose-response assays, which measured Rev-RRE function, and in replication experiments. The early Rev and RRE populations in 6/8 patients were characterized by low diversity, most likely from single-variant transmissions. Patient Rev populations were more diverse and underwent more divergence than corresponding RRE populations in 5/8 patients. Patient RRE function was more sensitive to sequence variation than Rev function, and matched or exceeded control RRE activity from the NL4-3 molecular clone in all patients studied. Conversely, Rev variants from 4/5 patients were significantly less active than the control Rev. In three patients, cognate Rev-RRE activities changed significantly between the time points. Importantly, the activities of Rev-RRE cognate pairs could not be predicted using only the activities determined individually with the control NL4-3 partner. The in viva phosphorylation state of NL4-3 Rev was also examined. Mass spectrometry revealed that two serines, S8 and S54, are phosphorylated in 293T cells. Mutations at S8, but not S54, affected Rev-RRE activity in dose-response and replication assays, suggesting that S8 may be an important regulatory residue for Rev function. Together, these studies show that rather than acting as a simple on-off switch, or maintaining a constant level of activity, Rev-RRE activity can fluctuate, presumably to control replication, like a molecular rheostat. The Rev-RRE system should thus be viewed as a dynamic regulatory system with the potential to impact HIV replication and pathogenesis.

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