Item Details

Print View

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

Sloan, Emily A
Thesis/Dissertation; Online
Sloan, Emily A
Rekosh, David
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. Note: Abstract extracted from PDF file via OCR
University of Virginia, Department of Microbiology, Immunology, and Cancer Biology, PHD, 2012
Published Date
All rights reserved (no additional license for public reuse)
Libra ETD Repository


Read Online