HIV replication is a highly regulated process, and binding of the viral Rev protein to the Rev Response Element (RRE) is essential for the nucleocytoplasmic export of mRNAs that retain introns. Previous studies have shown that there is substantial variation in Rev and RRE sequences. We previously observed activity variation of the Rev/RRE system in subtype B viruses isolated from patients. In the present study, we examine Rev/RRE functional variation in different HIV subtypes and seek to determine driving factors behind that variation.
A functional assay was developed which utilizes an HIV vector system that packages HIV vector RNA into particles in a Rev/RRE-dependent fashion. The virus that is produced transduces hygromycin resistance to target cells, allowing Rev/RRE activity to be read-out by counting resistant colonies. Rev and RRE sequences can be tested in this system to measure the function of naturally occurring or artificial Rev-RRE pairs. Twelve sequences were selected representing two subtype A, four AG, two G, and four B isolates. Variation in Rev levels was assayed by Western blots.
There was a 24-fold difference in functional activity between the most and least active naturally occurring Rev/RRE combinations. In a linear regression, there was low correlation of RRE activity (R²=0.32) but high correlation of Rev activity (R²=0.95) with the functional activity of the original cognate pair. There was no substantial correlation between levels of Rev and either cognate pair (R²=0.11) or Rev/NL4-3 RRE functional activity (R²=0.12.)
Rev/RRE functional activity can vary dramatically between naturally occurring cognate pairs. This functional variation is driven by differences in Rev sequence. Taken together, our results demonstrate a substantial variation in the Rev/RRE activity of circulating viruses that cannot explained by differences in steady-state Rev protein levels. This variation may play a role in viral fitness and replication dynamics and may be reflective of immune pressure. Identification of the most efficient Rev/RRE combination would also likely be of use in optimizing lentiviral vector systems for gene therapy.
D. Rekosh, None
M. L. Hammarskjold, None