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NIH Research Festival

September 13 – 15, 2017

A single nucleotide change in a functionally undefined region of the RRE impairs HIV replication through an RRE conformational switch

Wednesday, September 13, 2017 – Poster Session II
3:30 – 5:00 p.m.

FAES Terrace

NCI

VIROL-1

Authors

  • C Sherpa
  • P Jackson
  • ML Hammarksjold
  • D Rekosh
  • S LeGrice

Abstract

The HIV-1 Rev Response Element (RRE) is a cis-acting RNA element with multiple stem-loops. Binding and subsequent multimerization of the HIV-1 Rev on the RRE are essential steps in HIV replication. Most of our understanding of the Rev-RRE regulatory axis comes from studies on a few lab-adapted HIV clones. However, from a therapeutic standpoint, in a rapidly evolving virus like HIV, mechanistic studies of naturally occurring Rev and RRE sequences are very critical. A recent study on Rev-RRE variation in serum samples from HIV infected individuals reported that, in one patient, the predominant RRE sequence at around seroconversion (early SC3) and after AIDS onset (late SC3) differed only by 4 isolated nucleotide changes in functionally undefined regions of the RRE. The viral construct with late SC3 was found more active than that containing the early SC3 by a Gag-Pol reporter assay and viral growth assays. Interestingly, the Rev sequence remained unchanged between the two time-points, suggesting RRE variation as the driver of this change in activity. To understand the mechanism behind this functional difference between the two RREs, these RRE RNAs were in-vitro transcribed and their secondary structure determined using CE-SHAPE (capillary electrophoresis based selective 2' -hydroxyl acylation analyzed by primer extension) and SHAPE-MaP (high throughput sequencing based SHAPE-mutational profiling) technologies. The late RRE assumed a canonical 5 stem-loop structure whereas the early RRE formed a structure with differential folding in stem-loops I, IV, and V. To further investigate the contribution of each nucleotide change in RRE folding, early SC3 RRE mutants, carrying different combination of the four signature late RRE nucleotides sequence, were created and their RNA structure determined by SHAPE-MaP. We found that only one nucleotide change (G to C) in the functionally undefined central loop region of the RRE was responsible for the structural switch between the early and late RRE. We also have preliminary data suggesting that the same mutation alone restores the RRE activity of the early RRE to the late RRE level. Thus, using naturally occurring RRE variants, we show for the first time, that a single nucleotide change in the functionally undefined central loop region of the RRE can affect global RRE structure. Studies investigating the effect of these RRE mutations on Rev binding and multimerization are underway.

Scientific Focus Area: Virology

This page was last updated on Friday, March 26, 2021

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Current Research Festival

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