The RNA binding protein HuR does not interact directly with HIV-1 reverse transcriptase and does not affect reverse transcription in vitro

Jinwoo Ahn,Kelly Huber,In-Ja L Byeon,Sanjeewa Dharmasena,Nicolas Sluis-Cremer,Angela M Gronenborn,Jason Concel

doi:10.1186/1742-4690-7-40

Jinwoo Ahn, Kelly Huber + Show 5 more

Open Access

https://doi.org/10.1186/1742-4690-7-40

Copy DOI

Journal: Retrovirology	Publication Date: May 7, 2010
Citations: 39	License type: cc-by

Affiliation: University of Pittsburgh

Abstract

BackgroundLemay et al recently reported that the RNA binding protein HuR directly interacts with the ribonuclease H (RNase H) domain of HIV-1 reverse transcriptase (RT) and influences the efficiency of viral reverse transcription (Lemay et al., 2008, Retrovirology 5:47). HuR is a member of the embryonic lethal abnormal vision protein family and contains 3 RNA recognition motifs (RRMs) that bind AU-rich elements (AREs). To define the structural determinants of the HuR-RT interaction and to elucidate the mechanism(s) by which HuR influences HIV-1 reverse transcription activity in vitro, we cloned and purified full-length HuR as well as three additional protein constructs that contained the N-terminal and internal RRMs, the internal and C-terminal RRMs, or the C-terminal RRM only.ResultsAll four HuR proteins were purified and characterized by biophysical methods. They are well structured and exist as monomers in solution. No direct protein-protein interaction between HuR and HIV-1 RT was detected using NMR titrations with 15N labeled HuR variants or the 15N labeled RNase H domain of HIV-1 RT. Furthermore, HuR did not significantly affect the kinetics of HIV-1 reverse transcription in vitro, even on RNA templates that contain AREs.ConclusionsOur results suggest that HuR does not impact HIV-1 replication through a direct protein-protein interaction with the viral RT.

Highlights

Lemay et al recently reported that the RNA binding protein HuR directly interacts with the ribonuclease H (RNase H) domain of HIV-1 reverse transcriptase (RT) and influences the efficiency of viral reverse transcription (Lemay et al, 2008, Retrovirology 5:47)
The viral proteins nucleocapsid, matrix, integrase, tat, nef and vif may participate in the regulation and/or efficiency of reverse transcription [1,2,3,4,5,6], synthesis of the nascent HIV-1 DNA is entirely carried-out by the DNA polymerase and ribonuclease H (RNase H) activities of HIV-1 reverse transcriptase (RT)
Purification and characterization of HuR HuR belongs to the Hu family of mRNA stabilizing proteins that interact with AU-rich elements (ARE), sharing significant sequence similarity with the Drosophila RNAbinding protein ELAV [13]

Summary

Introduction

Lemay et al recently reported that the RNA binding protein HuR directly interacts with the ribonuclease H (RNase H) domain of HIV-1 reverse transcriptase (RT) and influences the efficiency of viral reverse transcription (Lemay et al, 2008, Retrovirology 5:47). The viral proteins nucleocapsid, matrix, integrase, tat, nef and vif may participate in the regulation and/or efficiency of reverse transcription [1,2,3,4,5,6], synthesis of the nascent HIV-1 DNA is entirely carried-out by the DNA polymerase and ribonuclease H (RNase H) activities of HIV-1 reverse transcriptase (RT). The p66/p51 HIV-1 RT heterodimer contains one DNA polymerization active site and one RNase H active site, which both reside in the p66 subunit in spatially distinct regions [7]

Methods

Results

Conclusion