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


RESULTS
All 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.


CONCLUSIONS
Our results suggest that HuR does not impact HIV-1 replication through a direct protein-protein interaction with the viral RT.
Ahn
Jinwoo
J
Department of Structural Biology, Division of Infectious Diseases, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261, USA. jia12@pitt.edu
Byeon
In-Ja L
IJ
Dharmasena
Sanjeewa
S
Huber
Kelly
K
Concel
Jason
J
Gronenborn
Angela M
AM
Sluis-Cremer
Nicolas
N
D000954:Antigens, Surface / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D003001:Cloning, Molecular; D051959:ELAV Proteins; D000067780:ELAV-Like Protein 1; D054303:HIV Reverse Transcriptase / Q000378:metabolism*; D015497:HIV-1 / Q000502:physiology*; D006801:Humans; D019906:Nuclear Magnetic Resonance, Biomolecular; D025941:Protein Interaction Mapping; D055503:Protein Multimerization; D012367:RNA, Viral / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D048348:Reverse Transcription
D000954:Antigens, Surface; D051959:ELAV Proteins; D000067780:ELAV-Like Protein 1; C098770:ELAVL1 protein, human; D012367:RNA, Viral; D016601:RNA-Binding Proteins; D011994:Recombinant Proteins; C514824:reverse transcriptase, Human immunodeficiency virus 1; D054303:HIV Reverse Transcriptase
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