Published in:
Open Access 01-12-2015 | research
Binding of the eukaryotic translation elongation factor 1A with the 5’UTR of HIV-1 genomic RNA is important for reverse transcription
Published in: Virology Journal | Issue 1/2015
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Background
The cellular protein eukaryotic translation elongation factor 1A (eEF1A) binds to aminoacylated transfer RNAs and delivers them to the ribosome during translation. eEF1A also binds to RNA secondary structures present in genomes of several RNA viruses and plays important roles in their replication. As a RNA binding protein, whether eEF1A can bind with HIV-1 genomic RNA has not been investigated and was the aim of the study.
Methods
RNA-protein interaction was determined by reversible crosslink co-immunoprecipitation (RC-Co-IP) and biolayer Interferometry assay (BLI). eEF1A binding region within RNA was mapped by deletion and mutation analysis. Virus with genomic RNA mutations were examined for eEF1A-RT interaction by proximity ligation assay, for reverse transcription by qPCR and for replication by CAp24 ELISA in cells.
Results
The interaction of eEF1A with 5’UTR of HIV-1 genomic RNA was detected in cells and in vitro. Truncation and substitution mutations in the 5’UTR RNA demonstrated that a stem-loop formed by nucleotides 142 to 170, which encompass a reported tRNA anticodon-like-element, binds to eEF1A. Mutations that altered the stem-loop structure by changing two highly conserved sequence clusters in the stem-loop region result in reduction of the interaction with eEF1A in vitro. HIV-1 virus harbouring the same 5’UTR mutations significantly reduced the interaction of eEF1A with HIV-1 reverse transcription complex (RTC), reverse transcription and replication.
Conclusion
eEF1A interacts with 5’UTR of HIV-1 genomic RNA and the interaction is important for late DNA synthesis in reverse transcription.