title: Binding of the eukaryotic translation elongation factor 1A with the 5'UTR of HIV-1 genomic RNA is important for reverse transcription.
abstract: 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.
  
  
  CONCLUSIONS
  eEF1A interacts with 5'UTR of HIV-1 genomic RNA and the interaction is important for late DNA synthesis in reverse transcription.
authors.affiliation: Department of Cell and Molecular Biology, QIMR Berghofer Medical Research Institute, Herston, QLD, 4029, Australia.
authors.forename: Dongsheng
authors.initials: D
authors.lastname: Li
authors.affiliation: Department of Cell and Molecular Biology, QIMR Berghofer Medical Research Institute, Herston, QLD, 4029, Australia.
authors.forename: Ting
authors.initials: T
authors.lastname: Wei
authors.affiliation: Department of Cell and Molecular Biology, QIMR Berghofer Medical Research Institute, Herston, QLD, 4029, Australia.
authors.forename: Hongping
authors.initials: H
authors.lastname: Jin
authors.affiliation: Department of Cell and Molecular Biology, QIMR Berghofer Medical Research Institute, Herston, QLD, 4029, Australia.
authors.forename: Amanda
authors.initials: A
authors.lastname: Rose
authors.affiliation: Department of Cell and Molecular Biology, QIMR Berghofer Medical Research Institute, Herston, QLD, 4029, Australia.|Present address: Centre of Infectious Diseases, Beijing Youan Hospital, Capital Medical University, No. 8 Xitoutiao Youanmenwai, Fengtai District, Beijing, 100069, China.
authors.forename: Rui
authors.initials: R
authors.lastname: Wang
authors.affiliation: Department of Cell and Molecular Biology, QIMR Berghofer Medical Research Institute, Herston, QLD, 4029, Australia.
authors.forename: Min-Hsuan
authors.initials: MH
authors.lastname: Lin
authors.affiliation: School of Biomedical Science, Queensland University of Technology, Brisbane, QLD, 4001, Australia.
authors.forename: Kirsten
authors.initials: K
authors.lastname: Spann
authors.affiliation: Department of Cell and Molecular Biology, QIMR Berghofer Medical Research Institute, Herston, QLD, 4029, Australia. david.harrich@qimrberghofer.edu.au.
authors.forename: David
authors.initials: D
authors.lastname: Harrich
mesh_terms: D020121:5' Untranslated Regions; D002460:Cell Line; D016679:Genome, Viral; D015497:HIV-1 / Q000235:genetics*; D006801:Humans; D055029:Inverted Repeat Sequences; D009154:Mutation; D009690:Nucleic Acid Conformation; D020648:Peptide Elongation Factor 1 / Q000378:metabolism*; D011485:Protein Binding; D012367:RNA, Viral / Q000737:chemistry / Q000235:genetics; D048348:Reverse Transcription
chemical_list: D020121:5' Untranslated Regions; C094814:EEF1A1 protein, human; D020648:Peptide Elongation Factor 1; D012367:RNA, Viral