Translation elongation factor-1 alpha interacts with the 3' stem-loop region of West Nile virus genomic RNA.
The conserved 3'-terminal stem-loop (3' SL) of the West Nile virus (WNV) genomic RNA was previously used to probe for cellular proteins that may be involved in flavivirus replication and three cellular proteins were detected that specifically interact with the WNV 3' SL RNA (J. L. Blackwell and M. A. Brinton, J. Virol. 69:5650-5658, 1995). In this study, one of these cellular proteins was purified to apparent homogeneity by ammonium sulfate precipitation and liquid chromatography. Amino acid sequence Western blotting, and supershift analyses identified the cellular protein as translation elongation factor-1 alpha (EF-1 alpha). Competition gel mobility shift assays demonstrated that the interaction between EF-1 alpha and WNV 3' SL RNA was specific. Dephosphorylation of EF-1 alpha by calf intestinal alkaline phosphatase inhibited its binding to WNV 3' SL RNA. The apparent equilibrium dissociation constant for the interaction between EF-1 alpha and WNV 3' SL RNA was calculated to be 1.1 x 10(-9) M. Calculation of the stoichiometry of the interaction indicated that one molecule of EF-1 alpha binds to each molecule of WNV 3' SL RNA. Using RNase footprinting and nitrocellulose filter binding assays, we detected a high-activity binding site on the main stem of the WNV 3' SL RNA. Interaction with EF-1 alpha at the high-activity binding site was sequence specific, since nucleotide substitution in this region reduced the binding activity of the WNV 3' SL RNA for EF-1 alpha by approximately 60%. Two low-activity binding sites were also detected, and each accounted for approximately 15 to 20% of the binding activity. Intracellular association between the host protein and the viral RNA was suggested by coimmunoprecipitation of WNV genomic RNA and EF-1 alpha, using an anti-EF-1 alpha antibody.
Blackwell
J L
JL
Department of Biology, Georgia State University, Atlanta 30302-4010, USA.
Brinton
M A
MA
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002460:Cell Line; D006224:Cricetinae; D016679:Genome, Viral; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D020648:Peptide Elongation Factor 1; D010445:Peptide Elongation Factors / Q000378:metabolism*; D014176:Protein Biosynthesis; D012367:RNA, Viral / Q000737:chemistry / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000378:metabolism*; D012260:Ribonucleases / Q000378:metabolism; D017384:Sequence Deletion; D014764:Viral Proteins / Q000302:isolation & purification / Q000378:metabolism; D014902:West Nile virus / Q000235:genetics*
D020648:Peptide Elongation Factor 1; D010445:Peptide Elongation Factors; D012367:RNA, Viral; D016601:RNA-Binding Proteins; D014764:Viral Proteins; D012260:Ribonucleases