Binding of Epstein-Barr virus small RNA EBER-1 to the double-stranded RNA-activated protein kinase DAI.
Epstein-Barr virus encodes two small RNAs, EBER-1 and -2, that are abundantly expressed in latently infected cells. Recent evidence suggests a role for EBER-1 in regulation of translation since this RNA is able to prevent the inhibition of protein synthesis by double-stranded RNA in rabbit reticulocyte lysates. We show here that EBER-1 that has been synthesized in vitro forms a complex with the dsRNA-activated inhibitor of protein synthesis DAI, a protein kinase that specifically phosphorylates polypeptide chain initiation factor eIF-2. Gel retardation assays and UV crosslinking experiments indicate that complex formation is specific for EBER-1 and requires the presence of some secondary structure in the molecule. RNA competition studies show that EBER-1-DAI complex formation is not inhibited in the presence of other small RNA species, heparin or the synthetic double-stranded RNA, poly(I).poly(C). SDS gel analysis reveals the existence of two forms of the crosslinked complex, of 64-68kDa and 46-53kDa, both of which are recognized by anti-DAI antibodies in immunoprecipitation experiments. These data suggest that EBER-1 regulates protein synthesis through its ability to interact with DAI.
Clarke
P A
PA
Department of Cellular and Molecular Sciences, St George's Hospital Medical School, London, UK.
Schwemmle
M
M
Schickinger
J
J
Hilse
K
K
Clemens
M J
MJ
D015153:Blotting, Western; D003432:Cross-Linking Reagents; D004591:Electrophoresis, Polyacrylamide Gel; D004854:Herpesvirus 4, Human / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D010957:Plasmids; D011233:Precipitin Tests; D014176:Protein Biosynthesis; D011494:Protein Kinases / Q000378:metabolism*; D012320:RNA Polymerase III / Q000235:genetics; D012330:RNA, Double-Stranded / Q000235:genetics / Q000378:metabolism*; D012367:RNA, Viral / Q000235:genetics / Q000378:metabolism*; D014466:Ultraviolet Rays; D019892:eIF-2 Kinase
D003432:Cross-Linking Reagents; D012330:RNA, Double-Stranded; D012367:RNA, Viral; D011494:Protein Kinases; D019892:eIF-2 Kinase; D012320:RNA Polymerase III