BHK cell proteins that bind to the 3' stem-loop structure of the West Nile virus genome RNA.
The first 83 3' nucleotides of the genome RNA of the flavivirus West Nile encephalitis virus (WNV) form a stable stem-loop (SL) structure which is followed in the genome by a smaller SL. These 3' structures are highly conserved among divergent flaviviruses, suggesting that they may function as cis-acting signals for RNA replication and as such might specifically bind to cellular or viral proteins. Cellular proteins from uninfected and WNV-infected BHK-21 S100 cytoplasmic extracts formed three distinct complexes with the WNV plus-strand 3' SL [(+)3'SL] RNA in a gel mobility shift assay. Subsequent competitor gel shift analyses showed that two of these RNA-protein complexes, complexes 1 and 2, contained cell proteins that specifically bound to the WNV (+)3'SL RNA. UV-induced cross-linking and Northwestern blotting analyses detected WNV (+)3'SL RNA-binding proteins of 56, 84, and 105 kDa. When the S100 cytoplasmic extracts were partially purified by ion-exchange chromatography, a complex that comigrated with complex 1 was detected in fraction 19, while a complex that comigrated with complex 2 was detected in fraction 17. UV-induced cross-linking experiments indicated that an 84-kDa cell protein in fraction 17 and a 105-kDa protein in fraction 19 bound specifically to the WNV (+)3'SL RNA. In addition to binding to the (+)3'SL RNA, the 105-kDa protein bound to the SL structure located at the 3' end of the WNV minus-strand RNA. Initial mapping studies indicated that the 84- and 105-kDa proteins bind to different regions of the (+)3'SL RNA. The 3'-terminal SL RNA of another flavivirus, dengue virus type 3, specifically competed with the WNV (+)3'SL RNA in gel shift assays, suggesting that the host proteins identified in this study are flavivirus specific.
Blackwell
J L
JL
Department of Biology, Georgia State University, Atlanta 30303, USA.
Brinton
M A
MA
D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D002852:Chromatography, Ion Exchange; D003001:Cloning, Molecular; D006224:Cricetinae; D003593:Cytoplasm / Q000378:metabolism; D017931:DNA Primers; D003716:Dengue Virus / Q000235:genetics; D016679:Genome, Viral; D015151:Immunoblotting; D007668:Kidney; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009690:Nucleic Acid Conformation; D016133:Polymerase Chain Reaction; D015347:RNA Probes; D012367:RNA, Viral / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000302:isolation & purification / Q000378:metabolism*; D014158:Transcription, Genetic; D014902:West Nile virus / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*
D017931:DNA Primers; D015347:RNA Probes; D012367:RNA, Viral; D016601:RNA-Binding Proteins