Cellular proteins from human monocytes bind to dengue 4 virus minus-strand 3' untranslated region RNA.
The synthesis of plus and minus RNA strands of several RNA viruses requires as a first step the interaction of some viral regulatory sequences with cellular and viral proteins. The dengue 4 virus genome, a single-stranded, positive-polarity RNA, is flanked by two untranslated regions (UTR) located in the 5' and 3' ends. The 3'UTR in the minus-strand RNA [3'UTR (-)] has been thought to function as a promoter for the synthesis of plus-strand RNA. To study the initial interaction between this 3'UTR and cellular and viral proteins, mobility shift assays were performed, and four ribonucleoprotein complexes (I through IV) were formed when uninfected and infected U937 cells (human monocyte cell line) interacted with the 3'UTR (-) of dengue 4 virus. Cross-linking assays with RNAs containing the complete 3'UTR (-) (nucleotides [nt] 101 to 1) or a partial sequence from nt 101 to 45 and nt 44 to 1 resulted in specific binding of some cellular proteins. Supermobility shift and immunoprecipitation assays demonstrated that the La protein forms part of these complexes. To determine the region in the 3' UTR that interacted with the La protein, two deletion mutants were generated. The mutant (del-96), with a deletion of nt 96 to 101, was unable to interact with the La protein, suggesting that La interacted with the 5' portion of the 3'UTR (-). Complex I, which was the main ribonucleoprotein complex formed with the 3'UTR (-) and which had the fastest electrophoretic migration, contained proteins such as calreticulin and protein disulfide isomerase, which constitute important components of the endoplasmic reticulum.
Yocupicio-Monroy
Rosa Martha E
RM
Departamento de Patología Experimental, Centro de Investigación y de Estudios Avanzados del IPN, México City 07360, México.
Medina
Fernando
F
Reyes-del Valle
Jorge
J
del Angel
Rosa M
RM
D020413:3' Untranslated Regions / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001324:Autoantigens; D001483:Base Sequence; D001665:Binding Sites; D015153:Blotting, Western; D037282:Calreticulin / Q000378:metabolism*; D003432:Cross-Linking Reagents; D003716:Dengue Virus / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D019704:Protein Disulfide-Isomerases / Q000378:metabolism; D012367:RNA, Viral / Q000378:metabolism; D012261:Ribonucleoproteins / Q000378:metabolism*; D020298:U937 Cells; D000097585:SS-B Antigen
D020413:3' Untranslated Regions; D001324:Autoantigens; D037282:Calreticulin; D003432:Cross-Linking Reagents; D019704:Protein Disulfide-Isomerases; D012367:RNA, Viral; D012261:Ribonucleoproteins; D000097585:SS-B Antigen