Characterization of the nucleic acid-binding activity of the avian reovirus non-structural protein sigma NS.
The avian reovirus non-structural protein sigma NS has previously been shown to bind single-stranded (ss) RNA in vitro in a sequence-independent manner. The results of the present study further reveal that sigma NS binds poly(A), poly(U) and ssDNA, but not poly(C), poly(G) or duplex nucleic acids, suggesting that sigma NS has some nucleotide-sequence specificity for ssRNA binding. The current findings also show that sigma NS is present in large ribonucleoprotein complexes in the cytoplasm of avian reovirus-infected cells, indicating that it exists in intimate association with ssRNAs in vivo. Removal of RNA from the complexes generates a sigma NS protein form that sediments between 4.5 and 7 S, suggesting that RNA-free sigma NS associates into small oligomers. Expression and purification of recombinant sigma NS in insect cells allowed us to generate specific antibodies and to perform a variety of assays. The results of these assays revealed that: (i) RNA-free sigma NS exists as homodimers and homotrimers; (ii) the minimum RNA size for sigma NS binding is between 10 and 20 nt; (iii) sigma NS does not have a preference for viral mRNA sequences; and (iv) its RNA-binding activity is conformation-dependent. Baculovirus expression of point and deletion sigma NS mutants in insect cells showed that the five conserved basic amino acids that are important for RNA binding and ribonucleoprotein-complex formation are dispersed throughout the entire sigma NS sequence, suggesting that this protein binds ssRNA through conformational domains. Finally, the properties of the avian reovirus protein sigma NS are compared with those of its mammalian reovirus counterpart.
Tourís-Otero
Fernando
F
Departamento de Bioquímica y Biología Molecular, Facultad de Farmacia, Universidad de Santiago de Compostela, 15782-Santiago de Compostela, Spain.
Martínez-Costas
José
J
Departamento de Bioquímica y Biología Molecular, Facultad de Farmacia, Universidad de Santiago de Compostela, 15782-Santiago de Compostela, Spain.
Vakharia
Vikram N
VN
Center for Biosystems Research, University of Maryland Biotechnology Institute and VA-MD Regional College of Veterinary Medicine, University of Maryland, College Park, MD 20742, USA.
Benavente
Javier
J
Departamento de Bioquímica y Biología Molecular, Facultad de Farmacia, Universidad de Santiago de Compostela, 15782-Santiago de Compostela, Spain.
D000595:Amino Acid Sequence; D000818:Animals; D002478:Cells, Cultured; D002642:Chick Embryo; D005347:Fibroblasts; D008969:Molecular Sequence Data; D016296:Mutagenesis; D030101:Orthoreovirus, Avian / Q000235:genetics / Q000378:metabolism / Q000472:pathogenicity*; D012367:RNA, Viral / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012261:Ribonucleoproteins / Q000378:metabolism; D018411:Spodoptera; D014764:Viral Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D054334:Viral Regulatory and Accessory Proteins
D012367:RNA, Viral; D011994:Recombinant Proteins; D012261:Ribonucleoproteins; D014764:Viral Proteins; D054334:Viral Regulatory and Accessory Proteins; C031657:sigma NS protein, Reovirus