1887

Abstract

The avian reovirus non-structural protein NS has previously been shown to bind single-stranded (ss) RNA in a sequence-independent manner. The results of the present study further reveal that NS binds poly(A), poly(U) and ssDNA, but not poly(C), poly(G) or duplex nucleic acids, suggesting that NS has some nucleotide-sequence specificity for ssRNA binding. The current findings also show that NS is present in large ribonucleoprotein complexes in the cytoplasm of avian reovirus-infected cells, indicating that it exists in intimate association with ssRNAs . Removal of RNA from the complexes generates a NS protein form that sediments between 4·5 and 7 S, suggesting that RNA-free NS associates into small oligomers. Expression and purification of recombinant 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 NS exists as homodimers and homotrimers; (ii) the minimum RNA size for NS binding is between 10 and 20 nt; (iii) 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 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 NS sequence, suggesting that this protein binds ssRNA through conformational domains. Finally, the properties of the avian reovirus protein NS are compared with those of its mammalian reovirus counterpart.

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2005-04-01
2024-03-29
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