Characterization of the nucleic acid-binding activity of the avian reovirus non-structural protein σNS

Publisher Website
Google Scholar
Abstract
The avian reovirus non-structural proteinσNS has previously been shown to bind single-stranded (ss) RNAin vitroin a sequence-independent manner. The results of the present study further reveal thatσNS binds poly(A), poly(U) and ssDNA, but not poly(C), poly(G) or duplex nucleic acids, suggesting thatσNS has some nucleotide-sequence specificity for ssRNA binding. The current findings also show thatσNS is present in large ribonucleoprotein complexes in the cytoplasm of avian reovirus-infected cells, indicating that it exists in intimate association with ssRNAsin vivo. Removal of RNA from the complexes generates aσNS protein form that sediments between 4·5 and 7 S, suggesting that RNA-freeσNS associates into small oligomers. Expression and purification of recombinantσNS 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-freeσNS exists as homodimers and homotrimers; (ii) the minimum RNA size forσNS binding is between 10 and 20 nt; (iii)σNS does not have a preference for viral mRNA sequences; and (iv) its RNA-binding activity is conformation-dependent. Baculovirus expression of point and deletionσNS 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 entireσNS sequence, suggesting that this protein binds ssRNA through conformational domains. Finally, the properties of the avian reovirus proteinσNS are compared with those of its mammalian reovirus counterpart.