Identification of a short domain within the non-structural protein NS2 of epizootic haemorrhagic disease virus that is important for single strand RNA-binding activity.
The role that a conserved amino acid motif, found in the non-structural protein NS2 of orbiviruses, plays in the interaction of this protein with single stranded (ss) RNA was investigated by mutation analysis of the NS2 of epizootic haemorrhagic disease virus. An NS2 mutant in which this motif (amino acids 75 to 83) was deleted was expressed in Spodoptera frugiperda cells by a recombinant baculovirus and found to be unable to bind to poly(U)-Sepharose. The deletion mutant also differed from wild-type NS2 in that it did not appear to be complexed with ssRNA in cells infected with the baculovirus recombinant. Furthermore, the deletion exerted an adverse effect on the ability of NS2 to form inclusion bodies in the cytoplasm of baculovirus-infected insect cells. To further characterize the role of this motif in RNA-binding, specific residues within the region were substituted by site-directed mutagenesis and the mutants were expressed in Escherichia coli as fusion proteins. Analysis of the different mutant proteins indicated that in each case ssRNA-binding was impaired relative to that of the wild-type NS2 control. The degree of impairment corresponded to the number of amino acid substitutions and the largest effects were associated with non-conserved substitutions. It is suggested that the conserved motif is an important structural determinant in the interaction of NS2 with ssRNA.
Theron
J
J
Department of Microbiology, University of Pretoria, South Africa.
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D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002460:Cell Line; D004279:DNA, Viral / Q000235:genetics; D004926:Escherichia coli; D018164:Hemorrhagic Disease Virus, Epizootic / Q000737:chemistry* / Q000235:genetics / Q000648:ultrastructure; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017924:Nucleopolyhedroviruses / Q000235:genetics / Q000648:ultrastructure; D011072:Poly U / Q000378:metabolism; D011485:Protein Binding; D012313:RNA / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017384:Sequence Deletion; D018411:Spodoptera; D017361:Viral Nonstructural Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D004279:DNA, Viral; C091614:NS2 protein, epizootic hemorrhagic disease virus; D016601:RNA-Binding Proteins; D011993:Recombinant Fusion Proteins; D017361:Viral Nonstructural Proteins; D011072:Poly U; D012313:RNA