Deletion and mutational analyses of bluetongue virus NS2 protein indicate that the amino but not the carboxy terminus of the protein is critical for RNA-protein interactions.
Genome segment 8 (S8) of bluetongue virus serotype 10 (BTV-10) encodes the nonstructural protein NS2. This protein, which has single-stranded RNA (ssRNA) binding capacity, is found in BTV-infected cells in the form of virus inclusion bodies (VIBs). To identify the domain(s) important for RNA binding and oligomerization of the protein, a number of deletions were made in regions of the gene that code for either the amino or carboxy terminus of the protein. The modified genes were cloned into and expressed from baculovirus vectors based on Autographa californica nuclear polyhedrosis virus. Truncated NS2 proteins were individually analyzed for the ability to bind ssRNA and to form VIBs. The results indicated that the carboxy terminus of the protein is involved neither in RNA binding nor in the formation of VIBs. The amino terminus of NS2 was shown to be essential for ssRNA binding but not for NS2 protein oligomerization. Point mutations that involved the substitution of various charged residues at the amino terminus of NS2 were generated and tested for the ability to bind ssRNA. The results showed that the arginines at amino acid residues 6 and 7 and the lysine at residue 4, but not the glutamic acid at residue 2, are involved in ssRNA binding.
Zhao
Y
Y
Department of Biochemistry, University of Oxford, United Kingdom.
Thomas
C
C
Bremer
C
C
Roy
P
P
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001820:Bluetongue virus / Q000235:genetics* / Q000378:metabolism; D002478:Cells, Cultured; D004252:DNA Mutational Analysis; D007181:Inclusion Bodies, Viral / Q000378:metabolism / Q000648:ultrastructure; D008969:Molecular Sequence Data; D009036:Moths / Q000166:cytology; D017924:Nucleopolyhedroviruses / Q000235:genetics; D017354:Point Mutation; D011485:Protein Binding; D015347:RNA Probes; D012367:RNA, Viral / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D017384:Sequence Deletion; D013329:Structure-Activity Relationship; D017361:Viral Nonstructural Proteins / Q000235:genetics* / Q000378:metabolism*
D015347:RNA Probes; D012367:RNA, Viral; D011994:Recombinant Proteins; D017361:Viral Nonstructural Proteins