Porcine respiratory and reproductive syndrome virus nucleocapsid protein and RNA interactions

To characterize the RNA-binding regions of PRRSV (porcine reproductive and respiratory syndrome virus) nucleocapsid (N) protein, the interactions between deletion constructs of N protein with the 5′ leader RNAs were studied by northwestern blot and gel mobility shift assays. Deletion constructs of N protein differing by approximately 20 amino acid residues were generated by polymerase chain reaction, and expressed as histidine tagged fusion proteins in E. coli. RNA probes generated by in vitro transcription were used to localize the RNA domain that is essential for the binding. The domain of PRRSV N protein (amino acid residues 34 to 53) that is involved in specific RNA-binding activity was localized and identified the regions of PRRSV leader RNAs participating in protein binding to the first 91 nucleotides (nt) of 5′ (+) leader RNA. The in vitro interactions between the N protein and leader RNAs were further demonstrated in vivo by immunoprecipitaion-RNA RT-PCR method. Two-dimensional proton NMR spectroscopy was used to characterize the secondary structure of the N protein RNA-binding peptide. NMR chemical shift index values and NOE connectivities indicate that the PRRSV N protein RNA binding domain forms an α-helix in solution. The formation of α-helices is a common property of RNA-binding domains rich in basic amino acids, particularly lysine and arginine rich domains.