Colocalization and interaction of the porcine arterivirus nucleocapsid protein with the small nucleolar RNA-associated protein fibrillarin.
Porcine reproductive and respiratory syndrome virus (PRRSV) replicates in the cytoplasm of infected cells, but its nucleocapsid (N) protein localizes specifically to the nucleus and nucleolus. The mechanism of nuclear translocation and whether N associates with particular nucleolar components are unknown. In the present study, we show by confocal microscopy that the PRRSV N protein colocalizes with the small nucleolar RNA (snoRNA)-associated protein fibrillarin. Direct and specific interaction of N with fibrillarin was demonstrated in vivo by the mammalian two-hybrid assay in cells cotransfected with the N and fibrillarin genes and in vitro by the glutathione S-transferase pull-down assay using the expressed fibrillarin protein. Using a series of deletion mutants, the interactive domain of N with fibrillarin was mapped to a region of amino acids 30 to 37. For fibrillarin, the first 80 amino acids, which contain the glycine-arginine-rich region (the GAR domain), was determined to be the domain interactive with N. The N protein was able to bind to the full-length genomic RNA of PRRSV, and the RNA binding domain was identified as the region overlapping with the nuclear localization signal situated at positions 41 to 47. These results suggest that the N protein nuclear transport may be controlled by the binding of RNA to N. The PRRSV N protein was also able to bind to both 28S and 18S ribosomal RNAs. The protein-protein interaction between N and fibrillarin was RNA dependent but independent of N protein phosphorylation. Taken together, our studies demonstrate a specific interaction of the PRRSV nucleocapsid protein with the host cell protein fibrillarin in the nucleolus, and they imply a potential linkage of viral strategies for the modulation of host cell functions, possibly through rRNA precursor processing and ribosome biogenesis.
Yoo
Dongwan
D
Department of Pathobiology, Ontario Veterinary College, University of Guelph, Guelph, Ontario N1G 2W1, Canada. dyoo@uoguelph.ca
Wootton
Sarah K
SK
Li
Gang
G
Song
Cheng
C
Rowland
Raymond R
RR
D000818:Animals; D002460:Cell Line; D002466:Cell Nucleolus / Q000737:chemistry*; D002868:Chromosomal Proteins, Non-Histone / Q000032:analysis* / Q000378:metabolism; D006801:Humans; D019590:Nucleocapsid Proteins / Q000032:analysis* / Q000378:metabolism; D019316:Porcine respiratory and reproductive syndrome virus / Q000737:chemistry* / Q000472:pathogenicity; D012335:RNA, Ribosomal / Q000378:metabolism; D012367:RNA, Viral / Q000378:metabolism; D011817:Rabbits; D013552:Swine
D002868:Chromosomal Proteins, Non-Histone; D019590:Nucleocapsid Proteins; D012335:RNA, Ribosomal; D012367:RNA, Viral; C047458:fibrillarin