Disulfide linkages mediating nucleocapsid protein dimerization are not required for porcine arterivirus infectivity.
The nucleocapsid (N) proteins of the North American (type II) and European (type I) genotypes of porcine reproductive and respiratory syndrome virus (PRRSV) share only approximately 60% genetic identity, and the functionality of N in both genotypes, especially its role in virion assembly, is still poorly understood. In this study, we demonstrated that the ORF7 3' untranslated region or ORF7 of type I is functional in the type II PRRSV background. Based on these results, we postulated that the cysteine at position 90 (Cys90) of the type II N protein, which corresponds to an alanine in the type I protein, is nonessential for virus infectivity. The replacement of Cys90 with alanine confirmed this hypothesis. We then hypothesized that all of the cysteines in the N protein could be replaced by alanines. Mutational analysis revealed that, in contradiction to previously reported findings, the replacement of all of the cysteines, either singly or in combination, did not impair the growth of either type II or type I PRRSV. Treatment with the alkylating agent N-ethylmaleimide inhibited cysteine-mediated N dimerization in living cells but not in released virions. Additionally, bimolecular fluorescence complementation assays revealed noncovalent interactions in living cells among the N and C termini and between the N-terminal and C-terminal regions of the N proteins of both genotypes of PRRSV. These results demonstrate that the disulfide linkages mediating the N dimerization are not required for PRRSV viability and help to promote our understanding of the mechanism underlying arterivirus particle assembly.
Department of Swine Infectious Diseases, Shanghai Veterinary Research Institute, The Chinese Academy of Agricultural Sciences, Shanghai 200241, China.
Rong
R
Zhang
Chunyan
C
Chen
Zhi
Z
Sun
Feifei
F
Tan
Jinshan
J
Zhuang
Debin
D
Tian
Guangzhi
G
Tong
Shishan
S
Yuan
D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D003545:Cysteine / Q000737:chemistry / Q000378:metabolism; D005033:Ethylmaleimide / Q000494:pharmacology; D023061:Gene Order; D008969:Molecular Sequence Data; D009154:Mutation; D019590:Nucleocapsid Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D019316:Porcine respiratory and reproductive syndrome virus / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D055503:Protein Multimerization / Q000187:drug effects; D016415:Sequence Alignment; D014771:Virion / Q000187:drug effects / Q000378:metabolism
D019590:Nucleocapsid Proteins; D003545:Cysteine; D005033:Ethylmaleimide