Open AccessDisulfide Linkages Mediating Nucleocapsid Protein Dimerization Are Not Required for Porcine Arterivirus Infectivity
Author(s) -
Rong Zhang,
Chunyan Chen,
Zhoutong Sun,
Feiquan Tan,
Jing Zhuang,
Debin Tian,
Guangzhi Tong,
Sheng Yuan
Publication year - 2012
Publication title -
journal of virology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 2.617
H-Index - 292
eISSN - 1070-6321
pISSN - 0022-538X
DOI - 10.1128/jvi.06709-11
Subject(s) - biology , porcine reproductive and respiratory syndrome virus , arterivirus , infectivity , cysteine , virology , nidovirales , complementation , wild type , alanine , nucleoprotein , genetics , amino acid , microbiology and biotechnology , virus , biochemistry , gene , mutant , medicine , disease , covid-19 , pathology , infectious disease (medical specialty) , enzyme
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 agentN -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.