Disulfide Linkages Mediating Nucleocapsid Protein Dimerization Are Not Required for Porcine Arterivirus Infectivity

Abstract

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. P orcine reproductive and respiratory syndrome virus (PRRSV)is an enveloped, single-stranded, positive-sense RNA virus belonging to the family Arteriviridae in the order Nidovirales, which also includes the families Coronaviridae and Roniviridae (7,8,16,47). Other members of the family Arteriviridae include equine arteritis virus (EAV), lactate dehydrogenase-elevating virus (LDV), and simian hemorrhagic fever virus (SHFV) (47). The PRRSV genome is about 15 kb in length, and the coding region is flanked by untranslated regions (UTRs) at each end (the 5= and 3=UTR, respectively) (36). The genome contains at least 10 open reading frames (ORFs), of which ORF1a and ORF1b encode the two long nonstructural polyproteins pp1a and pp1ab and ORF2 to ORF7 encode at least eight structural proteins, glycoprotein 2 (GP2), small envelope (E), GP3, GP4, GP5, ORF5a, membrane (M), and nucleocapsid (N) (13,25,35,36,47,56,61). The structural proteins are expressed from a set of coterminal subgenomic mRNAs (sgmRNAs), which are synthesized by a unique discontinuous transcription strategy (40). The transcription-regulating sequence (TRS) of the leader (5=UTR) and the downstream body TRS (TRS-B) preceding the ORF coding region of each structural protein in the viral genome are believed to play key roles in mediating this process.Based on genetic and antigenic differences, PRRSV strains are classified into two distinct genotypes, North American (t...

The Nsp12-coding region of type 2 PRRSV is required for viral subgenomic mRNA synthesis

“…(Roche). Dimer detection was performed as previously described [22]. Proteins in the cell lysates were separated by SDS-PAGE, transferred to PVDF membranes (Millipore, Germany) and probed with the indicated antibodies.…”

“…Cell supernatants were collected and clarified at 5,000 × g for 1 h at 4°C to remove nonadherent cells and cellular debris. The supernatants were collected and pelleted twice through a 30% sucrose cushion as previously reported [22]; the purified virions were applied to 10% to 35% sucrose cushions and centrifuged. Typically, 12 fractions were collected from the top to the bottom and analyzed by Western blot.…”

Cysteine residues of the porcine reproductive and respiratory syndrome virus ORF5a protein are not essential for virus viability

“…The BiFC assays were conducted as previously described with minor modifications (Zhang et al, 2012). The coding region for the individual structural proteins were amplified by PCR using gene-specific primers (Table 1) …”

“…To explore the status of the interaction between ORF5a proteins in cultured cells, we employed a newly developed technique, known as bimolecular fluorescence complementation (BiFC), which allow for the visualization and localization of specific protein-protein interactions within living cells (Hu and Kerppola, 2003;Zhang et al, 2012) The fluorescence produced by these BiFC pairs demonstrated that there is an intermolecular interaction between ORF5a proteins regardless of the absence of cysteines, while the mutation of Cys30 to glycine affected the interaction between ORF5a proteins. A further study was conducted to examine whether the ORF5a protein forms a cysteine-linked homodimerization.…”

“…Type 2 PRRSV N proteins contain three highly conserved cysteine residues at amino acid positions 23, 75 and 90. By mutational analysis using the expressed protein and an infectious PRRSV cDNA clone, the cysteine at position 23 has been shown to be capable of forming N-N disulfide linkages (Wootton and Yoo, 2003), but the disulfide linkages mediating the N dimerization are not required for PRRSV viability (Zhang et al, 2012). GP5 and M proteins form a disulfide linked heterodimer, which is an essential requirement for infectivity in LDV and EAV (Faaberg et al, 1995;Snijder et al, 2003).…”