Correction: Porcine Reproductive and Respiratory Syndrome Virus Nonstructural Protein 4 Induces Apoptosis Dependent on Its 3C-Like Serine Protease Activity.

[This corrects the article DOI: 10.1371/journal.pone.0069387.].

Porcine reproductive and respiratory syndrome virus nonstructural protein 4 antagonizes beta interferon expression by targeting the NF-κB essential modulator.

UNLABELLED: Porcine reproductive and respiratory syndrome virus (PRRSV) is a highly infectious pathogen that causes severe diseases in pigs and great economic losses to the swine industry worldwide. Type I interferons (IFNs) play a crucial role in antiviral immunity. In the present study, we demonstrated that infection with the highly pathogenic PRRSV strain JXwn06 antagonized type I IFN expression induced by poly(I·C) in both porcine alveolar macrophages (PAMs) and blood monocyte-derived macrophages (BMo). Subsequently, we showed that the inhibition of poly(I·C)-induced IFN-ß production by PRRSV was dependent on the blocking of NF-κB signaling pathways. By screening PRRSV nonstructural and structural proteins, we demonstrated that nonstructural protein 4 (nsp4), a viral 3C-like serine protease, significantly suppressed IFN-ß expression. Moreover, we verified that nsp4 inhibited NF-κB activation induced by signaling molecules, including RIG-I, VISA, TRIF, and IKKß. nsp4 was shown to target the NF-κB essential modulator (NEMO) at the E349-S350 site to mediate its cleavage. Importantly, nsp4 mutants with defective protease activity abolished its ability to cleave NEMO and inhibit IFN-ß production. These findings might have implications for our understanding of PRRSV pathogenesis and its mechanisms for evading the host immune response. IMPORTANCE: Porcine reproductive and respiratory syndrome virus (PRRSV) is a major agent of respiratory diseases in pigs. Like many other viruses, PRRSV has evolved a variety of strategies to evade host antiviral innate immunity for survival and propagation. In this study, we show that PRRSV nsp4 is a novel antagonist of the NF-κB signaling pathway, which is responsible for regulating the expression of type I interferons and other crucial cytokines. We then investigated the underlying mechanism used by nsp4 to suppress NF-κB-mediated IFN-ß production. We found that nsp4 interfered with the NF-κB signaling pathway through the cleavage of NEMO (a key regulator of NF-κB signaling) at the E349-S350 site, leading to the downregulation of IFN-ß production induced by poly(I·C). The data presented here may help us to better understand PRRSV pathogenesis.

Porcine reproductive and respiratory syndrome virus nonstructural protein 4 induces apoptosis dependent on its 3C-like serine protease activity.

Porcine reproductive and respiratory syndrome (PRRS) is a highly contagious disease in pigs caused by PRRS virus (PRRSV). Although PRRSV infection-induced cell apoptosis has been established, the related viral protein is still unknown. Here, we reported that PRRSV nonstructural protein 4 (nsp4) was a critical apoptosis inducer. Nsp4 could activate caspase-3, -8, and -9. Using truncated constructs without different domains in nsp4, we demonstrated that the full-length of nsp4 structure was required for its apoptosis-inducing activity. Furthermore, using site-directed mutagenesis to inactivate the 3C-like serine protease activity of nsp4, we showed that nsp4-induced apoptosis was dependent on its serine protease activity. The ability of nsp4 to induce apoptosis was significantly impaired by His39, Asp64, and Ser118 mutations, suggesting that His39, Asp64, and Ser118 were essential for nsp4 to trigger apoptosis. In conclusion, our present work showed that PRRSV nsp4 could induce apoptosis in host cells and might be partially responsible for the apoptosis induced by PRRSV infection. PRRSV 3C-like protease-mediated apoptosis represents the first report in the genus Arterivirus, family Arteriviridae.

A novel dendrimeric peptide induces high level neutralizing antibodies against classical swine fever virus in rabbits.

The amino acid sequence (TAVSPTTLR, 829-837aa) on the glycoprotein E2 of classical swine fever virus (CSFV) is a conserved and linear neutralizing epitope. In the present study, two peptides were constructed based the core sequence of this neutralizing epitope, the dendrimeric peptide (Th-B(4)) containing four copies of B cell epitope fused to one copy of promiscuous T helper (Th) cell epitope and the peptide Th-B containing a single copy of B cell epitope fused to one copy of Th cell epitope. The dendrimeric peptide Th-B(4) elicited high titers of neutralizing antibodies as detected in an indirect ELISA, blocking ELISA and neutralization test and induced a complete protection against CSFV C strain in rabbits. The Th-B elicited low titers of neutralizing antibodies and did not induce a protection in rabbits. These results suggest that the dendrimeric peptide Th-B(4) may be a promising marker vaccine candidate against CSFV and the multimerization is a requirement for development of a peptide vaccine.

Identification of immunodominant T-cell epitopes in membrane protein of highly pathogenic porcine reproductive and respiratory syndrome virus.

The development of cell-mediated immunity has been known extremely important in clearing porcine reproductive and respiratory syndrome virus (PRRSV) in infected pigs. However, the PRRS immunology regarding the interaction of T-cells and PRRSV proteins is poorly understood. To identify the T-cell immunodominant epitopes on the membrane (M) protein of PRRSV, a series of 31 overlapping pentadecapeptides covering the entire M protein were designed and synthesized. These peptides were screened by ELIspot analysis for their capabilities to elicit interferon-gamma (IFN-γ) responses in the peripheral blood mononuclear cells (PBMCs), which were collected from pigs immunized with attenuated PRRSV HuN4-F112 strain and challenged with highly pathogenic HuN4 strain. After three rounds of screening, 4 peptides (M3, M6, M8 and M12) were shown to elicit high expression of IFN-γ. The stimulation of high IFN-γ transcription in PBMCs by these 4 peptides was further confirmed in real-time PCR. The sequence alignment revealed that the epitope represented by peptide M6 was fully conserved in all of examined 42 North American genotype II PRRSV isolates and the epitopes represented by peptides M3, M8 and M12 showed 2-4 amino acid replacements. The finding of 4 T-cell immunodominant epitopes in the M protein of PRRSV will be beneficial to the understanding of the development of cell-mediated immunity.

Characterization of the biochemical properties and identification of amino acids forming the catalytic center of 3C-like proteinase of porcine reproductive and respiratory syndrome virus.

PURPOSE OF WORK: The non-structural protein 4 (Nsp4) of porcine reproductive and respiratory syndrome virus (PRRSV) functions as a 3C-like proteinase (3CLpro) and plays a pivotal role in gene expression and replication. We have examined the biochemical properties of PRRSV 3CLpro and identified those amino acid residues involved in its catalytic activity as a prelude to developing anti-PRRSV strategies. The 3C-like proteinase (3CLpro) of porcine reproductive and respiratory syndrome virus (PRRSV) was expressed in Escherichia coli and characterized. The optimal temperature and pH for its proteolytic activity were 8°C and 7.5, respectively. Na(+) (1000 mM) and K(+) (500 mM) were not inhibitory to its activity but Cu(2+), Zn(2+), PMSF and EDTA were significantly inhibitory. His(39), Asp(64) and Ser(118) residues were identified to form the catalytic triad of PRRSV 3CLpro by a series of site-directed mutagenesis analysis.

Genetic diversity of the ORF5 gene of porcine reproductive and respiratory syndrome virus isolates in China from 2006 to 2008.

Since April 2006, swine herds have experienced the outbreaks of a highly pathogenic porcine reproductive and respiratory syndrome (PRRS) in China. To explore the possible mechanism of the emergence of the highly pathogenic PRRS and more fully understand the extent of genetic diversity of PRRSV in China, we analyzed the ORF5 gene sequences of 159 representative PRRSV isolates in 16 provinces from 2006 to 2008. Sequence and phylogenetic analyses showed that all these 159 isolates belonged to the North American genotype and were further divided into six subgenotypes; 140 of 159 isolates were closely related to the highly pathogenic PRRSV with 98.5-100% nucleotide and 98.3-100% amino acid sequence identities and belonged to Subgenotype I; and 3, 8, 4, 3, 1 of 159 isolates were part of Subgenotypes II-VI, respectively. Amino acid analysis of the GP5 protein revealed that all the isolates in Subgenotypes I-III were found to be highly variable in the primary neutralizing epitope; most of the isolates in Subgenotypes I and IV had more glycosylation sites than those in Subgenotypes II, III, V and VI; and 1, 5, and 9 unique amino acid mutations were observed in Subgenotypes I, IV and VI, respectively. In conclusion, our study provides the evidence of coexistence of six different subgenotype isolates in pigs in China from 2006 to 2008, and emphasizes the importance of reinforcing PRRSV surveillance, especially after the emergence of highly pathogenic PRRS in China.