Phosphorylation of the porcine reproductive and respiratory syndrome virus nucleocapsid protein

Porcine reproductive and respiratory syndrome virus (PRRSV) is a cytoplasmic RNA virus with the unique or unusual feature of having a nucleocapsid (N) protein that is specifically transported to the nucleolus of virus-infected cells. In this communication, we show that the N protein is a phosphoprotein. Phosphoamino acid analysis of authentic and recombinant N proteins demonstrated that serine residues were exclusively phosphorylated. The pattern of phosphorylated N protein cellular distribution in comparison with that of [(35)S]methionine-labeled N protein suggested that phosphorylation does not influence subcellular localization of the protein. Time course studies showed that phosphorylation occurred during, or shortly after, synthesis of the N protein and that the protein remained stably phosphorylated throughout the life cycle of the virus to the extent that phosphorylated N protein was found in the mature virion. Two-dimensional electrophoresis and acid-urea gel electrophoresis showed that one species of the N protein is predominant in virus-infected cells, suggesting that multiple phosphorylated isoforms of N do not exist.     

Structure of the nucleocapsid protein of porcine reproductive and respiratory syndrome virus

Porcine reproductive and respiratory syndrome virus (PRRSV) is an enveloped RNA virus of the Arteriviridae family, genomically related to the coronaviruses. PRRSV is the causative agent of both severe and persistent respiratory disease and reproductive failure in pigs worldwide. The PRRSV virion contains a core made of the 123 amino acid nucleocapsid (N) protein, a product of the ORF7 gene. We have determined the crystal structure of the capsid-forming domain of N. The structure was solved to 2.6 A resolution by SAD methods using the anomalous signal from sulfur. The N protein exists in the crystal as a tight dimer forming a four-stranded beta sheet floor superposed by two long alpha helices and flanked by two N- and two C-terminal alpha helices. The structure of N represents a new class of viral capsid-forming domains, distinctly different from those of other known enveloped viruses, but reminiscent of the coat protein of bacteriophage MS2.     

Genetic variability of the nucleocapsid protein of the virus of the porcine reproductive and respiratory syndrome

The porcine reproductive and respiratory syndrome (PRRS) is a contagious viral pathology caused by PRRS virus. There are 2 types of the above virus--the European and American ones. Distribution patterns of the PRRS virus were studied for Russia and Byelorussia. Above 700 porcine sera obtained from 32 households of 21 Russia's administrative regions and from 19 households of 6 Byelorussia's administrative regions were tested for presence of antibodies to the PRRS virus. Simultaneously, the samples were tested for virus presence by polymerase chain reaction (PCR). It was proven serologically that the PRRS virus is widespread in the territories of Russia and Byelorussia. Noteworthily, all field isolates found in Russia and Byelorussia belong to the European type. Not a single viral isolate of the American PRRS type was found. The nucleocapsid (N) recombinant protein was obtained on the basis of the Russian field isolate of the PRRS virus by using the E. coli. expression system. Finally, it was shown as possible to use the recombinant protein in indirect immune enzyme assay for the sake of detecting the antibodies to the PRRS virus.     

Biophysical characterisation of the nucleocapsid protein from a highly pathogenic porcine reproductive and respiratory syndrome virus strain

The arterivirus nucleocapsid (N) protein is a multifunctional protein that binds viral RNA for encapsidation and has potential roles in host cell processes. This study characterised the N protein from a highly virulent North American strain of porcine reproductive and respiratory syndrome virus (PRRSV). The association with viral RNA was mapped to defined motifs on the N protein. The results indicated that disulphide bridge formation played a key role in RNA binding, offering an explanation why infectious virus cannot be rescued if cysteine residues are mutated, and that multiple sites may promote RNA binding.     

RNA interference targeting nucleocapsid protein inhibits porcine reproductive and respiratory syndrome virus replication in Marc-145 cells

Porcine reproductive and respiratory syndrome (PRRS) is an important disease, which leads to severe economic losses in swine-producing areas of the world. However, current antiviral strategies cannot provide highly effective protection. In this study, three theoretically effective interference target sites (71–91, 144–164, 218–238) targeting the nucleocapsid (N) gene of PRRSV were designed and selected, and then three siRNA-expressing plasmids were constructed, respectively named p2.1-N71, p2.1-N144, and p2.1-N218. The recombinant siRNA-expressing plasmids were transfected into Marc-145 cells; then the cells were infected with PRRSV (JL07SW strain); finally, after incubation for 48 h, the antiviral activity of those siRNA-expressing plasmids in Marc-145 cells was assessed by cytopathic effects, virus titers, indirect immunofluorescence, and quantitative real-time PCR. Experimental results demonstrated that these three siRNA-expressing plasmids could effectively and significantly inhibit the replication of PRRSV by 93.2%, 83.6%, and 89.2% in Marc-145 cells, respectively. Among these three siRNA-expressing plasmids, p2.1-N71 was found to be most effective, while p2.1-N144 and p2.1-N218 displayed relatively weak inhibition of virus replication. The results indicated that siRNA-expressing plasmids targeting the N gene of PRRSV could significantly inhibit PRRSV replication in Marc-145 cells. Based on our experimental results and previous reports, the 71–91, 179–197, and 234–252 sites of the N gene are good choices to effectively inhibit the replication of PRRSV, and this RNA interference technique can be a potential anti-PRRSV strategy.     

The dimer interface of the SARS coronavirus nucleocapsid protein adapts a porcine respiratory and reproductive syndrome virus-like structure

We have employed NMR to investigate the structure of SARS coronavirus nucleocapsid protein dimer. We found that the secondary structure of the dimerization domain consists of five alpha helices and a beta-hairpin. The dimer interface consists of a continuous four-stranded beta-sheet superposed by two long alpha helices, reminiscent of that found in the nucleocapsid protein of porcine respiratory and reproductive syndrome virus. Extensive hydrogen bond formation between the two hairpins and hydrophobic interactions between the beta-sheet and the alpha helices render the interface highly stable. Sequence alignment suggests that other coronavirus may share the same structural topology.     

Overview: Replication of porcine reproductive and respiratory syndrome virus

Porcine reproductive and respiratory syndrome virus (PRRSV), an arterivirus that causes significant losses in the pig industry, is one of the most important animal pathogens of global significance. Since the discovery of the virus, significant progress has been made in understanding its epidemiology and transmission, but no adequate control measures are yet available to eliminate infection with this pathogen. The genome replication of PRRSV is required to reproduce, within a few hours of infection, the millions of progeny virions that establish, disseminate, and maintain infection. Replication of the viral RNA genome is a multistep process involving a replication complex that is formed not only from components of viral and cellular origin but also from the viral genomic RNA template; this replication complex is embedded within particular virus-induced membrane vesicles. PRRSV RNA replication is directed by at least 14 replicase proteins that have both common enzymatic activities, including viral RNA polymerase, and also unusual and poorly understood RNA-processing functions. In this review, we summarize our current understanding of PRRSV replication, which is important for developing a successful strategy for the prevention and control of this pathogen.     

Divergence time of porcine reproductive and respiratory syndrome virus subtypes

Porcine reproductive and respiratory syndrome virus (PRRSV) recently emerged in domestic pigs of Western Europe and North America. Although time of emergence was identical on the two continents, genetic composition was markedly different with a clear geographical subtype structure, indicating that subtypes diverged in separate reservoirs prior to emergence. Genetic analyses have shown that the most recent common ancestor (MRCA) of Western European isolates existed around 1980 and that these originate from Eastern European pigs. These findings are challenged by a study of Hanada et al. who place the MRCA of all PRRSV isolates around 1980 and find that no significant subtype divergence occurred before emergence. Here, I discuss problems of information content, methodology, and biological plausibility associated with this study. Using alternative methodology, I reanalyze the existing data and conclude that the MRCA of all PRRSV isolates existed around 1880, 100 years before the date estimated by Hanada et al.     

Development and validation of a recombinant nucleocapsid protein-based ELISA for detection of the antibody to porcine reproductive and respiratory syndrome virus

Three indirect enzyme-linked immunosorbent assays (iELISA) based on the North American like (NA-like), European like (EU-like) and co-expressed NA- and EU-like recombinant nucleocapsid proteins (N-protein) of porcine reproductive and respiratory syndrome virus (PRRSV) were validated for the detection of the antibodies in porcine sera. A total of 422 serum samples from unvaccinated pigs were tested. The cut-off value was optimized by a two-graph receiver operating characteristics analysis at a 95% confidence level. This assay was validated with Western blot analysis and IDEXX HerdChek™ ELISA. Cross-reactivity results showed that iELISA was PRRSV-specific. Repeatability tests revealed that the coefficients of variation of positive sera within and between runs were less than 10%. The results indicate that iELISA is simpler to produce and perform, time-saving and suitable for large scale surveys of PRRSV infection at low cost, and is potentially useful to evaluate the efficiency of various vaccines against PRRSV.     

Intracellularly expressed nanobodies against non-structural protein 4 of porcine reproductive and respiratory syndrome virus inhibit virus replication

Objective

To isolate specific nanobodies to porcine reproductive and respiratory syndrome virus (PRRSV) non-structural protein 4 (Nsp4) and investigate their potential antiviral activities.

Results

Three PRRSV Nsp4-specific nanobodies were isolated from a phage display library of the variable domains of camelid heavy chain-only antibodies. Nanobody genes were introduced into MARC-145 cells using lentivirus vectors to establish cell lines stably expressing nanobodies. These intracellularly expressed nanobodies were tested for interaction with PRRSV-encoded Nsp4 within PRRSV-infected MARC-145 cells. Nb41 and Nb43 intrabodies each potently inhibited PRRSV replication, protected MARC-145 cells from PRRSV-induced cytopathic effect and fully blocked PRRSV replication at an MOI of 0.001 or lower.

Conclusion

Intracellularly expressed Nb41 and Nb43 potently suppressed PRRSV replication in MARC-145 cells. Nanobodies hold great potential for development as novel antiviral treatments for PRRSV infection.

     

Characterization of interaction between porcine reproductive and respiratory syndrome virus and porcine dendritic cells

The porcine reproductive and respiratory syndrome Virus (PRRSV) is an infectious disease that causes abortions and respiratory disorders in swine. In this study, the interaction between PRRSV and porcine dendritic cells generated from CD14(+) monocytes in the presence of GM-CSF and IL-4 was examined. As a result, it was shown that immature and mature dendritic cells can be productively infected with PRRSV. When the expression of surface MHC molecules on infected dendritic cells was determined, MHC classes I and II were found to be downregulated when compared with uninfected dendritic cells. With the exception of the IL-4 and IFN-gamma cytokines, the induction of the IL-10, IL-12, and TNF-alpha cytokines all increased in dendritic cells infected with PRRSV. A mixed lymphocyte reaction showed that peripheral blood mononuclear cells cocultured with PRRSVinfected dendritic cells were less stimulated than peripheral blood mononuclear cells cocultured with dendritic cells treated with PBS, LPS, or UV-inactivated PRRSV. Therefore, these results suggest that PRRSV would appear to modulate the immune stimulatory function of porcine dendritic cells.     

Antigen-specific B-cell responses to porcine reproductive and respiratory syndrome virus infection

Porcine reproductive and respiratory syndrome virus (PRRSV) causes an acute, viremic infection of 4 to 6 weeks, followed by a persistent infection lasting for several months. We characterized antibody and B-cell responses to viral proteins in acute and persistent infection to better understand the immunological basis of the prolonged infection. The humoral immune response to PRRSV was robust overall and varied among individual viral proteins, with the important exception of a delayed and relatively weak response to envelope glycoprotein 5 (GP5). Memory B cells were in secondary lymphoid organs, not in bone marrow or Peyer's patches, in contrast to the case for many mammalian species. Potent anti-PRRSV memory responses were elicited to recall antigen in vitro, even though a second infection did not increase the B-cell response in vivo, suggesting that productive reinfection does not occur in vivo. Antibody titers to several viral proteins decline over time, even though abundant antigen is known to be present in lymphoid tissues, possibly indicating ineffective antigen presentation. The appearance of antibodies to GP5 is delayed relative to the resolution of viremia, suggesting that anti-GP5 antibodies are not crucial for resolving viremia. Lastly, viral infection had no immunosuppressive effect on the humoral response to a second, unrelated antigen. Taking these data together, the active effector and memory B-cell responses to PRRSV are robust, and over time the humoral immune response to PRRSV is effective. However, the delayed response against GP5 early in infection may contribute to the prolonged acute infection and the establishment of persistence.     

Application of GP5 protein to develop monoclonal antibody against porcine reproductive and respiratory syndrome virus

In this study,a panel of monoclonal antibodies (mAbs) against Porcine reproductive and respiratory syndrome virus(PRRSV),named as 8C9 and4B4,were produced by fusing SP2/0 myeloma cells and spleen cells of BALB/c mice immunized with the PRRSV (TCID50=5.5),screened by the indirect ELISA and subjected to several limiting dilutions.mAbs were then identified by biological characterization.Among the two fusion cell strains,8C9 belonged to the IgG1 subclass and 4B4 belonged to the IgG2a subclass.The titers in cell culture supernatant and abdomen liquor reached to 1:104and 1:105,respectively.The specificity test indicated that the two cells had specific reactions for the PRRSV and GP5 protein respectively,and no reaction with Classical swine fever virus (CSFV) or Swine vesicular disease virus (SVDV).The molecular weights of the heavy chain and light chain were about 45.0 kDa and 25.0 kDa,respectively.In neutralization activity tests,the results showed that the prepared mAb 4B4 can protect 50% of cells with no CPE in dilution up to 1:512,but mAB 8C9 has no neutralization activities to PRRSV.     

重组猪肺表面活性蛋白A在体外可抑制PRRSV感染宿主细胞

【目的】研究重组猪肺表面活性蛋白A(SP-A)在体外对猪繁殖与呼吸综合征病毒(PRRSV)感染的抑制作用。【方法】采用PCR方法从含有猪SP-A基因的质粒中扩增SP-A基因,并将其插入到含有人CD5信号肽序列的真核表达载体pcDNA3.1A-CD5中,构建成SP-A基因的真核分泌型表达载体pcDNA-CD5-SPA/MH。将重组表达载体通过磷酸钙介导转染HEK293T细胞进行瞬时表达,通过Western blot方法鉴定表达产物,采用Ni-NTA琼脂糖凝胶亲和层析法从培养基中分离和纯化重组SP-A蛋白,通过ELISA方法检测SP-A蛋白与PRRSV的结合活性。将SP-A蛋白与PRRSV孵育,然后感染MARC-145细胞和猪肺泡巨噬细胞,感染72 h后测定病毒滴度,分析重组SP-A蛋白对PRRSV感染的抑制作用。【结果】结果表明构建的真核表达载体能够介导SP-A基因在HEK293T细胞中进行分泌表达;表达的重组猪SP-A蛋白能够与PRRSV进行剂量依赖性结合;用重组猪SP-A蛋白与PRRSV进行孵育,然后感染MARC-145细胞和猪肺泡巨噬细胞,结果显示SP-A处理的PRRSV感染细胞后的病变程度明显低于对照组。感染72 h后,SP-A处理组的PRRSV在MARC-145细胞和猪肺泡巨噬细胞的滴度明显低于SP-A非处理组。【结论】重组猪SP-A在体外对PRRSV的感染有明显的抑制作用,揭示SP-A具有抗PRRSV的活性。     

Nonstructural protein 2 of porcine reproductive and respiratory syndrome virus inhibits the antiviral function of interferon-stimulated gene 15

Type I interferon (alpha/beta interferon [IFN-α/β]) stimulates the expression of interferon-stimulated gene 15 (ISG15), which encodes a ubiquitin-like protein, ISG15. Free ISG15 and ISG15 conjugates function in diverse cellular pathways, particularly regulation of antiviral innate immune responses. In this study, we demonstrate that ISG15 overexpression inhibits porcine reproductive and respiratory syndrome virus (PRRSV) replication in cell culture and that the antiviral activity of interferon is reduced by inhibition of ISG15 conjugation. PRRSV nonstructural protein 2 (nsp2) was previously identified as a potential antagonist of ISG15 production and conjugation. The protein contains a papain-like protease domain (PLP2) that plays a crucial role in the proteolytic cleavage of the PRRSV replicase polyproteins. PLP2 was also proposed to belong to the ovarian tumor domain-containing superfamily of deubiquitinating enzymes (DUBs), which is capable of inhibiting ISG15 production and counteracting ISG15 conjugation to cellular proteins. To determine whether this immune antagonist function could be selectively inactivated, we engineered a panel of mutants with deletions and/or mutations at the N-terminal border of the nsp2 PLP2-DUB domain. A 23-amino-acid deletion (amino acids 402 to 424 of the ORF1a-encoded protein) largely abolished the inhibitory effect of nsp2 on ISG15 production and conjugation, but no viable recombinant virus was recovered. A 19-amino-acid deletion (amino acids 402 to 420), in combination with a downstream point mutation (S465A), partially relieved the ISG15 antagonist function and yielded a viable recombinant virus. Taken together, our data demonstrate that ISG15 and ISGylation play an important role in the response to PRRSV infection and that nsp2 is a key factor in counteracting the antiviral function of ISG15.