PRRSV Replication Research Articles

Generation of murine macrophage-derived cell lines expressing porcine CD163 that support porcine reproductive and respiratory syndrome virus infection

BackgroundPorcine reproductive and respiratory syndrome virus (PRRSV) exhibits a highly restricted tropism for cells of the monocyte-macrophage lineage, utilizing porcine CD163 (pCD163) as an indispensable cellular receptor for infection. Transfection the gene of pCD163 into several non-permissive cell lines followed by protein expression confers susceptibility to PRRSV. A lack of specialized porcine antibody tools for use with existing porcine-derived primary cells and cell lines has hampered studies of both PRRSV pathogenesis and virus triggering of immune response cascades. Therefore, we constructed PRRSV-susceptible murine alveolar macrophage-derived MH-S and peritoneal macrophage-like RAW264.7 cell lines by achieving pCD163 cell surface expression in these cells. We then evaluated PRRSV susceptibility and cytokine expression patterns induced upon PRRSV infection of these pCD163-expressing cell lines.ResultsGrowth of MH-SCD163 and RAW264.7CD163 cells was indistinguishable from growth of un-transfected parental cell lines. Meanwhile, various stages of the PRRSV replication cycle, including viral particle attachment, internalization, disassembly and infection were confirmed in both pCD163-transfected cell lines. Analysis of PRRSV replication using immunofluorescence staining of virus and viral titration of cell lysates demonstrated that both MH-SCD163 and RAW264.7CD163 cells supported replication of various genotype 2 PRRSV isolates. Moreover, PRRSV replication in MH-SCD163 cells was similar to that observed in porcine alveolar macrophages (PAMs) and was more efficient than in RAW264.7CD163 cells. However, peak virus titers in MH-SCD163 cells were attained at 60 h post-infection (pi) versus 48 hpi in PAMs. Analysis of cytokine expression showed that post-PRRSV infection, mRNA expression patterns of anti-inflammatory cytokines (IL-4 and IL-10) and pro-inflammatory cytokines (TNF-α and IFN-γ) in MH-SCD163 cells were more similar to those observed in PAMs versus levels in RAW264.7CD163 cells.ConclusionsMH-S and RAW264.7 cells were not susceptible to PRRSV infection until transfection and subsequent expression of pCD163 were achieved in these cell lines. The PRRSV-susceptible MH-SCD163 cell line efficiently supported viral replication of various genotype 2 PRRSV isolates and exhibited similar cytokine expression patterns as observed in PAMs. In conclusion, this work describes the development of new tools to further understand PRRSV pathogenesis and immune response mechanisms to PRRSV infection.

Heparanase Upregulation Contributes to Porcine Reproductive and Respiratory Syndrome Virus Release.

Porcine reproductive and respiratory syndrome virus (PRRSV) continues to cause substantial economic losses to the pig industry worldwide. Heparan sulfate (HS) is used by PRRSV for initial attachment to target cells. However, the role of HS in the late phase of PRRSV infection and the mechanism of virus release from host cells remain largely unknown. In this study, we showed that PRRSV infection caused a decrease in HS expression and upregulated heparanase, the only known enzyme capable of degrading HS. We subsequently demonstrated that the NF-κB signaling pathway and cathepsin L protease were involved in regulation of PRRSV infection-induced heparanase. In addition, we found that ablation of heparanase expression using small interfering RNA duplexes increased cell surface expression of HS and suppressed PRRSV replication and release, whereas overexpression of heparanase reduced HS surface expression and enhanced PRRSV replication and release. These data suggest that PRRSV activates NF-κB and cathepsin L to upregulate and process heparanase, and then the active heparanase cleaves HS, resulting in viral release. Our findings provide new insight into the molecular mechanism of PRRSV egress from host cells, which might help us to further understand PRRSV pathogenesis.IMPORTANCE Porcine reproductive and respiratory syndrome virus (PRRSV) causes great economic losses each year to the pig industry worldwide. The molecular mechanism of PRRSV release from host cells largely remains a mystery. In this study, we demonstrate that PRRSV activates NF-κB and cathepsin L to upregulate and process heparanase, and then the active heparanase is released to the extracellular space and exerts enzymatic activity to cleave heparan sulfate, resulting in viral release. Our findings provide new insight into the molecular mechanism of PRRSV egress from host cells, which might help us to further understand PRRSV pathogenesis.

Interleukin-2 enhancer binding factor 2 interacts with the nsp9 or nsp2 of porcine reproductive and respiratory syndrome virus and exerts negatively regulatory effect on the viral replication

BackgroundPorcine reproductive and respiratory syndrome virus (PRRSV) causes reproductive failures in sows and respiratory diseases in growing pigs, resulting in huge economic loss for the pig production worldwide. The nonstructural protein 9 (nsp9) and nonstructural protein 2 (nsp2) of PRRSV are known to play important roles in viral replication. Cellular interleukin-2 enhancer binding factor 2 (ILF2) participates in many cellular pathways and involves in life cycle of some viruses. In the present study, we analyzed the interaction of cellular ILF2 with the nsp9 and nsp2 of PRRSV in vitro and explored the effect of ILF2 on viral replication.MethodsThe interaction of ILF2 with the nsp9 or nsp2 of PRRSV was analyzed in 293FT cells and MARC-145 cells by co-immunoprecipitation (Co-IP) and the co-localization of ILF2 with the nsp9 or nsp2 of PRRSV in MARC-145 cell and pulmonary alveolar macrophages (PAMs) was examined by confocal immunofluorescence assay. The effect of ILF2 knockdown and over-expression on PRRSV replication was explored in MARC-145 cells by small interfering RNA (siRNA) and lentivirus transduction, respectively.ResultsThe interaction of ILF2 with nsp9 or nsp2 was first demonstrated in 293FT cells co-transfected with ILF2-expressing plasmid and nsp9-expressing plasmid or nsp2-expressing plasmid. The interaction of endogenous ILF2 with the nsp9 or nsp2 of PRRSV was further confirmed in MARC-145 cells transduced with GFP-nsp9-expressing lentiviruses or infected with PRRSV JXwn06. The RdRp domain of nsp9 was shown to be responsible for its interaction with ILF2, while three truncated nsp2 were shown to interact with ILF2. Moreover, we observed that ILF2 partly translocated from the nucleus to the cytoplasm and co-localized with nsp9 and nsp2 in PRRSV-infected MARC-145 cells and PAMs. Finally, our analysis indicated that knockdown of ILF2 favored the replication of PRRSV, while over-expression of ILF2 impaired the viral replication in MARC-145 cells.ConclusionOur findings are the first to confirm that the porcine ILF2 interacts with the nsp9 and nsp2 of PRRSV in vitro, and exerts negatively regulatory effect on the replication of PRRSV. Our present study provides more evidence for understanding the roles of the interactions between cellular proteins and viral proteins in the replication of PRRSV.

Identification of host cellular proteins that interact with the M protein of a highly pathogenic porcine reproductive and respiratory syndrome virus vaccine strain

BackgroundThe highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV) continues to pose one of the greatest threats to the swine industry. M protein is the most conserved and important structural protein of PRRSV. However, information about the host cellular proteins that interact with M protein remains limited.MethodsHost cellular proteins that interact with the M protein of HP-PRRSV were immunoprecipitated from MARC-145 cells infected with PRRSV HuN4-F112 using the M monoclonal antibody (mAb). The differentially expressed proteins were identified by LC-MS/MS. The screened proteins were used for bioinformatics analysis including Gene Ontology, the interaction network, and the enriched KEGG pathways. Some interested cellular proteins were validated to interact with M protein by CO-IP.ResultsThe PRRSV HuN4-F112 infection group had 10 bands compared with the control group. The bands included 219 non-redundant cellular proteins that interact with M protein, which were identified by LC-MS/MS with high confidence. The gene ontology and Kyoto encyclopedia of genes and genomes (KEGG) pathway bioinformatic analyses indicated that the identified proteins could be assigned to several different subcellular locations and functional classes. Functional analysis of the interactome profile highlighted cellular pathways associated with protein translation, infectious disease, and signal transduction. Two interested cellular proteins—nuclear factor of activated T cells 45 kDa (NF45) and proliferating cell nuclear antigen (PCNA)—that could interact with M protein were validated by Co-IP and confocal analyses.ConclusionsThe interactome data between PRRSV M protein and cellular proteins were identified and contribute to the understanding of the roles of M protein in the replication and pathogenesis of PRRSV. The interactome of M protein will aid studies of virus/host interactions and provide means to decrease the threat of PRRSV to the swine industry in the future.

Carbon Monoxide Inhibits Porcine Reproductive and Respiratory Syndrome Virus Replication by the Cyclic GMP/Protein Kinase G and NF-κB Signaling Pathway.

PRRSV causes great economic losses each year to the swine industry worldwide. Carbon monoxide (CO), a metabolite of HO-1, has been shown to have antimicrobial and antiviral activities in infected cells. Our previous research demonstrated that HO-1 can suppress PRRSV replication. Here we show that endogenous CO produced through HO-1 catalysis mediates the antiviral effect of HO-1. CO inhibits PRRSV replication by activating the cellular cGMP/PKG signaling pathway and by negatively regulating cellular NF-κB signaling. These findings not only provide new insights into the molecular mechanism of HO-1 inhibition of PRRSV replication but also suggest potential new control measures for future PRRSV outbreaks.

Genetic resistance - an alternative for controlling PRRS?

PRRS is one of the most challenging diseases for world-wide pig production. Attempts for a sustainable control of this scourge by vaccination have not yet fully satisfied. With an increasing knowledge and methodology in disease resistance, a new world-wide endeavour has been started to support the combat of animal diseases, based on the existence of valuable gene variants with regard to any host-pathogen interaction. Several groups have produced a wealth of evidence for natural variability in resistance/susceptibility to PRRS in our commercial breeding lines. However, up to now, exploiting existing variation has failed because of the difficulty to detect the carriers of favourable and unfavourable alleles, especially with regard to such complex polygenic traits like resistance to PRRS. New hope comes from new genomic tools like next generation sequencing which have become extremely fast and low priced. Thus, research is booming world-wide and the jigsaw puzzle is filling up – slowly but steadily. On the other hand, knowledge from virological and biomedical basic research has opened the way for an “intervening way”, i.e. the modification of identified key genes that occupy key positions in PRRS pathogenesis, like CD163. CD163 was identified as the striking receptor in PRRSV entry and its knockout from the genome by gene editing has led to the production of pigs that were completely resistant to PRRSV – a milestone in modern pig breeding. However, at this early step, concerns remain about the acceptance of societies for gene edited products and regulation still awaits upgrading to the new technology. Further questions arise with regard to upcoming patents from an ethical and legal point of view. Eventually, the importance of CD163 for homeostasis, defence and immunity demands for more insight before its complete or partial silencing can be answered. Whatever path will be followed, even a partial abolishment of PRRSV replication will lead to a significant improvement of the disastrous herd situation, with a significant impact on welfare, performance, antimicrobial consumption and consumer protection. Genetics will be part of a future solution.

MicroRNA let-7f-5p Inhibits Porcine Reproductive and Respiratory Syndrome Virus by Targeting MYH9

Porcine reproductive and respiratory syndrome virus (PRRSV) is one of the most important viral pathogens in the swine industry. Current antiviral strategies do not effectively prevent and control PRRSV. Recent reports show that microRNAs (miRNAs) play vital roles in viral infections by post transcriptionally regulating the expression of viral or host genes. Our previous research showed that non-muscle myosin heavy chain 9 (MYH9) is an essential factor for PRRSV infection. Using bioinformatic prediction and experimental verification, we demonstrate that MYH9 expression is regulated by the miRNA let-7f-5p, which binds to the MYH9 mRNA 3′UTR and may play an important role during PRRSV infection. To understand how let-7f-5p regulates PRRSV infection, we analyzed the expression pattern of both let-7f-5p and MYH9 in porcine alveolar macrophages (PAMs) after infection with either highly pathogenic PRRSV (HP-PRRSV) or classical type PRRSV (N-PRRSV) using a deep sequencing approach with quantitative real-time PCR validation. Our results showed that both HP-PRRSV and N-PRRSV infection reduced let-7f-5p expression while also inducing MYH9 expression. Furthermore, let-7f-5p significantly inhibited PRRSV replication through suppression of MYH9 expression. These findings not only provide new insights into the pathogenesis of PRRSV, but also suggest potential new antiviral strategies against PRRSV infection.

In Vitro Antiviral Activity of Germacrone Against Porcine Reproductive and Respiratory Syndrome Virus.

Porcine reproductive and respiratory syndrome (PRRS) is one of the most serious diseases affecting the swine industry worldwide; however, there is no efficient control strategies against PRRSV at present. Therefore, development of new antiviral treatment strategies is urgently needed. As reported, germacrone can efficiently impair influenza virus replication. In this study, we exploited whether germacrone has the potential to inhibit PRRSV infection. Our results showed that the germacrone significantly inhibited replication of PRRSV in vitro and repressed the synthesis of viral RNA and protein. However, it did not block PRRSV binding and entry. Further studies confirmed that germacrone impaired PRRSV replication at an early stage, and inhibited infection of both classic and highly pathogenic type II PRRSV strains. Collectively, our findings imply that the germacrone has the potential to be used as an anti-PRRSV drug.

Autophagy postpones apoptotic cell death in PRRSV infection through Bad-Beclin1 interaction

Autophagy and apoptosis play significant roles in PRRSV infection and replication. However, the interaction between these 2 processes in PRRSV replication is still far from been completely understood. In our studies, the exposure of MARC-145 cells to PRRSV confirmed the activation of autophagy and subsequent induction of apoptosis. The inhibition of autophagy by 3-methyladenine (3-MA) caused a significant increase in PRRSV-induced apoptosis, showing a potential connection between both mechanisms. Moreover, we observed an increase in Bad expression (a pro-apoptotic protein) and Beclin1 (an autophagy regulator) in virus-infected cells up to 36h. Co-immunoprecipitation assays showed the formation of Bad and Beclin1 complex in PRRSV infected cells. Accordingly, Bad co-localized with Beclin1 in MARC-145 infected cells. Knockdown of Beclin1 significantly decreased PRRSV replication and PRRSV-induced autophagy, while Bad silencing resulted in increased autophagy and enhanced viral replication. Furthermore, PRRSV infection phosphorylated Bad (Ser112) to promote cellular survival. These results demonstrate that autophagy can favor PRRSV replication by postponing apoptosis through the formation of a Bad-Beclin1 complex.

Interplay of autophagy and apoptosis during PRRSV infection of Marc145 cell

Autophagy and apoptosis play essential roles 'in virus infection. Our study was performed to investigate the interplay between autophagy and apoptosis in PRRSV replication. In our present study, autophagy and apoptosis were induced by PRRSV infection. Viral replication was dampened/attenuated by autophagy deficient and potentiated by apoptosis inhibition. Furthermore, PRRSV replication was restored by apoptosis inhibition in autophagy deficient cells. Taken together, our findings unveil the functional relationship between autophagy and apoptosis during PRRSV replication.

HP-PRRSV is attenuated by de-optimization of codon pair bias in its RNA-dependent RNA polymerase nsp9 gene

There is an urgent need to develop new vaccines against highly pathogenic PRRS virus (HP-PRRSV) variant in China. The actual use of each codon pairs is more or less frequent than that of the statistical prediction and codon pair bias (CPB) usage affects gene translation. We “shuffled” the existing codons in HP-PRRSV genes GP5, M, nsp2 and nsp9, so that the CPB of these genes could be more negative. De-optimization of nsp9, the RNA-dependent RNA polymerase, significantly decreased PRRSV replication in porcine alveolar macrophages (PAMs). In vitro study showed that HV-nsp9min and HV-nsp29min were remarkably attenuated in PAMs, and inoculation of pigs with 2ml⁎105.0 TCID50/ml of HV-nsp9min or HV-nsp29min did not cause PRRS. Importantly, pigs immunized with HV-nsp29min were fully protected against different HP-PRRSV strains׳ lethal challenges. Our results imply that the CPB de-optimized HV-nsp29min has the potential to be used as a live vaccine candidate against HP-PRRSV.

MicroRNA miR-24-3p promotes porcine reproductive and respiratory syndrome virus replication through suppression of heme oxygenase-1 expression.

Porcine reproductive and respiratory syndrome virus (PRRSV) is one of the most economically important viruses affecting the swine industry worldwide. Our previous research showed that PRRSV downregulates the expression of heme oxygenase-1 (HO-1), a pivotal cytoprotective enzyme, postinfection and that overexpression of HO-1 inhibits PRRSV replication. MicroRNAs regulate gene expression at the posttranscriptional level and have recently been demonstrated to play vital roles in pathogen-host interactions. The present study sought to determine whether microRNAs modulate HO-1 expression and, by doing so, regulate PRRSV replication. Using bioinformatic prediction and experimental verification, we demonstrate that HO-1 expression is regulated by miR-24-3p. A direct interaction between miR-24-3p and HO-1 mRNA was confirmed using a number of approaches. Overexpression of miR-24-3p significantly decreased HO-1 mRNA and protein levels. PRRSV infection induced miR-24-3p expression to facilitate viral replication. The suppressive effect of HO-1 induction by protoporphyrin IX cobalt chloride (CoPP; a classical inducer of HO-1 expression) on PRRSV replication in MARC-145 cells and primary porcine alveolar macrophages could also be reversed by overexpression of miR-24-3p. Collectively, these results suggested that miR-24-3p promotes PRRSV replication through suppression of HO-1 expression, which not only provides new insights into virus-host interactions during PRRSV infection but also suggests potential new antiviral strategies against PRRSV infection. MicroRNAs (miRNAs) play vital roles in viral infections by regulating the expression of viral or host genes at the posttranscriptional level. Heme oxygenase-1 (HO-1), a pivotal cytoprotective enzyme, has antiviral activity for a number of viruses, such as Ebola virus, hepatitis C virus, human immunodeficiency virus, and our focus, PRRSV, which causes great economic losses each year in the swine industry worldwide. Here, we show that PRRSV infection induces host miRNA miR-24-3p expression and that miR-24-3p regulates HO-1 expression through both mRNA degradation and translation repression. Suppression of HO-1 expression by miR-24-3p facilitates PRRSV replication. This work lends credibility to the hypothesis that an arterivirus can manipulate cellular miRNAs to enhance virus replication by regulating antiviral responses following viral infection. Therefore, our findings provide new insights into the pathogenesis of PRRSV.

Identification of a single nucleotide promoter polymorphism regulating the transcription of ubiquitin specific protease 18 gene related to the resistance to porcine reproductive and respiratory syndrome virus infection

Porcine reproductive and respiratory syndrome (PRRS), characterized by reproductive failure in sows and respiratory disease and mortality in piglets, is a major infectious disease that causes great economic loss throughout the world. Previous studies revealed that the overexpression of porcine ubiquitin specific protease 18 (USP18) gene inhibits PRRSV replication in vitro. The objective of this study is to compare the promoter activity of USP18 in Chinese indigenous Dapulian (DPL) pigs and Duroc×Landrace×Yorkshire (DLY) commercial pigs and screen single nucleotide polymorphism (SNP) affecting porcine USP18 transcription. We found that the promoter activity was significantly higher in DPL pigs than DLY commercial pigs (p<0.05), deletion of the promoter from –1790 to –1314bp decreased the transcriptional activity by roughly 60% (p<0.05) and a SNP G–1533A in this region increased the mRNA expression both prior to and post PRRSV infection in MARC-145 cells. Population genetics analysis showed that allele A was only detected in Chinese pig breeds which are generally resistant to PRRSV. These results suggest that the SNP G–1533A polymorphism in the promoter region of porcine USP18 gene is a potential DNA marker for the resistance to PRRSV.

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.

Up-regulation of pro-inflammatory factors by HP-PRRSV infection in microglia: Implications for HP-PRRSV neuropathogenesis

Atypical porcine reproductive and respiratory syndrome (PRRS) caused by a highly pathogenic PRRS virus (HP-PRRSV) is characterized by high fever, high morbidity, high mortality, and associated with severe neurological symptoms. Microglia are the resident innate immune cells in central nervous system (CNS), and their activation has been implicated as an important contributor to the pathogenesis of CNS diseases. In the present study, we successfully cultured porcine microglia and demonstrated that microglia could support PRRSV infection and replication in vitro. We further showed that HP-PRRSV infection significantly up-regulated the key inflammatory factors including IL-1β, TNF-α, IL-6, IL-12, IL-8, CXCL10, MCP-1, CCL3, CCL4, and CCL5 in cultured microglia as well as in the CNS of HP-PRRSV-infected pigs. The transcription factors NF-κB and AP-1, which are widely reported to regulate cytokine and chemokine productions, were activated by HP-PRRSV infection in microglia. Meanwhile, we found that HP-PRRSV induced cellular ROS formation in microglia and ROS scavenger was proved to significantly abolish the activation of pro-inflammatory cytokines (IL-1β, TNF-α, IL-6, and IL-8), suggesting that ROS are crucial for pro-inflammatory gene production. Importantly, incubation with supernatants from HP-PRRSV-infected microglia cell culture remarkably induced SH-SY5Y neuroblastoma cell death. Collectively, these results showed that PRRSV infection induced cytokine and ROS up-regulation in microglia, which might contribute to neurotoxicity. These data have implications for us to understand the neuropathogenesis of HP-PRRSV in pigs.

A brief review of microRNA and its role in PRRSV infection and replication

A brief review of microRNA and its role in PRRSV infection and replication

MicroRNA 181 Suppresses Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) Infection by Targeting PRRSV Receptor CD163

We previously showed that microRNA 181 (miR-181) can inhibit PRRSV replication by directly targeting its genomic RNA. Here, we report that miR-181 can downregulate the PRRSV receptor CD163 in blood monocytes and porcine alveolar macrophages (PAMs) through targeting the 3' untranslated region (UTR) of CD163 mRNA. Downregulation of CD163 leads to the inhibition of PRRSV entry into PAMs and subsequently suppresses PRRSV infection. Our findings indicate that delivery of miR-181 can be used as antiviral therapy against PRRSV infection.

Porcine reproductive and respiratory syndrome virus induces autophagy to promote virus replication

An increasing number of studies demonstrate that autophagy, an intrinsic mechanism that can degrade cytoplasmic components, is involved in the infection processes of a variety of pathogens. It can be hijacked by various viruses to facilitate their replication. In this study, we found that PRRSV infection significantly increases the number of double- or single-membrane vesicles in the cytoplasm of host cells in ultrastructural analysis. Our results showed the LC3-I was converted into LC3-II after virus infection, suggesting the autophagy machinery was activated. We further used pharmacological agents and shRNAs to confirm that autophagy promoted the replication of PRRSV in host cells. Confocal microscopy analysis showed that PRRSV inhibited the fusion between autophagosomes and lysosomes, suggesting that PRRSV induced incomplete autophagy. This suppression caused the accumulation of autophagosomes which may serve as replication site to enhance PRRSV replication. It has been shown that NSP2 and NSP3 of arterivirus are two components of virus replication complex. We also found in our studies that NSP2 colocalized with LC3 in MARC-145 cells by performing confocal microscopy analysis and continuous density gradient centrifugation. Our studies presented here indicated that autophagy was activated during PRRSV infection and enhanced PRRSV replication in host cells by preventing autophagosome and lysosome fusion.

Poly(A)-binding protein interacts with the nucleocapsid protein of porcine reproductive and respiratory syndrome virus and participates in viral replication

Interactions between host factors and the viral protein play important roles in host adaptation and regulation of virus replication. Poly(A)-binding protein (PABP), a host cellular protein that enhances translational efficiency by circularizing mRNAs, was identified by yeast two-hybrid screening as a cellular partner for PRRSV nucleocapsid (N) protein in porcine alveolar macrophages. The specific interaction of PRRSV N protein with PABP was confirmed in infected cells by co-immunoprecipitation and in vitro by GST pull-down assay. We showed by confocal microscopy that the PABP co-localized with the PRRSV N protein. Using a series of deletion mutants, the interactive domain of N protein with PABP was mapped to a region of amino acids 52–69. For PABP, C-terminal half, which interestingly interacts other translation regulators, was determined to be the domain interactive with N protein. Short hairpin RNA (shRNA)-mediated silencing of PABP in cells resulted in significantly reduced PRRSV RNA synthesis, viral encoded protein expression and viral titer. Overall, the results presented here point toward an important role for PABP in regulating PRRSV replication.

Role of non-structural protein 2 in the regulation of the replication of the porcine reproductive and respiratory syndrome virus in MARC-145 cells: Effect of gene silencing and over expression

Porcine reproductive and respiratory syndrome (PRRS) is an economically important disease in swine-producing areas. Many vaccine strategies have been developed to control the disease, but none have yet been completely successful. The development of a cell line that can produce large yields of PRRSV vaccine is very necessary. In order to determine the role of Nsp2 in the replication of the highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV) in MARC-145 cells, we used an RNA interference-based short hairpin RNA of Nsp2 and constructed cell lines expressing the HP-PRRSV Nsp2 gene. Conserved HP-PRRSV Nsp2 sequences were used to design short interfering RNAs and test their ability to silence PRRSV transcript expression and replication in cells in vitro transfection. Nsp2, ORF7, and β-actin mRNA expression were determined using semi-quantitative real-time PCR. Infection with siRNA targeting Nsp2 was found to reduce the Nsp2 expression in MARC-145 cells infected with PRRSV. Both MARC-145-TJ Nsp2 and MARC-145-TJM Nsp2 cell lines were screened by G418, which were infected with HP-PRRSV, normal MARC-145 cells for mock, and then virus titers were calculated by TCID50 after the CPE showing up. The downregulation of Nsp2 induced a remarkable decrease in PRRSV replication, causing the reduction of structural protein. The Nsp2-targeted siRNA was found to downregulate the expression of Nsp2 in MARC-145 cells and inducing replication reduce of PRRSV in MARC-145 cells. The shRNA vectors S-1 and S-2 could effectively induce the inhibition of viral replication in MARC-145. Results showed that cells expressing the Nsp2 gene of the highly pathogenic PRRSV TJ and attenuated TJM remained stable. PRRSV replication was faster in these cells than in MARC-145 cells, especially during the early stage. This shows that Nsp2 plays a positive role in PRRSV proliferation.