Inhibited Porcine Reproductive And Respiratory Syndrome Virus Infection Research Articles

Swine NONO promotes IRF3-mediated antiviral immune response by Detecting PRRSV N protein.

Non-POU domain-containing octamer-binding protein (NONO) is a multi-functional nuclear protein which belongs to the Drosophila behavior/human splicing (DBHS) protein family. NONO is known to regulate multiple important biological processes including host antiviral immune response. However, whether NONO can inhibit porcine reproductive and respiratory syndrome virus (PRRSV) replication is less well understood. In this study, we demonstrated that swine NONO (sNONO) inhibited PRRSV replication, via increasing expression of IFN-β, whereas NONO knockdown or knockout in PAM-KNU cells was more susceptible to PRRSV infection. As an IRF3 positive regulation factor, NONO promoted IFN-β expression by enhancing activation of IRF3. During PRRSV infection, NONO further up-regulated IRF3-mediated IFN-β expression by interacting with PRRSV N protein. Mechanistically, NONO functioned as a scaffold protein to detect PRRSV N protein and formed N-NONO-IRF3 complex in the nucleus. Interestingly, it was found that the NONO protein reversed the inhibitory effect of PRRSV N protein on type I IFN signaling pathway. Taken together, our study provides a novel mechanism for NONO to increase the IRF3-mediated IFN-β activation by interacting with the viral N protein to inhibit PRRSV infection.

Antiviral activity and underlying mechanisms of baicalin against porcine reproductive and respiratory syndrome virus in vitro

Porcine reproductive and respiratory syndrome (PRRS) is a major challenge for the global swine industry, causing huge economic losses worldwide. To date, there are no effective measures to prevent and control the spread of PRRS virus (PRRSV). Baicalin (BA) is a natural flavonoid with various pharmacological effects, including antiviral, anti-inflammatory, antioxidant and immunomodulatory. Here, we demonstrate that BA exhibits potent anti-PRRSV activity in vitro, BA concentrations in the range of 5–20 μg/mL significantly inhibited PRRSV infection in a dose-dependent manner and were independent of PRRSV strain. Mechanistically, BA inhibited PRRSV replication by directly interacting with virions, thereby affecting multiple stages of the virus life cycle. Meanwhile, the preventive effect of BA on PRRSV could be realized by inhibiting CD151 and CD163 expression. Furthermore, BA reduced the PRRSV-induced expression of PAMs cytokines (IFN-α, IL-6, IL-8, and TNF-α), suggesting that BA-induced antiviral cytokines may help BA inhibit PRRSV infection. Taken together, BA can be used as an inhibitor of PRRSV infection in vitro, which provides a theoretical basis for the clinical application of BA and the prevention and control of PRRSV infection, which is worthy of further in vivo studies in swine.

FADD promotes type I interferon production to suppress porcine reproductive and respiratory syndrome virus infection.

Porcine reproductive and respiratory syndrome (PRRS) is an epidemic animal infectious disease worldwide, causing huge economic losses to the global swine industry. Fas-associated death domain (FADD) was previously reported to be an adaptor protein that functions in transferring the apoptotic signals regulated by the death receptors. In the current study, we unravel its unidentified role in promoting type I interferon (IFN) production during PRRS virus (PRRSV) infection. We identified that FADD inhibited PRRSV infection via promotion of type I IFN transcription. Overexpression of FADD suppressed the replication of PRRSV, while knockout of FADD increased viral titer and nucleocapsid protein expression. Mechanistically, FADD promoted mitochondrial antiviral signaling protein (MAVS)-mediated production of IFN-β and some IFN-stimulated genes (ISGs). Furthermore, FADD exerted anti-PRRSV effects in a MAVS-dependent manner and increased the type I IFN signaling during PRRSV infection. This study highlights the importance of FADD in PRRSV replication, which may have implications for the future control of PRRS.

The natural compound Sanggenon C inhibits PRRSV infection by regulating the TRAF2/NF-κB signalling pathway

Porcine reproductive and respiratory syndrome (PRRS) is a serious infectious disease and one of the major causes of death in the global pig industry. PRRS virus (PRRSV) strains have complex and diverse genetic characteristics and cross-protection between strains is low, which complicates vaccine selection; thus, the current vaccination strategy has been greatly compromised. Therefore, it is necessary to identify effective natural compounds for the clinical treatment of PRRS. A small molecule library composed of 720 natural compounds was screened in vitro, and we found that Sanggenon C (SC) was amongst the most effective natural compound inhibitors of PRRSV infection. Compared with ribavirin, SC more significantly inhibited PRRSV infection at both the gene and protein levels and reduced the viral titres and levels of protein expression and inflammatory cytokine secretion to more effectively protect cells from PRRSV infection and damage. Mechanistically, SC inhibits activation of the NF-κB signalling pathway by promoting TRAF2 expression, thereby reducing PRRSV replication. In conclusion, by screening natural compounds, we found that SC suppresses PRRSV infection by regulating the TRAF2/NF-κB signalling pathway. This study contributes to a deeper understanding of the therapeutic targets and pathogenesis of PRRSV infection. More importantly, our results demonstrate that SC has potential as a candidate for the treatment of PRRS.

The shape-dependent inhibitory effect of rhein/silver nanocomposites on porcine reproductive and respiratory syndrome virus

Traditional Chinese medicines (TCMs)/nanopreparations as viral antagonists exhibited a structure–function correlation, i.e., the differences in surface area/volume ratio caused by the variations in shape and size could result in different biochemical properties and biological activities, suggesting an important impact of morphology and structure on the antiviral activity of TCM-based nanoparticles. However, few studies paid attention to this aspect. Here, the effect of TCM-based nanoparticles with different morphologies on their antiviral activity was explored by synthesizing rhein/silver nanocomposites (Rhe@AgNPs) with spherical (S-Rhe/Ag) and linear (L-Rhe/Ag) morphologies, using rhein (an active TCM ingredient) as a reducing agent and taking its self-assembly advantage. Using porcine reproductive and respiratory syndrome virus (PRRSV) as a model virus, the inhibitory effects of S-Rhe/Ag and L-Rhe/Ag on PRRSV were compared. Results showed that the product morphology could be regulated by varying pH values, and both S- and L-Rhe/Ag exhibited good dispersion and stability, but with a smaller size for L-Rhe/Ag. Antiviral experiments revealed that Rhe@AgNPs could effectively inhibit PRRSV infection, but the antiviral effect was morphology-dependent. Compared with L-Rhe/Ag, S-Rhe/Ag could more effectively inactivate PRRSV in vitro and antagonize its adsorption, invasion, replication, and release stages. Mechanistic studies indicated that Rhe@AgNPs could reduce the production of reactive oxygen species (ROS) induced by PRRSV infection, and S-Rhe/Ag also had stronger ROS inhibitory effect. This work confirmed the inhibitory effect of Rhe@AgNPs with different morphologies on PRRSV and provided useful information for treating PRRSV infection with metal nanoparticles synthesized from TCM ingredients.Graphical abstract

MARCO Inhibits Porcine Reproductive and Respiratory Syndrome Virus Infection through Intensifying Viral GP5-Induced Apoptosis.

Studying viral glycoprotein-host membrane protein interactions contributes to the discovery of novel cell receptors or entry facilitators for viruses. Glycoprotein 5 (GP5), which is a major envelope protein of porcine reproductive and respiratory syndrome virus (PRRSV) virions, is a key target for the control of the virus. Here, the macrophage receptor with collagenous structure (MARCO), which is a member of the scavenger receptor family, was identified as one of the host interactors of GP5 through a DUALmembrane yeast two-hybrid screening. MARCO was specifically expressed on porcine alveolar macrophages (PAMs), and PRRSV infection downregulated MARCO expression both in vitro and in vivo. MARCO was not involved in viral adsorption and internalization processes, indicating that MARCO may not be a PRRSV-entry facilitator. Contrarily, MARCO served as a host restriction factor for PRRSV. The knockdown of MARCO in PAMs enhanced PRRSV proliferation, whereas overexpression suppressed viral proliferation. The N-terminal cytoplasmic region of MARCO was responsible for its inhibitory effect on PRRSV. Further, we found that MARCO was a proapoptotic factor in PRRSV-infected PAMs. MARCO knockdown weakened virus-induced apoptosis, whereas overexpression aggravated apoptosis. MARCO aggravated GP5-induced apoptosis, which may result in its proapoptotic function in PAMs. The interaction between MARCO and GP5 may contribute to the intensified apoptosis induced by GP5. Additionally, the inhibition of apoptosis during PRRSV infection weakened the antiviral function of MARCO, suggesting that MARCO inhibits PRRSV through the regulation of apoptosis. Taken together, the results of this study reveal a novel antiviral mechanism of MARCO and suggest a molecular basis for the potential development of therapeutics against PRRSV. IMPORTANCE Porcine reproductive and respiratory syndrome virus (PRRSV) has been one of the most serious threats to the global swine industry. Glycoprotein 5 (GP5) exposed on the surface of PRRSV virions is a major glycoprotein, and it is involved in viral entry into host cells. A macrophage receptor with collagenous structure (MARCO), which is a member of the scavenger receptor family, was identified to interact with PRRSV GP5 in a DUALmembrane yeast two-hybrid screening. Further investigation demonstrated that MARCO may not serve as a potential receptor to mediate PRRSV entry. Instead, MARCO was a host restriction factor for the virus, and the N-terminal cytoplasmic region of MARCO was responsible for its anti-PRRSV effect. Mechanistically, MARCO inhibited PRRSV infection through intensifying virus-induced apoptosis in PAMs. The interaction between MARCO and GP5 may contribute to GP5-induced apoptosis. Our work reveals a novel antiviral mechanism of MARCO and advances the development of control strategies for the virus.

Porcine reproductive and respiratory syndrome virus non-structural protein 4 cleaves guanylate-binding protein 1 via its cysteine proteinase activity to antagonize GBP1 antiviral effect

Porcine reproductive and respiratory syndrome (PRRS) is a highly infectious disease caused by PRRS virus (PRRSV) that causes great economic losses to the swine industry worldwide. PRRSV has been recognized to modulate the host antiviral interferon (IFN) response and downstream interferon-stimulated gene expression to intercept the antiviral effect of host cells. Guanylate-binding proteins (GBPs) are IFN-inducible GTPases that exert broad antiviral activity against several DNA and RNA viruses, of which GBP1 is considered to play a pivotal role. However, the role of GBP1 in PRRSV replication remains unknown. The present study showed that overexpression of GBP1 notably inhibited PRRSV infection, while the knockdown of endogenous GBP1 promoted PRRSV infection. The K51 and R48 residues of GBP1 were essential for the suppression of PRRSV replication. Furthermore, GBP1 abrogated PRRSV replication by disrupting normal fibrous actin structures, which was indispensable for effective PRRSV replication. By using a co-immunoprecipitation assay, we found that GBP1 interacted with the non-structural protein 4 (nsp4) protein of PRRSV, and this interaction was mapped to the N-terminal globular GTPase domain of GBP1 and amino acids 1–69 of nsp4. PRRSV infection significantly downregulated GBP1 protein expression in Marc-145 cells, and nsp4, a 3C-like serine proteinase, was responsible for GBP1 cleavage, and the cleaved site was located at glutamic acid 338 of GBP1. Additionally, the anti-PRRSV activity of GBP1 was antagonized by nsp4. Taken together, these findings expand our understanding of the sophisticated interaction between PRRSV and host cells, PRRSV pathogenesis and its mechanisms of evading the host immune response.

A synthetic toll-like receptor 7 agonist inhibits porcine reproductive and respiratory syndrome virus replication in piglets

Toll-like receptor 7 (TLR7) agonists have been shown to exert therapeutic effects against several viruses. However, antiviral potential of TLR7 agonist in inhibiting porcine reproductive and respiratory syndrome virus (PRRSV) infection has not been assessed in vivo. In our previous study, a synthetic TLR7 agonist, SZU101, was confirmed to inhibit PRRSV infection of porcine alveolar macrophages (PAMs). Here, antiviral effects of SZU101 were evaluated in PRRSV-challenged piglets based on assessments of rectal temperature, viremia, gross and microscopic lung lesions, PRRSV-specific antibodies, PRRSV-specific lymphocyte proliferation and serum IFN-β level. Our results revealed that SZU101 treatment alleviated PRRSV-induced rectal temperature spikes, pulmonary pathologic changes, and serum viral load. Meanwhile, administration of SZU101 led to increased proliferation of PRRSV-specific lymphocytes and serum IFN-β levels, but did not enhance PRRSV-specific antibody production. These results demonstrate that SZU101 has potential as a therapeutic treatment for PRRS.

MiR-142-3p suppresses porcine reproductive and respiratory syndrome virus (PRRSV) infection by directly targeting Rac1

Porcine reproductive and respiratory syndrome (PRRS) caused by PRRS virus (PRRSV) has been recognized as one of the severest epidemics in pigs worldwide. microRNAs (miRNAs) play important roles in a variety of biological processes, including cell differentiation, proliferation and death, as well as viral infections and antiviral immune responses. In this study, we found that miR-142–3p was expressed lower in cells susceptible to PRRSV infection than in cells less or no permissive to PRRSV infection. Subsequently, we showed that overexpression of miR-142–3p remarkably inhibited PRRSV infection in PAMs, while blockage of endogenous miR-142–3p significantly enhanced PRRSV replication. Then, we demonstrated that miR-142–3p directly targeted Ras-related C3 botulinum toxin substrate 1 (Rac1), a member of Rho GTPases family, by using luciferase reporter assay and UV cross-linking and immunoprecipitation (CLIP) assay. Importantly, we verified that miR-142–3p inhibited PRRSV entry into PAMs and accordingly suppressed PRRSV infection by downregulating Rac1 expression. These findings reveal an important role of miR-142–3p in modulating PRRSV infection and provide us with some ideas for developing novel antiviral therapy against PRRSV infection.

Exostosin glycosyltransferase 1 reduces porcine reproductive and respiratory syndrome virus infection through proteasomal degradation of nsp3 and nsp5

Porcine reproductive and respiratory syndrome virus (PRRSV) continues to be a serious threat to the swine industry worldwide. Exostosin glycosyltransferase 1 (EXT1), an enzyme involved in the biosynthesis of heparin sulfate, has also been reported to be a host factor essential for a wide variety of pathogens. However, the role of EXT1 in PRRSV infection remains uncharted. Here, we identified that PRRSV infection caused an increase of EXT1 expression. EXT1 knockdown promoted virus infection, whereas its overexpression inhibited virus infection, suggesting an inhibitory function of EXT1 to PRRSV infection. We found that EXT1 had no effects on the attachment, internalization, or release of PRRSV but did restrict viral RNA replication. EXT1 was determined to interact with viral nonstructural protein 3 (nsp3) and nsp5 via its N-terminal cytoplasmic tail and to enhance K48-linked polyubiquitination of these two nsps to promote their degradation. Furthermore, the C-terminal glycosyltransferase activity domain of EXT1 was necessary for nsp3 and nsp5 degradation. We also found that EXT2, a EXT1 homolog, interacted with EXT1 and inhibited PRRSV infection. Similarly, EXT1 effectively restricted porcine epidemic diarrhea virus and porcine enteric alphacoronavirus infection in Vero cells. Taken together, this study reveals that EXT1 may serve as a broad-spectrum host restriction factor and suggests a molecular basis for the potential development of therapeutics against PRRSV infection.

Nanobody Nb6 fused with porcine IgG Fc as the delivering tag to inhibit porcine reproductive and respiratory syndrome virus replication in porcine alveolar macrophages

Porcine reproductive and respiratory syndrome virus (PRRSV) is a highly contagious virus that has led to enormous economic loss worldwide because of ineffective prevention and treatment. In view of their minimized size, high target specificity and affinity, nanobodies have been extensively investigated as diagnostic tools and treatments of many diseases. Previously, a PRRSV Nsp9-specific nanobody (Nb6) was identified as a PRRSV replication inhibitor. When it was fused with cell-penetrating peptide (CPP) TAT, Nb6-TAT could enter the cells for PRRSV suppression. However, delivery of molecules by CPP lack cell specificity and have a short duration of action. PRRSV has a tropism for monocyte/macrophage lineage, which expresses high levels of Fcγ receptors. Herein, we designed a nanobody containing porcine IgG Fc (Fcγ) to inhibit PRRSV replication in PRRSV permissive cells. Fcγ fused Nb6 chimeric antibody (Nb6-pFc) was assembled into a dimer with interchain disulfide bonds and expressed in a Pichia pastoris system. The results show that Nb6-pFc exhibits a well-binding ability to recombinant Nsp9 or PRRSV-encoded Nsp9 and that FcγR-mediated endocytosis of Nb6-pFc into porcine alveolar macrophages (PAM) was in a dose-dependent manner. Nb6-pFc can inhibit PRRSV infection efficiently not only by binding with Nsp9 but also by upregulating proinflammatory cytokine production in PAM. Together, this study proposes the design of a porcine IgG Fc-fused nanobody that can enter PRRSV susceptible PAM via FcγR-mediated endocytosis and inhibit PRRSV replication. This research reveals that nanobody-Fcγ chimeric antibodies might be effective for the control and prevention of monocyte/macrophage lineage susceptible pathogeneses.

Small molecule screening identified cepharanthine as an inhibitor of porcine reproductive and respiratory syndrome virus infection in vitro by suppressing integrins/ILK/RACK1/PKCα/NF-κB signalling axis

Porcine Reproductive and Respiratory Syndrome (PRRS) is a devastating disease among the most notorious threats to the swine industry worldwide and is characterized by respiratory distress and reproductive failure. Highly evolving porcine reproductive and respiratory syndrome virus (PRRSV) strains with complicated genetic diversity make the current vaccination strategy far from cost-effective and thus urge identification of potent lead candidates to provide prevention and treatment approaches. From an in vitro small molecule screening with the TargetMol Natural Compound Library comprising 623 small molecules, cytopathic effect (CPE) observations and RT-qPCR analysis of viral ORF7 gene expression identified cepharanthine (CEP) to be one of the most protent inhibitors of PRRSV infection in Marc-145 cells. When compared with tilmicosin, which is one of the most commonly used antibiotics in swine industry to inhibit infections, CEP more prominently inhibited PRRSV infection represented by both RNA and protein levels, further reduced the TCID50 by 5.6 times, and thus more remarkably protected Marc-145 cells against PRRSV infection. Mechanistically, western blot analyses of the Marc-145 cells and the porcine alveolar macrophages (PAMs) with or without CEP treatment and PRRSV infection at various time points revealed that CEP can inhibit the expression of integrins β1 and β3, integrin-linked kinase (ILK), RACK1 and PKCα, leading to NF-κB suppression and consequent alleviation of PRRSV infection. Collectively, our small molecule screening identified cepharanthine as an inhibitor of PRRSV infection in vitro by suppressing Integrins/ILK/RACK1/PKCα/NF-κB signalling axis, which may enlighten the deeper understanding of the molecular pathogenesis of PRRSV infection and more importantly, suggested CEP as a potential promising drug for PRRS control in veterinary clinics.

Antiviral Mechanism of Tea Polyphenols against Porcine Reproductive and Respiratory Syndrome Virus.

Neither inactivated nor attenuated vaccines can effectively prevent and control the infection and spread of porcine reproductive and respiratory syndrome virus (PRRSV). Therefore, it is necessary to broaden new horizons and to conceive effective preventive strategies. The main components of Tea polyphenol (TPP) are catechins and their derivatives. TPP has many physiological activities and has certain antiviral and antifungal effects. However, whether TPP shows anti-PRRSV activity remains unclear. We found that TPP effectively inhibited PRRSV infection in Marc-145 cells by suppressing the stages of viral attachment, internalization, replication, and release. TPP exhibited a potent anti-PRRSV effect regardless of pre-treatment or post-treatment. In addition, we demonstrated that TPP restrained PRRSV-induced p65 entry into the nucleus to suppress the activation of the NF-κB signaling pathway, which ultimately leads to the inhibition of the expression of inflammatory cytokines. Furthermore, TPP limited the synthesis of viral non-structural protein 2 (nsp2), the core component of viral replication transcription complexes, which may contribute to the inhibition of viral RNA replication. TPP has the potential to develop into an effective antiviral agent for PRRSV prevention and control in the future.

Downregulation of miR-218 by porcine reproductive and respiratory syndrome virus facilitates viral replication via inhibition of type I interferon responses

Porcine reproductive and respiratory syndrome virus (PRRSV) is a devastating pathogen in the swine industry worldwide. miRNAs are reported to be involved in virus–host interaction. Here, we used high-throughput sequencing and miRNA inhibitors to screen possible miRNAs that can inhibit PRRSV infection on its target cell, porcine alveolar macrophages. We observed that miR-218 was downregulated upon virus infection, and knockdown of miR-218 significantly enhanced PRRSV replication. Overexpression of miR-218 resulted in a decrease in PRRSV replication, and this overexpression did not alter viral genomic RNA levels, but rather increased antiviral interferon signaling. Further analysis revealed that miR-218 regulated PRRSV replication by directly targeting porcine suppressor of cytokine signaling 3 (SOCS3), a JAK2 kinase inhibitor. Knockdown of the endogenous SOCS3 expression led to augmentation of type I interferon genes and resulted in decreased PRRSV replication, and vice versa. During PRRSV infection in vivo and in vitro, cellular miR-218 expression was downregulated and SOCS3 expression was upregulated, further supporting the inverse correlation between miR-218 and SOCS3 expression. The data on SOCS3 depletion in combination with miR-218 inhibition suggested that the antiviral activity of miR-218 required the SOCS3-mediated signaling pathway. Similarly, miR-218 negatively regulated PRRSV replication in Marc-145 cells, as well as the replication of porcine epidemic diarrhea virus and transmissible gastroenteritis virus in Vero and ST cells respectively. Taken together, these results demonstrate that PRRSV-induced miR-218 downregulation serves to inhibit the type I interferon response and may provide a novel therapeutic target for treatment of PRRSV and other viral infections.

Integrin β3, a RACK1 interacting protein, is critical for porcine reproductive and respiratory syndrome virus infection and NF-κB activation in Marc-145 cells

Porcine reproductive and respiratory syndrome virus (PRRSV) is the pathogen of porcine reproductive and respiratory syndrome (PRRS), which is one of the most economically harmful diseases in modern pig production worldwide. Receptor of activated protein C kinase 1 (RACK1) was previously shown to be indispensable for the PRRSV replication and NF-κB activation in Marc-145 cells. Here we identified a membrane protein, integrin β3 (ITGB3), as a RACK1-interacting protein. PRRSV infection in Marc-145 cells upregulated the ITGB3 expression. Abrogation of ITGB3 by siRNA knockdown or antibody blocking inhibited PRRSV infection and NF-κB activation, while on the other hand, overexpression of ITGB3 enhanced PRRSV infection and NF-κB activation. Furthermore, inhibition of ITGB3 alleviated the cytopathic effects and reduced the TCID50 titer in Marc-145 cells. We also showed that RACK1 and ITGB3 were NF-κB target genes during PRRSV infection, and that they regulated each other. Our data indicated that ITGB3, presumably as a co-receptor, played an imperative role during PRRSV infection and NF-κB activation in Marc-145 cells. PRRSV infection activates a positive feedback loop involving the activation of NF-κB and upregulation of ITGB3 and RACK1 in Marc-145 cells. The findings would advance our elaborated understanding of the molecular host–pathogen interaction mechanisms underlying PRRSV infection in swine and suggest ITGB3 and NF-κB signaling pathway as potential therapeutic targets for PRRS control.

Lipopolysaccharide Downregulates CD163 Expression to Inhibit PRRSV Infection via TLR4-NF-κB Pathway.

Porcine reproductive and respiratory syndrome virus (PRRSV) has been recognized to induce proinflammatory cytokine production and modulate the host interferon (IFN) system. Proinflammatory cytokines and type I IFNs contribute to the prevention of viral infection. Lipopolysaccharide (LPS), a specific agonist to Toll-like receptor 4 (TLR4), provokes signal transduction and activates immune response in vivo and in vitro. Here we identified LPS inhibited PRRSV infection in porcine alveolar macrophages (PAMs) and in Marc-145 cells. To investigate the possible mechanism, we found TLR4-NF-κB pathway was obviously activated in LPS-treated PAMs at the early stage of PRRSV infection. As a result, the expression of proinflammatory cytokines was strongly induced following LPS and PRRSV co-treatment. Due to the enhanced proinflammatory response, CD163 expression was significantly reduced and a disintegrin and metalloproteinase 17 was activated, which promotes the cleavage of membrane CD163. Ultimately, CD163 down-regulation led to the suppression of PRRSV replication. Our data demonstrate that LPS has an impact on PRRSV infection via inflammation response, which provides a new insight of inflammation-mediated antiviral immunity and a new strategy to control PRRSV infection.

TRIM59 inhibits porcine reproductive and respiratory syndrome virus (PRRSV)-2 replication in vitro

Porcine reproductive and respiratory syndrome (PRRS) caused by PRRS virus (PRRSV), has ranked among the major economically significant pathogen in the global swine industry. The PRRSV nonstructural protein (nsp)11 possesses nidovirus endoribonuclease (NendoU) activity, which is important for virus replication and suppression of the host innate immunity system. Recent proteomic study found that TRIM59 (tripartite motif-containing 59) interacted with the nsp11, albeit the exact role it plays in PRRSV infection remains enigmatic. Herein, we first confirmed the interaction between nsp11 and TRIM59 in co-transfected HEK293T cells and PRRSV-infected pulmonary alveolar macrophages (PAMs). The interacting domains between TRIM59 and nsp11 were further determined to be the N-terminal RING domain in TRIM59 and the C-terminal NendoU domain in nsp11, respectively. Moreover, we reported that overexpression of TRIM59 inhibited PRRSV infection in Marc-145 cells. Conversely, small interfering RNA (siRNA) depletion of TRIM59 resulted in enhanced production of PRRSV in PAMs. Together, these data add TRIM59 as a crucial antiviral component against PRRSV and provide new insights for development of new compounds to reduce PRRSV infection.

Multiple antiviral approaches of (–)-epigallocatechin-3-gallate (EGCG) against porcine reproductive and respiratory syndrome virus infection in vitro

Porcine reproductive and respiratory syndrome virus (PRRSV) remains an economically important pathogen in the global pig industry, effective measures to control the virus are still lacking. (-)-Epigallocatechin-3-gallate (EGCG), the most abundant and bioactive catechin in green tea, has been reported to have antiviral effect against the diverse groups of viruses. In this study, the comprehensive anti-PRRSV activity of EGCG was investigated using various in vitro assays. EGCG effectively inhibited PRRSV infection and replication in porcine alveolar macrophages (PAMs), regardless of whether it was administrated pre- or post-infection, and the cytotoxicity to PAMs was low. Next, anti-PRRSV approaches of EGCG were characterized in MARC-145 cells. EGCG was demonstrated to be able to significantly prevent PRRSV from infecting MARC-145 cells either through blocking of EGCG-treated viruses docking to susceptible cells involving a direct virus-EGCG interaction or by blocking of the infective virus binding to EGCG pre-treated cells via triggering down-regulation of viral receptors and/or related proteins required for infection. In addition, PRRSV replication was suppressed in MARC-145 cells treated with EGCG post-infection, likely because of down-regulation of pro-inflammatory cytokines, such as TNF-α, IL-6 and IL-8. Taken together, these data showed that treatment of primary PAMs with EGCG can inhibit PRRSV infection and revealed that multiple antiviral approaches of EGCG operate in PRRSV-susceptible MARC-145 cells.

Cholesterol 25-hydroxylase is an interferon-inducible factor that protects against porcine reproductive and respiratory syndrome virus infection

Porcine reproductive and respiratory syndrome virus (PRRSV), a single-stranded, positive-sense RNA virus of the Arteriviridae family, has become a global health threat for swine. Cholesterol 25-hydroxylase (CH25H) is an enzyme that catalyzes oxidation of cholesterol to 25-hydroxycholesterol (25HC). The purpose of this study was to explore the antiviral activity of CH25H against PRRSV infection. We found that CH25H was induced by interferon-α and PRRSV in Marc-145 monkey kidney cells. In addition, CH25H and 25HC significantly inhibited PRRSV infection by preventing virus entry. A CH25H mutant that exhibited decreased catalytic activity had an antiviral effect against PRRSV. Treatment with 25HC pre-infection or post-infection significantly inhibited PRRSV infection in primary porcine alveolar macrophages. Our results reveal that CH25H is an interferon-stimulated gene and its production of 25HC can be used as a natural antiviral agent to combat PRRSV infection.

Cholesterol 25-Hydroxylase Inhibits Porcine Reproductive and Respiratory Syndrome Virus Replication through Enzyme Activity-Dependent and -Independent Mechanisms.

Cholesterol 25-hydroxylase (CH25H) has recently been identified as a host restriction factor that exerts antiviral effects by catalyzing the production of 25-hydroxycholesterol (25HC). CH25H can be rapidly induced upon infection with some viruses. Porcine reproductive and respiratory syndrome virus (PRRSV), an arterivirus, has ranked among the most important swine pathogens since it was discovered in the late 1980s. In this study, we found that PRRSV infection significantly downregulated the expression of CH25H in cells by a so-far unknown mechanism, suggesting that CH25H exerts antiviral activity against PRRSV. Indeed, overexpression of CH25H inhibited PRRSV replication, whereas knockdown of CH25H by short interfering RNA (siRNA) promoted PRRSV infection. The anti-PRRSV effect of 25HC operates via inhibition of viral penetration. Interestingly, a CH25H mutant (CH25H-M) lacking hydroxylase activity still inhibited PRRSV infection. Screening using a yeast two-hybrid system followed by coimmunoprecipitation and immunofluorescence colocalization analyses confirmed that both CH25H and CH25H-M interact with the nonstructural protein 1 alpha (nsp1α) of PRRSV. Unexpectedly, the expression of nsp1α decreased following coexpression with CH25H or CH25H-M. Detailed analyses demonstrated that CH25H/CH25H-M could degrade nsp1α through the ubiquitin-proteasome pathway and that site K169 in the nsp1α protein is the key site of ubiquitination. Taken together, our findings demonstrate that CH25H restricts PRRSV replication by targeting viral penetration as well as degrading nsp1α, revealing a novel antiviral mechanism used by CH25H.IMPORTANCE PRRSV has been a continuous threat to the global swine industry, and current vaccines are insufficient to provide sustainable control. CH25H has been found to exert a broad antiviral effect; thus, it is an attractive target for the development of anti-PRRSV drugs. Here, we demonstrate that CH25H is an interferon-stimulated gene that is highly expressed in porcine alveolar macrophages. CH25H exerts its anti-PRRSV effect not only via the production of 25HC to inhibit viral penetration but also by degrading viral protein through the ubiquitin-proteasome pathway, suggesting that CH25H is a candidate for the development of antiviral therapeutics. However, PRRSV infection appears to actively decrease CH25H expression to promote viral replication, highlighting the complex game between PRRSV and its host.