Porcine Reproductive And Respiratory Syndrome Virus Nonstructural Protein Research Articles

Porcine reproductive and respiratory syndrome virus degrades TANK-binding kinase 1 via chaperon-mediated autophagy to suppress type I interferon production and facilitate viral proliferation

Porcine reproductive and respiratory syndrome virus (PRRSV) has led to significant economic losses in the global swine industry. Type I interferon (IFN-I) plays a crucial role in the host’s resistance to PRRSV infection. Despite extensive research showing that PRRSV employs multiple strategies to antagonise IFN-I induction, the underlying mechanisms remain to be fully elucidated. In this study, we have discovered that PRRSV inhibits the production of IFN-I by degrading TANK-binding kinase 1 (TBK1) through chaperon-mediated autophagy (CMA). From a mechanistic standpoint, PRRSV nonstructural protein 2 (Nsp2) increases the interaction between the heat shock protein member 8 (HSPA8) and TBK1. This interaction leads to the translocation of TBK1 into lysosomes for degradation, mediated by lysosomal-associated membrane protein 2A (LAMP2A). As a result, the downstream activation of IFN regulatory factor 3 (IRF3) and the production of IFN-I are hindered. Together, these results reveal a new mechanism by which PRRSV suppresses host innate immunity and contribute to the development of new antiviral strategies against the virus.

PRRSV non-structural protein 5 inhibits antiviral innate immunity by degrading multiple proteins of RLR signaling pathway through FAM134B-mediated ER-phagy.

Viruses employ various evasion strategies to establish prolonged infection, with evasion of innate immunity being particularly crucial. Porcine reproductive and respiratory syndrome virus (PRRSV) is a significant pathogen in swine industry, characterized by reproductive failures in sows and respiratory distress in pigs of all ages, leading to substantial economic losses globally. In this study, we found that the non-structural protein 5 (Nsp5) of PRRSV antagonizes innate immune responses via inhibiting the expression of type I interferon (IFN-I) and IFN-stimulated genes (ISGs), which is achieved by degrading multiple proteins of RIG-I-like receptor (RLR) signaling pathway (RIG-I, MDA5, MAVS, TBK1, IRF3, and IRF7). Furthermore, we showed that PRRSV Nsp5 is located in endoplasmic reticulum (ER), where it promotes accumulation of RLR signaling pathway proteins. Further data demonstrated that Nsp5 activates reticulophagy (ER-phagy), which is responsible for the degradation of RLR signaling pathway proteins and IFN-I production. Mechanistically, Nsp5 interacts with one of the ER-phagy receptor family with sequence similarity 134 member B (FAM134B), promoting the oligomerization of FAM134B. These findings elucidate a novel mechanism by which PRRSV utilizes FAM134B-mediated ER-phagy to elude host antiviral immunity.IMPORTANCEInnate immunity is the first line of host defense against viral infections. Therefore, viruses developed numerous mechanisms to evade the host innate immune responses for their own benefit. PRRSV, one of the most important endemic swine viruses, poses a significant threat to the swine industry worldwide. Here, we demonstrate for the first time that PRRSV utilizes its non-structural protein Nsp5 to degrade multiple proteins of RLR signaling pathways, which play important roles in IFN-I production. Moreover, FAM134B-mediated ER-phagy was further proved to be responsible for the protein's degradation. Our study highlights the critical role of ER-phagy in immune evasion of PRRSV to favor replication and provides new insights into the prevention and control of PRRSV.

PDCD4 restricts PRRSV replication in an eIF4A-dependent manner and is antagonized by the viral nonstructural protein 9.

As obligate parasites, viruses have evolved multiple strategies to evade the host immune defense. Manipulation of the host proteasome system to degrade specific detrimental factors is a common viral countermeasure. To identify host proteins targeted for proteasomal degradation by porcine reproductive and respiratory syndrome virus (PRRSV), we conducted a quantitative proteomics screen of PRRSV-infected Marc-145 cells under the treatment with proteasome inhibitor MG132. The data revealed that the expression levels of programmed cell death 4 (PDCD4) were strongly downregulated by PRRSV and significantly rescued by MG132. Further investigation confirmed that PRRSV infection induced the translocation of PDCD4 from the nucleus to the cytoplasm, and the viral nonstructural protein 9 (Nsp9) promoted PDCD4 proteasomal degradation in the cytoplasm by activating the Akt-mTOR-S6K1 pathway. The C-terminal domain of Nsp9 was responsible for PDCD4 degradation. As for the role of PDCD4 during PRRSV infection, we demonstrated that PDCD4 knockdown favored viral replication, while its overexpression significantly attenuated replication, suggesting that PDCD4 acts as a restriction factor for PRRSV. Mechanistically, we discovered eukaryotic translation initiation factor 4A (eIF4A) was required for PRRSV. PDCD4 interacted with eIF4A through four sites (E249, D253, D414, and D418) within its two MA3 domains, disrupting eIF4A-mediated translation initiation in the 5'-untranslated region of PRRSV, thereby inhibiting PRRSV infection. Together, our study reveals the antiviral function of PDCD4 and the viral strategy to antagonize PDCD4. These results will contribute to our understanding of the immune evasion strategies employed by PRRSV and offer valuable insights for developing new antiviral targets.IMPORTANCEPorcine reproductive and respiratory syndrome virus (PRRSV) infection results in major economic losses in the global swine industry and is difficult to control effectively. Here, using a quantitative proteomics screen, we identified programmed cell death 4 (PDCD4) as a host protein targeted for proteasomal degradation by PRRSV. We demonstrated that PDCD4 restricts PRRSV replication by interacting with eukaryotic translation initiation factor 4A, which is required for translation initiation in the viral 5'-untranslated region. Additionally, four sites within two MA3 domains of PDCD4 are identified to be responsible for its antiviral function. Conversely, PRRSV nonstructural protein 9 promotes PDCD4 proteasomal degradation in the cytoplasm by activating the Akt-mTOR-S6K1 pathway, thus weakening the anti-PRRSV function. Our work unveils PDCD4 as a previously unrecognized host restriction factor for PRRSV and reveals that PRRSV develops countermeasures to overcome PDCD4. This will provide new insights into virus-host interactions and the development of new antiviral targets.

Highly pathogenic PRRSV upregulates IL-13 production through nonstructural protein 9–mediated inhibition of N6-methyladenosine demethylase FTO

Porcine reproductive and respiratory syndrome virus (PRRSV), a highly infectious and causes severe losses in the swine industry by regulating the inflammatory response, inducing tissue damage, suppressing the innate immune response, and promoting persistent infection in hosts. Interleukin-13 (IL-13) is a cytokine that plays a critical role in regulating immune responses and inflammation, particularly in immune-related disorders, certain types of cancer, and numerous bacterial and viral infections; however, the underlying mechanisms of IL-13 regulation during PRRSV infection are not well understood. In this study, we demonstrated that PRRSV infection elevates IL-13 levels in porcine alveolar macrophages (PAMs). PRRSV enhances m6A methylated RNA levels while reducing the expression of fat mass and obesity associated protein (FTO, an m6A demethylase), thereby augmenting IL-13 production. PRRSV nonstructural protein 9 (nsp9) was a key factor for this modulation. Furthermore, we found that the residues Asp567, Tyr586, Leu593, and Asp595 were essential for nsp9 to induce IL-13 production via attenuation of FTO expression. These insights delineate PRRSV nsp9's role in FTO-mediated IL-13 release, advancing our understanding of PRRSV's impact on host immune and inflammatory responses.

Ubiquitin-specific proteinase 1 stabilizes PRRSV nonstructural protein Nsp1β to promote viral replication by regulating K48 ubiquitination.

The porcine reproductive and respiratory syndrome virus (PRRSV) can lead to severe reproductive problems in sows, pneumonia in weaned piglets, and increased mortality, significantly negatively impacting the economy. Post-translational changes are essential for the host-dependent replication and long-term infection of PRRSV. Uncertainty surrounds the function of the ubiquitin network in PRRSV infection. Here, we screened 10 deubiquitinating enzyme inhibitors and found that the ubiquitin-specific proteinase 1 (USP1) inhibitor ML323 significantly inhibited PRRSV replication in vitro. Importantly, we found that USP1 interacts with nonstructural protein 1β (Nsp1β) and deubiquitinates its K48 to increase protein stability, thereby improving PRRSV replication and viral titer. Among them, lysine at position 45 is essential for Nsp1β protein stability. In addition, deficiency of USP1 significantly reduced viral replication. Moreover, ML323 loses antagonism to PRRSV rSD16-K45R. This study reveals the mechanism by which PRRSV recruits the host factor USP1 to promote viral replication, providing a new target for PRRSV defense.IMPORTANCEDeubiquitinating enzymes are critical factors in regulating host innate immunity. The porcine reproductive and respiratory syndrome virus (PRRSV) nonstructural protein 1β (Nsp1β) is essential for producing viral subgenomic mRNA and controlling the host immune system. The host inhibits PRRSV proliferation by ubiquitinating Nsp1β, and conversely, PRRSV recruits the host protein ubiquitin-specific proteinase 1 (USP1) to remove this restriction. Our results demonstrate the binding of USP1 to Nsp1β, revealing a balance of antagonism between PRRSV and the host. Our research identifies a brand-new PRRSV escape mechanism from the immune response.

Tubercidin inhibits PRRSV replication via RIG-I/NF-κB pathways and interrupting viral nsp2 synthesis.

Porcine reproductive and respiratory syndrome (PRRS) is one of the most important swine diseases, which causes huge economic loss worldwide. However, there is no effective therapeutic method for PRRS prevention and control. Here, we found that tubercidin, a naturally extracted adenosine analog, exhibited strong anti-porcine reproductive and respiratory syndrome virus (PRRSV) activity. Mechanically, tubercidin inhibited viral binding, replication, and release. Tubercidin suppressed PRRSV non-structural protein 2 expression, which is important for the formation of replication and transcription complex, leading to the block of viral RNA synthesis and PRRSV replication. Moreover, tubercidin could activate retinoic acid-inducible gene I/nuclear factor kappa-light-chain-enhancer of activated B cell innate immune signaling pathway and increased the expression of interferons and proinflammatory cytokines, which was the other way to inhibit PRRSV replication. Our work evaluated the potential value of tubercidin as an antiviral agent on PRRSV replication and provided a new way to prevent PRRSV replication in vitro.

The Papain-Like Protease of Porcine Reproductive and Respiratory Syndrome Virus Impedes STING Translocation from the Endoplasmic Reticulum to the Golgi Apparatus by Deubiquitinating STIM1.

Stimulator of interferon (IFN) genes (STING) was recently pinpointed as an antiviral innate immune factor during the infection of RNA viruses. Porcine reproductive and respiratory syndrome virus (PRRSV), the swine arterivirus, is an enveloped RNA virus which has evolved many strategies to evade innate immunity. To date, the interactive network between PRRSV and STING remains to be fully established. Herein, we report that STING suppresses PRRSV replication through type I interferon signaling. However, PRRSV impedes STING trafficking from the endoplasmic reticulum (ER) to the Golgi apparatus, leading to the decreased phosphorylation of TANK-binding kinase 1 (TBK1) and interferon regulatory factor 3 (IRF3). Furthermore, PRRSV nonstructural protein 2 (Nsp2) colocalizes with STING, blocks STING translocation, and disrupts the STING-TBK1-IRF3 complex. Mechanistically, PRRSV Nsp2 retains STING at the ER by increasing the level of Ca2+ sensor stromal interaction molecule 1 (STIM1) protein. Functional analysis reveals that PRRSV Nsp2 deubiquitinates STIM1 by virtue of its papain-like protease 2 (PLP2) deubiquitinating (DUB) activity. Finally, we demonstrate that loss of STIM1 is associated with an elevated IFN response and restricts PRRSV replication. This work delineates the relationship between PRRSV infection and STING signaling and the importance of papain-like proteases (PLPs) in interfering in this axis. IMPORTANCE Porcine reproductive and respiratory syndrome virus (PRRSV), a member of the family Arteriviridae, is responsible for reproductive disorders in pregnant sows and respiratory problems in piglets, resulting in huge losses in the swine industry worldwide. Of note, PRRSV infection causes immunosuppression, of which the mechanism is not completely understood. Here, we demonstrate for the first time that STING, a protein typically associated with the antiviral response in DNA viruses, plays a critical role in controlling PRRSV infection. However, PRRSV utilizes its encoded protein Nsp2 to inhibit STING activity by blocking its translocation from the ER to the Golgi apparatus. In particular, Nsp2 retains STING at the ER by interacting with and further deubiquitinating STIM1. For this process, the activity of the viral PLP2 DUB enzyme is indispensable. The study describes a novel mechanism by which PLP2 plays a critical role in suppressing the innate immune response against arteriviruses and potentially other viruses that encode similar proteases.

PRRSV nonstructural protein 11 degrades swine ISG15 by its endoribonuclease activity to antagonize antiviral immune response

Porcine reproductive and respiratory syndrome virus (PRRSV) is an enveloped positive-stranded RNA virus which causes serious economic losses to pig industry worldwide. Type I IFN induces expression of interferon-stimulated genes 15 (ISG15) to inhibit virus replication. To survive in the host, PRRSV has evolved to antagonize the antiviral response of ISGylation. Previous studies have reported that nonstructural protein 2 of PRRSV inhibits the ISGylation and antiviral function of ISG15 depending on its ovarian tumor (OTU) domain/papain-like protease domain (PLP2). However, whether there are other PRRSV proteins inhibiting ISGylation of cellular proteins is less well understood. In this study, we first found that PRRSV Nsp11 decreased ISGylation of cellular proteins. Meanwhile, the expression level of ISG15 was significantly inhibited by Nsp11. Further mechanistic studies demonstrated that the transcription of ISG15 was reduced by endoribonuclease activity of Nsp11. Finally, we found that the Nsp11-induced degradation of ISG15 was partially relied on autophagy-lysosome system. Taken together, PRRSV Nsp11 antagonizes the antiviral response of ISG15 by its endoribonuclease activity to promote PRRSV replication. Our results reveal a novel mechanism that PRRSV inhibits ISGylation of cellular proteins and impairs host innate immune response.

Porcine Reproductive and Respiratory Syndrome Virus nsp5 Induces Incomplete Autophagy by Impairing the Interaction of STX17 and SNAP29.

Porcine reproductive and respiratory syndrome virus (PRRSV) is an economically important pathogen that has devastated the worldwide swine industry for over 30 years. Autophagy is an evolutionarily conserved intracellular lysosomal degradation pathway, and previous studies have documented that PRRSV infection prompts autophagosome accumulation. However, whether PRRSV induces complete or incomplete autophagy remains controversial. Here, we demonstrated that overexpression of PRRSV nonstructural protein 5 (nsp5) induced the accumulation of autophagosomes, and a similar scenario was observed in PRRSV-infected cells. Moreover, both PRRSV infection and nsp5 overexpression activated incomplete autophagy, as evidenced by the blockage of autophagosome-lysosome fusion. Mechanistically, nsp5 overexpression, as well as PRRSV infection, inhibited the interaction of syntaxin 17 (STX17) with synaptosomal-associated protein 29 (SNAP29), two SNARE proteins that mediate autophagosome fusion with lysosomes, to impair the formation of autolysosomes. We further confirmed that nsp5 interacted with STX17, rather than SANP29, and the interacting domains of STX17 were the N-terminal motif and SNARE motif. Taken together, the findings of our study suggest a mechanism by which PRRSV induces incomplete autophagy by blocking autophagosome degradation and provide insights into the development of new therapeutics to combat PRRSV infection. IMPORTANCE A substantial number of viruses have been demonstrated to utilize or hijack autophagy to benefit their replication. In the case of porcine reproductive and respiratory syndrome virus (PRRSV), previous studies have demonstrated the proviral effects of autophagy on PRRSV proliferation. Thus, an investigation of the mechanism by which PRRSV regulates the autophagy processes can provide new insight into viral pathogenesis. Autophagic flux is a dynamic process that consists of autophagosome formation and subsequent lysosomal degradation. However, the exact effect of PRRSV infection on the autophagic flux remains disputed. In this study, we demonstrated that PRRSV infection, as well as PRRSV nsp5 overexpression, inhibited the interaction of STX17 with SNAP29 to impair the fusion of autophagosomes with lysosomes, thereby blocking autophagic flux. This information will help us to understand PRRSV-host interactions and unravel new targets for PRRS prevention and control.

N-Acetyltransferase 9 Inhibits Porcine Reproductive and Respiratory Syndrome Virus Proliferation by N-Terminal Acetylation of the Structural Protein GP5.

Porcine reproductive and respiratory syndrome virus (PRRSV) is a serious threat to the global swine industry. As a typical immunosuppressive virus, PRRSV has developed a variety of complex mechanisms to escape the host innate immunity. In this study, we uncovered a novel immune escape mechanism of PRRSV infection. Here, we demonstrate for the first time that the endoplasmic reticulum (ER)-resident N-acetyltransferase Nat9 is an important host restriction factor for PRRSV infection. Nat9 inhibited PRRSV proliferation in an acetyltransferase activity-dependent manner. Mechanistically, glycoprotein 5 (GP5) of PRRSV was identified as interacting with Nat9 and being N-terminally acetylated by it, which generates a GP5 degradation signal, promoting the K27-linked-ubiquitination degradation of GP5 to decrease virion assembly. Meanwhile, the expression of Nat9 was inhibited during PRRSV infection. In detail, two transcription factors, ETV5 and SP1, were screened out as the key transcription factors binding to the core promoter region of Nat9, and the PRRSV nonstructural protein 1β (Nsp1β), Nsp4, Nsp9, and nucleocapsid (N) proteins were found to interfere significantly with the expression of ETV5 and SP1, thereby regulating the transcription activity of Nat9 and inhibiting the expression of Nat9. The findings suggest that PRRSV decreases the N-terminal acetylation of GP5 to support virion assembly by inhibiting the expression of Nat9. Taken together, our findings showed that PRRSV has developed complex mechanisms to inhibit Nat9 expression and trigger virion assembly. IMPORTANCE To ensure efficient replication, a virus must hijack or regulate multiple host factors for its own benefit. Understanding virus-host interactions and the molecular mechanisms of host resistance to PRRSV infection is necessary to develop effective strategies to control PRRSV. The N-acetyltransferase Nat9 plays important roles during virus infection. Here, we demonstrate that Nat9 exhibits an antiviral effect on PRRSV proliferation. The GP5 protein of PRRSV is targeted specifically by Nat9, which mediates GP5 N-terminal acetylation and degradation via a ubiquitination-dependent proteasomal pathway. However, PRRSV manipulates the transcription factors ETV5 and SP1 to inhibit the expression of Nat9 and promote virion assembly. Thus, we report a novel function of Nat9 in PRRSV infection and elucidate a new mechanism by which PRRSV can escape the host innate immunity, which may provide novel insights for the development of antiviral drugs.

Paraoxonase-1 Facilitates PRRSV Replication by Interacting with Viral Nonstructural Protein-9 and Inhibiting Type I Interferon Pathway.

Paraoxonase-1 (PON1), an esterase with specifically paraoxonase activity, has been proven to be involved in inflammation and infection. Porcine reproductive and respiratory syndrome virus (PRRSV) is still a major concern in pigs and causes severe economic losses to the swine industry worldwide. In this study, the role of PON1 was investigated in porcine alveolar macrophages (PAMs) during PRRSV infection. The results showed that PRRSV replication downregulated PON1, and the knockdown of PON1 significantly decreased PRRSV replication. Similarly, PON1 overexpression could enhance PRRSV replication. Interestingly, we observed that PON1 interacted with PRRSV nonstructural protein 9 (Nsp9), the RNA-dependent RNA polymerase, and the knockdown of PON1 lowered the RNA binding ability of Nsp9, suggesting that PON1 can facilitate Nsp9 function in viral replication. In addition, the knockdown of PON1 expression led to the amplification of type I interferon (IFN) genes and vice versa. In summary, our data demonstrate that PON1 facilitates PRRSV replication by interacting with Nsp9 and inhibiting the type I IFN signaling pathway. Hence, PON1 may be an additional component of the anti-PRRSV defenses.

Research Progress in Porcine Reproductive and Respiratory Syndrome Virus-Host Protein Interactions.

Simple SummaryPRRSV–host protein interactions are of great significance in finding new antiviral targets. We summarize the effects of all the PRRSV structural and nonstructural protein interactions with host proteins and on the protein interactions of the virus itself. These results provide a theoretical foundation for the therapeutic targeting of the intrinsic immune factors of the host protein and exploring viral replication mechanisms.Porcine reproductive and respiratory syndrome (PRRS) is a highly contagious disease caused by porcine reproductive and respiratory syndrome virus (PRRSV), which has been regarded as a persistent challenge for the pig industry in many countries. PRRSV is internalized into host cells by the interaction between PRRSV proteins and cellular receptors. When the virus invades the cells, the host antiviral immune system is quickly activated to suppress the replication of the viruses. To retain fitness and host adaptation, various viruses have evolved multiple elegant strategies to manipulate the host machine and circumvent against the host antiviral responses. Therefore, identification of virus–host interactions is critical for understanding the host defense against viral infections and the pathogenesis of the viral infectious diseases. Most viruses, including PRRSV, interact with host proteins during infection. On the one hand, such interaction promotes the virus from escaping the host immune system to complete its replication. On the other hand, the interactions regulate the host cell immune response to inhibit viral infections. As common antiviral drugs become increasingly inefficient under the pressure of viral selectivity, therapeutic agents targeting the intrinsic immune factors of the host protein are more promising because the host protein has a lower probability of mutation under drug-mediated selective pressure. This review elaborates on the virus–host interactions during PRRSV infection to summarize the pathogenic mechanisms of PRRSV, and we hope this can provide insights for designing effective vaccines or drugs to prevent and control the spread of PRRS.

Molecular characterization of the RNA-protein complex directing −2/−1 programmed ribosomal frameshifting during arterivirus replicase expression

Programmed ribosomal frameshifting (PRF) is a mechanism used by arteriviruses like porcine reproductive and respiratory syndrome virus (PRRSV) to generate multiple proteins from overlapping reading frames within its RNA genome. PRRSV employs −1 PRF directed by RNA secondary and tertiary structures within its viral genome (canonical PRF), as well as a noncanonical −1 and −2 PRF that are stimulated by the interactions of PRRSV nonstructural protein 1β (nsp1β) and host protein poly(C)-binding protein (PCBP) 1 or 2 with the viral genome. Together, nsp1β and one of the PCBPs act as transactivators that bind a C-rich motif near the shift site to stimulate −1 and −2 PRF, thereby enabling the ribosome to generate two frameshift products that are implicated in viral immune evasion. How nsp1β and PCBP associate with the viral RNA genome remains unclear. Here, we describe the purification of the nsp1β:PCBP2:viral RNA complex on a scale sufficient for structural analysis using small-angle X-ray scattering and stochiometric analysis by analytical ultracentrifugation. The proteins associate with the RNA C-rich motif as a 1:1:1 complex. The monomeric form of nsp1β within the complex differs from previously reported homodimer identified by X-ray crystallography. Functional analysis of the complex via mutational analysis combined with RNA-binding assays and cell-based frameshifting reporter assays reveal a number of key residues within nsp1β and PCBP2 that are involved in complex formation and function. Our results suggest that nsp1β and PCBP2 both interact directly with viral RNA during formation of the complex to coordinate this unusual PRF mechanism.

Wnt/β-catenin signaling pathway inhibits porcine reproductive and respiratory syndrome virus replication by enhancing the nuclear factor-κB-dependent innate immune response

The Wnt/β-catenin signaling pathway is an evolutionarily highly conserved signaling pathway related to the replication of various viruses. However, the interaction between the Wnt/β-catenin pathway and porcine reproductive and respiratory syndrome virus (PRRSV) is unknown. In the present study, we showed that PRRSV-infected Marc-145 and PAM cells expressed high levels of c-myc and cyclinD1 mRNA and accumulation of β-catenin in the nucleus. PRRSV nonstructural proteins (Nsps) 1α, 1β, 3, 4, 7, 10, and 12, and proteins encoded by open reading frames (ORFs) 2b, 3, and 5 induced the activation of the Wnt pathway according to TOP/FOP luciferase reporter assay. But, Nsp5 inhibited the activation of the Wnt pathway. Pre-treatment with Wnt3a inhibited PRRSV replication in Marc-145 cells in a dose-dependent manner. Over-expression of β-catenin also inhibited PRRSV replication, while silencing of β-catenin by small hairpin RNA increased its replication in Marc-145 cells. Over-expression of β-catenin increased interferon regulatory factor (IRF)-3 expression and nuclear factor (NF)-κB phosphorylation, NF-κB and interferon-stimulated response element promoter activities, and interferon-β, DExD/H-box helicase 58 (DDX58), interferon-induced protein with tetratricopeptide repeats 1 (IFIT1), interleukin (IL)-1β, tumor necrosis factor (TNF)-α, and IL-8 mRNA expression. Conversely, silencing β-catenin decreased phosphorylated IRF-3 and NF-κB, NF-κB and IFIT1 promoter activities, and IFN-β, DDX58, IFIT1, IL-1β, TNF-α, and IL-8 mRNA levels in Marc-145 cells. Co-immunoprecipitation and immunofluorescence colocalization analyses confirmed that β-catenin interacted with NF-κB in Marc-145 cells. In conclusion, PRRSV infection activates the Wnt/β-catenin signaling pathway via Nsps 1α, 1β, 3, 4, 7, 10, and 12, and proteins encoded by ORFs 2b, 3, and 5. The Wnt/β-catenin pathway then inhibits PRRSV replication by enhancing the NF-κB-dependent innate immune response. These findings further our understanding of the role of the Wnt/β-catenin signaling pathway in regulating PRRSV replication and provide new insights into virus–host interactions.

Porcine reproductive and respiratory syndrome virus Nsp4 cleaves ZAP to antagonize its antiviral activity

Porcine reproductive and respiratory syndrome virus (PRRSV) is one of the most economically important pathogens impacting the global swine industry. PRRSV has been recognized to modulate the host immune response through a number of mechanisms. In our previous study, we found that over-expression of ZAP, a zinc finger antiviral protein of host, could suppress PRRSV replication, but how PRRSV escape the restriction of ZAP under natural conditions was still unknown. In this study, We found PRRSV infection significantly down-regulate the endogenous ZAP protein expression in Marc-145 cells. And PRRSV nonstructural protein 4 (Nsp4), a 3C-like serine proteinase, was screened to be responsible for ZAP reduction. Nsp4 could cleave ZAP, depending on its protease activity. The anti-PRRSV activity of ZAP was antagonized by Nsp4 in Marc-145 cells. In addition, we identified a unique amino acid, serine 180 of Nsp4 was required for efficient degradation of ZAP, and the mutation at residue 180 could decrease the ability of recombinant PRRSV to degrade ZAP. Those findings reveal a manner of PRRSV Nsp4 antagonizing the antiviral activity of ZAP, and shed light on a new strategy evolved by PRRSV to escape the host defense.

Proteasomal degradation of nonstructural protein 12 by RNF114 suppresses porcine reproductive and respiratory syndrome virus replication

Porcine reproductive and respiratory syndrome virus (PRRSV) poses a significant threat to the swine industry worldwide, and the development of effective and sustainable measures to control PRRSV transmission remains a pressing problem. The function of PRRSV nonstructural protein 12 (Nsp12), which might play essential roles in viral replication and production, remains unknown. In this study, we identified a new host-restricted factor, porcine RING finger protein 114 (RNF114), as an inhibitor of PRRSV replication through its degradation of viral Nsp12. Western blot, quantitative real-time polymerase chain reaction, and viral titer assays indicated that RNF114 overexpression suppressed PRRSV replication, whereas RNF114 knockdown increased viral titer and nucleocapsid protein levels. Additionally, we observed that PPRSV infection led to increased RNF114 levels during the middle and late phases of infection in both porcine alveolar macrophages and MARC-145 cells. Moreover, screening of PRRSV Nsps showed that RNF114 interacted with viral Nsp12, and that RNF114-specific anti-PRRSV effects were associated with its ubiquitin ligase activity, which involves K27-linked polyubiquitination and degradation of Nsp12 through a proteasome-dependent pathway. These findings identified RNF114 as a critical regulator of PRRSV replication and offer insights into the roles of Nsp12 in PRRSV pathogenesis.

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.

Porcine Reproductive and Respiratory Syndrome Virus nsp11 Antagonizes Type I Interferon Signaling by Targeting IRF9.

Porcine reproductive and respiratory syndrome virus (PRRSV) is an arterivirus from the Nidovirales order that causes reproductive failure and respiratory disease in pigs and poses a constant threat to the global pig industry. The PRRSV-encoded nonstructural protein 11 (nsp11) is a nidovirus-specific endoribonuclease (NendoU) that is conserved throughout the Arteriviridae and Coronaviridae families. Previously, our research and that of others demonstrated that PRRSV nsp11 inhibits type I interferon (IFN) production through NendoU activity-dependent mechanisms. Here, we found that PRRSV nsp11 also inhibited IFN-stimulated response element (ISRE) promoter activity and subsequent transcription of IFN-stimulated genes (ISGs). Detailed analysis showed that nsp11 targeted interferon regulatory factor 9 (IRF9), but not transducer and activator of transcription 1 (STAT1) or STAT2, key molecules in the type I IFN signaling pathway. Furthermore, the nsp11-IRF9 interaction impaired the formation and nuclear translocation of the transcription factor complex IFN-stimulated gene factor 3 (ISGF3) in both nsp11-overexpressed and PRRSV-infected cells. Importantly, nsp11 mutations (H129A, H144A, and K173A) that ablate NendoU activity or its cell cytotoxicity also interacted with IRF9 and retained the ability to block IFN signaling, indicating that the nsp11-IRF9 interaction is independent of NendoU activity or cell cytotoxicity of nsp11. Taking the results together, our study demonstrated that PRRSV nsp11 antagonizes type I IFN signaling by targeting IRF9 via a NendoU activity-independent mechanism, and this report describes a novel strategy evolved by PRRSV to counteract host innate antiviral responses, revealing a potential new function for PRRSV nsp11 in type I IFN signaling.IMPORTANCE The nidovirus-specific endoribonuclease (NendoU) encoded by PRRSV nonstructural protein 11 (nsp11) is a unique NendoU of nidoviruses that infect vertebrates; thus, it is an attractive target for the development of antinidovirus drugs. Previous studies have revealed that the NendoU of nidoviruses, including porcine reproductive and respiratory syndrome virus (PRRSV) and human coronavirus 229E (HCoV-229E), acts as a type I interferon (IFN) antagonist. Here, for the first time, we demonstrated that overexpression of PRRSV nsp11 also inhibits IFN signaling by targeting the C-terminal interferon regulatory factor (IRF) association domain of IRF9. This interaction impaired the ability of IRF9 to form the transcription factor complex IFN-stimulated gene factor 3 (ISGF3) and to act as a signaling protein of IFN signaling. Collectively, our data identify IRF9 as a natural target of PRRSV NendoU and reveal a novel mechanism evolved by an arterivirus to counteract innate immune signaling.

Nucleotide-binding oligomerization domain-like receptor X1 restricts porcine reproductive and respiratory syndrome virus-2 replication by interacting with viral Nsp9

Porcine reproductive and respiratory syndrome virus (PRRSV) causes one of the most economically important diseases of swine worldwide. Current antiviral strategies provide only limited protection. Nucleotide-binding oligomerization domain-like receptor (NLR) X1 is unique among NLR proteins in its functions as a pro-viral or antiviral factor to different viral infections. To date, the impact of NLRX1 on PRRSV infection remains unclear. In this study, we found that PRRSV infection promoted the expression of NLRX1 gene. In turn, ectopic expression of NLRX1 inhibited PRRSV replication in Marc-145 cells, whereas knockdown of NLRX1 enhanced PRRSV propagation in porcine alveolar macrophages (PAMs). Mechanistically, NLRX1 was revealed to impair intracellular viral subgenomic RNAs accumulation. Finally, Mutagenic analyses indicated that the LRR (leucine-rich repeats) domain of NLRX1 interacted with PRRSV Nonstructural Protein 9 (Nsp9) RdRp (RNA-dependent RNA Polymerase) domain and was necessary for antiviral activity. Thus, our study establishes the role of NLRX1 as a new host restriction factor in PRRSV infection.

E3 ligase ASB8 promotes porcine reproductive and respiratory syndrome virus proliferation by stabilizing the viral Nsp1α protein and degrading host IKKβ kinase

Porcine reproductive and respiratory syndrome virus (PRRSV) can potently suppress type I interferon production and escape from innate immune responses. PRRSV nonstructural protein 1α (Nsp1α) can inhibit IFN-β and NF-κB gene promoter activities, but the precise mechanisms are largely unclear. In this study, we demonstrated that PRRSV Nsp1α interacted with the host E3 ubiquitin ligase ankyrin repeat and SOCS box–containing 8 (ASB8). Specifically, porcine ASB8 promoted K63–linked ubiquitination and increased stability of Nsp1α and boosted PRRSV replication. Moreover, we found that ASB8 was phosphorylated at the N-terminal Ser-31 by host IκB kinase β (IKKβ). In turn, ASB8 facilitated K48–linked ubiquitination and degradation of IKKβ via the ubiquitin–proteasome pathway, resulting in remarkable inhibition of I-kappa-B-alpha (IκBα) and of p65 phosphorylation, consequently suppressing NF-κB activity. Our results provide evidence that PRRSV Nsp1α hijacks up-regulated host ASB8 to escape from intrinsic antiviral immunity.