GTPase activity of porcine Mx1 plays a dominant role in inhibiting the N-Nsp9 interaction and thus inhibiting PRRSV replication.

Porcine Mx1 is a type of interferon-induced GTPase that inhibits the replication of certain RNA viruses. However, the antiviral effects and the underlying mechanism of porcine Mx1 for porcine reproductive and respiratory syndrome virus (PRRSV) remain unknown. In this study, we demonstrated that porcine Mx1 could significantly inhibit PRRSV replication in MARC-145 cells. By Mx1 segment analysis, it was indicated that the GTPase domain (68-341aa) was the functional area to inhibit PRRSV replication and that Mx1 interacted with the PRRSV-N protein through the GTPase domain (68-341aa) in the cytoplasm. Amino acid residues K295 and K299 in the G domain of Mx1 were the key sites for Mx1-N interaction while mutant proteins Mx1(K295A) and Mx1(K299A) still partially inhibited PRRSV replication. Furthermore, we found that the GTPase activity of Mx1 was dominant for Mx1 to inhibit PRRSV replication but was not essential for Mx1-N interaction. Finally, mechanistic studies demonstrated that the GTPase activity of Mx1 played a dominant role in inhibiting the N-Nsp9 interaction and that the interaction between Mx1 and N partially inhibited the N-Nsp9 interaction. We propose that the complete anti-PRRSV mechanism of porcine Mx1 contains a two-step process: Mx1 binds to the PRRSV-N protein and subsequently disrupts the N-Nsp9 interaction by a process requiring the GTPase activity of Mx1. Taken together, the results of our experiments describe for the first time a novel mechanism by which porcine Mx1 evolves to inhibit PRRSV replication. IMPORTANCE: Mx1 protein is a key mediator of the interferon-induced antiviral response against a wide range of viruses. How porcine Mx1 affects the replication of porcine reproductive and respiratory syndrome virus (PRRSV) and its biological function has not been studied. Here, we show that Mx1 protein inhibits PRRSV replication by interfering with N-Nsp9 interaction. Furthermore, the GTPase activity of porcine Mx1 plays a dominant role and the Mx1-N interaction plays an assistant role in this interference process. This study uncovers a novel mechanism evolved by porcine Mx1 to exert anti-PRRSV activities.

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.

Porcine reproductive and respiratory syndrome virus (PRRSV) up-regulates IL-8 expression through TAK-1/JNK/AP-1 pathways.

The acute phase of respiratory distress caused by porcine reproductive and respiratory syndrome virus (PRRSV) is likely a consequence of the release of inflammatory cytokines in the lung. IL-8, the main chemokine and activator of neutrophils, might be related to the lung injury upon PRRSV infection. In this study, we showed that PRRSV induced IL-8 expression in vivo and in vitro. Subsequently, we demonstrated that JNK and NF-κB pathways were activated upon PRRSV infection and required for the enhancement of IL-8 expression. We further verified that PRRSV-activated TAK-1 was essential for the activation of JNK and NF-κB pathways and IL-8 expression. Moreover, we revealed an AP-1 binding motif in the cloned porcine IL-8 (pIL-8) promoter, and deletion of this motif abolished the pIL-8 promoter activity. Finally, we found that the JNK-activated AP-1 subunit c-Jun was critical for the up-regulation of IL-8 expression by PRRSV. These data suggest that PRRSV-induced IL-8 production is likely through the TAK-1/JNK/AP-1 pathways.

Overexpression of Histone Deacetylase 6 Enhances Resistance to Porcine Reproductive and Respiratory Syndrome Virus in Pigs.

Porcine reproductive and respiratory syndrome virus (PRRSV) is one of the most economically relevant viral pathogens in pigs and causes substantial losses in the pig industry worldwide each year. At present, PRRSV vaccines do not effectively prevent and control this disease. Consequently, it is necessary to develop new antiviral strategies to compensate for the inefficacy of the available vaccines. Histone deacetylase 6 (HDAC6) is an important member of the histone deacetylase family that is responsible for regulating many important biological processes. Studies have shown that HDAC6 has anti-viral activities during the viral life cycle. However, whether HDAC6 overexpression enhances resistance to PRRSV in pigs remains unknown. In this study, we used a somatic cell cloning method to produce transgenic (TG) pigs that constitutively overexpress porcine HDAC6. These TG pigs showed germ line transmission with continued overexpression of HDAC6. In vitro, virus-challenged porcine alveolar macrophages (PAMs) overexpressed HDAC6, which suppressed viral gene expression and PRRSV production. In vivo, resistance to PRRSV in TG pigs was evaluated by direct or cohabitation mediated infection with a highly pathogenic PRRSV (HP-PRRSV) strain. Compared with non-TG (NTG) siblings, TG pigs showed a significantly lower viral load in the lungs and an extended survival time after infection with HP-PRRSV via intramuscular injection. In the cohabitation study, NTG pigs housed with challenged NTG pigs exhibited significantly worse clinical symptoms than the other three in-contact groups. These results collectively suggest that HDAC6 overexpression enhances resistance to PRRSV infection both in vitro and in vivo. Our findings suggest the potential involvement of HDAC6 in the response to PRRSV, which will facilitate the development of novel therapies for PRRSV.

Porcine reproductive and respiratory syndrome virus envelope (E) protein interacts with mitochondrial proteins and induces apoptosis.

Porcine reproductive and respiratory syndrome virus (PRRSV) causes significant economic losses for the swine industry worldwide. The PRRSV E protein, encoded by ORF 2b, is one of the non-glycosylated minor structural proteins. In this study, we present evidence for the interaction of the E protein with mitochondrial proteins ATP5A (part of ATP synthase complex), prohibitin, and ADP/ATP translocase. We additionally demonstrate partial mitochondrial localization of the E protein in transfected cells. To functionally investigate these interactions, we infected MARC-145 cells with PRRSV or alphavirus replicon particles (VRPs) expressing PRRSV E protein. In infected cells, production of ATP was significantly reduced. The E protein also induced apoptosis by activating caspase-3, which results in PARP cleavage. Taken together, these data suggest that the PRRSV E protein interacts with mitochondrial proteins and induces apoptosis by inhibiting ATP production.

Downregulation of protein kinase PKR activation by porcine reproductive and respiratory syndrome virus at its early stage infection.

The interferon-induced double-strand RNA activated protein kinase (PKR) plays an important role in antiviral response. The objective of this study was to assess the effect of porcine reproductive and respiratory syndrome virus (PRRSV) on PKR activation. Here we report that PRRSV inhibited PKR activation during its early stage infection of primary pulmonary alveolar macrophages (PAMs). PRRSV infection led to lower level of phosphorylated PKR in comparison with mock-infected cells. The PKR inhibition was sustained until 10h post infection in the presence of polyI:C, a synthetic analog of double-stranded RNA activating PKR. PKR-mediated phosphorylation of the eukaryotic translation initiation factor eIF2α was also lower in the PRRSV-infected PAMs during the early stage infection. Interestingly, inactivated PRRSV was capable to inhibit the PKR activation until 6h post infection. This suggests that structural components of PRRSV virions were responsible for the inhibition, although PRRSV replication was needed for longer inhibition. These results indicate that the downregulation of PKR activation during early infection stage should be essential for PRRSV to avoid the antiviral response to initiate replication. This finding contributes to our understanding on PRRSV interaction with host innate immune response and reveal a target for control of PRRSV infection.

Elevated plasma-soluble CD16 levels in porcine reproductive and respiratory syndrome virus-infected pigs: correlation with ADAM17-mediated shedding.

Soluble CD16 (sCD16) is closely correlated with chronic diseases in humans. Here, plasma sCD16 levels in pigs were increased by infection with porcine reproductive and respiratory syndrome virus (PRRSV) but not with porcine epidemic diarrhea virus, porcine circovirus type 2 and pseudorabies virus. Of interest, PRRSV attached to blood neutrophils and reduced surface CD16 expression on neutrophils. In vitro data confirmed that PRRSV caused CD16 shedding in neutrophils. Further analyses revealed that ADAM17 was involved in porcine CD16 shedding. Thus, our findings suggest that increase in sCD16 levels may be an indicator of PRRSV infection.

The involvement of FAK-PI3K-AKT-Rac1 pathway in porcine reproductive and respiratory syndrome virus entry.

CD163 and sialoadhesin had been reported as the two receptors for porcine reproductive and respiratory syndrome virus (PRRSV) infection. The signaling pathway activated by PRRSV entry was seldom reported. In our studies, we demonstrated that PRRSV entry triggers FAK, PI3K, AKT and Rac1 activation. The signaling pathway FAK-PI3K-AKT-Rac1 is essential for PRRSV entry. Blocking FAK by PF573228 attenuates the activation of PI3K, AKT, Rac1 and the cytoskeleton remodeling induced by virus entry. Inhibitors to FAK, PI3K, AKT and Rac1 can significantly inhibit the virus entry. In conclusion, our observations reveal that PRRSV triggers the activation of FAK-PI3K-AKT-Rac1 signaling pathway to facilitate its entry into cells.

Porcine reproductive and respiratory syndrome virus counteracts the porcine intrinsic virus restriction factors-IFITM1 and Tetherin in MARC-145 cells.

Porcine reproductive and respiratory syndrome virus (PRRSV) has been recognized to modulate the innate immune response of host. However, little is known about the effects of PRRSV infection on porcine intrinsic virus-restriction factors. This study presents the first demonstration that the nonstructural protein 3 (Nsp3) or envelope (E) protein of PRRSV interacted with porcine intrinsic virus-restriction factor IFITM1 or Tetherin. Next, in PRRSV-infected MARC-145 cells and the transfected cells with the IFITM1- or Tetherin-expressing plasmid, IFITM1 was shown to be mainly distributed perinuclear, and Tetherin was proposed to be partially removed away from cell surface. Moreover, the overexpression of IFITM1 and Tetherin were shown to have no obvious effects on the replication of PRRSV in MARC-145 cells. The Nsp3 of PRRSV was demonstrated to induce the proteasome-dependent degradation of IFITM1 upon PRRSV infection. These findings suggest that PRRSV might counteract the antiviral functions of IFITM1 and Tetherin by the interaction of the Nsp3 with IFITM1 and the E protein with Tetherin, providing a novel clue for exploring possible mechanisms associated with the evasion of PRRSV from immune recognition of host.

Modulation of CD163 expression by metalloprotease ADAM17 regulates porcine reproductive and respiratory syndrome virus entry.

UNLABELLED: As a consequence of their effects on ectodomain shedding, members of the A disintegrin and metalloprotease (ADAM) family have been implicated in the control of various cellular processes. Although ADAM family members are also involved in cancer, inflammation, and other pathologies, it is unclear whether they affect porcine reproductive and respiratory syndrome virus (PRRSV) infection. Here, we demonstrate for the first time that inhibition of ADAM17 enhances PRRSV entry in Marc-145 and porcine alveolar macrophages (PAMs). We also demonstrate that the inhibition of ADAM17 upregulates membrane CD163 expression, a putative PRRSV receptor that is exogenously expressed in BHK-21 and endogenously expressed in Marc-145 and PAMs. Furthermore, overexpression of ADAM17 induced downregulation of CD163 expression and a reduction in PRRSV infection, whereas ablation of ADAM17 expression using specific small interfering RNA resulted in upregulation of CD163 expression with a corresponding increase in PRRSV infection. These ADAM17-mediated effects were confirmed with PRRSV nonpermissive BHK-21 cells transfected with CD163 cDNA. Overall, these findings indicate that ADAM17 downregulates CD163 expression and hinders PRRSV entry. Hence, downregulation of ADAM17 particular substrates may be an additional component of the anti-infection defenses. IMPORTANCE: ADAM17 is one of the important membrane-associated metalloproteases that mediate various cellular events, as well as inflammation, cancer, and other pathologies. Here, we investigate for the first time the role of the metalloprotease ADAM17 in PRRSV infection. By using inhibitor and genetic modification methods, we demonstrate that ADAM17 negatively regulate PRRSV entry by regulating its substrate(s). More specifically, ADAM 17 mediates the downregulation of the PRRSV cellular receptor CD163. The reduction in CD163 expression represents another component of the anti-infection response initiated by ADAM17.

Role of phosphatidylinositol 3-kinase (PI3K) and Akt1 kinase in porcine reproductive and respiratory syndrome virus (PRRSV) replication.

We have previously reported that inhibition of phosphatidylinositol 3-kinase (PI3K) reduces porcine reproductive and respiratory syndrome (PRRSV) replication. Here, we further investigate the mechanism by which PI3K inhibition affects virus replication and the role of Akt1 kinase in virus replication. We found that PI3K inhibition reduced viral gene transcription by approximately 1.5-fold. Accordingly, viral protein synthesis was significantly reduced by PI3K inhibition. Interestingly, cells overexpressing the dominant negative mutant Akt1 exhibited a significant reduction in viral gene transcription compared to cells overexpressing the constitutively active Akt1. Viral protein synthesis was also enhanced in cells overexpressing the constitutively active Akt1. Overall, our data show that both PI3K and Akt1 play a role in viral gene expression, leading to an increase in virus replication.

Blood antioxidant enzymes (SOD, GPX), biochemical and haematological parameters in pigs naturally infected with porcine reproductive and respiratory syndrome virus.

Porcine reproductive and respiratory syndrome (PRRS) has become one of the most economically important diseases for the swine industry worldwide. The objective of the study was to determine selected blood antioxidant enzymes (glutathione peroxidase (GPX), superoxide dismutase (SOD)), biochemical and haematological parameters in PRRS positive and negative pigs of three different categories, mainly to test oxidative stress hypothesis in pigs naturally infected with PRRS virus. Ninety PRRS positive and 90 PRRS negative pigs were included in the study. The presence of PRRS was confirmed by serological detection of antibodies against PRRS virus (PRRSV) and detection of PRRS viral RNA by RT-PCR. Pigs were further divided into three groups of 30: piglets just before weaning (weaners), fatteners and finishers. Blood samples for determining selected blood parameters were collected from the vena cava cranialis. Significantly (P < 0.05) higher activities of SOD in weaners and fatteners and of GPX in weaners were determined in PRRS positive pigs than in corresponding groups of PRRS negative pigs. In contrast, significantly (P < 0.05) lower GPX activity was observed in finishers of PRRS positive pigs than in the corresponding group of PRRS negative pigs. Concentrations of serum total protein in PRRS positive weaners and fatteners were significantly (P < 0.05) higher than those found in PRRS negative pigs. Leukopenia was observed in all three groups of PRRS positive pigs. It has been demonstrated, for the first time, that oxidative stress might be increased in PRRSV naturally infected pigs, especially in weaners.

Porcine reproductive and respiratory syndrome virus (PRRSV) infection activates chemokine RANTES in MARC-145 cells.

RANTES (regulated upon activation, normally T-cell expressed and presumably secreted), a CC chemokine, plays an important role in the inflammatory response associated with viral infections. Previous studies have demonstrated that infection with porcine reproductive and respiratory syndrome virus (PRRSV) induces RANTES transcription in vitro and in vivo. However, the molecular mechanism remains unclear. In this study, real-time RT-PCR and promoter luciferase reporter assays showed that PRRSV infection significantly upregulates RANTES gene transcription in both a time- and dose-dependent manner and this induction requires viral replication in MARC-145 cells. Promoter mutagenesis experiments found that the nuclear factor (NF-κB) binding sites play an important role in PRRSV-induced RANTES transcription, while the interferon-stimulated responsive element (ISRE) site is not essential. PRRSV-induced RANTES transcription was dramatically inhibited by administration of a dominant-negative mutant of IκB kinase alpha (mIκBα), NF-κB inhibitor BAY11-7082 or ERK1/2 inhibitor U0126. In addition, the use of dominant-negative mutants of various adaptor molecules of the Toll-like receptor (TLR) or RIG-I-like receptor (RLR) signaling pathways demonstrated that PRRSV upregulated RANTES transcription is dependent on myeloid differentiation primary response gene 88 (MyD88), TIR domain-containing adaptor inducing IFN-ß (TRIF) and TNF receptor-associated factor 6 (TRAF6), indicating that the TLR signaling pathway is involved in PRRSV-induced RANTES activation.

Porcine reproductive and respiratory syndrome virus replication is suppressed by inhibition of the extracellular signal-regulated kinase (ERK) signaling pathway.

Viruses are known to develop the ability to manipulate a variety of host cell signal transduction pathways in order to facilitate successful virus survival. However, to date, little is known about the intracellular signaling mechanisms involved in porcine reproductive and respiratory syndrome virus (PRRSV) replication. The extracellular signal-regulated kinase (ERK) pathway that transduces signals to modulate a wide range of cellular functions has been shown to regulate a number of viral infections. The present study therefore aimed to determine the role of this pathway during PRRSV infection in porcine alveolar macrophages. We found that the PRRSV infection induces early robust but transient activation of ERK1/2 by 6h postinfection and thereafter the progressive decrease of its phosphorylation. However, the maximal induction of phosphorylated ERK1/2 seen at 6h postinfection was inconsistent with synthesis of a viral nucleocapsid protein that was first evident by 12h postinfection. These results indicate that ERK1/2 activation is mediated independently of viral gene expression during PRRSV replication. Notably, infection with UV-irradiated, inactivated virus, which is capable of receptor binding and internalization but prevents viral gene synthesis, was sufficient to trigger ERK1/2 phosphorylation, suggesting that the viral entry process may be responsible for early ERK activation. Treatment of cells with U0126, a selective ERK1/2 inhibitor, markedly diminished PRRSV infection and its inhibitory effect on PRRSV replication was exerted at the early stage in virus infection. Furthermore, inhibition of ERK activation resulted in significant suppression of subgenomic RNA transcription, viral protein translation, and progeny virus production. Taken together, the findings in this study suggest that the ERK signaling pathway plays an important role in postentry steps of the PRRSV replication cycle and beneficially contributes to viral infection.

Increased proteolytic activity and matrix metalloprotease expression in lungs during infection by porcine reproductive and respiratory syndrome virus.

The local increase in the secretion of extracellular proteases, allowing cleavage of the extracellular matrix and thereby facilitating the infiltration of T cells, monocytes and neutrophils, is a hallmark of chronic inflammation and autoimmunity. In pulmonary genetic diseases, such as emphysema and cystic fibrosis, proteases can also favour the development of local immunodeficiency by degrading key regulators of the immune response, such as CD4, CD8, IgG, ICAM-1 and C3b receptors. Since several infectious agents can give rise to severe pulmonary disorders associated with opportunistic infections, we sought to determine whether an increase in proteolytic activity occurred during infection with porcine reproductive and respiratory syndrome virus (PRRSV), the causative agent of a new disease in swine characterized by severe respiratory problems in young pigs. Piglets were infected with the virus and bronchoalveolar lavages were collected at various times post-infection to measure the net proteolytic activity. It was shown that PRRSV infection leads to a significant increase in proteolytic activity in pulmonary fluids. Maximal activity was found at 7 and 14 days post-infection, with a return towards normal levels at day 42. Zymographic analyses showed a significant increase in the secretion of matrix metalloproteases (MMPs) 2 and 9, two enzymes involved in tissue remodelling. Histological analyses showed a correlation between the increase in proteolytic activity and the appearance of lesions that were characterized by massive lymphomononuclear cell infiltration. These results suggest that virus infection of the lungs can lead to a transient increase in proteolytic activity that could favour opportunistic infection.

Inducible nitric oxide synthase expression in porcine immune cells.

Porcine immune cells were examined for the ability to produce inducible nitric oxide synthase following in vitro or in vivo stimulation. Enzyme activity and product formation were not detected following stimulation of porcine peripheral blood mononuclear cells (PBMC), splenocytes, or alveolar macrophages with a combination of ConA and lipopolysaccharide (LPS) or recombinant porcine interferon gamma and LPS. In vitro engulfment of Haemophilus parasuis by macrophages also failed to induce inducible nitric oxide synthase (iNOS) activity or nitrite formation. Swine Herpes Virus infection led to a small but significant increase in level of nitrite detected in lung lavage fluid, whereas the infection of pigs with Porcine Respiratory and Reproductive Syndrome Virus did not alter the lavage fluid nitrite levels. iNOS mRNA was detected in both stimulated and unstimulated porcine immune cells and in macrophages from both control and infected animals suggesting that it is constitutively expressed with little or no upregulation following cellular stimulation. The results presented in this paper indicate that the reactive nitrogen intermediate pathway is not an vital innate immune response in the pig.