Seneca valley virus 3C protease blocks EphA2-Mediated mTOR activation to facilitate viral replication.

The Seneca Valley virus (SVV) is a recently discovered porcine pathogen that causes vesicular diseases and poses a significant threat to the pig industry worldwide. Erythropoietin-producing hepatoma receptor A2 (EphA2) is involved in the activation of the AKT/mTOR signaling pathway, which is involved in autophagy. However, the regulatory relationship between SVV and EphA2 remains unclear. In this study, we demonstrated that EphA2 is proteolysed in SVV-infected BHK-21 and PK-15 cells. Overexpression of EphA2 significantly inhibited SVV replication, as evidenced by decreased viral protein expression, viral titers, and viral load, suggesting an antiviral function of EphA2. Subsequently, viral proteins involved in the proteolysis of EphA2 were screened, and the SVV 3C protease (3Cpro) was found to be responsible for this cleavage, depending on its protease activity. However, the protease activity sites of 3Cpro did not affect the interactions between 3Cpro and EphA2. We further determined that EphA2 overexpression inhibited autophagy by activating the mTOR pathway and suppressing SVV replication. Taken together, these results indicate that SVV 3Cpro targets EphA2 for cleavage to impair its EphA2-mediated antiviral activity and emphasize the potential of the molecular interactions involved in developing antiviral strategies against SVV infection.

When DNA-damage responses meet innate and adaptive immunity.

When cells proliferate, stress on DNA replication or exposure to endogenous or external insults frequently results in DNA damage. DNA-Damage Response (DDR) networks are complex signaling pathways used by multicellular organisms to prevent DNA damage. Depending on the type of broken DNA, the various pathways, Base-Excision Repair (BER), Nucleotide Excision Repair (NER), Mismatch Repair (MMR), Homologous Recombination (HR), Non-Homologous End-Joining (NHEJ), Interstrand Crosslink (ICL) repair, and other direct repair pathways, can be activated separately or in combination to repair DNA damage. To preserve homeostasis, innate and adaptive immune responses are effective defenses against endogenous mutation or invasion by external pathogens. It is interesting to note that new research keeps showing how closely DDR components and the immune system are related. DDR and immunological response are linked by immune effectors such as the cyclic GMP-AMP synthase (cGAS)-Stimulator of Interferon Genes (STING) pathway. These effectors act as sensors of DNA damage-caused immune response. Furthermore, DDR components themselves function in immune responses to trigger the generation of inflammatory cytokines in a cascade or even trigger programmed cell death. Defective DDR components are known to disrupt genomic stability and compromise immunological responses, aggravating immune imbalance and leading to serious diseases such as cancer and autoimmune disorders. This study examines the most recent developments in the interaction between DDR elements and immunological responses. The DDR network's immune modulators' dual roles may offer new perspectives on treating infectious disorders linked to DNA damage, including cancer, and on the development of target immunotherapy.

The Role of Inflammatory Biomarkers in Mediating the Effect of Inflammatory Bowel Disease on nonmalignant Digestive System Diseases: A Multivariable Mendelian Randomized Study.

Background: While observation studies have shown a positive correlation between inflammatory bowel disease (IBD) and the risk of nonmalignant digestive system diseases, a definitive causal relationship has not yet been clearly established. Methods: Mendelian randomization (MR) was employed to investigate the potential causal association between genetic susceptibility to IBD and nonmalignant gastrointestinal diseases. Genetic variants were extracted as instrumental variables (IVs) from a genome-wide association study (GWAS) meta-analysis, which included 12,194 cases of Crohn's disease (CD) and 28,072 control cases of European ancestry. The GWAS for ulcerative colitis (UC) included 12,366 UC and 33,609 control cases of European ancestry. All IVs reached genome-wide significance (GWAS p value <5 × 10-8). Summary-level data for acute pancreatitis (AP), irritable bowel syndrome (IBS), gastroesophageal reflux disease, cholelithiasis, and CeD (celiac disease) were obtained from the GWAS meta-analysis and the FinnGen dataset. Summary-level data on relevant inflammatory factors were provided by the International Genetic Consortium. Univariate MR analysis was conducted using inverse variance weighting as the primary method for estimating causal effects. Multivariate MR analyses were also performed to detect possible mediators. Results: Genetic susceptibility to UC was associated with an increased risk of AP (OR = 1.08; 95% CI = 1.03-1.13; p=0.002) and IBS odds ratio (OR] = 1.07; 95% confidence interval (CI] = 1.03-1.11; (p < 0.001). In terms of potential mediators, interleukin 6 (IL-6) had a driving effect on the association between UC and AP. There was no apparent evidence of increased risk with CD. Meanwhile, genetic susceptibility to CD increases the risk of CeD (OR = 1.14; 95% CI = 1.03-1.25; p=0.01). Conclusions: The evidence suggests that UC is associated with an elevated risk of AP and IBS, and IL-6 may be responsible in AP. CD is associated with an increased risk of developing CeD. Implementing a proactive monitoring program for assessing the risk of gastrointestinal diseases in UC patients, particularly those with elevated IL-6 levels, may be of interest. In addition, the presence of AP and IBS may indicate the presence of UC. Preventing CeD is an essential consideration in the therapeutic management of patients with CD.

The association between tea consumption and non-malignant digestive system diseases: A Mendelian randomized study.

BACKGROUND: Tea consumption might be closely related to non-malignant digestive diseases. Nevertheless, this correlation remains inadequately comprehended. Therefore, our objective was to elucidate the essence of these connections. METHODS: This study employed a Mendelian randomization approach to investigate the impact of tea consumption on specific digestive disorders. Genetic data associated with tea consumption were obtained from the UK Biobank (UKB), encompassing 447,485 participants. We chose a gene-wide association study with no sample overlap and UKB as our data source for all outcomes. The primary analytical method utilized was inverse variance weighting, and multiple analytical models were employed to enhance the analysis's reliability and ensure robust results. RESULT: Our investigation revealed that tea consumption was linked to an elevated susceptibility to gastroesophageal reflux disease (GERD). However, there was a lack of substantial evidence suggesting an association between tea intake and Crohn's disease (CD), ulcerative colitis (UC), or non-alcoholic fatty liver disease (NAFLD). CONCLUSIONS: Our study suggests that the excessive consumption of tea may heighten the likelihood of GERD. These results hold potential significance in guiding dietary pattern modifications for individuals with GERD. Furthermore, there may be value in implementing GERD monitoring and preventive measures in populations with elevated tea consumption.

Seneca Valley virus 3C protease cleaves OPTN (optineurin) to Impair selective autophagy and type I interferon signaling.

Seneca Valley virus (SVV) causes vesicular disease in pigs, posing a threat to global pork production. OPTN (optineurin) is a macroautophagy/autophagy receptor that restricts microbial propagation by targeting specific viral or bacterial proteins for degradation. OPTN is degraded and cleaved at glutamine 513 following SVV infection via the activity of viral 3C protease (3C[pro]), resulting in N-terminal and a C-terminal OPTN fragments. Moreover, OPTN interacts with VP1 and targets VP1 for degradation to inhibit viral replication. The N-terminal cleaved OPTN sustained its interaction with VP1, whereas the degradation capacity targeting VP1 decreased. The inhibitory effect of N-terminal OPTN against SVV infection was significantly reduced, C-terminal OPTN failed to inhibit viral replication, and degradation of VP1 was blocked. The knockdown of OPTN resulted in reduced TBK1 activation and phosphorylation of IRF3, whereas overexpression of OPTN led to increased TBK1-IRF3 signaling. Additionally, the N-terminal OPTN diminished the activation of the type I IFN (interferon) pathway. These results show that SVV 3C[pro] targets OPTN because its cleavage impairs its function in selective autophagy and type I IFN production, revealing a novel model in which the virus develops diverse strategies for evading host autophagic machinery and type I IFN response for survival.Abbreviations: Co-IP: co-immunoprecipitation; GFP-green fluorescent protein; hpi: hours post-infection; HRP: horseradish peroxidase; IFN: interferon; IFNB/IFN-ß: interferon beta; IRF3: interferon regulatory factor 3; LIR: LC3-interacting region; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MOI: multiplicity of infection; OPTN: optineurin; PBS: phosphate-buffered saline; SVV: Seneca Valley virus; SQSTM1: sequestosome 1; TAX1BP1: Tax1 binding protein 1; TBK1: TANK binding kinase 1; TCID50: 50% tissue culture infectious doses; UBAN: ubiquitin binding in TNIP/ABIN (TNFAIP3/A20 and inhibitor of NFKB/NF-kB) and IKBKG/NEMO; UBD: ubiquitin-binding domain; ZnF: zinc finger.

Seneca Valley virus 3Cpro antagonizes type I interferon response by targeting STAT1-STAT2-IRF9 and KPNA1 signals.

IMPORTANCE: Type I interferon (IFN) signaling plays a principal role in host innate immune responses against invading viruses. Viruses have evolved diverse mechanisms that target the Janus kinase-signal transducer and activator of transcription (STAT) signaling pathway to modulate IFN response negatively. Seneca Valley virus (SVV), an emerging porcine picornavirus, has received great interest recently because it poses a great threat to the global pork industry. However, the molecular mechanism by which SVV evades host innate immunity remains incompletely clear. Our results revealed that SVV proteinase (3Cpro) antagonizes IFN signaling by degrading STAT1, STAT2, and IRF9, and cleaving STAT2 to escape host immunity. SVV 3Cpro also degrades karyopherin 1 to block IFN-stimulated gene factor 3 nuclear translocation. Our results reveal a novel molecular mechanism by which SVV 3Cpro antagonizes the type I IFN response pathway by targeting STAT1-STAT2-IRF9 and karyopherin α1 signals, which has important implications for our understanding of SVV-evaded host innate immune responses.

Characterization of avain metapneumovirus subgroup C isolated from chickens in Beijing, China.

Avian metapneumovirus (aMPV) is an important causative agent that causes acute respiratory disease and egg-dropping in chickens and turkeys. Here, we characterized an aMPV subgroup C (aMPV/C) from 320-day-old broiler breeder chickens with severe respiratory diseases in Beijing, China, as evidenced by RT-PCR typing and confirmation of the nucleoprotein (N) gene sequence. The N gene sequence of the aMPV/C strain (designated BJ17) exhibited no deletions or insertions and possessed 94.6% to 99.6% identity to those of published aMPV/C isolates. The phylogenetic tree of the nucleotide sequences constructed using the neighbor-joining clustering method showed that the BJ17 strain formed one cluster with other aMPV/C viruses and formed one subcluster with published Chinese aMPV/C isolates regardless of Muscovy duck or chicken origins. Comparative analysis of the N proteins showed that a unique amino acid residue D at position 110 might be associated with regional distribution due to its occurrence in all the Chinese aMPV/C isolates only. Strain BJ17 was successfully isolated by cultured Vero cell passage and further inoculated in 3-wk-old specific-pathogen-free chickens for the examination of pathogenicity. Animal experimental results showed that BJ17-inoculated chickens had severe respiratory diseases and inflammatory lesions, as demonstrated by pathological changes and aMPV antigen in the nasal turbinate, tracheae, and lung tissues. These results enrich the available information regarding the epidemiology and pathogenicity of aMPV/C in chickens, which may facilitate the development of effective measures against aMPV/C infection in China.

Causal relationship between tea intake and cardiovascular diseases: A Mendelian randomization study.

Background: Although studies suggest that tea consumption is associated with a reduced risk of cardiovascular disease (CVD). There is no unified conclusion about the potential relationship between tea drinking and CVD. We used a two-sample Mendelian randomized (MR) analysis to systematically explore the causal relationship between tea intake and CVD subtypes for the first time. Furthermore the mediating effect of hypertension was also explored by a two-step MR. Methods: Genetic instruments for tea intake were identified from a genome-wide association studies (GWAS) involving 447,485 people. Summary data on cardio-vascular disease came from different GWAS meta-analysis studies. In the first step we explored the causal effect of tea intake and CVD. In the second step, we examined the association of hypertension with heart failure and ischemic stroke and estimated the mediating effect of hypertension. Inverse variance weighted MR analysis was used as the primary method for causal analysis. A further sensitivity analysis was performed to ensure robustness of the results. Results: One standard deviation increase in tea intake was associated with a 25% (OR = 0.75, 95%CI = 0.61-0.91, p = 0.003) lower risk of hypertension, a 28% (OR = 0.72, 95%CI = 0.58-0.89, p = 0.002) lower risk of heart failure, and a 29% (OR = 0.71, 95%CI = 0.55-0.92, p = 0.008) lower risk of ischemic stroke, respectively. And the association between tea drinking and the risk of heart failure and ischemic stroke may be mediated by hypertension. Sensitivity analyses found little evidence of pleiotropy. Conclusion: Our two-sample MR analysis provided genetic evidence that tea intake was significantly associated with a reduced risk of hypertension, heart failure, and ischemic stroke, and that hypertension may be a potential mediator. Further large randomized controlled trials should be conducted to confirm the causal effect of tea consumption on cardiovascular disease risk.

False lumen patency status and outcomes after endovascular repair of uncomplicated chronic type B dissection.

BACKGROUND: Thoracic endovascular aortic repair (TEVAR) remains a controversial treatment for uncomplicated chronic type B aortic dissection (cTBAD). This study was performed to investigate the postoperative outcomes of TEVAR, such as survival and reintervention, and the risk factors for prognoses. METHODS: In total, 41 patients with uncomplicated cTBAD who underwent TEVAR from 2014 to 2021 were reviewed. The patients were divided into two groups: those with false lumen complete thrombosis (FLCT) and false lumen partial thrombosis (FLPT) based on computed tomography angiography (CTA) images. Kaplan-Meier analysis was performed to estimate survival and freedom from reintervention. Binary logistic analysis was performed to estimate risk factors for partial thrombosis. RESULTS: During a mean follow-up of 31 (1-78) months, five deaths and six reinterventions had occurred at 5 years. By 1 week, thoracic FLCT had occurred in 23 (56.1%) patients and thoracic FLPT had occurred in 18 (43.9%). The rate of freedom from reintervention was significantly lower in the FLCT than in the FLPT group (p = 0.04). The 5-year survival rate of the two groups was not statistically significant (p = 0.14). Risk factors for thoracic FLPT were the distance between the re-entry site and the graft (p = 0.02) and the proximal oversizing ratio (p = 0.04). CONCLUSIONS: TEVAR is an effective and safe treatment for uncomplicated cTBAD and has a low mortality rate. Thoracic FLCT is associated with less reintervention, but overall survival is not impacted by this difference. Patients treated with TEVAR without certain risk factors can have a good prognosis.

Seneca Valley Virus 3C pro Cleaves Heterogeneous Nuclear Ribonucleoprotein K to Facilitate Viral Replication.

Seneca Valley virus (SVV) has emerged as an important pathogen that is associated with idiopathic vesicular infection in pigs, causing a potential threat to the global swine industry. The heterogeneous nuclear ribonucleoprotein K (hnRNP K) that shuttles between the nucleus and cytoplasm plays an important role in viral infection. In this study, we observed that infection with SVV induced cleavage, degradation, and cytoplasmic redistribution of hnRNP K in cultured cells, which was dependent on the activity of viral 3Cpro protease. Also, the 3Cpro induced degradation of hnRNP K via the caspase pathway. Further studies demonstrated that SVV 3Cpro cleaved hnRNP K at residue Q364, and the expression of the cleavage fragment hnRNP K (aa.365-464) facilitates viral replication, which is similar to full-length hnRNP K, whereas hnRNP K (aa.1-364) inhibits viral replication. Additionally, hnRNP K interacts with the viral 5' untranslated region (UTR), and small interfering RNA (siRNA)-mediated knockdown of hnRNP K results in significant inhibition of SVV replication. Overall, our results demonstrated that the hnRNP K positively regulates SVV replication in a protease activity-dependent fashion in which the cleaved C-terminal contributes crucially to the upregulation of SVV replication. This finding of the role of hnRNP K in promoting SVV propagation provides a novel antiviral strategy to utilize hnRNP K as a potential target for therapy.

Seneca Valley Virus 3Cpro Mediates Cleavage and Redistribution of Nucleolin To Facilitate Viral Replication.

Seneca Valley virus (SVV) is a recently discovered pathogen that poses a significant threat to the global pig industry. It has been shown that many viruses are reliant on nucleocytoplasmic trafficking of nucleolin (NCL) for their own replication. Here, we demonstrate that NCL, a critical protein component of the nucleolus, is cleaved and translocated out of the nucleoli following SVV infection. Furthermore, our data suggest that SVV 3C protease (3Cpro) is responsible for this cleavage and subsequent delocalization from the nucleoli, and that inactivation of this protease activity abolished this cleavage and translocation. SVV 3Cpro cleaved NCL at residue Q545, and the cleavage fragment (aa 1 to 545) facilitated viral replication, which was similar to the activities described for full-length NCL. Small interfering RNA-mediated knockdown indicated that NCL is required for efficient viral replication and viral protein expression. In contrast, lentivirus-mediated overexpression of NCL significantly enhanced viral replication. Taken together, these results indicate that SVV 3Cpro targets NCL for its cleavage and redistribution, which contributes to efficient viral replication, thereby emphasizing the potential target of antiviral strategies for the control of SVV infection. IMPORTANCE The nucleolus is a subnuclear cellular compartment, and nucleolin (NCL) resides predominantly in the nucleolus. NCL participates in viral replication, translation, internalization, and also serves as a receptor for virus entry. The interaction between NCL and SVV is still unknown. Here, we demonstrate that SVV 3Cpro targets NCL for its cleavage and nucleocytoplasmic transportation, which contributes to efficient viral replication. Our results reveal novel function of SVV 3Cpro and provide further insight into the mechanisms by which SVV utilizes nucleoli for efficient replication.

The effect of in situ laser fenestration for total endovascular arch repair in redo aortic dissection.

OBJECTIVE: Treatment of aortic arch pathologies in redo cases is technically challenging. In this study, we assessed early and mid-term outcomes of total endovascular arch repair combined with a new method of in situ laser fenestration. METHODS: Between January 2018 and March 2019, five patients with a history of cardiovascular surgery underwent in situ laser fenestration procedures using the "squid capture technique" for aortic arch pathologies with dissection. All patients were followed up regularly and imaging examinations were performed. The technical success, procedural complications, as well as the early and mid-term mortality and morbidity rates were evaluated. RESULTS: All patients survived the operation and fenestration was technically successful in all of the patients. There was no in-hospital mortality. No patients developed major complications, such as peri-operative strokes, transient ischemic attacks, or spinal cord ischemia. The 11-22 months follow-up (mean, 17 months) was completed by all patients. No endoleaks were discovered; false lumen thromboses and subsequent positive remodeling of the aorta were demonstrated and all in situ laser-fenestrated arteries were patent. CONCLUSIONS: In situ laser fenestration combined with "squid capture technique" was shown to may be an effective and safe option for reconstruction of aortic arch during thoracic endovascular aortic repair. In situ laser fenestration combined with "squid capture technology" was shown to be an effective treatment option for patients with prior history of cardiovascular surgery and who are at high risk for redo open operations.

Synergetic Contributions of Viral VP1, VP3, and 3C to Activation of the AKT-AMPK-MAPK-MTOR Signaling Pathway for Seneca Valley Virus-Induced Autophagy.

Seneca Valley virus (SVV), a member of the Picornaviridae family, can activate autophagy via the PERK and ATF6 unfolded protein response pathways and facilitate viral replication; however, the precise molecular mechanism that regulates SVV-induced autophagy remains unclear. Here, we revealed that SVV infection inhibited the phosphorylation of mechanistic target of rapamycin kinase (MTOR) and activated phosphorylation of the serine/threonine kinase AKT. We observed that activating AMP-activated protein kinase (AMPK), extracellular signal-regulated kinase (ERK), mitogen-activated protein kinase (MAPK), and p38 MAPK signaling by SVV infection promoted autophagy induction and viral replication; additionally, the SVV-induced autophagy was independent of the ULK1 complex. We further evaluated the role of viral protein(s) in the AKT-AMPK-MAPK-MTOR pathway during SVV-induced autophagy and found that VP1 induced autophagy, as evidenced by puncta colocalization with microtubule-associated protein 1 light chain 3 (LC3) in the cytoplasm and enhanced LC3-II levels. This might be associated with the interaction of VP1 with sequestosome 1 and promoting its degradation. In addition, the expression of VP1 enhanced AKT phosphorylation and AMPK phosphorylation, while MTOR phosphorylation was inhibited. These results indicate that VP1 induces autophagy by the AKT-AMPK-MTOR pathway. Additionally, expression of VP3 and 3C was found to activate autophagy induction via the ERK1/2 MAPK-MTOR and p38 MAPK-MTOR pathway. Taken together, our data suggest that SVV-induced autophagy has finely tuned molecular mechanisms in which VP1, VP3, and 3C contribute synergistically to the AKT-AMPK-MAPK-MTOR pathway. IMPORTANCE Autophagy is an essential cellular catabolic process to sustain normal physiological processes that are modulated by a variety of signaling pathways. Invading virus is a stimulus to induce autophagy that regulates viral replication. It has been demonstrated that Seneca Valley virus (SVV) induced autophagy via the PERK and ATF6 unfolded protein response pathways. However, the precise signaling pathway involved in autophagy is still poorly understood. In this study, our results demonstrated that viral proteins VP1, VP3, and 3C contribute synergistically to activation of the AKT-AMPK-MAPK-MTOR signaling pathway for SVV-induced autophagy. These findings reveal systemically the finely tuned molecular mechanism of SVV-induced autophagy, thereby facilitating deeper insight into the development of potential control strategies against SVV infection.

Seneca Valley virus 3Cpro degrades heterogeneous nuclear ribonucleoprotein A1 to facilitate viral replication.

Seneca Valley virus (SVV) is a recently-identified important pathogen that is closely related to idiopathic vesicular disease in swine. Infection of SVV has been shown to induce a variety of cellular factors and their activations are essential for viral replication, but whether heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) involved in SVV replication is unknown. The cytoplasmic redistribution of hnRNP A1 is considered to play an important role in the virus life cycle. Here, we demonstrated that SVV infection can promote redistribution of the nucleocytoplasmic shuttling RNA-binding protein hnRNP A1 to the cytoplasm from the nucleus, whereas hnRNP A1 remained mainly in the nucleus of mock-infected cells. siRNA-mediated knockdown of the gene encoding hnRNP A1 attenuated viral replication as evidenced by decreased viral protein expression and virus production, whereas its overexpression enhanced replication. Moreover, infection with SVV induced the degradation of hnRNP A1, and viral 3 C protease (3 Cpro) was found to be responsible for its degradation and translocation. Further studies demonstrated that 3 Cpro induced hnRNP A1 degradation through its protease activity, via the proteasome pathway. This degradation could be attenuated by a proteasome inhibitor (MG132) and inactivation of the conserved catalytic box in 3 Cpro. Taken together, these results presented here reveal that SVV 3 C protease targets cellular hnRNP A1 for its degradation and translocation, which is utilized by SVV to aid viral replication, thereby highlighting the control potential of strategies for infection of SVV.

Avian Metapneumovirus Subgroup C Induces Mitochondrial Antiviral Signaling Protein Degradation through the Ubiquitin-Proteasome Pathway.

The mitochondrial antiviral signaling (MAVS) protein, a critical adapter, links the upstream recognition of viral RNA to downstream antiviral signal transduction. However, the interaction mechanism between avian metapneumovirus subgroup C (aMPV/C) infection and MAVS remains unclear. Here, we confirmed that aMPV/C infection induced a reduction in MAVS expression in Vero cells in a dose-dependent manner, and active aMPV/C replication was required for MAVS decrease. We also found that the reduction in MAVS occurred at the post-translational level rather than at the transcriptional level. Different inhibitors were used to examine the effect of proteasome or autophagy on the regulation of MAVS. Treatment with a proteasome inhibitor MG132 effectively blocked MAVS degradation. Moreover, we demonstrated that MAVS mainly underwent K48-linked ubiquitination in the presence of MG132 in aMPV/C-infected cells, with amino acids 363, 462, and 501 of MAVS being pivotal sites in the formation of polyubiquitin chains. Finally, E3 ubiquitin ligases for MAVS degradation were screened and identified and RNF5 targeting MAVS at Lysine 363 and 462 was shown to involve in MAVS degradation in aMPV/C-infected Vero cells. Overall, these results reveal the molecular mechanism underlying aMPV/C infection-induced MAVS degradation by the ubiquitin-proteasome pathway.

Nucleolar Phosphoprotein NPM1 Interacts With Porcine Circovirus Type 3 Cap Protein and Facilitates Viral Replication.

Porcine circovirus type 3 (PCV3) is a recently discovered virus with potentially significant implications on the global swine industry. PCV3 replication involves the entry of the viral capsid (Cap) protein with nucleolar localization signals into the nucleus. Using liquid chromatography-mass spectrometry analysis, nucleolar phosphoprotein NPM1 was identified as one of the cellular proteins bound to PCV3 Cap. Co-immunoprecipitation demonstrated that PCV3 Cap interacts directly with NPM1, where the region binding with NPM1 is mapped to amino acid residues 1-38 of Cap. Upon co-transfection, the expression of Cap protein promoted the redistribution of NPM1, which translocated from the nucleus to the cytoplasm and colocalized with Cap in cultured PK15 cells. NPM1 expression was upregulated and translocated from the nucleus to the cytoplasm in PCV3-infected cells, upon siRNA-mediated depletion, or upon treatment with NPM1 inhibitor in PK15 cells with impaired PCV3 replication, as evidenced by decreased levels of viral DNA synthesis and protein expression. By contrast, the replication of PCV3 was enhanced in stably NPM1-expressing cells via a lentivirus-delivered system. Taken together, these findings indicate that NPM1 interacts with PCV3 Cap and plays a crucial role in PCV3 replication.

Porcine Circovirus Type 3 Enters Into PK15 Cells Through Clathrin- and Dynamin-2-Mediated Endocytosis in a Rab5/Rab7 and pH-Dependent Fashion.

Porcine circovirus type 3 (PCV3) invades multiple tissues and organs of pigs of different ages and are widely spread throughout pig farms, emerging as an important viral pathogen that can potentially damage the pig industry worldwide. Since PCV3 is a newly discovered virus, many aspects of its life cycle remain unknown. Porcine kidney epithelial cells are important host targets for PCV3. Here, we used systematic approaches to dissect the molecular mechanisms underlying the cell entry and intracellular trafficking of PCV3 in PK15 cells, a cell line of porcine kidney epithelial origin. A large number of PCV3 viral particles were found to colocalize with clathrin but not caveolin-1 after entry, and PCV3 infection was significantly decreased when treated with chlorpromazine, dynasore, knockdown of clathrin heavy chain expression via RNA interference, or overexpression of a dominant-negative mutant of EPS15 in PCV3-infected cells. After internalization, the viral particles were further observed to colocalize with Rab5 and Rab7, and knockdown of both expression by RNA interference significantly inhibited PCV3 replication. We also found that PCV3 infection was impeded by ammonium chloride treatment, which indicated the requirement of an acidic environment for viral entry. Taken together, our findings demonstrate that PCV3 enters PK15 cells through a clathrin- and dynamin-2-mediated endocytic pathway, which requires early and late endosomal trafficking, as well as an acidic environment, providing an insightful theoretical basis for further understanding the PCV3 life cycle and its pathogenesis.

Reprogramming the unfolded protein response for replication by porcine reproductive and respiratory syndrome virus.

The unfolded protein response (UPR) in the endoplasmic reticulum (ER) constitutes a critical component of host innate immunity against microbial infections. In this report, we show that porcine reproductive and respiratory syndrome virus (PRRSV) utilizes the UPR machinery for its own benefit. We provide evidence that the virus targets the UPR central regulator GRP78 for proteasomal degradation via a mechanism that requires viral glycoprotein GP2a, while both IRE1-XBP1s and PERK-eIF2α-ATF4 signaling branches of the UPR are turned on at early stage of infection. The activated effector XBP1s was found to enter the nucleus, but ATF4 was unexpectedly diverted to cytoplasmic viral replication complexes by means of nonstructural proteins nsp2/3 to promote viral RNA synthesis. RNAi knockdown of either ATF4 or XBP1s dramatically attenuated virus titers, while overexpression caused increases. These observations reveal attractive host targets (e.g., ATF4 and XBP1s) for antiviral drugs and have implications in vaccine development.

Characterizing the PRRSV nsp2 Deubiquitinase Reveals Dispensability of Cis-Activity for Replication and a Link of nsp2 to Inflammation Induction.

The papain-like cysteine protease 2 (PLP2) within the N-terminus of the porcine reproductive and respiratory syndrome virus (PRRSV) nsp2 replicase protein specifies a deubiquitinating enzyme (DUB), but its biochemical properties and the role in infection have remained poorly defined. By using in vitro assays, we found that the purified PLP2 could efficiently cleave K63 and K48 linked polyubiquitin chains Ub3-7 in vitro although displaying a differential activity in converting the respective ubiquitin dimers to monomer. The subsequent mutagenesis analyses revealed that the requirement for PLP2 DUB activity surprisingly resembled that for cis-cleavage activity, as several mutations (e.g., D91R, D85R, etc.) that largely ablated the DUB function also blocked the cis- but not trans-proteolytic cleavage of nsp2/3 polyprotein. Moreover, the analyses identified key mutations that could differentiate DUB from PLP2 cis- and trans-cleavage activities. Further reverse genetics analyses revealed the following findings: (i) mutations that largely blocked the DUB activity were all lethal to the virus, (ii) a point mutation T88G that selectively blocked the cis-cleavage activity of PLP2 did not affect viral viability in cell culture, and (iii) an E90Q mutation that did not affect either of the PLP2 activities led to rescue of WT-like virus but displayed significantly reduced ability to induce TNF-α production. Our findings support the possibility that the PLP2 DUB activity, but not cis-cleavage activity, is essential for PRRSV replication. The data also establish a strong link of nsp2 to pro-inflammatory cytokine induction during infection that operates in a manner independent of PLP2 DUB activity.

The nsp2 Hypervariable Region of Porcine Reproductive and Respiratory Syndrome Virus Strain JXwn06 Is Associated with Viral Cellular Tropism to Primary Porcine Alveolar Macrophages.

Porcine reproductive and respiratory syndrome virus (PRRSV) poses a major threat to global pork production and has been notorious for its rapid genetic evolution in the field. The nonstructural protein 2 (nsp2) replicase protein represents the fastest evolving region of PRRSV, but the underlying biological significance has remained poorly understood. By deletion mutagenesis, we discovered that the nsp2 hypervariable region plays an important role in controlling the balance of genomic mRNA and a subset of subgenomic mRNAs. More significantly, we revealed an unexpected link of the nsp2 hypervariable region to viral tropism. Specifically, a mutant of the Chinese highly pathogenic PRRSV strain JXwn06 carrying a deletion spanning nsp2 amino acids 323 to 521 (nsp2Δ323-521) in its hypervariable region was found to lose infectivity in primary porcine alveolar macrophages (PAMs), although it could replicate relatively efficiently in the supporting cell line MARC-145. Consequently, this mutant failed to establish an infection in piglets. Further dissection of the viral life cycle revealed that the mutant had a defect (or defects) lying in the steps between virus penetration and negative-stranded RNA synthesis. Taken together, our results reveal novel functions of nsp2 in the PRRSV life cycle and provide important insights into the mechanisms of PRRSV RNA synthesis and cellular tropism.IMPORTANCE The PRRSV nsp2 replicase protein undergoes rapid and broad genetic variations in its middle region in the field, but the underlying significance has remained enigmatic. Here, we demonstrate that the nsp2 hypervariable region not only plays an important regulatory role in maintaining the balance of different viral mRNA species but also regulates PRRSV tropism to primary PAMs. Our results reveal novel functions for PRRSV nsp2 and have important implications for understanding the mechanisms of PRRSV RNA synthesis and cellular tropism.