Cholesterol transport through lysosome-peroxisome membrane contacts.

Cholesterol is dynamically transported among organelles, which is essential for multiple cellular functions. However, the mechanism underlying intracellular cholesterol transport has remained largely unknown. We established an amphotericin B-based assay enabling a genome-wide shRNA screen for delayed LDL-cholesterol transport and identified 341 hits with particular enrichment of peroxisome genes, suggesting a previously unappreciated pathway for cholesterol transport. We show dynamic membrane contacts between peroxisome and lysosome, which are mediated by lysosomal Synaptotagmin VII binding to the lipid PI(4,5)P2 on peroxisomal membrane. LDL-cholesterol enhances such contacts, and cholesterol is transported from lysosome to peroxisome. Disruption of critical peroxisome genes leads to cholesterol accumulation in lysosome. Together, these findings reveal an unexpected role of peroxisome in intracellular cholesterol transport. We further demonstrate massive cholesterol accumulation in human patient cells and mouse model of peroxisomal disorders, suggesting a contribution of abnormal cholesterol accumulation to these diseases.

Pseudorabies virus inhibits progesterone-induced inactivation of TRPML1 to facilitate viral entry.

Viral infection is a significant risk factor for fertility issues. Here, we demonstrated that infection by neurotropic alphaherpesviruses, such as pseudorabies virus (PRV), could impair female fertility by disrupting the hypothalamus-pituitary-ovary axis (HPOA), reducing progesterone (P4) levels, and consequently lowering pregnancy rates. Our study revealed that PRV exploited the transient receptor potential mucolipin 1 (TRPML1) and its lipid activator, phosphatidylinositol 3,5-bisphosphate (PI(3,5)P2), to facilitate viral entry through lysosomal cholesterol and Ca2+. P4 antagonized this process by inducing lysosomal storage disorders and promoting the proteasomal degradation of TRPML1 via murine double minute 2 (MDM2)-mediated polyubiquitination. Overall, the study identifies a novel mechanism by which PRV hijacks the lysosomal pathway to evade P4-mediated antiviral defense and impair female fertility. This mechanism may be common among alphaherpesviruses and could contribute significantly to their impact on female reproductive health, providing new insights for the development of antiviral therapies.

Porcine reproductive and respiratory syndrome virus 2 hijacks CMA-mediated lipolysis through upregulation of small GTPase RAB18.

RAB GTPases (RABs) control intracellular membrane trafficking with high precision. In the present study, we carried out a short hairpin RNA (shRNA) screen focused on a library of 62 RABs during infection with porcine reproductive and respiratory syndrome virus 2 (PRRSV-2), a member of the family Arteriviridae. We found that 13 RABs negatively affect the yield of PRRSV-2 progeny virus, whereas 29 RABs have a positive impact on the yield of PRRSV-2 progeny virus. Further analysis revealed that PRRSV-2 infection transcriptionally regulated RAB18 through RIG-I/MAVS-mediated canonical NF-κB activation. Disrupting RAB18 expression led to the accumulation of lipid droplets (LDs), impaired LDs catabolism, and flawed viral replication and assembly. We also discovered that PRRSV-2 co-opts chaperone-mediated autophagy (CMA) for lipolysis via RAB18, as indicated by the enhanced associations between RAB18 and perlipin 2 (PLIN2), CMA-specific lysosomal associated membrane protein 2A (LAMP2A), and heat shock protein family A (Hsp70) member 8 (HSPA8/HSC70) during PRRSV-2 infection. Knockdown of HSPA8 and LAMP2A impacted on the yield of PRRSV-2 progeny virus, implying that the virus utilizes RAB18 to promote CMA-mediated lipolysis. Importantly, we determined that the C-terminal domain (CTD) of HSPA8 could bind to the switch II domain of RAB18, and the CTD of PLIN2 was capable of associating with HSPA8, suggesting that HSPA8 facilitates the interaction between RAB18 and PLIN2 in the CMA process. In summary, our findings elucidate how PRRSV-2 hijacks CMA-mediated lipid metabolism through innate immune activation to enhance the yield of progeny virus, offering novel insights for the development of anti-PRRSV-2 treatments.

Pseudorabies virus upregulates low-density lipoprotein receptors to facilitate viral entry.

Pseudorabies virus (PRV) is the causative agent of Aujeszky's disease in pigs. The low-density lipoprotein receptor (LDLR) is a transcriptional target of the sterol-regulatory element-binding proteins (SREBPs) and participates in the uptake of LDL-derived cholesterol. However, the involvement of LDLR in PRV infection has not been well characterized. We observed an increased expression level of LDLR mRNA in PRV-infected 3D4/21, PK-15, HeLa, RAW264.7, and L929 cells. The LDLR protein level was also upregulated by PRV infection in PK-15 cells and in murine lung and brain. The treatment of cells with the SREBP inhibitor, fatostatin, or with SREBP2-specific small interfering RNA prevented the PRV-induced upregulation of LDLR expression as well as viral protein expression and progeny virus production. This suggested that PRV activated SREBPs to induce LDLR expression. Furthermore, interference in LDLR expression affected PRV proliferation, while LDLR overexpression promoted it. This indicated that LDLR was involved in PRV infection. The study also demonstrated that LDLR participated in PRV invasions. The overexpression of LDLR or inhibition of proprotein convertase subtilisin/kexin type 9 (PCSK9), which binds to LDLR and targets it for lysosomal degradation, significantly enhanced PRV attachment and entry. Mechanistically, LDLR interacted with PRV on the plasma membrane, and pretreatment of cells with LDLR antibodies was able to neutralize viral entry. An in vivo study indicated that the treatment of mice with the PCSK9 inhibitor SBC-115076 promoted PRV proliferation. The data from the study indicate that PRV hijacks LDLR for viral entry through the activation of SREBPs.IMPORTANCEPseudorabies virus (PRV) is a herpesvirus that primarily manifests as fever, pruritus, and encephalomyelitis in various domestic and wild animals. Owing to its lifelong latent infection characteristics, PRV outbreaks have led to significant financial setbacks in the global pig industry. There is evidence that PRV variant strains can infect humans, thereby crossing the species barrier. Therefore, gaining deeper insights into PRV pathogenesis and developing updated strategies to contain its spread are critical. This study posits that the low-density lipoprotein receptor (LDLR) could be a co-receptor for PRV infection. Hence, strategies targeting LDLR may provide a promising avenue for the development of effective PRV vaccines and therapeutic interventions.

TMEM41B Is an Interferon-Stimulated Gene That Promotes Pseudorabies Virus Replication.

Pseudorabies virus (PRV) is a double-stranded DNA virus that causes Aujeszky's disease and is responsible for economic loss worldwide. Transmembrane protein 41B (TMEM41B) is a novel endoplasmic reticulum (ER)-localized regulator of autophagosome biogenesis and lipid mobilization; however, the role of TMEM41B in regulating PRV replication remains undocumented. In this study, PRV infection was found to upregulate TMEM41B mRNA and protein levels both in vitro and in vivo. For the first time, we found that TMEM41B could be induced by interferon (IFN), suggesting that TMEM41B is an IFN-stimulated gene (ISG). While TMEM41B knockdown suppressed PRV proliferation, TMEM41B overexpression promoted PRV proliferation. We next studied the specific stages of the virus life cycle and found that TMEM41B knockdown affected PRV entry. Mechanistically, we demonstrated that the knockdown of TMEM41B blocked PRV-stimulated expression of the key enzymes involved in lipid synthesis. Additionally, TMEM41B knockdown played a role in the dynamics of lipid-regulated PRV entry-dependent clathrin-coated pits (CCPs). Lipid replenishment restored the CCP dynamic and PRV entry in TMEM41B knockdown cells. Together, our results indicate that TMEM41B plays a role in PRV infection via regulating lipid homeostasis. IMPORTANCE PRV belongs to the alphaherpesvirus subfamily and can establish and maintain a lifelong latent infection in pigs. As such, an intermittent active cycle presents great challenges to the prevention and control of PRV disease and is responsible for serious economic losses to the pig breeding industry. Studies have shown that lipids play a crucial role in PRV proliferation. Thus, the manipulation of lipid metabolism may represent a new perspective for the prevention and treatment of PRV. In this study, we report that the ER transmembrane protein TMEM41B is a novel ISG involved in PRV infection by regulating lipid synthesis. Therefore, our findings indicate that targeting TMEM41B may be a promising approach for the development of PRV vaccines and therapeutics.

Pseudorabies virus hijacks the Rab6 protein to promote viral assembly and egress.

Pseudorabies virus (PRV) is recognized as the aetiological agent responsible for Aujeszky's disease, or pseudorabies, in swine populations. Rab6, a member of the small GTPase family, is implicated in various membrane trafficking processes, particularly exocytosis regulation. Its involvement in PRV infection, however, has not been documented previously. In our study, we observed a significant increase in the Rab6 mRNA and protein levels in both PK-15 porcine kidney epithelial cells and porcine alveolar macrophages, as well as in the lungs and spleens of mice infected with PRV. The overexpression of wild-type Rab6 and its GTP-bound mutant facilitated PRV proliferation, whereas the GDP-bound mutant form of Rab6 had no effect on viral propagation. These findings indicated that the GTPase activity of Rab6 was crucial for the successful spread of PRV. Further investigations revealed that the reduction in Rab6 levels through knockdown significantly hampered PRV proliferation and disrupted virus assembly and egress. At the molecular level, Rab6 was found to interact with the PRV glycoproteins gB and gE, both of which are essential for viral assembly and egress. Our results collectively suggest that PRV exploits Rab6 to expedite its assembly and egress and identify Rab6 as a promising novel target for therapeutic treatment for PRV infection.

Alphaherpesvirus upregulates NDRG1 expression to facilitate the nuclear import of viral UL31 and UL34 proteins.

Proteins UL31 and UL34 encoded by alphaherpesvirus are critical for viral primary envelopment and nuclear egress. We report here that pseudorabies virus (PRV), a useful model for research on herpesvirus pathogenesis, uses N-myc downstream regulated 1 (NDRG1) to assist the nuclear import of UL31 and UL34. PRV promoted NDRG1 expression through DNA damage-induced P53 activation, which was beneficial to viral proliferation. PRV induced the nuclear translocation of NDRG1, and its deficiency resulted in the cytosolic retention of UL31 and UL34. Therefore, NDRG1 assisted the nuclear import of UL31 and UL34. Furthermore, in the absence of the nuclear localization signal (NLS), UL31 could still translocate to the nucleus, and NDRG1 lacked an NLS, thus suggesting the existence of other mediators for the nuclear import of UL31 and UL34. We demonstrated that heat shock cognate protein 70 (HSC70) was the key factor in this process. UL31 and UL34 interacted with the N-terminal domain of NDRG1 and the C-terminal domain of NDRG1 bound to HSC70. Replenishment of HSC70ΔNLS in HSC70-knockdown cells, or interference in importin α expression, abolished the nuclear translocation of UL31, UL34, and NDRG1. These results indicated that NDRG1 employs HSC70 to facilitate viral proliferation in the nuclear import of PRV UL31 and UL34.

RhoA suppresses pseudorabies virus replication in vitro.

The porcine pseudorabies virus (PRV) is one of the most devastating pathogens and brings great economic losses to the swine industry worldwide. Viruses are intracellular parasites that have evolved numerous strategies to subvert and utilize different host processes for their life cycle. Among the different systems of the host cell, the cytoskeleton is one of the most important which not only facilitate viral invasion and spread into neighboring cells, but also help viruses to evade the host immune system. RhoA is a key regulator of cytoskeleton system that may participate in virus infection. In this study, we characterized the function of RhoA in the PRV replication by chemical drugs treatment, gene knockdown and gene over-expression strategy. Inhibition of RhoA by specific inhibitor and gene knockdown promoted PRV proliferation. On the contrary, overexpression of RhoA or activation of RhoA by chemical drug inhibited PRV infection. Besides, our data demonstrated that PRV infection induced the disruption of actin stress fiber, which was consistent with previous report. In turn, the actin specific inhibitor cytochalasin D markedly disrupted the normal fibrous structure of intracellular actin cytoskeleton and decreased the PRV replication, suggesting that actin cytoskeleton polymerization contributed to PRV replication in vitro. In summary, our data displayed that RhoA was a host restriction factor that inhibited PRV replication, which may deepen our understanding the pathogenesis of PRV and provide further insight into the prevention of PRV infection and the development of anti-viral drugs.

Inhibition of PARP1 Dampens Pseudorabies Virus Infection through DNA Damage-Induced Antiviral Innate Immunity.

Pseudorabies virus (PRV) is the causative pathogen of Aujeszky's disease in pigs. Although vaccination is currently applied to prevent the morbidity of PRV infection, new applications are urgently needed to control this infectious disease. Poly(ADP-ribose) polymerase 1 (PARP1) functions in DNA damage repair. We report here that pharmacological and genetic inhibition of PARP1 significantly influenced PRV replication. Moreover, we demonstrate that inhibition of PARP1 induced DNA damage response and antiviral innate immunity. Mechanistically, PARP1 inhibition-induced DNA damage response resulted in the release of double-stranded DNA (dsDNA) into the cytosol, where dsDNA interacted with cyclic GMP-AMP (cGAMP) synthase (cGAS). cGAS subsequently catalyzed cGAMP production to activate the STING/TBK1/IRF3 innate immune signaling pathway. Furthermore, challenge of mice with PARP1 inhibitor stimulated antiviral innate immunity and protected mice from PRV infection in vivo. Our results demonstrate that PARP1 inhibitors may be used as a new strategy to prevent Aujeszky's disease in pigs. IMPORTANCE Aujeszky's disease is a notifiable infectious disease of pigs and causes economic losses worldwide in the pig industry. The causative pathogen is PRV, which is a member of the subfamily Alphaherpesvirinae of the family Herpesviridae. PRV has a wide range of hosts, such as ruminants, carnivores, and rodents. More seriously, recent reports suggest that PRV can cause human endophthalmitis and encephalitis, which indicates that PRV may be a potential zoonotic pathogen. Although vaccination is currently the major strategy used to control the disease, new applications are also urgently needed for the pig industry and public health. We report here that inhibition of PARP1 induces DNA damage-induced antiviral innate immunity through the cGAS-STING signaling pathway. Therefore, PARP1 is a therapeutic target for PRV infection as well as alphaherpesvirus infection.

BRD4 inhibition exerts anti-viral activity through DNA damage-dependent innate immune responses.

Chromatin dynamics regulated by epigenetic modification is crucial in genome stability and gene expression. Various epigenetic mechanisms have been identified in the pathogenesis of human diseases. Here, we examined the effects of ten epigenetic agents on pseudorabies virus (PRV) infection by using GFP-reporter assays. Inhibitors of bromodomain protein 4 (BRD4), which receives much more attention in cancer than viral infection, was found to exhibit substantial anti-viral activity against PRV as well as a range of DNA and RNA viruses. We further demonstrated that BRD4 inhibition boosted a robust innate immune response. BRD4 inhibition also de-compacted chromatin structure and induced the DNA damage response, thereby triggering the activation of cGAS-mediated innate immunity and increasing host resistance to viral infection both in vitro and in vivo. Mechanistically, the inhibitory effect of BRD4 inhibition on viral infection was mainly attributed to the attenuation of viral attachment. Our findings reveal a unique mechanism through which BRD4 inhibition restrains viral infection and points to its potent therapeutic value for viral infectious diseases.

Engineered Antibody Fragment against the Urokinase Plasminogen Activator for Fast Delineation of Triple-Negative Breast Cancer by Positron Emission Tomography.

The urokinase plasminogen activator (uPA) and its cofactors are important regulators of tumor initiation and progression (including metastasis), and its overexpression is associated with unfavorable situations in cancer patients. We have previously used positron emission tomography (PET) imaging with a radiolabeled monoclonal antibody against the uPA (named ATN-291) to detect the uPA signaling activity in various cancer types; however, good tumor contrast can only be observed 24 h postinjection. To shorten the antibody circulation time and decrease interactions of ATN-291 with the mononuclear phagocyte system (MPS), our goal in this study is to develop an engineered antibody fragment (F(ab')2) from the parent antibody. By pepsin digestion and chromatography purification, ATN-291 F(ab')2 was obtained and characterized. Subsequently, it was conjugated with NOTA-Bn-NCS or fluorescein isothiocyanate (FITC) for PET imaging and fluorescence-mediated cellular analysis (i.e., flow cytometry or fluorescence microscopy). We confirmed that ATN-291 F(ab')2 still maintained a good targeting efficacy for the uPA in MDA-MB-231 cells (uPA+) and it had a faster blood clearance speed compared with ATN-291, while its interaction with MPS has been significantly decreased. In rodent tumor xenografts, radiolabeled ATN-291 F(ab')2 had a selective and persistent uptake in MDA-MB-231 tumors, with an early tumor-to-blood ratio of 1.3 ± 0.8 (n = 4) at 2 h postinjection from PET imaging. During our observation, radiolabeled ATN-291 F(ab')2 was excreted from both renal and hepatobiliary pathways. Radiolabeled ATN-291 F(ab')2 was also used for detecting uPA fluctuation during the tumor treatment in test animals. We concluded that radiolabeled ATN-291 F(ab')2 could be used as fast as PET cancer diagnostics with versatile applicability.

CRISPR/Cas9-based generation of a recombinant double-reporter pseudorabies virus and its characterization in vitro and in vivo.

Pseudorabies, caused by pseudorabies virus (PRV) variants, has broken out among commercial PRV vaccine-immunized swine herds and resulted in major economic losses to the pig industry in China since late 2011. However, the mechanism of virulence enhancement of variant PRV is currently unclear. Here, a recombinant PRV (rPRV HN1201-EGFP-Luc) with stable expression of enhanced green fluorescent protein (EGFP) and firefly luciferase as a double reporter virus was constructed on the basis of the PRV variant HN1201 through CRISPR/Cas9 gene-editing technology coupled with two sgRNAs. The biological characteristics of the recombinant virus and its lethality to mice were similar to those of the parental strain and displayed a stable viral titre and luciferase activity through 20 passages. Moreover, bioluminescence signals were detected in mice at 12 h after rPRV HN1201-EGFP-Luc infection. Using the double reporter PRV, we also found that 25-hydroxycholesterol had a significant inhibitory effect on PRV both in vivo and in vitro. These results suggested that the double reporter PRV based on PRV variant HN1201 should be an excellent tool for basic virology studies and evaluating antiviral agents.

Porcine Reproductive and Respiratory Syndrome Virus Activates Lipophagy To Facilitate Viral Replication through Downregulation of NDRG1 Expression.

Autophagy maintains cellular homeostasis by degrading organelles, proteins, and lipids in lysosomes. Autophagy is involved in the innate and adaptive immune responses to a variety of pathogens. Some viruses can hijack host autophagy to enhance their replication. However, the role of autophagy in porcine reproductive and respiratory syndrome virus (PRRSV) infection is unclear. Here, we show that N-Myc downstream-regulated gene 1 (NDRG1) deficiency induced autophagy, which facilitated PRRSV replication by regulating lipid metabolism. NDRG1 mRNA is expressed ubiquitously in most porcine tissues and most strongly in white adipose tissue. PRRSV infection downregulated the expression of NDRG1 mRNA and protein, while NDRG1 deficiency contributed to PRRSV RNA replication and progeny virus assembly. NDRG1 deficiency reduced the number of intracellular lipid droplets (LDs), but the expression levels of key genes in lipogenesis and lipolysis were not altered. Our results also show that NDRG1 deficiency promoted autophagy and increased the subsequent yields of hydrolyzed free fatty acids (FFAs). The reduced LD numbers, increased FFA levels, and enhanced PRRSV replication were abrogated in the presence of an autophagy inhibitor. Overall, our findings suggest that NDRG1 plays a negative role in PRRSV replication by suppressing autophagy and LD degradation.IMPORTANCE Porcine reproductive and respiratory syndrome virus (PRRSV), an enveloped single-positive-stranded RNA virus, causes acute respiratory distress in piglets and reproductive failure in sows. It has led to tremendous economic losses in the swine industry worldwide since it was first documented in the late 1980s. Vaccination is currently the major strategy used to control the disease. However, conventional vaccines and other strategies do not provide satisfactory or sustainable prevention. Therefore, safe and effective strategies to control PRRSV are urgently required. The significance of our research is that we demonstrate a previously unreported relationship between PRRSV, NDRG1, and lipophagy in the context of viral infection. Furthermore, our data point to a new role for NDRG1 in autophagy and lipid metabolism. Thus, NDRG1 and lipophagy will have significant implications for understanding PRRSV pathogenesis for developing new therapeutics.

PKM2 knockdown influences SREBP activation and lipid synthesis in bovine mammary-gland epithelial MAC-T cells.

OBJECTIVE: The purpose of the article is to evaluate the changes in lipid metabolism in bovine mammary-gland epithelial MAC-T cells after PKM2 knockdown. RESULTS: MAC-T cells stably expressing low levels of PKM2 were established with lentivirus-mediated small hairpin RNA. Although the knockdown of PKM2 had no effect on MAC-T cell growth, the reduced expression of PKM2 attenuated the mRNA and protein expression of key enzymes involved in sterol synthesis through the SREBP pathway. CONCLUSIONS: The downregulation of PKM2 significantly influenced lipid synthesis in bovine mammary-gland epithelial MAC-T cells. These findings extend our understanding of the crosstalk between glycolysis and lipid metabolism in bovine mammary-gland epithelial cells.

Cholesterol 25-hydroxylase acts as a host restriction factor on pseudorabies virus replication.

Cholesterol 25-hydroxylase (CH25H) catalyses the production of 25-hydroxycholesterol (25HC) from cholesterol by adding a second hydroxyl group at position 25. The aim of this study was to examine the antiviral effect of CH25H on pseudorabies virus (PRV), a swine pathogen that can cause devastating disease and economic losses worldwide. The results showed that porcine ch25h was induced by either interferon or PRV infection. PRV infection of porcine alveolar macrophages (3D4/21 cells) was attenuated by CH25H overexpression and enhanced by silencing of CH25H. Furthermore, treatment of 3D4/21 cells with 25HC inhibited the growth of PRV in vitro, suggesting that CH25H may restrict PRV replication by 25HC production. We further identified that the anti-PRV role of CH25H and 25HC was subject to their inhibitory effect on PRV attachment and entry. Collectively, these findings demonstrate that CH25H is an intrinsic host restriction factor in PRV infection of porcine alveolar macrophages.

Sus scrofa matrix attachment region enhances expression of the PiggyBac system transfected into HEK293T cells.

OBJECTIVES: To determine the effects of the Sus scrofa matrix attachment region (SusMAR) on transgene expression in HEK293T cells. RESULTS: Three expression vectors with the MAR at different sites in the PiggyBac (PB) transposon vector backbone were compared: two MARs flanking the ß-galactosidase (ß-gal) expression cassette, and one at the upstream or downstream site. Bos taurus MAR (BosMAR) and a ß-gal expression cassette without MARs were the positive and negative controls, respectively. Compared to the control, ß-gal activity of all SusMAR and BosMAR vectors was significantly improved in the presence of PB transposase (PBase). However, only the downstream SusMAR and upstream BosMAR vectors showed increased expression in the absence of PBase. Expression was significantly increased in all vectors with the PBase group compared to those without the PBase group. Gene copy numbers were not increased compared to the negative control. CONCLUSIONS: SusMAR enhanced recombinant gene expression levels and stability in HEK293T cells, was not increase transgene copy number. These results could facilitate the development of vectors for stable production of therapeutic proteins.

Comparation of the effects of different 5'-untranslated regions (UTRs) on gene expression in HEK293 cells.

OBJECTIVES: To evaluate four 5'-UTRs on GFP expression in HEK293T cells. RESULTS: The recombinant plasmids were constructed by restriction enzyme digestion, digestion and DNA sequencing. Quantitative real-time PCR and western blotting results showed that the transcription and translation level of PPRV-GFP mRNA was significantly lower than that of the other reporters. The transcription and translation level of ChEF1-GFP was the highest in HEK293T cells. CONCLUSIONS: Different UTRs can significantly affect protein expression. Additionally, the findings also will be useful in biological applications that require tuning of gene expression and system optimization.

Pseudorabies virus manipulates mitochondrial tryptophanyl-tRNA synthetase 2 for viral replication.

The pseudorabies virus (PRV) is identified as a double-helical DNA virus responsible for causing Aujeszky's disease, which results in considerable economic impacts globally. The enzyme tryptophanyl-tRNA synthetase 2 (WARS2), a mitochondrial protein involved in protein synthesis, is recognized for its broad expression and vital role in the translation process. The findings of our study showed an increase in both mRNA and protein levels of WARS2 following PRV infection in both cell cultures and animal models. Suppressing WARS2 expression via RNA interference in PK-15 â€‹cells led to a reduction in PRV infection rates, whereas enhancing WARS2 expression resulted in increased infection rates. Furthermore, the activation of WARS2 in response to PRV was found to be reliant on the cGAS/STING/TBK1/IRF3 signaling pathway and the interferon-alpha receptor-1, highlighting its regulation via the type I interferon signaling pathway. Further analysis revealed that reducing WARS2 levels hindered PRV's ability to promote protein and lipid synthesis. Our research provides novel evidence that WARS2 facilitates PRV infection through its management of protein and lipid levels, presenting new avenues for developing preventative and therapeutic measures against PRV infections.

The glycoprotein 5 of porcine reproductive and respiratory syndrome virus stimulates mitochondrial ROS to facilitate viral replication.

IMPORTANCE: Porcine reproductive and respiratory syndrome virus (PRRSV) presents a significant economic concern for the global swine industry due to its connection to serious production losses and increased mortality rates. There is currently no specific treatment for PRRSV. Previously, we had uncovered that PRRSV-activated lipophagy to facilitate viral replication. However, the precise mechanism that PRRSV used to trigger autophagy remained unclear. Here, we found that PRRSV GP5 enhanced mitochondrial Ca2+ uptake from ER by promoting ER-mitochondria contact, resulting in mROS release. Elevated mROS induced autophagy, which alleviated NLRP3 inflammasome activation for optimal viral replication. Our study shed light on a novel mechanism revealing how PRRSV exploits mROS to facilitate viral replication.