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Classical swine fever virus employs the PERK- and IRE1-dependent autophagy for viral replication in cultured cells.

ABSTRACT: Endoplasmic reticulum stress (ERS)-mediated autophagy is indispensable for modulation of replication and pathogenesis of numerous mammalian viruses. We have previously shown that classical swine fever virus (CSFV) infection induces ERS-mediated autophagy for maintaining viral replication both in vivo and in vitro, however, the underlying mechanism remains unclarified. Here we found that CSFV infection activates the PERK pathway-dependent complete autophagy to promote viral replication in cultured PK-15 and 3D4/2 cells. Likewise, our results also suggested the essential roles of the IRE1/GRP78-mediated complete autophagy in CSFV replication in vitro. Furthermore, we suggested that CSFV infection induces activation of the PERK and IRE1 pathway for potential immunoregulation via promoting transcription of proinflammatory cytokine (IFN-γ and TNF-α) genes in the CSFV-infected cells. Finally, pharmacological treatment of PERK- or IRE1-pathway regulators, and the corresponding SiRNAs interventions did not affect the viabilities of the cells, excluding the potential interference elicited by altered cell viabilities. Taken together, our results suggest that CSFV infection induces complete autophagy through activation of the PERK and IRE1 pathway to facilitate viral replication in cultured cells, and modulation of proinflammatory cytokines may be a potential mechanism involved in this event. Our findings will open new horizons for molecular mechanisms of sustainable replication and pathogenesis of CSFV, and lay a theoretical foundation for the development of ERS-autophagy-targeting therapeutic strategies for clinical control of CSF.

SUBMITTER: Zhu E

PROVIDER: S-EPMC7781608 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Endoplasmic reticulum (ER) stress-mediated autophagy plays significant roles in replication and pathogenesis of many animal viruses. However, the relationship between ER stress, autophagy, and viral replication during in vivo and in vitro infection of classical swine fever virus (CSFV) remains unclear. In this study, we established a pig model for CSFV infection and found that viral loads of CSFV differed in 10 kinds of infected organs, and that the degree of tissue lesions was to some extent positively correlated with CSFV replication in vivo. Next, we found that CSFV infection not only induced ER stress and subsequently activated three unfolded protein responses (UPR) pathways including protein kinase R-like ER kinase (PERK), inositol requiring enzyme 1 (IRE1), and activating transcription factor-6 (ATF-6) pathways, but also triggered complete autophagy in main immune organs and partial nonimmune organs exhibiting severer pathological injuries and higher viral loads. However, only the IRE1 pathway and no autophagy were activated in some other nonimmune organs with slighter pathologies and lower viral loads. These results indicate a potential link between CSFV-induced ER stress and autophagy, both of which are associated with the CSFV replication in vivo. We further performed in vitro experiments and found that CSFV infection activates the PERK and IRE1 pathways and autophagy in cultured porcine kidney cell lines (PK-15) and macrophage cell lines (3D4/2), and pharmacological regulation of ER stress remarkably changed autophagic activities induced by CSFV, suggesting that CSFV-induced autophagy can be mediated by ER stress possibly via the PERK and IRE1 pathway. Furthermore, treatment with ER stress regulators significantly altered copy numbers of NS5B genes, expression of Npro proteins, and viral titers in CSFV-infected cells or in cells treated with autophagy regulators prior to CSFV infection, suggesting the requirement of ER stress-mediated autophagy for CSFV replication in vitro. Collectively, our data demonstrate that CSFV induces ER stress-mediated autophagy to sustain its replication in vivo and in vitro, which may be one of the potential strategies exploited by CSFV for immune evasion. This finding will provide new insights into mechanisms of replication and pathogenesis of CSFV, and development of new strategies for controlling CSF.

Project description:Recently moderate-virulence classical swine fever virus (CSFV) strains have been proven capable of generating postnatal persistent infection (PI), defined by the maintenance of viremia and the inability to generate CSFV-specific immune responses in animals. These animals also showed a type I interferon blockade in the absence of clinical signs. In this study, we assessed the infection generated in 7-week-old CSFV PI wild boars after infection with the African swine fever virus (ASFV). The wild boars were divided in two groups and were infected with ASFV. Group A comprised boars who were CSFV PI in a subclinical form and Group B comprised pestivirus-free wild boars. Some relevant parameters related to CSFV replication and the immune response of CSFV PI animals were studied. Additionally, serum soluble factors such as IFN-α, TNF-α, IL-6, IL-10, IFN-γ and sCD163 were analysed before and after ASFV infection to assess their role in disease progression.After ASFV infection, only the CSFV PI wild boars showed progressive acute haemorrhagic disease; however, the survival rates following ASFV infection was similar in both experimental groups. Notwithstanding, the CSFV RNA load of CSFV PI animals remained unaltered over the study; likewise, the ASFV DNA load detected after infection was similar between groups. Interestingly, systemic type I FN-α and IL-10 levels in sera were almost undetectable in CSFV PI animals, yet detectable in Group B, while detectable levels of IFN-γ were found in both groups. Finally, the flow cytometry analysis showed an increase in myelomonocytic cells (CD172a+) and a decrease in CD4+ T cells in the PBMCs from CSFV PI animals after ASFV infection.Our results showed that the immune response plays a role in the progression of disease in CSFV subclinically infected wild boars after ASFV infection, and the immune response comprised the systemic type I interferon blockade. ASFV does not produce any interference with CSFV replication, or vice versa. ASFV infection could be a trigger factor for the disease progression in CSFV PI animals, as their survival after ASFV was similar to that of the pestivirus-free ASFV-infected group. This fact suggests a high resistance in CSFV PI animals even against a virus like ASFV; this may mean that there are relevant implications for CSF control in endemic countries. The diagnosis of ASFV and CSFV co-infection in endemic countries cannot be ruled out and need to be studied in greater depth.

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Classical swine fever virus employs the PERK- and IRE1-dependent autophagy for viral replication in cultured cells.

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