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PKR activation enhances replication of classical swine fever virus in PK-15 cells.

ABSTRACT: Classical swine fever (CSF) is a highly contagious swine disease that is responsible for economic losses worldwide. Protein kinase R (PK)R is an important protein in the host viral response; however, the role of PKR in CSFV infection remains unknown. This issue was addressed in the present study using the PK-15 swine kidney cell line. We found that CSFV infection increased the phosphorylation of eukaryotic translation initiation factor (eIF)2? and its kinase PKR. However, the expression of viral proteins continued to increase. Furthermore, PKR overexpression enhanced CSFV replication, while PKR inhibition resulted in reduced CSFV replication and an increase in interferon (IFN) induction. In addition, PKR was responsible for eIF2? phosphorylation in CSFV-infected cells. These results suggest that the activation of PKR during CSFV infection is beneficial to the virus. The virus is able to commandeer the host cell's translation machinery for viral protein synthesis while evading innate immune defenses.

SUBMITTER: Liu WJ

PROVIDER: S-EPMC7114430 | biostudies-literature | 2015 Jun

REPOSITORIES: biostudies-literature

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PKR activation enhances replication of classical swine fever virus in PK-15 cells.

Liu Wen-Jun WJ Yang You-Tian YT Zhao Ming-Qiu MQ Dong Xiao-Ying XY Gou Hong-Chao HC Pei Jing-Jing JJ Chen Jin-Ding JD

Virus research 20150418

Classical swine fever (CSF) is a highly contagious swine disease that is responsible for economic losses worldwide. Protein kinase R (PK)R is an important protein in the host viral response; however, the role of PKR in CSFV infection remains unknown. This issue was addressed in the present study using the PK-15 swine kidney cell line. We found that CSFV infection increased the phosphorylation of eukaryotic translation initiation factor (eIF)2α and its kinase PKR. However, the expression of viral ...[more]

PMID: 25899421

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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.

Project description:Two groups with three wild boars each were used: Group A (animals 1 to 3) served as the control, and Group B (animals 4 to 6) was postnatally persistently infected with the Cat01 strain of CSFV (primary virus). The animals, six weeks old and clinically healthy, were inoculated with the virulent strain Margarita (secondary virus). For exclusive detection of the Margarita strain, a specific qRT-PCR assay was designed, which proved not to have cross-reactivity with the Cat01 strain. The wild boars persistently infected with CSFV were protected from superinfection by the virulent CSFV Margarita strain, as evidenced by the absence of clinical signs and the absence of Margarita RNA detection in serum, swabs and tissue samples. Additionally, in PBMCs, a well-known target for CSFV viral replication, only the primary infecting virus RNA (Cat01 strain) could be detected, even after the isolation in ST cells, demonstrating SIE at the tissue level in vivo. Furthermore, the data analysis of the Margarita qRT-PCR, by means of calculated ΔCt values, supported that PBMCs from persistently infected animals were substantially protected from superinfection after in vitro inoculation with the Margarita virus strain, while this virus was able to infect naive PBMCs efficiently. In parallel, IFN-α values were undetectable in the sera from animals in Group B after inoculation with the CSFV Margarita strain. Furthermore, these animals were unable to elicit adaptive humoral (no E2-specific or neutralising antibodies) or cellular immune responses (in terms of IFN-γ-producing cells) after inoculation with the second virus. Finally, a sequence analysis could not detect CSFV Margarita RNA in the samples tested from Group B. Our results suggested that the SIE phenomenon might be involved in the evolution and phylogeny of the virus, as well as in CSFV control by vaccination. To the best of our knowledge, this study was one of the first showing efficient suppression of superinfection in animals, especially in the absence of IFN-α, which might be associated with the lack of innate immune mechanisms.

Project description:UnlabelledClassical swine fever virus (CSFV), a member of the genus Pestivirus within the family Flaviviridae, is a small, enveloped, positive-strand RNA virus. Due to its economic importance to the pig industry, the biology and pathogenesis of CSFV have been investigated extensively. However, the mechanisms of CSFV entry into cells are not well characterized. In this study, we used systematic approaches to dissect CSFV cell entry. We first observed that CSFV infection was inhibited by chloroquine and NH4Cl, suggesting that viral entry required a low-pH environment. By using the specific inhibitor dynasore, or by expressing the dominant negative (DN) K44A mutant, we verified that dynamin is required for CSFV entry. CSFV particles were observed to colocalize with clathrin at 5 min postinternalization, and CSFV infection was significantly reduced by chlorpromazine treatment, overexpression of a dominant negative form of the EPS15 protein, or knockdown of the clathrin heavy chain by RNA interference. These results suggested that CSFV entry depends on clathrin. Additionally, we found that endocytosis of CSFV was dependent on membrane cholesterol, while neither the overexpression of a dominant negative caveolin mutant nor the knockdown of caveolin had an effect. These results further suggested that CSFV entry required cholesterol and not caveolae. Importantly, the effect of DN mutants of three Rab proteins that regulate endosomal traffic on CSFV infection was examined. Expression of DN Rab5 and Rab7 mutants, but not the DN Rab11 mutant, significantly inhibited CSFV replication. These results were confirmed by silencing of Rab5 and Rab7. Confocal microscopy showed that virus particles colocalized with Rab5 or Rab7 during the early phase of infection within 45 min after virus entry. These results indicated that after internalization, CSFV moved to early and late endosomes before releasing its RNA. Taken together, our findings demonstrate for the first time that CSFV enters cells through the endocytic pathway, providing new insights into the life cycle of pestiviruses.ImportanceBovine viral diarrhea virus (BVDV), a single-stranded, positive-sense pestivirus within the family Flaviviridae, is internalized by clathrin-dependent receptor-mediated endocytosis. However, the detailed mechanism of cell entry is unknown for other pestiviruses, such as classical swine fever (CSF) virus (CSFV). CSFV is the etiological agent of CSF, a highly contagious disease of swine that causes numerous deaths in pigs and enormous economic losses in China. Understanding the entry pathway of CSFV will not only advance our knowledge of CSFV infection and pathogenesis but also provide novel drug targets for antiviral intervention. Based on this objective, we used systematic approaches to dissect the pathway of entry of CSFV into PK-15 cells. This is the first report to show that the entry of CSFV into PK-15 cells requires a low-pH environment and involves dynamin- and cholesterol-dependent, clathrin-mediated endocytosis that requires Rab5 and Rab7.

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PKR activation enhances replication of classical swine fever virus in PK-15 cells.

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PKR activation enhances replication of classical swine fever virus in PK-15 cells.

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