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Poly(C)-binding protein 1, a novel N(pro)-interacting protein involved in classical swine fever virus growth.

ABSTRACT: N(pro) is a multifunctional autoprotease unique to pestiviruses. The interacting partners of the N(pro) protein of classical swine fever virus (CSFV), a swine pestivirus, have been insufficiently defined. Using a yeast two-hybrid screen, we identified poly(C)-binding protein 1 (PCBP1) as a novel interacting partner of the CSFV N(pro) protein and confirmed this by coimmunoprecipitation, glutathione S-transferase (GST) pulldown, and confocal assays. Knockdown of PCBP1 by small interfering RNA suppressed CSFV growth, while overexpression of PCBP1 promoted CSFV growth. Furthermore, we showed that type I interferon was downregulated by PCBP1, as well as N(pro). Our results suggest that cellular PCBP1 positively modulates CSFV growth.

SUBMITTER: Li D

PROVIDER: S-EPMC3571455 | biostudies-literature | 2013 Feb

REPOSITORIES: biostudies-literature

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Poly(C)-binding protein 1, a novel N(pro)-interacting protein involved in classical swine fever virus growth.

Li Dan D Li Su S Sun Yuan Y Dong Hong H Li Yongfeng Y Zhao Bibo B Guo Dongwei D Weng Changjiang C Qiu Hua-Ji HJ

Journal of virology 20121205 4

N(pro) is a multifunctional autoprotease unique to pestiviruses. The interacting partners of the N(pro) protein of classical swine fever virus (CSFV), a swine pestivirus, have been insufficiently defined. Using a yeast two-hybrid screen, we identified poly(C)-binding protein 1 (PCBP1) as a novel interacting partner of the CSFV N(pro) protein and confirmed this by coimmunoprecipitation, glutathione S-transferase (GST) pulldown, and confocal assays. Knockdown of PCBP1 by small interfering RNA supp ...[more]

PMID: 23221550

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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:The mitogen-activated protein kinase kinase/extracellular regulated kinase (MEK1/2/ERK1/2) cascade is involved in the replication of several members of the Flaviviridae family, including hepatitis C virus and dengue virus. The effects of the cascade on the replication of classical swine fever virus (CSFV), a fatal pestivirus of pigs, remain unknown. In this study, MEK2 was identified as a novel binding partner of the E2 protein of CSFV using yeast two-hybrid screening. The E2-MEK2 interaction was confirmed by glutathione S-transferase pulldown, coimmunoprecipitation, and laser confocal microscopy assays. The C termini of E2 (amino acids [aa] 890 to 1053) and MEK2 (aa 266 to 400) were mapped to be crucial for the interaction. Overexpression of MEK2 significantly promoted the replication of CSFV, whereas knockdown of MEK2 by lentivirus-mediated small hairpin RNAs dramatically inhibited CSFV replication. In addition, CSFV infection induced a biphasic activation of ERK1/2, the downstream signaling molecules of MEK2. Furthermore, the replication of CSFV was markedly inhibited in PK-15 cells treated with U0126, a specific inhibitor for MEK1/2/ERK1/2, whereas MEK2 did not affect CSFV replication after blocking the interferon-induced Janus kinase-signal transducer and activator of transcription (JAK-STAT) signaling pathway by ruxolitinib, a JAK-STAT-specific inhibitor. Taken together, our results indicate that MEK2 positively regulates the replication of CSFV through inhibiting the JAK-STAT signaling pathway. IMPORTANCE Mitogen-activated protein kinase kinase 2 (MEK2) is a kinase that operates immediately upstream of extracellular regulated kinase 1/2 (ERK1/2) and links to Raf and ERK via phosphorylation. Currently, little is known about the role of MEK2 in the replication of classical swine fever virus (CSFV), a devastating porcine pestivirus. Here, we investigated the roles of MEK2 and the MEK2/ERK1/2 cascade in the growth of CSFV for the first time. We show that MEK2 positively regulates CSFV replication. Notably, we demonstrate that MEK2 promotes CSFV replication through inhibiting the interferon-induced JAK-STAT signaling pathway, a key antiviral pathway involved in innate immunity. Our work reveals a novel role of MEK2 in CSFV infection and sheds light on the molecular basis by which pestiviruses interact with the host cell.

Dataset Information

Poly(C)-binding protein 1, a novel N(pro)-interacting protein involved in classical swine fever virus growth.

Publications

Poly(C)-binding protein 1, a novel N(pro)-interacting protein involved in classical swine fever virus growth.

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