In vitro inhibition of the replication of classical swine fever virus by porcine Mx1 protein
Introduction
Classical swine fever virus (CSFV), a member of the genus Pestivirus within the family Flaviviridae, is a small enveloped positive-strand RNA virus (Meyers et al., 1989, Wengler, 1991). Classical swine fever (CSF) caused by CSFV is an acute and highly contagious disease of swine. Although conventional vaccines, including the attenuated lapinized Chinese strain (C strain), provide complete protection to control CSF (Graham et al., 2012), they do not adhere to the principle of differentiating infected from vaccinated animals (DIVA) and consequently they are not often used in areas where CSF has been eradicated. However, DIVA-adherent vaccines, including subunit vaccines, provide incomplete protection compared with conventional vaccines (Ahrens et al., 2000). Vaccination for CSF control is a complicated issue (Newcomer and Givens, 2013). It is, therefore, necessary to develop novel antiviral treatments against CSFV infection.
Type I interferon (IFN) is produced abundantly in the virus-infected cells soon after infection. Mx proteins are interferon-induced dynamin-like GTPases that are present in all vertebrates, such as mammals, birds and fish (Chaleston and Stewart, 1993, Hailer and Kochs, 2002, Haller et al., 2007), and have a broad range of antiviral activities (Mitchell et al., 2013). Murine Mx1 is a nuclear protein with specific activity against Orthomyxoviruses, whereas human MxA is a cytoplasmic protein and has a broad antiviral spectrum against many RNA viruses, including Orthomyxoviridae, Rhabdoviridae and Bunyaviridae (Haller et al., 2007). Porcine Mx was firstly isolated from German Landrace breed and the existence of two Mx genes, Mx1and Mx2, was reported (Müller et al., 1992). The porcine Mx1 (poMx1) gene was mapped on chromosome 13 (Rettenberger et al., 1996) and the full-length poMx1 gene is 1992 bp. Previous studies have shown that poMx1 can confer resistance to vesicular stomatitis virus (VSV) (Asano et al., 2002, Zhang et al., 2013) and influenza virus (Nakajima et al., 2007, Palm et al., 2010), suggesting that poMx1 is an important mediator of the IFN-induced antiviral state. Endogenous Mx genes are not expressed without IFN (Haller et al., 1998). Previous studies have demonstrated that the lack of IFN-α/β in CSFV-infected cells leads to the absence of expression of dozens of genes responsible for executing antiviral effects, including the porcine endogenous Mx1 (Seago et al., 2007, Durand et al., 2009, Xia et al., 2005). Therefore, we strengthened the expression of Mx1 in CSFV-infected cells to investigate whether exogenous Mx1 harbors an antiviral activity against CSFV. In this study, under G418 selection, a mammalian cell line PK-15 stably expressing EGFP-poMx1 fusion protein was developed and the anti-CSFV activity of EGFP–poMx1 was determined by means of a series of assays. Moreover, PTD–poMx1 fusion protein expressed previously was used to treat the CSFV-infected PK-15 cells in order to confirm the anti-CSFV ability of poMx1. The results provided a support that poMx1 could inhibit CSFV replication and be a novel antiviral therapeutic agent against CSFV infection.
Section snippets
Cells, virus and plasmid
PK-15 cells were purchased from ATCC (CCL-33), and propagated in DMEM supplemented with 10% fetal bovine serum, 100 μg/ml streptomycin and 100 IU/ml penicillin (GIBCO, Invitrogen). Virulent CSFV Shimen strain was obtained from the National Institute of Veterinary Drug Control (Beijing, China; GenBank accession number: AF092448). Plasmid pT-poMx1 which contains porcine Mx1 gene was reported previously (Zhang et al., 2013).
To construct poMx1-expressing plasmid, the 1992 bp of poMx1 gene was
The loss of endogenous poMx1 in CSFV-infected cells
As shown in Fig. 1, under the stimulation of IFN-α, PK-15 cells could produce the endogenous poMx1 protein (lane 2 and 3), but no inner poMx1 protein was produced in CSFV-infected PK-15 cells (lane 4). The results demonstrated that high virulent CSFV Shimen strain could inhibit the pathway of IFN-α, causing the loss of endogenous interferon-induced proteins, such as poMx1.
Stable expression of EGFP-poMx1 fusion protein
The constructed pEGFP–poMx1 plasmid was transfected into the PK-15 cells, cultured in the presence of G418, and
Discussion
This is the first report that poMx1 protein constitutively expressed in PK-15 cell has antiviral activities against CSFV infection. Moreover, purified PTD-poMx1 fusion protein can inhibit CSFV replication in a dose dependent manner. At present, CSF is still an epidemic disease that could severely threaten the swine industry worldwide. To our knowledge, although the C strain is still considered one of the best CSFV vaccines, the inability to serologically differentiate vaccinated from infected
Acknowledgements
This work was supported by a Grant from the National Natural Science Foundation of China (31001062) and A Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD). Also, the authors show tremendous gratitude towards Dr. Kui Yang and Ms. Elizabeth G. Wills, both from department of microbiology and immunology at Cornell University, for critically reading the manuscript and revising it.
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