Porcine Mx1 Protein In Vivo Inhibits Classical Swine Fever Virus Infection And The Antiviral Activity Analysis Of Its Mutants

The interferon I-induced Mx proteins of vertebrates are dynamin-like GTPases and have a very broad antiviral spectrum.Classical swine fever(CSF),which caused by classical swine fever virus(CSFV),is a highly contagious disease causing important economic losses in pig industry almost worldwide.As a number of Mx protein family,our previous studies have reported that the purified PTD-poMxl fusion proteins capable of blocking the vesicular stomatitis virus(VSV)have been identified,the expression of EGFP-poMxl fusion protein could inhibit the replication of CSFV on PK-15 cells and progeny virus production was decreased.Here,we confirmed the antiviral activity of purified fusion proteins in VSV-infected Vero cells,two porcine cell lines,ST and 3D4/21,were first used to investigate the antiviral activities of PTD-poMxl against CSFV in vitro.Furthermore,pigs were used to further investigate the antiviral activity of PTD-poMx1 against CSFV in vivo.In addition,a series of experiments we did suggested that GTPase activity and oligomerization were not essential for porcine Mxl to inhibit CSFV replication.1.In vitro inhibition of vesicular stomatitis virus replication by purified porcine Mxl protein fused to HIV-1 Tat protein transduction domain(PTD)We applied the pCold system to express the fusion protein(PTD-poMx1),which consisted of an N-terminal HIV-1 Tat protein transduction domain(PTD)and the full-length porcine Mx1,and investigated its effects on the replication of VSV in Vero cells.The results showed that the purified PTD-poMx1 fusion proteins could transduct into cells after incubated for 5 h and had no cytotoxic.Furthermore,plaque reduction assay,determination of TCID50,real-time RT-PCR and Western blot analyses were carried out to confirm the antiviral activity of purified fusion protein in VSV-infected Vero cells.Altogether,these data suggested that PTD-poMxl fusion proteins might be applicable to inhibit VSV replication as a novel antiviral therapeutic agent.2.PTD-poMxl inhibits classical swine fever virus infection in vitro and in vivoTwo porcine cell lines,ST and 3D4/21,were first used to further investigate the antiviral activities of PTD-poMxl against CSFV in vitro.The results showed that PTD-poMxl both transduced into two cell lines within 3 h but did not affect CSFV binding and uptake,and maintained in the cells for 20 h.qRT-PCR data and viral titers showed that PTD-poMxl effectively inhibited CSFV replication in two cell lines,further revealing that the antiviral activity occurred during the period after infection.Significantly,in vivo results of lmg PTD-poMxl/dose injected into the CSFV-challenged pigs demonstrated that PTD-poMxl could attenuate the clinical expression and viremia of the CSFV-challenged pigs depending on the number of doses given but not completely impede virus replication in vivo,suggesting that PTD-poMx1 conferred the partial protection against a lethal challenge.Overall,our finding strengthened previous in vitro studies and further counseled the possibility of a clinical application in vivo.3.GTPase activity and oligomerization are not essential for porcine Mxl to inhibit the replication of classical swine fever virusWe constructed GTPase defective mutants(K83A,T103A and L612K)and a mutant with the GTPase activity(E646R)and investigated its effects on the replication of CSFV in PK-15 cells.The results demonstrated that all proteins are located in the endoplasmic reticulum.Furthermore,real-time RT-PCR and determination of TCID50 were carried out to show that only L612K and WT had the antiviral activity in CSFV-infected PK-15 cells.T103A exhibited a dominant-negative effect on the antiviral activity of WT rendering Mx1-expressing cells susceptible to infection with CSFV.Altogether,these data suggested that GTPase activity and oligomerization are not essential for porcine Mxl to inhibit CSFV replication.