The Roles Of NS5A Protein In Pathogenesis Of Classical Swine Fever Virus

Classical swine fever (CSF) is a fatal disease of pigs worldwide and classified as anotifiable (previously list A) disease by the World Organization for Animal Health (OIE) dueto its potential for rapid spread across national borders and the considerable socio-economicimpact on the pig industry. The effects of CSFV nonstructural protein5A (NS5A) protein onhost cells are still unknown by now. To elucidate the function of CSFV NS5A, the CSFVNS5A protein was expressed in the swine umbilical vein endothelial cells (SUVEC) cells inthis present study. Then subcellular localization of CSFV NS5A protein, the effect of CSFVNS5A protein on the oxidative stress in SUVEC, the regulation of CSFV NS5A protein on theinflammatory response and the intracellular proteins interacting with CSFV NS5A wereextensively investigated. The results were shown as fellow:(1) The CSFV NS5A gene was successfully amplified from the total RNA of CSFVinfected cells by reverse transcription polymerase chain reaction (RT-PCR). The analysis ofthe obtained sequences showed that, CSFV NS5A gene was1491bp encoding497aminoacids (aa). Bioinformatics analysis showed that there were no signal sequences and CSFVNS5A probably attached to the membranes by the sequences between the226th aa and247thaa. The analysis also showed that a low complexity region (LCR) VVVDTTDVTVTVV wasfound, it indicated that CSFV NS5A protein probably has two domains. Besides, CSFVNS5A protein was a phosphorylated protein and the position of Ser15, Ser81, S9er2, Thr27and Thr401showed great possibility of phosphorylation.(2) CSFV NS5A gene was cloned into the pEGFP-C1and pEGFP-N1expression vectoraccording the vector information. the plasmids pEGFP-NS5A, pNS5A-EGFP and pEGFP-C1vector were transfected into SUVEC, then the resulting stably transfected cell linesexpressing fusion proteins GFPNS5A, NS5AGFP and GFP were established by adding theselective media containing1500μg/ml G418to each well. RT-PCR and western blot analysisshowed that CSFV NS5A gene is successfully expressed in SUVEC and the molecular weightof CSFV NS5A is approximately60ku. Co-localization suggested that CSFV NS5A proteinlocalizes in the endoplasmic reticulum (ER) by confocal laser scanning microscope detection. (3) On the basic of the establishing of SUVEC cells expressing CSFV NS5A, the role ofCSFV NS5A on oxidative stress was explored. CSFV NS5A induces oxidative stressassociated with enhanced reactive oxygen species (ROS) production. The expression of CSFVNS5A protein exerts different effects on the three major antioxidants. Particularly, it exhibits asignificant increase in transcriptional activities of antioxidant proteins thioredoxin (Trx) andperoxiredoxin-6(PRDX-6), but accompanied by a concomitant decrease of antioxidantprotein heme oxygenase-1(HO-1). Further studies showed that cyclooxygenase-2(COX-2), apro-inflammatory protein related to oxidative stress, is up-regulated while anti-inflammatoryprotein peroxisome proliferator-activated receptor-γ (PPAR-γ), an important mediator invascular functional regulation, is down-regulated in CSFV NS5A expressing cells. This studysuggested that CSFV NS5A plays important roles in the induction of oxidative stress andinflammatory response in vascular endothelial cells.(4) Five cDNAs of the NS5A gene were amplified by the PCR deletion method andcloned into a eukaryotic expression vector, which was transfected into SUVEC. Subcellularlocalization of the NS5A protein was characterized by confocal microscopy, and westernblots were carried out to analyze protein expression. It showed that the peptide NS5A/1-84was in charge of the subcellular localization of CSFV NS5A. and the domain NS5A/1-804were the peptide which play a key role of oxidative stress induced in SUVEC.(5) The intracellular proteins of SUVEC interacted with CSFV NS5A protein wereascertained by the yeast two-hybrid technique. The result showed that CSFV NS5A couldinteract with28proteins in SUVEC,19of them are known proteins, includingATP-dependent RNA helicase (DDX5), PHD finger protein5A, heat shock70, RNApolymerase I subunit39(hRPA39), G protein, neuron navigator1(NAV1) and so on, theseintracellular proteins may play roles in the cell cycle, RNA transcription, protein folding,angiopoiesis and cellular permeability.(6) CSFV itself induces oxidative stress in SUVEC after the infection of CSFV onSUVEC. The level of ROS was significantly enhanced and the antioxidant proteins wereregulated by CSFV as the time went on after the infection. And it is found that the addition ofthe antioxidant NAC, GSH, BHA, and curcumin in the media of CSFV inhibit the replicationof CSFV.In conclusion, we firstly found that the CSFV NS5A protein was located to ERmembrance and the level of ROS and antioxidants were significantly elevated in SUVECafter being transfected with NS5A expressing plasmids. The domain which plays a key rolein oxidative stress was indentified and28intracellular proteins interacted with CSFV NS5Awere explored. This is the first time to reveal the interaction of CSFV NS5A and its host cell, which might provide new evidences to the effect of NS5A protein in the pathogenesis andmechanism of CSFV.