Studies on the cellular localization of nonstructural protein 2 of Classical swine fever virus

• Main Authors: Hui-Hsuan Tien, 田卉萱
• Other Authors: Ching-Hsiu Tsai
• Format: Others
• Language: en_US
• Published: 2010
• Online Access: http://ndltd.ncl.edu.tw/handle/79946907692095858559

碩士 === 國立中興大學 === 生物科技學研究所 === 98 === Classical swine fever virus (CSFV) the causative agent of swine fever belongs to the pestivirus genus of the family Flaviviridae. Clinical symptoms include anorexia, depression, hyperpyrexia and hemorrhage. Most of the countries in which this disease breaks choose to exterminate all the pigs, leading to huge economic losses. The single strand positive sense CSFV RNA genome contains one single open reading frame that encodes a 3,983 polypeptide which is processed to 12 different proteins. Previous studies indicate that, Jiv protein (J-domain protein interacting with viral protein) from BVDV infected cells acts as a regulating cofactor for the nonstructural protein 2 (NS2) autoprotease and initiates NS2-3 cleavage in trans releasing nonstructural protein 3 (NS3), one of the essential components of the viral RNA replicase complex. Recent studies show that after NS3 release the NS2 still binds to Jiv and prevents further rounds of cofactor function which; an event thought to be the switch to stop RNA replication and start packaging. Though NS2 influences CSFV RNA replication it does not participate in it directly. Whether NS2 involved in other functions of CSFV life cycle is still unknown. Therefore, the distribution of NS2 in porcine kidney cells (PK-15) could be the starting point in understanding its role in CSFV life cycle. In order to pursue localization studies NS2 and uncleavable NS2/3 were fused with T7 and FLAG tags on their N- and C-terminus, respectively. Constructs were transfected using Lipofectamine 2000 in PK-15 cells and the distribution of NS2 was examined 24 h post-transfection using ImmunoFluorescence Assay (IFA) and confocal imaging. Preliminary results using EGFP-ER marker showed that NS2 co-localizes with endoplasmic reticulum (ER). Interestingly this localization corresponds to a cellular host factor Jiv that binds to and influences pestiviral NS2 as mentioned in previous studies. However, the amount of NS2 expressed in cells is low and reports on Hepatitis C Virus (HCV) NS2 suggest that NS2 degrades rapidly in infected cells. To investigate further, we will focus on the distribution of NS2 protein in PK-15 cells using different NS2 mutants to determine the ER localization signal and the putative Jiv binding domain.