Characterization of RNase activity and translation regulation of Pseudorabies virus VHS protein

• Main Authors: Pei-Yun Tsai, 蔡佩芸
• Other Authors: Wei-Li Hsu
• Format: Others
• Language: zh-TW
• Published: 2011
• Online Access: http://ndltd.ncl.edu.tw/handle/16236442387553742231

碩士 === 國立中興大學 === 微生物暨公共衛生學研究所 === 99 === Pseudorabies virus (PRV) belongs to the alpha-herpesvirinae; the best characterized virus is herpes simplex virus type 1 (HSV-1). UL41 gene encodes vhs (virion host shutoff) protein that is highly conserved in alpha -herpesvirinae. Previous studies showed HSV-1 vhs harbors ribonuclease activity that contributes to the shutoff of protein synthesis in infected cells via mRNA degradation. HSV-1 vhs selectively decays the region near translation initiation and proceeds in an overall 5- to -3 direction. Moreover, vhs-induced RNA cleavage events is also affected by RNA structure; HSV-1 vhs cleavages RNA at sequences immediately 3 to the Internal Ribosome Entry Site (IRES). Reads and Smiley groups showed that HSV-1 vhs selectively targets actively translated mRNAs through interactions with eIF4F components, i.e. eIF4H, eIF4B, and eIF4A. Up to now the function of PRV vhs is poorly understood. Due to the low sequence similarity (as low as 39.3%), vhs proteins of HSV and PRV might share different biochemistry characteristics. Hence, the main goal of our study was set to explore the mechanisms of PRV vhs on the RNase hydrolysis and translational regulation. To do so, vhs was expressed and purified from E. coli. In vitro vhs assay indicated that PRV vhs indeed exerted RNase activity, 70% RNA was degraded after 40 min incubation and also degraded IRES structure. Interestingly, not only mRNA but also rRNA, PRV vhs degraded RNA/DNA hybrid that was shown for the first time. Different from HSV-1, RNA decay mediated by PRV vhs didn’t show specific direction and didn’t preferentially target at the region at 3 end of IRES. As HSV, PRV vhs ribonuclease activity was significant enhanced by addition of eIF4H and 4B proteins. Moreover in vivo assay demonstrated that each of four highly conserved boxes of vhs contributed to shutoff of protein synthesis via degradation of reporter RNA. Moreover, the dominant negative residue T172 (found in HSV-1 214), and 352, and 356 residues in box IV was the key site for PRV vhs ribonulease activity.