Functional studies of coronavirus M protein in viral RNA replication

• Main Author: Lim, Yvonne Xinyi
• Other Authors: Liu Ding Xiang
• Format: Final Year Project
• Language: English
• Published: 2015
• Subjects: DRNTU::Science::Biological sciences
• Online Access: http://hdl.handle.net/10356/65386
• Institution: Nanyang Technological University

Coronavirus M protein is an integral membrane glycoprotein that plays pivotal roles in viral assembly. Our recent studies show that its amino terminus may also participate in the regulation of viral RNA replication. In particular, the K19 residue at the amino terminus is hypothesized to be crucial in this regulation. In this study, we aim to characterize the involvement of K19 residue on M protein in viral RNA replication. A full-length cDNA of infectious bronchitis virus (IBV), a prototypic coronavirus, was constructed and used as a template for in vitro transcription. The RNA transcripts generated were transfected into Vero cells. The effects of K19 mutations were analyzed by RT-PCR and Western blot. Additionally, we attempted to develop a transcription system in this study by transfecting full-length IBV cDNA into Vero cells infected with vaccinia virus expressing T7 RNA polymerase. Our findings show that substitution of K19 residue was detrimental to subgenomic RNA transcription, viral protein expression and virus recovery. Collectively, this study provides a novel perspective to the possible functional role of coronavirus M protein in viral RNA replication. It also serves as a reference for alternative strategies of delivering full-length coronavirus genome for future research.