Structural analysis and biochemical characterization of human metapneumovirus L protein

• Main Author: Yeo, Tiong Han
• Other Authors: Julien Lescar
• Format: Theses and Dissertations
• Language: English
• Published: 2018
• Subjects: DRNTU::Science::Biological sciences
• Online Access: https://hdl.handle.net/10356/89014; http://hdl.handle.net/10220/46071
• Institution: Nanyang Technological University

Human metapneumovirus (HMPV) is a nonsegmented negative sense RNA virus (NNS), first described in 2001, that causes acute respiratory tract infection in children and elderly. As HMPV is relatively new, most of our current understanding on this virus has been inferred from homologous viruses of the NNS family. The large (L) protein is a multi-domain protein involved in replication and transcription of viral RNA. The replication and transcription process is mediated by the viral (P) phosphoprotein. The L protein is expressed in low abundance in the infected cells due to the position of the cognate gene on the viral genome and little is known about the L protein. Our study shows that HMPV L protein interacts with P protein to form a properly folded and active HMPV polymerase. Using recombinant protein expression techniques, we describe the expression and purification of functional HMPV polymerase complex. The purified polymerase complex was found to be functional in de novo RNA synthesis and to recognize HMPV leader (le) sequence for replication. Structural analysis of HMPV polymerase complex using electron microscopy reveals ring like structures presumably representing the RdRp and capping domains. Due to the recent advances in cryo-electron microscopy techniques, the recombinant HMPV polymerase complex as obtained here can be used for high-resolution cryo-electron microscopy analysis to determine the structure at atomic resolution. Knowledge gained will assist the development of anti-HMPV L therapeutics targeting the replication and transcription of the virus.