A Crystal Structure of the Dengue Virus NS5 Protein Reveals a Novel Inter-domain Interface Essential for Protein Flexibility and Virus Replication

PLoS Pathogens

Pei-Yong Shi¹  Moon Y. F. Tay²  Siew Pheng Lim³  Tingjin Sherryl Soh³  Kitti Wing Ki Chan⁴  Subhash G. Vasudevan⁴  Dahai Luo⁴  Kunchithapadam Swaminathan⁵  Tobias C. Cornvik⁵  Chin Chin Lee⁵  Yongqian Zhao⁵  Julien Lescar⁶  Jie Zheng⁶  Wint Wint Phoo⁶ 

[1] Department of Biological Sciences, National University of Singapore, Singapore;Lee Kong Chian School of Medicine, Nanyang Technological University, Singapore;NUS Graduate School for Integrative Sciences and Engineering, National University of Singapore, Singapore;Novartis Institute for Tropical Diseases, Singapore;Program in Emerging Infectious Diseases, DUKE-NUS Graduate Medical School, Singapore;School of Biological Sciences, Nanyang Technological University, Singapore

关键词: Crystal structure;    Viral replication;    Deuterium;    Polymerases;    Flaviviruses;    Protein structure;    RNA synthesis;    RNA viruses;   

DOI  :  10.1371/journal.ppat.1004682

学科分类：生物科学（综合）

来源: Public Library of Science

【 摘 要 】

Flavivirus RNA replication occurs within a replication complex (RC) that assembles on ER membranes and comprises both non-structural (NS) viral proteins and host cofactors. As the largest protein component within the flavivirus RC, NS5 plays key enzymatic roles through its N-terminal methyltransferase (MTase) and C-terminal RNA-dependent-RNA polymerase (RdRp) domains, and constitutes a major target for antivirals. We determined a crystal structure of the full-length NS5 protein from Dengue virus serotype 3 (DENV3) at a resolution of 2.3 Å in the presence of bound SAH and GTP. Although the overall molecular shape of NS5 from DENV3 resembles that of NS5 from Japanese Encephalitis Virus (JEV), the relative orientation between the MTase and RdRp domains differs between the two structures, providing direct evidence for the existence of a set of discrete stable molecular conformations that may be required for its function. While the inter-domain region is mostly disordered in NS5 from JEV, the NS5 structure from DENV3 reveals a well-ordered linker region comprising a short 310 helix that may act as a swivel. Solution Hydrogen/Deuterium Exchange Mass Spectrometry (HDX-MS) analysis reveals an increased mobility of the thumb subdomain of RdRp in the context of the full length NS5 protein which correlates well with the analysis of the crystallographic temperature factors. Site-directed mutagenesis targeting the mostly polar interface between the MTase and RdRp domains identified several evolutionarily conserved residues that are important for viral replication, suggesting that inter-domain cross-talk in NS5 regulates virus replication. Collectively, a picture for the molecular origin of NS5 flexibility is emerging with profound implications for flavivirus replication and for the development of therapeutics targeting NS5.

【 授权许可】

CC BY