Mapping the Interactions between the NS4B and NS3 Proteins of Dengue Virus Jing Zou, Le Tian Lee, Qing Yin Wang, Xuping Xie, Siyan Lu, Yin Hoe Yau, Zhiming Yuan, Susana Geifman Shochat, Congbao Kang, Julien Lescar, and Pei-Yong Shi J. Virol. April 2015 89:7 3471-3483; Accepted manuscript posted online 14 January 2015, doi:10.1128/JVI.03454-14
Flavivirus RNA synthesis is mediated by a multi-protein complex associated with the endoplasmic reticulum membrane, named the replication complex (RC). Within the flavivirus RC, NS4B, an integral membrane protein with a role in virulence and regulation of the innate immune response, binds to the NS3 protease-helicase. NS4B modulates the RNA helicase activity of NS3, but the molecular details of their interaction remain elusive. Here we used dengue virus (DENV) to map the determinants for the NS3/NS4B interaction. Co-immunoprecipitation and in situ proximity ligation assay confirmed that NS3 colocalizes with NS4B both in DENV-infected cells and in cells co-expressing both proteins. Surface plasmon resonance demonstrated that subdomains 2 and 3 of the NS3 helicase region and the cytoplasmic loop of NS4B are required for binding. Using NMR, we found that the isolated cytoplasmic loop of NS4B is flexible with a tendency to form a α-helix and two short β-strands. Upon binding to the NS3 helicase, twelve amino acids within the cytoplasmic loop of NS4B exhibited line broadening, suggesting a participation in the interaction. Sequence alignment showed that four of these twelve residues are strictly conserved across different flaviviruses. Mutagenesis analysis showed that three (Q134, G140, and N144) of the four evolutionary conserved NS4B residues are essential for DENV replication. The mapping of the NS3/NS4B interacting regions described here can assist the design of inhibitors that disrupt their interface for antiviral therapy.