A conformation-based intra-molecular initiation factor identified in the flavivirus RNA-dependent RNA polymerase

Abstract

The flaviviruses pose serious threats to human health. Being a natural fusion of a methyltransferase (MTase) and an RNA-dependent RNA polymerase (RdRP), NS5 is the most conserved flavivirus protein and an important antiviral target. Previously reported NS5 structures represented by those from the Japanese encephalitis virus (JEV) and Dengue virus serotype 3 (DENV3) exhibit two apparently different global conformations, defining two sets of intra-molecular MTase-RdRP interactions. However, whether these NS5 conformations are conserved in flaviviruses and their specific functions remain elusive. Here we report two forms of DENV serotype 2 (DENV2) NS5 crystal structures representing two conformational states with defined analogies to the JEV-mode and DENV3-mode conformations, respectively, demonstrating the conservation of both conformation modes and providing clues for how different conformational states may be interconnected. Data from in vitro polymerase assays further demonstrate that perturbing the JEV-mode but not the DENV3-mode intramolecular interactions inhibits catalysis only at initiation, while the cell-based virological analysis suggests that both modes of interactions are important for virus proliferation. Our work highlights the role of MTase as a unique intra-molecular initiation factor specifically only through the JEV-mode conformation, providing an example of conformation-based crosstalk between naturally fused protein functional modules.

Crystal structure of a tick-borne flavivirus RNA-dependent RNA polymerase suggests a host adaptation hotspot in RNA viruses

“…Consistent with available flavivirus RdRP-only structures, part of the ring and pinky finger subdomains (residues 458–475 in ring and 408–416 in pinky for the TBEV structure) is disordered ( 20 , 41 ). The dynamics in the ring/pinky fingers, and in some cases also in index finger are also observed in full-length NS5 structures ( 18 , 39 ). To date, two types of full-length NS5 global conformations have been identified by crystallography.…”

“…Corresponding TBEV and JEV NS5 mutant constructs were then compared with WT enzymes in both primer-dependent and dinucleotide-driven RdRP assays to assess whether substitutions in region B-C affect polymerase catalytic activities. In previous studies characterizing flavivirus NS5 RdRP, DENV2 NS5 exhibited manganese ion (Mn 2+ )-independent activities in vitro , while JEV NS5 required Mn 2+ in both primer-dependent and dinucleotide-driven assays ( 38 , 39 ). It turned out that TBEV NS5 also needs Mn 2+ in our experimental settings.…”

“…The flavivirus NS5 harbors a 260-residue MTase that interacts with the RdRP through at least two modes of intra-molecular interactions ( 17 , 18 ). While both interaction modes revealed by crystallography are critical for virus proliferation, only one of them contributes to RdRP catalysis by improving NTP binding at the initiation stage of the synthesis ( 39 , 64 ). The coronavirus RdRP nsp12 contains a 250-residue NiRAN (nidovirus RdRP-associated nucleotidyltransferase) domain and a 120-residue interface domain that fold with the RdRP palm and fingers.…”

“…Enzyme-based preparation of the 33-nucleotide (nt) RNA template (T33) was as previously described ( 37 , 38 ). The 30-nt template (T30) and 10-nt RNA primer (P10) were chemically synthesized (Integrated DNA Technologies) and the T30 was purified as previously described ( 39 ). For primer-dependent polymerase assays, the annealing procedures of the T33/P10 construct were as previously described ( 38 ).…”

“…For primer-dependent polymerase assays, the annealing procedures of the T33/P10 construct were as previously described ( 38 ). For dinucleotide-driven polymerase assays, T30 was annealed with a GG dinucleotide primer bearing a 5′-phosphate (P2, Jena BioSciences) at a 1:5 molar ratio following previously described annealing procedures ( 39 ).…”