Crystal Structure of the Full-Length Japanese Encephalitis Virus NS5 Reveals a Conserved Methyltransferase-Polymerase Interface

Abstract

The flavivirus NS5 harbors a methyltransferase (MTase) in its N-terminal ≈265 residues and an RNA-dependent RNA polymerase (RdRP) within the C-terminal part. One of the major interests and challenges in NS5 is to understand the interplay between RdRP and MTase as a unique natural fusion protein in viral genome replication and cap formation. Here, we report the first crystal structure of the full-length flavivirus NS5 from Japanese encephalitis virus. The structure completes the vision for polymerase motifs F and G, and depicts defined intra-molecular interactions between RdRP and MTase. Key hydrophobic residues in the RdRP-MTase interface are highly conserved in flaviviruses, indicating the biological relevance of the observed conformation. Our work paves the way for further dissection of the inter-regulations of the essential enzymatic activities of NS5 and exploration of possible other conformations of NS5 under different circumstances.

The C-terminal 50 Amino Acid Residues of Dengue NS3 Protein Are Important for NS3-NS5 Interaction and Viral Replication

“…Its C-terminal domain contains ATPase/helicase activity for unwinding of the double-stranded RNA (dsRNA) intermediate (9 -14) and RNA 5Ј-triphosphatase activity for viral RNA 5Ј-capping reaction that is carried out together with the N-terminal domain of NS5, which has methyltransferase activity (3,9,13,(15)(16)(17)(18)(19)(20)(21)(22). The C-terminal domain of NS5 has RNA-dependent RNA polymerase (RdRP) activity, which is crucial for RNA replication (19,(23)(24)(25)(26). NS3 and NS5 have been shown to interact and colocalize in infected cells, and NS3 RNA 5Ј-triphosphatase activity has been reported to be stimulated by NS5 in vitro (9,(27)(28)(29).…”

Adaptive Diversification between Yellow Fever Virus West African and South American Lineages: A Genome-Wide Study

“…38 Because the overall structure of YFV protein NS5 is still lacking and only methyltransferase domain (aa 1-268) are well studied, 39 the functional divergence-related changes were mapped on the three-dimensional structures of the YFV methyltransferase domain and the full-length flavivirus NS5 from Japanese encephalitis virus. 40 We note that four out of six South American lineage-specific adaptive changes cluster in the methyltransferase domain and, in contrast, the majority (six out of seven) of the West African lineage-specific and all four phylogenetically ambiguous changes are distributed on the RdRp domain (aa 275-905, correspond to aa 276-905 of Japanese encephalitis virus NS5) (Figures 2 and 3). The inconsistent pattern between the lineages inferred that they underwent quite different evolutionary fates potentially constituting different functional changes.…”

“…39,40 Such structural distribution pattern, in combination with the evolutional co-occurrence within specific lineage at the early epidemic stage, implied coevolution and functional interaction of these identified sites. For the West African lineagespecific changes, except the substitution I229V lied in the methyltransferase domain ( Figure 3A), the others are distributed on several disparate regions of the RdRp domain (Figures 2 and 3C).…”

“…First, the changes at the identified sites might contribute to the alteration of interacting behavior between NS5 and putative function-related factors and (or) template. It has been reported that, during viral replication, the methyltransferase domains of the flavivirus NS5 proteins recruit cellular factors kinases (CK1 and PKG), 46,47 mitochondrial trifunctional protein (MTP), 48 and PSD-95/Dlg/ ZO-1 proteins, 49,50 while the RdRp domains can interact with the nuclear import receptors importin-α and importin-β, 43,51 cyclophilin A, 52 heat shock protein 70 (Hsp70), 53 protein kinase G (PKG), 40 L subunit of human eukaryotic translation initiation factor 3 (eIF3L), 54 and small nuclear ribonucleoprotein U1A. 55 These factors can enhance formation of the replication complex, RNA stability, genome translation, and/or posttranslational modification.…”

“…40 In (B), five sites identified specifically in the South American lineage are shown in the methyltransferase and RNA-dependent RNA polymerase domains. In (C), six sites identified specifically in the West African lineage and four phylogenetically ambiguous substitution sites are shown in the RNA-dependent RNA polymerase domain.…”