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Flavivirus methyltransferase catalyzes both guanine N7 and ribose 2′-OH methylations of the viral RNA cap (GpppA-RNA→m7GpppAm-RNA). The methyltransferase is physically linked to an RNA-dependent RNA polymerase (RdRp) in the flaviviral NS5 protein. Here, we report genetic interactions of West Nile virus (WNV) methyltransferase with the RdRp and the 5′-terminal stem-loop of viral genomic RNA. Genome-length RNAs, containing amino acid substitutions of D146 (a residue essential for both cap methylations) in the methyltransferase, were transfected into BHK-21 cells. Among the four mutant RNAs (D146L, D146P, D146R, and D146S), only D146S RNA generated viruses in transfected cells. Sequencing of the recovered viruses revealed that, besides the D146S change in the methyltransferase, two classes of compensatory mutations had reproducibly emerged. Class 1 mutations were located in the 5′-terminal stem-loop of the genomic RNA (a G35U substitution or U38 insertion). Class 2 mutations resided in NS5 (K61Q in methyltransferase and W751R in RdRp). Mutagenesis analysis, using a genome-length RNA and a replicon of WNV, demonstrated that the D146S substitution alone was lethal for viral replication; however, the compensatory mutations rescued replication, with the highest rescuing efficiency occurring when both classes of mutations were present. Biochemical analysis showed that a low level of N7 methylation of the D146S methyltransferase is essential for the recovery of adaptive viruses. The methyltransferase K61Q mutation facilitates viral replication through improved N7 methylation activity. The RdRp W751R mutation improves viral replication through an enhanced polymerase activity. Our results have clearly established genetic interactions among flaviviral methyltransferase, RdRp, and the 5′ stem-loop of the genomic RNA. [ABSTRACT FROM AUTHOR]

Mosquito-borne flaviviruses, which include many important human pathogens such as West Nile virus (WNV), Dengue virus (DENV) and Zika virus (ZIKV), had caused numerous emerging epidemics in recent years. Knowledge on the viral genome functions necessary for effective viral replication in mosquito and vertebrate hosts remained obscure. Here, using ZIKV as a model, we found that the conserved downstream of AUG region (DAR), which is known as an essential element for the genome cyclization, involved in viral replication in a host specific manner. Mutational analysis of the DAR element showed that a single-nucleotide mismatch between 5'DAR-3'DAR had little effect on ZIKV replication in mammalian cells, but dramatically impaired viral propagation in mosquito cells. The revertant viruses passaged in mosquito cells generated compensatory mutations restoring the base pairing of DAR, further confirming the importance of the complementarity of DAR in mosquito cells. We demonstrated that a single nucleotide mutation in DAR is sufficient to destroy long-range RNA interaction of ZIKV genome, and affects de novo RNA synthesis at 28 °C instead of 37 °C, resulting in the different replication efficiencies of the mutant viruses in mosquito and mammalian cells. Our results reveal a novel function of the circular form of flavivirus genome in host-specific viral replication, providing new ideas to further explore the functions of viral genome during host adaptation. [ABSTRACT FROM AUTHOR]

The 5′ end of the flavivirus plus-sense RNA genome contains a type 1 cap (m7 GpppAmG), followed by a conserved stem-loop structure. We report that nonstructural protein 5 (NS5) from four serocomplexes of flaviviruses specifically methylates the cap through recognition of the 5′ terminus of viral RNA. Distinct RNA elements are required for the methylations at guanine N-7 on the cap and ribose 2′-OH on the first transcribed nucleotide. In a West Nile virus (WNV) model, N-7 cap methylation requires specific nucleotides at the second and third positions and a 5′ stem-loop structure; in contrast, 2′-OH ribose methylation requires specific nucleotides at the first and second positions, with a minimum 5′ viral RNA of 20 nucleotides. The cap analogues GpppA and m7 GpppA are not active substrates for WNV methytransferase. Footprinting experiments using Gppp- and m7 Gppp-terminated RNAs suggest that the 5′ termini of RNA substrates interact with NS5 during the sequential methylation reactions. Cap methylations could be inhibited by an antisense oligomer targeting the first 20 nucleotides of WNV genome. The viral RNA-specific cap methylation suggests methyltransferase as a novel target for flavivirus drug discovery. [ABSTRACT FROM AUTHOR]