Mutagenesis of the Dengue Virus Type 2 NS5 Methyltransferase Domain

Abstract

The Flavivirus NS5 protein possesses both (guanine-N7)-methyltransferase and nucleoside-2 -O methyltransferase activities required for sequential methylation of the cap structure present at the 5 end of the Flavivirus RNA genome. Seventeen mutations were introduced into the dengue virus type 2 NS5 methyltransferase domain, targeting amino acids either predicted to be directly involved in S-adenosyl-L-methionine binding or important for NS5 conformation and/or charged interactions. The effects of the mutations on (i) (guanine-N7)-methyltransferase and nucleoside-2 -O methyltransferase activities using biochemical assays based on a bacterially expressed NS5 methyltransferase domain and (ii) viral replication using a dengue virus type 2 infectious cDNA clone were examined. Clustered mutations targeting the S-adenosyl-L-methionine binding pocket or an active site residue abolished both methyltransferase activities and viral replication, demonstrating that both methyltransferase activities utilize a single S-adenosyl-L-methionine binding pocket. Substitutions to single amino acids binding S-adenosyl-L-methionine decreased both methyltransferase activities by varying amounts. However, viruses that replicated at wild type levels could be recovered with mutations that reduced both activities by >75%, suggesting that only a threshold level of methyltransferase activity was required for virus replication in vivo. Mutation of residues outside of regions directly involved in S-adenosyl-L-methionine binding or catalysis also affected methyltransferase activity and virus replication. The recovery of viruses containing compensatory second site mutations in the NS5 and NS3 proteins identified regions of the methyltransferase domain important for overall stability of the protein or likely to play a role in virus replication distinct from that of cap methylation.Cellular and many viral mRNAs contain a modified 5Ј-terminal guanosine "cap" structure covalently linked to the 5Ј end of the mRNA. The study of mRNA cap formation using viral, eukaryotic, and protozoan systems has shown that the formation of the 5Ј-RNA cap structure requires three sequential enzymatic reactions. First, the 5Ј-terminal triphosphate of the nascent RNA is hydrolyzed to a diphosphate by the enzyme RNA triphosphatase. Second, the RNA is capped with GMP in a 5Ј-5Ј triphosphate linkage by mRNA guanyltransferase, and third, the guanosine is methylated at the N 7 position by a (guanine-N7)-methyltransferase (N7 MTase) 2 using S-adenosyl-Lmethionine (AdoMet) as a methyl donor to form a type 0 ( 7Me G 5Ј -ppp 5Ј N) cap structure (1, 2). Nucleotides adjacent to the cap structure may be further methylated by nucleoside-2Ј-O methyltransferases (2Ј-O MTase) to give type I ( 7Me G 5Ј -ppp 5Ј N Me ) or type II cap structures ( 7Me G 5Ј -ppp 5Ј N Me N Me ). In the simplest case, such as for yeast, each of the enzymatic activities required for RNA capping resides in an individual protein. However, studies on viral capping systems have revealed many interesting variations o...

Substitution of NS5 N-terminal Domain of Dengue Virus Type 2 RNA with Type 4 Domain Caused Impaired Replication and Emergence of Adaptive Mutants with Enhanced Fitness

“…The sequence alignment of the D2MT domain that was substituted with D4MT ( Fig. 1A) shows that all of these amino acid residues that were shown to be important for N-7 and 2Ј-OMe activities and viral replication (49) are conserved in the D2MT and D4MT domains. This observation may explain why the MT activities were not affected ( Fig.…”

“…Moreover, the clustered mutations targeting the S-adenosylmethionine binding pocket (K76A/D79A and G81A/G83A/G85A) or the mutation of MT active site residues (D146A/E149A) all either abolished or severely reduced both N-7 and 2Ј-OMe activities (Ͼ90%). Interestingly, the S46A and D131A mutations were unstable and reverted back to WT (49). The sequence alignment of the D2MT domain that was substituted with D4MT ( Fig.…”

A Crystal Structure of the Dengue Virus Non-structural Protein 5 (NS5) Polymerase Delineates Interdomain Amino Acid Residues That Enhance Its Thermostability and de Novo Initiation Activities

“…The wild-type and mutant plasmids were used to produce in vitro RNA transcripts as described previously (13). The in vitro transcripts were transfected into BHK-21 cells to recover infectious virus, and the virus was analyzed for the presence of the introduced mutation as described previously (14).…”

Structural and Functional Analyses of a Conserved Hydrophobic Pocket of Flavivirus Methyltransferase

“…Previous studies have established functional significance of conserved residues within the AdoMet-binding pocket (19,20,28,37). Those studies showed that mutations of residues predicted to be involved in AdoMet binding (such as Ser-56, Gly-81, Gly-83, Gly-85, Trp-87, Lys-105, His-110, Glu-111, Asp-131, Ile-147, Asp-146, and Glu-149) reduced, and in some cases abolished, either the N7 or the 2Ј-O-methylation, or both.…”

“…The differences between host and viral cap formation could potentially be used for development of antiviral therapy. Flavivirus MTase has been shown to be essential for WNV (20,27), Kunjin virus (35), YFV (36), and DENV replication (37). Furthermore, sinefungin (SIN), an AdoMet analog, was shown to inhibit both N7 and 2Ј-O-methylations of WNV MTase, as well as to inhibit WNV replication in cell culture (28).…”