The flavivirus NS5 protein is a true RNA guanylyltransferase that catalyzes a two-step reaction to form the RNA cap structure

Abstract

The 59-end of the flavivirus genome harbors a methylated m7 GpppA 29OMe cap structure, which is generated by the virus-encoded RNA triphosphatase, RNA (guanine-N7) methyltransferase, nucleoside 29-O-methyltransferase, and RNA guanylyltransferase. The presence of the flavivirus guanylyltransferase activity in NS5 has been suggested by several groups but has not been empirically proven. Here we provide evidence that the N-terminus of the flavivirus NS5 protein is a true RNA guanylyltransferase. We demonstrate that GTP can be used as a substrate by the enzyme to form a covalent GMP-enzyme intermediate via a phosphoamide bond. Mutational studies also confirm the importance of a specific lysine residue in the GTP binding site for the enzymatic activity. We show that the GMP moiety can be transferred to the diphosphate end of an RNA transcript harboring an adenosine as the initiating residue. We also demonstrate that the flavivirus RNA triphosphatase (NS3 protein) stimulates the RNA guanylyltransferase activity of the NS5 protein. Finally, we show that both enzymes are sufficient and necessary to catalyze the de novo formation of a methylated RNA cap structure in vitro using a triphosphorylated RNA transcript. Our study provides biochemical evidence that flaviviruses encode a complete RNA capping machinery.

Identification of a Novel Antiviral Inhibitor of the Flavivirus Guanylyltransferase Enzyme

“…Chlorines in the R2 and R3 positions appear to weaken the enzymatic inhibition capacity of BG-324 (IC 50 of 155 M), whereas chlorines in the R2 and R4 positions appear to increase enzymatic inhibition (IC 50 of 2.6 M), although the K i values of BG-324 and BG-330 are identical. How the capping enzyme forms the guanylated intermediate is unknown, as there is no recognizable guanylyltransferase active-site motif present in any flavivirus protein (15). Therefore, without understanding how the reaction occurs, it is unclear how the presence of chlorines at R2, R3, and R4 may differentially affect enzymatic activity.…”

“…The flavivirus capping enzyme has three distinct functions that can be targeted for therapeutic intervention: the N7/2=-O-methyltransferase reactions (2,3,7,10) and the recently discovered guanylyltransferase reaction (11,14,15). The formation of the 5= cap structure is critical to the survival of the virus for several reasons, including direction of viral polyprotein translation and protection of the 5= end of the genome from cellular exonucleases.…”

Characterization of Dengue Virus NS4A and NS4B Protein Interaction

“…NS3 serves as a protease (using NS2B as a cofactor) (4), RNA helicase, nucleotide triphosphatase, and RNA triphosphatase (5)(6)(7). NS5 functions as a methyltransferase (8)(9)(10), weak guanylyltransferase (11), and RNA-dependent RNA polymerase (12). The glycosylated NS1 protein is essential for viral replication (13,14).…”

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 first step in 5Ј-capping of the positive strand progeny viral RNA is hydrolysis of ␥-phosphate of the 5Ј-triphosphorylated RNA, which is catalyzed by NS3 (11,12). Then GT activity of NS5 adds a GMP, hydrolysis product of GTP, to the 5Ј-diphosphorylated RNA to yield GpppN-RNA (13). Finally, MT activity of NS5 transfers a methyl group from the S-adenosylmethionine (AdoMet) substrate to the N-7 position of the G in GpppN-RNA to yield 7-Me GpppN-RNA (cap 0 structure) (5) (for a review, see Ref.…”