Cellular mRNA of higher eukaryotes and many viral RNA are methylated at the N-7 and 2′-O positions of the 5′ guanosine cap by specific nuclear and cytoplasmic methyltransferases (MTases), respectively. Whereas N-7 methylation is essential for RNA translation and stability 1, the function of 2′-O methylation has remained uncertain since its discovery 35 years ago 2-4. Here, we show that a West Nile virus (WNV) mutant (E218A) that lacks 2′-O MTase activity was attenuated in wild type primary cells and mice but was pathogenic in the absence of type I interferon (IFN) signaling. 2′-O methylation of viral RNA did not affect IFN induction in WNV-infected fibroblasts but instead modulated the antiviral effects of IFN-induced proteins with tetratricopeptide repeats (IFIT), which are interferon-stimulated genes (ISG) implicated in regulation of protein translation. Poxvirus and coronavirus mutants that lacked 2′-O MTase activity similarly showed enhanced sensitivity to the antiviral actions of IFN and specifically, IFIT proteins. Our results demonstrate that the 2′-O methylation of the 5′ cap of viral RNA functions to subvert innate host antiviral responses through escape of IFIT-mediated suppression, and suggest an evolutionary explanation for 2′-O methylation of cellular mRNA: to distinguish self from non-self RNA. Differential methylation of cytoplasmic RNA likely serves as a paradigm for pattern recognition and restriction of propagation of foreign viral RNA in host cells.

The plus-strand RNA genome of flavivirus contains a 5 terminal cap 1 structure (m 7 GpppAmG). The flaviviruses encode one methyltransferase, located at the N-terminal portion of the NS5 protein, to catalyze both guanine N-7 and ribose 2-OH methylations during viral cap formation. Representative flavivirus methyltransferases from dengue, yellow fever, and West Nile virus (WNV) sequentially generate GpppA 3 m 7 GpppA 3 m 7 GpppAm. The 2-O methylation can be uncoupled from the N-7 methylation, since m 7 GpppA-RNA can be readily methylated to m 7 GpppAm-RNA. Despite exhibiting two distinct methylation activities, the crystal structure of WNV methyltransferase at 2.8 Å resolution showed a single binding site for S-adenosyl-L-methionine (SAM), the methyl donor. Therefore, substrate GpppA-RNA should be repositioned to accept the N-7 and 2-O methyl groups from SAM during the sequential reactions. Electrostatic analysis of the WNV methyltransferase structure showed that, adjacent to the SAM-binding pocket, is a highly positively charged surface that could serve as an RNA binding site during cap methylations. Biochemical and mutagenesis analyses show that the N-7 and 2-O cap methylations require distinct buffer conditions and different side chains within the K 61 -D 146 -K 182 -E 218 motif, suggesting that the two reactions use different mechanisms. In the context of complete virus, defects in both methylations are lethal to WNV; however, viruses defective solely in 2-O methylation are attenuated and can protect mice from later wild-type WNV challenge. The results demonstrate that the N-7 methylation activity is essential for the WNV life cycle and, thus, methyltransferase represents a novel target for flavivirus therapy.Eukaryotic mRNAs possess a 5Ј cap structure that is cotranscriptionally formed in the nucleus. mRNA capping is essential for mRNA stability and efficient translation (13, 39). Most animal viruses that replicate in cytoplasm encode their own capping machinery to produce capped RNAs. RNA capping generally consists of three steps in which the 5Ј triphosphate end of nascent RNA transcript is first hydrolyzed to a 5Ј diphosphate by an RNA triphosphatase, then capped with GMP by an RNA guanylyltransferase, and finally methylated at the N-7 position of guanine by an RNA guanine-methyltransferase (N-7 MTase) (15). Additionally, the first and second nucleotides of many cellular and viral mRNAs are further methylated at the ribose 2Ј-OH position by a nucleoside 2Ј-O MTase, to form cap 1 (m 7 GpppNm) and cap 2 (m 7 GpppNmNm) structures, respectively (13). Both N-7 and 2Ј-O MTases use S-adenosyl-L-methionine (SAM) as a methyl donor and generate S-adenosyl-L-homocysteine (SAH) as a by-product. The order of capping and methylation varies among cellular and viral RNAs (13).The genus Flavivirus comprises approximately 70 viruses, many of which are important human pathogens, including four serotypes of dengue virus (DENV), yellow fever virus (YFV), St. Louis encephalitis virus, and West Nile virus (WNV) (23).The flaviviru...

Most eukaryotic and viral mRNAs possess a 5Ј cap that is important for mRNA stability and efficient translation (11). The cap consists of an inverted guanosine, methylated at the N-7 position, linked to the first transcribed RNA nucleotide by a unique 5Ј-5Ј triphosphate bridge (m 7 GpppN; cap 0 structure) (32). The process of RNA capping generally consists of three steps, in which the 5Ј triphosphate end of the nascent RNA transcript is first hydrolyzed to a 5Ј diphosphate by an RNA triphosphatase, then capped with GMP by an RNA guanylyltransferase, and finally methylated at the N-7 position of guanine by an RNA guanine-methyltransferase (N-7 MTase) (13). Additionally, the first and second nucleotides of many cellular and viral mRNAs are further methylated at the ribose 2Ј-OH position by a nucleoside 2Ј-O MTase to form cap 1 (m 7 GpppNm) and cap 2 (m 7 GpppNmNm) structures, respectively (11). Both N-7 and 2Ј-O MTases use S-adenosyl-Lmethionine (AdoMet) as a methyl donor and generate Sadenosyl-L-homocysteine (AdoHcy) as a by-product. The order of the capping and methylation steps is variable among cellular and viral RNAs (11).Many members of the Flavivirus genus are arthropod-borne human pathogens, including West Nile virus (WNV), Yellow fever virus, four serotypes of Dengue virus (DENV), Japanese encephalitis virus, St. Louis encephalitis virus, Murray Valley encephalitis virus, and Tick-borne encephalitis virus (4). The flavivirus genome is a single-stranded, plus-sense RNA of about 11,000 nucleotides that contains a type 1 cap at its 5Ј end (5, 35) and terminates with 5Ј-CU OH -3Ј (35) (see Fig. 1A). 5Ј and 3Ј untranslated regions flank a single open reading frame which encodes a polyprotein that is co-and posttranslationally processed by viral and cellular proteases into three structural proteins (capsid [C], premembrane [prM] or membrane [M], and envelope [E]) and seven nonstructural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5) (4). Since flaviviruses replicate in the cytoplasm, they are expected to encode their own capping enzymes, rather than to use the host's capping apparatus located in the nucleus. Alternatively, since all host proteins have to be synthesized in the cytoplasm, it is possible that cellular capping components could be retained in the cytoplasm for viral RNA capping through specific interactions with a viral protein. Of the four enzymes required for flavivirus m 7 GpppAm-cap formation, only the RNA triphosphatase and 2Ј-O MTase have been mapped to NS3 (19,36) and NS5 (8), respectively, whereas the guanylyltransferase and N-7 MTase remain to be identified. The crystal structure of a ternary complex comprising the DENV-2 MTase domain, AdoHcy, and a GTP analogue suggested that, during 2Ј-O methylation, a specific cap-binding site holds the guanine cap to register the ribose 2Ј-OH of the first transcribed adenosine in close proximity to the AdoMet CH 3 donor (2, 8). Structure and sequence alignments of DENV, vaccinia virus VP39, and other 2Ј-O MTases indicate that a conserved K-D-K-E ...