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...

Flaviviruses encode a single methyltransferase domain that sequentially catalyzes two methylations of the viral RNA cap, GpppA-RNA3m 7 GpppA-RNA3m 7 GpppAm-RNA, by using S-adenosyl-L-methionine (SAM) as a methyl donor. Crystal structures of flavivirus methyltransferases exhibit distinct binding sites for SAM, GTP, and RNA molecules. Biochemical analysis of West Nile virus methyltransferase shows that the single SAMbinding site donates methyl groups to both N7 and 2-O positions of the viral RNA cap, the GTP-binding pocket functions only during the 2-O methylation, and two distinct sets of amino acids in the RNA-binding site are required for the N7 and 2-O methylations. These results demonstrate that flavivirus methyltransferase catalyzes two cap methylations through a substrate-repositioning mechanism. In this mechanism, guanine N7 of substrate GpppA-RNA is first positioned to SAM to generate m 7 GpppA-RNA, after which the m 7 G moiety is repositioned to the GTP-binding pocket to register the 2-OH of the adenosine with SAM, generating m 7 GpppAm-RNA. Because N7 cap methylation is essential for viral replication, inhibitors designed to block the pocket identified for the N7 cap methylation could be developed for flavivirus therapy.Eukaryotic mRNAs contain a 5Ј cap structure that is essential for RNA splicing, export, stability, and translation (12). In general, RNA capping consists of four steps. (i) The 5Ј-triphosphate end of the nascent RNA transcript is hydrolyzed to a 5Ј diphosphate by an RNA triphosphatase; (ii) the GMP moiety of GTP is transferred to the 5Ј diphosphate of RNA by an RNA guanylyltransferase; (iii) the N7 position of guanine is methylated by an RNA guanine-methyltransferase (N7 MTase), yielding a cap 0 structure (GpppN); and (iv) 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, so as to form cap 1 (m 7 GpppNm) and cap 2 (m 7 GpppNmNm) structures, respectively (12). S-Adenosyl-L-methionine (SAM) is the methyl donor for both the N7 and 2Ј-O methylations, generating Sadenosyl-L-homocysteine (SAH) as a by-product. Because host mRNA capping occurs in the nucleus, viruses replicating in the cytoplasm encode their own unique machineries for RNA capping. For example, the plus-strand RNA alphaviruses methylate GTP prior to the transfer of m 7 GMP to the 5Ј diphosphate of the RNA (2). The minus-strand RNA vesicular stomatitis virus (VSV) transfers GDP, rather than GMP, to the 5Ј monophosphate of the RNA (24). The differences between the host and viral cap formation processes could potentially be used for development of antiviral therapy.The genus Flavivirus contains a number of significant human pathogens, including the four serotypes of dengue virus (DENV), yellow fever virus (YFV), Japanese encephalitis virus, West Nile virus (WNV), and tick-borne encephalitis virus (6). Among those, DENV alone was estimated to cause 50 million human cases annually (38). Flaviviruses replicate in the cytoplasm. The viral ge...

The hepatitis B virus (HBV) core protein is essential for HBV replication and an important target for antiviral drug discovery. We report the first, to our knowledge, high-resolution crystal structure of an antiviral compound bound to the HBV core protein. The compound NVR-010-001-E2 can induce assembly of the HBV core wild-type and Y132A mutant proteins and thermostabilize the proteins with a T m increase of more than 10°C. NVR-010-001-E2 binds at the dimer-dimer interface of the core proteins, forms a new interaction surface promoting protein-protein interaction, induces protein assembly, and increases stability. The impact of naturally occurring core protein mutations on antiviral activity correlates with NVR-010-001-E2 binding interactions determined by crystallography. The crystal structure provides understanding of a drug efficacy mechanism related to the induction and stabilization of proteinprotein interactions and enables structure-guided design to improve antiviral potency and drug-like properties.HBV treatment | HBV inhibitor | core | capsid | protein-protein interaction