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 → m ⁷ GpppA → 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.

Many flaviviruses are globally important human pathogens. Their plus-strand RNA genome contains a 5′-cap structure that is methylated at the guanine N-7 and the ribose 2′-OH positions of the first transcribed nucleotide, adenine (m ⁷ GpppAm). Using West Nile virus (WNV), we demonstrate, for the first time, that the nonstructural protein 5 (NS5) mediates both guanine N-7 and ribose 2′-O methylations and therefore is essential for flavivirus 5′-cap formation. We show that a recombinant full-length and a truncated NS5 protein containing the methyltransferase (MTase) domain methylates GpppA-capped and m ⁷ GpppA-capped RNAs to m ⁷ GpppAm-RNA, using S -adenosylmethionine as a methyl donor. Furthermore, methylation of GpppA-capped RNA sequentially yielded m ⁷ GpppA- and m ⁷ GpppAm-RNA products, indicating that guanine N-7 precedes ribose 2′-O methylation. Mutagenesis of a K ₆₁ -D ₁₄₆ -K ₁₈₂ -E ₂₁₈ tetrad conserved in other cellular and viral MTases suggests that NS5 requires distinct amino acids for its N-7 and 2′-O MTase activities. The entire K ₆₁ -D ₁₄₆ -K ₁₈₂ -E ₂₁₈ motif is essential for 2′-O MTase activity, whereas N-7 MTase activity requires only D ₁₄₆ . The other three amino acids facilitate, but are not essential for, guanine N-7 methylation. Amino acid substitutions within the K ₆₁ -D ₁₄₆ -K ₁₈₂ -E ₂₁₈ motif in a WNV luciferase-reporting replicon significantly reduced or abolished viral replication in cells. Additionally, the mutant MTase-mediated replication defect could not be trans complemented by a wild-type replicase complex. These findings demonstrate a critical role for the flavivirus MTase in viral reproduction and underscore this domain as a potential target for antiviral therapy.

The recent outbreak of coronavirus disease 2019 (COVID-19) highlights an urgent need for therapeutics. Through a series of drug repurposing screening campaigns, niclosamide, an FDA-approved anthelminthic drug, was found to be effective against various viral infections with nanomolar to micromolar potency such as SARS-CoV, MERS-CoV, ZIKV, HCV and human adenovirus, indicating its potential as an antiviral agent. In this brief article, we summarize the broad antiviral activities of niclosamide and highlight its potential clinical use for treatment of COVID-19.

▪ Abstract Superantigens (SAGs) are a class of immunostimulatory and disease-causing proteins of bacterial or viral origin with the ability to activate large fractions (5–20%) of the T cell population. Activation requires simultaneous interaction of the SAG with the Vβ domain of the T cell receptor (TCR) and with major histocompatibility complex (MHC) class II molecules on the surface of an antigen-presenting cell. Recent advances in knowledge of the three-dimensional structure of bacterial SAGs, and of their complexes with MHC class II molecules and the TCR β chain, provide a framework for understanding the molecular basis of T cell activation by these potent mitogens. These structures along with those of TCR-peptide/MHC complexes reveal how SAGs circumvent the normal mechanism for T cell activation by peptide/MHC and how they stimulate T cells expressing TCR β chains from a number of different families, resulting in polyclonal T cell activation. The crystal structures also provide insights into the basis for the specificity of different SAGs for particular TCR β chains, and for the observed influence of the TCR α chain on SAG reactivity. These studies open the way to the design of SAG variants with altered binding properties for TCR and MHC for use as tools in dissecting structure-activity relationships in this system.