Hongmin Li

West Nile Virus 5′-Cap Structure Is Formed by Sequential Guanine N-7 and Ribose 2′-O Methylations by Nonstructural Protein 5

ABSTRACT 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 (m7GpppAm). 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 m7GpppA-capped RNAs to m7GpppAm-RNA, using S-adenosylmethionine as a methyl donor. Furthermore, methylation of GpppA-capped RNA sequentially yielded m7GpppA- and m7GpppAm-RNA products, indicating that guanine N-7 precedes ribose 2′-O methylation. Mutagenesis of a K61-D146-K182-E218 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 K61-D146-K182-E218 motif is essential for 2′-O MTase activity, whereas N-7 MTase activity requires only D146. The other three amino acids facilitate, but are not essential for, guanine N-7 methylation. Amino acid substitutions within the K61-D146-K182-E218 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.

Structure and function of flavivirus NS5 methyltransferase

ABSTRACT The plus-strand RNA genome of flavivirus contains a 5′ terminal cap 1 structure (m7GpppAmG). 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 → m7GpppA → m7GpppAm. The 2′-O methylation can be uncoupled from the N-7 methylation, since m7GpppA-RNA can be readily methylated to m7GpppAm-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 K61-D146-K182-E218 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.

Three-Dimensional Structure of the Complex between a T Cell Receptor β Chain and the Superantigen Staphylococcal Enterotoxin B

Abstract Superantigens (SAGs) are a class of immunostimulatory proteins of bacterial or viral origin that activate T cells by binding to the Vβ domain of the T cell antigen receptor (TCR). The three-dimensional structure of the complex between a TCR β chain (mouse Vβ8.2) and the SAG staphylococcal enterotoxin B (SEB) at 2.4 A resolution reveals why SEB recognizes only certain Vβ families, as well as why only certain SAGs bind mouse Vβ8.2. Models of the TCR-SEB-peptide/MHC class II complex indicate that Vα interacts with the MHC β chain in the TCR-SAG-MHC complex. The extent of the interaction is variable and is largely determined by the geometry of Vα/Vβ domain association. This variability can account for the preferential expression of certain Vα regions among T cells reactive with SEB.

X-ray snapshots of the maturation of an antibody response to a protein antigen

The process whereby the immune system generates antibodies of higher affinities during a response to antigen (affinity maturation) is a prototypical example of molecular evolution. Earlier studies have been confined to antibodies specific for small molecules (haptens) rather than for proteins. We compare the structures of four antibodies bound to the same site on hen egg white lysozyme (HEL) at different stages of affinity maturation. These X-ray snapshots reveal that binding is enhanced, not through the formation of additional hydrogen bonds or van der Waals contacts or by an increase in total buried surface, but by burial of increasing amounts of apolar surface at the expense of polar surface, accompanied by improved shape complementarity. The increase in hydrophobic interactions results from highly correlated rearrangements in antibody residues at the interface periphery, adjacent to the central energetic hot spot. This first visualization of the maturation of antibodies to protein provides insights into the evolution of high affinity in other protein–protein interfaces.

Crystal Structure of a Superantigen Bound to the High-Affinity, Zinc-Dependent Site on MHC Class II

MHC class II molecules possess two binding sites for bacterial superantigens (SAGs): a low-affinity site on the alpha chain and a high-affinity, zinc-dependent site on the beta chain. Only the former has been defined crystallographically. We report the structure of streptococcal pyrogenic exotoxin C (SPE-C) complexed with HLA-DR2a (DRA*0101, DRB5*0101) bearing a self-peptide from myelin basic protein (MBP). SPE-C binds the beta chain through a zinc bridge that links the SAG and class II molecules. Surprisingly, SPE-C also makes extensive contacts with the MBP peptide, such that peptide accounts for one third of the surface area of the MHC molecule buried in the complex, similar to TCR-peptide/MHC complexes. Thus, SPE-C may optimize T cell responses by mimicking the peptide dependence of conventional antigen presentation and recognition.

Structures of two streptococcal superantigens bound to TCR beta chains reveal diversity in the architecture of T cell signaling complexes

Superantigens (SAGs) crosslink MHC class II and TCR molecules, resulting in an overstimulation of T cells associated with human disease. SAGs interact with several different surfaces on MHC molecules, necessitating the formation of multiple distinct MHC-SAG-TCR ternary signaling complexes. Variability in SAG-TCR binding modes could also contribute to the structural heterogeneity of SAG-dependent signaling complexes. We report crystal structures of the streptococcal SAGs SpeA and SpeC in complex with their corresponding TCR beta chain ligands that reveal distinct TCR binding modes. The SpeC-TCR beta chain complex structure, coupled with the recently determined SpeC-HLA-DR2a complex structure, provides a model for a novel T cell signaling complex that precludes direct TCR-MHC interactions. Thus, highly efficient T cell activation may be achieved through structurally diverse strategies of TCR ligation.

Immunoassay Targeting Nonstructural Protein 5 To Differentiate West Nile Virus Infection from Dengue and St. Louis Encephalitis Virus Infections and from Flavivirus Vaccination

ABSTRACT West Nile virus (WNV) is an emerging flavivirus that has caused frequent epidemics since 1996. Besides natural transmission by mosquitoes, WNV can also be transmitted through blood transfusion and organ transplantation, thus heightening the urgency of development of a specific and rapid serologic assay of WNV infection. The current immunoassays lack specificity because they are based on detection of antibodies against WNV structural proteins and immune responses to structural proteins among flaviviruses cross-react to each other. Here, we describe microsphere immunoassays that detect antibodies to nonstructural proteins 3 and 5 (NS3 and NS5). In contrast to immunoassays based on viral envelope and NS3 proteins, the NS5-based assay (i) reliably discriminates between WNV infections and dengue virus or St. Louis encephalitis virus infections, (ii) differentiates between flavivirus vaccination and natural WNV infection, and (iii) indicates recent infections. These unique features of the NS5-based immunoassay will be very useful for both clinical and veterinary diagnosis of WNV infection.

Structure of the Vdelta domain of a human gammadelta T-cell antigen receptor.

Antigen recognition by T lymphocytes is mediated by cell-surface glycoproteins known as T-cell antigen receptors (TCRs). These are composed of α and β, or γ and δ, polypeptide chains with variable (V) and constant (C) regions. In contrast to αβ TCRs, which recognize antigen only as peptide fragments bound to molecules of the major histocompatibility complex (MHC), γδ TCRs appear to recognize proteins directly, without antigen processing, and to recognize MHC molecules independently of the bound peptide. Moreover, small phosphate-containing non-peptide compounds have also been identified as ligands for certainγδ T cells,. These studies indicate that antigen recognition by γδ TCRs may be fundamentally different from that by αβ TCRs. The three-dimensional structures of several αβ TCRs and TCR fragments, and their complexes with peptide–MHC or superantigens, have been determined. Here we report the crystal structure of the Vδ domain of a human γδ TCR at 1.9u2009Å resolution. A comparison with antibody and αβ TCR V domains reveals that the framework structure of Vδ more closely resembles that of VHthan of Vα, Vβ or VL(where H and L refer to heavy and light chains), whereas therelative positions and conformations of its complementarity-determining regions (CDRs) share features of both Vα and VH. These results provide the first direct evidence that γδ TCRs are structurally distinct from αβ TCRs and, together with the observation that the CDR3 length distribution of TCR δ chains is similar to that of immunoglobulin heavy chains, are consistent with functional studies suggesting that recognition of certain antigens by γδ TCRs may resemble antigen recognition by antibodies.

Distinct RNA Elements Confer Specificity to Flavivirus RNA Cap Methylation Events

ABSTRACT The 5′ end of the flavivirus plus-sense RNA genome contains a type 1 cap (m7GpppAmG), followed by a conserved stem-loop structure. We report that nonstructural protein 5 (NS5) from four serocomplexes of flaviviruses specifically methylates the cap through recognition of the 5′ terminus of viral RNA. Distinct RNA elements are required for the methylations at guanine N-7 on the cap and ribose 2′-OH on the first transcribed nucleotide. In a West Nile virus (WNV) model, N-7 cap methylation requires specific nucleotides at the second and third positions and a 5′ stem-loop structure; in contrast, 2′-OH ribose methylation requires specific nucleotides at the first and second positions, with a minimum 5′ viral RNA of 20 nucleotides. The cap analogues GpppA and m7GpppA are not active substrates for WNV methytransferase. Footprinting experiments using Gppp- and m7Gppp-terminated RNAs suggest that the 5′ termini of RNA substrates interact with NS5 during the sequential methylation reactions. Cap methylations could be inhibited by an antisense oligomer targeting the first 20 nucleotides of WNV genome. The viral RNA-specific cap methylation suggests methyltransferase as a novel target for flavivirus drug discovery.

West Nile Virus Methyltransferase Catalyzes Two Methylations of the Viral RNA Cap through a Substrate-Repositioning Mechanism

ABSTRACT Flaviviruses encode a single methyltransferase domain that sequentially catalyzes two methylations of the viral RNA cap, GpppA-RNA→m7GpppA-RNA→m7GpppAm-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 SAM-binding 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 m7GpppA-RNA, after which the m7G moiety is repositioned to the GTP-binding pocket to register the 2′-OH of the adenosine with SAM, generating m7GpppAm-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.