Shee-Mei Lok

The Flavivirus Precursor Membrane-Envelope Protein Complex: Structure and Maturation

Many viruses go through a maturation step in the final stages of assembly before being transmitted to another host. The maturation process of flaviviruses is directed by the proteolytic cleavage of the precursor membrane protein (prM), turning inert virus into infectious particles. We have determined the 2.2 angstrom resolution crystal structure of a recombinant protein in which the dengue virus prM is linked to the envelope glycoprotein E. The structure represents the prM-E heterodimer and fits well into the cryo–electron microscopy density of immature virus at neutral pH. The pr peptide β-barrel structure covers the fusion loop in E, preventing fusion with host cell membranes. The structure provides a basis for identifying the stages of its pH-directed conformational metamorphosis during maturation, ending with release of pr when budding from the host.

Binding of a neutralizing antibody to dengue virus alters the arrangement of surface glycoproteins.

The monoclonal antibody 1A1D-2 has been shown to strongly neutralize dengue virus serotypes 1, 2 and 3, primarily by inhibiting attachment to host cells. A crystal structure of its antigen binding fragment (Fab) complexed with domain III of the viral envelope glycoprotein, E, showed that the epitope would be partially occluded in the known structure of the mature dengue virus. Nevertheless, antibody could bind to the virus at 37 °C, suggesting that the virus is in dynamic motion making hidden epitopes briefly available. A cryo-electron microscope image reconstruction of the virus:Fab complex showed large changes in the organization of the E protein that exposed the epitopes on two of the three E molecules in each of the 60 icosahedral asymmetric units of the virus. The changes in the structure of the viral surface are presumably responsible for inhibiting attachment to cells.

Structure of the thermally stable Zika virus

Zika virus (ZIKV), formerly a neglected pathogen, has recently been associated with microcephaly in fetuses, and with Guillian–Barré syndrome in adults. Here we present the 3.7 Å resolution cryo-electron microscopy structure of ZIKV, and show that the overall architecture of the virus is similar to that of other flaviviruses. Sequence and structural comparisons of the ZIKV envelope (E) protein with other flaviviruses show that parts of the E protein closely resemble the neurovirulent West Nile and Japanese encephalitis viruses, while others are similar to dengue virus (DENV). However, the contribution of the E protein to flavivirus pathobiology is currently not understood. The virus particle was observed to be structurally stable even when incubated at 40 °C, in sharp contrast to the less thermally stable DENV. This is also reflected in the infectivity of ZIKV compared to DENV serotypes 2 and 4 (DENV2 and DENV4) at different temperatures. The cryo-electron microscopy structure shows a virus with a more compact surface. This structural stability of the virus may help it to survive in the harsh conditions of semen, saliva and urine. Antibodies or drugs that destabilize the structure may help to reduce the disease outcome or limit the spread of the virus.

The Structural Basis for Serotype-Specific Neutralization of Dengue Virus by a Human Antibody

The mechanism of action of a serotype-specific natural human antibody against dengue virus has been identified. Defeating Dengue Dengue virus is a major mosquito-borne viral pathogen that is transmitted through the bite of an infected mosquito. Infection can be asymptomatic, cause a self-limiting fever, or result in potentially fatal hemorrhage. There are no approved vaccines or antiviral therapies for dengue, and current treatment is restricted to fluid replacement. Thus, there is an urgent need for new treatment options for this disease. Dengue virus consists of four related but distinct serotypes, and infection is thought to elicit lifelong immunity to the infecting serotype in patients who recover but only short-term immunity against the other serotypes. Immunity is mediated by serotype-specific antibodies, but little is known about their specificity or mode of action. Now, Teoh et al. characterize a neutralizing human monoclonal antibody induced by natural dengue infection. This antibody is specific for dengue virus serotype 1 and shows little or no binding or neutralizing activity for serotypes 2, 3, and 4. The authors demonstrate that the antibody binds across two adjacent viral envelope proteins and identify the amino acids that comprise the binding site. The antiviral activity of this antibody is linked principally to a blockade of virus binding to target host cells. Treatment with this antibody results in increased survival in a mouse model of dengue virus infection. This human antibody represents a new therapeutic candidate for treating dengue serotype 1 infection. These findings also provide a structural and molecular context for understanding the nature of durable, serotype-specific immunity to dengue infection and thus have implications for the design and evaluation of vaccines against dengue. Dengue virus (DENV) is a mosquito-borne flavivirus that affects 2.5 billion people worldwide. There are four dengue serotypes (DENV1 to DENV4), and infection with one elicits lifelong immunity to that serotype but offers only transient protection against the other serotypes. Identification of the protective determinants of the human antibody response to DENV is a vital requirement for the design and evaluation of future preventative therapies and treatments. Here, we describe the isolation of a neutralizing antibody from a DENV1-infected patient. The human antibody 14c10 (HM14c10) binds specifically to DENV1. HM14c10 neutralizes the virus principally by blocking virus attachment; at higher concentrations, a post-attachment step can also be inhibited. In vivo studies show that the HM14c10 antibody has antiviral activity at picomolar concentrations. A 7 Å resolution cryoelectron microscopy map of Fab fragments of HM14c10 in a complex with DENV1 shows targeting of a discontinuous epitope that spans the adjacent surface of envelope protein dimers. As found previously, a human antibody specific for the related West Nile virus binds to a similar quaternary structure, suggesting that this could be an immunodominant epitope. These findings provide a structural and molecular context for durable, serotype-specific immunity to DENV infection.

Structural basis for the preferential recognition of immature flaviviruses by a fusion‐loop antibody

Flaviviruses are a group of human pathogens causing severe encephalitic or hemorrhagic diseases that include West Nile, dengue and yellow fever viruses. Here, using X‐ray crystallography we have defined the structure of the flavivirus cross‐reactive antibody E53 that engages the highly conserved fusion loop of the West Nile virus envelope glycoprotein. Using cryo‐electron microscopy, we also determined that E53 Fab binds preferentially to spikes in noninfectious, immature flavivirions but is unable to bind significantly to mature virions, consistent with the limited solvent exposure of the epitope. We conclude that the neutralizing impact of E53 and likely similar fusion‐loop‐specific antibodies depends on its binding to the frequently observed immature component of flavivirus particles. Our results elucidate how fusion‐loop antibodies, which comprise a significant fraction of the humoral response against flaviviruses, can function to control infection without appreciably recognizing mature virions. As these highly cross‐reactive antibodies are often weakly neutralizing they also may contribute to antibody‐dependent enhancement and flavi virus pathogenesis thereby complicating development of safe and effective vaccines.

A highly potent human antibody neutralizes dengue virus serotype 3 by binding across three surface proteins

Dengue virus (DENV) infects ~400 million people annually. There is no licensed vaccine or therapeutic drug. Only a small fraction of the total DENV-specific antibodies in a naturally occurring dengue infection consists of highly neutralizing antibodies. Here we show that the DENV-specific human monoclonal antibody 5J7 is exceptionally potent, neutralizing 50% of virus at nanogram-range antibody concentration. The 9 Å resolution cryo-electron microscopy structure of the Fab 5J7–DENV complex shows that a single Fab molecule binds across three envelope proteins and engages three functionally important domains, each from a different envelope protein. These domains are critical for receptor binding and fusion to the endosomal membrane. The ability to bind to multiple domains allows the antibody to fully coat the virus surface with only 60 copies of Fab, that is, half the amount compared with other potent antibodies. Our study reveals a highly efficient and unusual mechanism of molecular recognition by an antibody.

Structural Changes in Dengue Virus When Exposed to a Temperature of 37°C

ABSTRACT Previous binding studies of antibodies that recognized a partially or fully hidden epitope suggest that insect cell-derived dengue virus undergoes structural changes at an elevated temperature. This was confirmed by our cryo-electron microscopy images of dengue virus incubated at 37°C, where viruses change their surface from smooth to rough. Here we present the cryo-electron microscopy structures of dengue virus at 37°C. Image analysis showed four classes of particles. The three-dimensional (3D) map of one of these classes, representing half of the imaged virus population, shows that the E protein shell has expanded and there is a hole at the 3-fold vertices. Fitting E protein structures into the map suggests that all of the interdimeric and some intradimeric E protein interactions are weakened. The accessibility of some previously found cryptic epitopes on this class of particles is discussed.

Cryo-Em Structure of an Antibody that Neutralizes Dengue Virus Type 2 by Locking E Protein Dimers.

An antibody to lock dengue virus out Mosquito-borne dengue virus (DENV) is a growing public health threat. Nearly 400 million people are infected annually, and no vaccine is currently available. Fibriansah et al. report that a human antibody (2D22) specific for DENV serotype 2, when given therapeutically, can protect mice from a lethal form of this virus. Structural analysis revealed that 2D22 binds across multiple DENV envelope proteins, which probably blocks the ability of these proteins to assemble into the orientation necessary for viral entry. The epitope where 2D22 binds to the virus may therefore represent a potential vaccine target. Science, this issue p. 88 A human antibody specific for dengue virus locks viral envelope proteins into a conformation that prohibits viral entry. There are four closely-related dengue virus (DENV) serotypes. Infection with one serotype generates antibodies that may cross-react and enhance infection with other serotypes in a secondary infection. We demonstrated that DENV serotype 2 (DENV2)–specific human monoclonal antibody (HMAb) 2D22 is therapeutic in a mouse model of antibody-enhanced severe dengue disease. We determined the cryo–electron microscopy (cryo-EM) structures of HMAb 2D22 complexed with two different DENV2 strains. HMAb 2D22 binds across viral envelope (E) proteins in the dimeric structure, which probably blocks the E protein reorganization required for virus fusion. HMAb 2D22 “locks” two-thirds of or all dimers on the virus surface, depending on the strain, but neutralizes these DENV2 strains with equal potency. The epitope defined by HMAb 2D22 is a potential target for vaccines and therapeutics.

A potent anti‐dengue human antibody preferentially recognizes the conformation of E protein monomers assembled on the virus surface

Dengue virus (DENV), which consists of four serotypes (DENV1‐4), infects over 400 million people annually. Previous studies have indicated most human monoclonal antibodies (HMAbs) from dengue patients are cross‐reactive and poorly neutralizing. Rare neutralizing HMAbs are usually serotype‐specific and bind to quaternary structure‐dependent epitopes. We determined the structure of DENV1 complexed with Fab fragments of a highly potent HMAb 1F4 to 6 Å resolution by cryo‐EM. Although HMAb 1F4 appeared to bind to virus and not E proteins in ELISAs in the previous study, our structure showed that the epitope is located within an envelope (E) protein monomer, and not across neighboring E proteins. The Fab molecules bind to domain I (DI), and DI‐DII hinge of the E protein. We also showed that HMAb 1F4 can neutralize DENV at different stages of viral entry in a cell type and receptor dependent manner. The structure reveals the mechanism by which this potent and specific antibody blocks viral infection.

2'-O methylation of internal adenosine by flavivirus NS5 methyltransferase.

RNA modification plays an important role in modulating host-pathogen interaction. Flavivirus NS5 protein encodes N-7 and 2′-O methyltransferase activities that are required for the formation of 5′ type I cap (m7GpppAm) of viral RNA genome. Here we reported, for the first time, that flavivirus NS5 has a novel internal RNA methylation activity. Recombinant NS5 proteins of West Nile virus and Dengue virus (serotype 4; DENV-4) specifically methylates polyA, but not polyG, polyC, or polyU, indicating that the methylation occurs at adenosine residue. RNAs with internal adenosines substituted with 2′-O-methyladenosines are not active substrates for internal methylation, whereas RNAs with adenosines substituted with N6-methyladenosines can be efficiently methylated, suggesting that the internal methylation occurs at the 2′-OH position of adenosine. Mass spectroscopic analysis further demonstrated that the internal methylation product is 2′-O-methyladenosine. Importantly, genomic RNA purified from DENV virion contains 2′-O-methyladenosine. The 2′-O methylation of internal adenosine does not require specific RNA sequence since recombinant methyltransferase of DENV-4 can efficiently methylate RNAs spanning different regions of viral genome, host ribosomal RNAs, and polyA. Structure-based mutagenesis results indicate that K61-D146-K181-E217 tetrad of DENV-4 methyltransferase forms the active site of internal methylation activity; in addition, distinct residues within the methyl donor (S-adenosyl-L-methionine) pocket, GTP pocket, and RNA-binding site are critical for the internal methylation activity. Functional analysis using flavivirus replicon and genome-length RNAs showed that internal methylation attenuated viral RNA translation and replication. Polymerase assay revealed that internal 2′-O-methyladenosine reduces the efficiency of RNA elongation. Collectively, our results demonstrate that flavivirus NS5 performs 2′-O methylation of internal adenosine of viral RNA in vivo and host ribosomal RNAs in vitro.