Moh Lan Yap

Structure of human enterovirus 71 in complex with a capsid-binding inhibitor

Human enterovirus 71 is a picornavirus causing hand, foot, and mouth disease that may progress to fatal encephalitis in infants and small children. As of now, no cure is available for enterovirus 71 infections. Small molecule inhibitors binding into a hydrophobic pocket within capsid viral protein 1 were previously shown to effectively limit infectivity of many picornaviruses. Here we report a 3.2-Å-resolution X-ray structure of the enterovirus 71 virion complexed with the capsid-binding inhibitor WIN 51711. The inhibitor replaced the natural pocket factor within the viral protein 1 pocket without inducing any detectable rearrangements in the structure of the capsid. Furthermore, we show that the compound stabilizes enterovirus 71 virions and limits its infectivity, probably through restricting dynamics of the capsid necessary for genome release. Thus, our results provide a structural basis for development of antienterovirus 71 capsid-binding drugs.

Structure and function of bacteriophage T4

Bacteriophage T4 is the most well-studied member of Myoviridae, the most complex family of tailed phages. T4 assembly is divided into three independent pathways: the head, the tail and the long tail fibers. The prolate head encapsidates a 172 kbp concatemeric dsDNA genome. The 925 Å-long tail is surrounded by the contractile sheath and ends with a hexagonal baseplate. Six long tail fibers are attached to the baseplates periphery and are the host cells recognition sensors. The sheath and the baseplate undergo large conformational changes during infection. X-ray crystallography and cryo-electron microscopy have provided structural information on protein-protein and protein-nucleic acid interactions that regulate conformational changes during assembly and infection of Escherichia coli cells.

Role of bacteriophage T4 baseplate in regulating assembly and infection.

Significance This study examines how the high-energy, dome-shaped infectious form of the bacteriophage T4 baseplate assembles as opposed to how it assembles in the low-energy, star-shaped form that occurs after infection. Normal expectations would be that a molecular assembly occurs as a result of loss of energy. However, a virus has to be poised in a high-energy form to fight its way into a host. Our investigations of T4 have now shown how bacteriophage T4 can assemble into a high-energy form and how the structure of the components directs the sequential conformational changes that gain access to the host, an Escherichia coli bacterium. Bacteriophage T4 consists of a head for protecting its genome and a sheathed tail for inserting its genome into a host. The tail terminates with a multiprotein baseplate that changes its conformation from a “high-energy” dome-shaped to a “low-energy” star-shaped structure during infection. Although these two structures represent different minima in the total energy landscape of the baseplate assembly, as the dome-shaped structure readily changes to the star-shaped structure when the virus infects a host bacterium, the dome-shaped structure must have more energy than the star-shaped structure. Here we describe the electron microscopy structure of a 3.3-MDa in vitro-assembled star-shaped baseplate with a resolution of 3.8 Å. This structure, together with other genetic and structural data, shows why the high-energy baseplate is formed in the presence of the central hub and how the baseplate changes to the low-energy structure, via two steps during infection. Thus, the presence of the central hub is required to initiate the assembly of metastable, high-energy structures. If the high-energy structure is formed and stabilized faster than the low-energy structure, there will be insufficient components to assemble the low-energy structure.

Obstruction of Dengue Virus Maturation by Fab Fragments of the 2H2 Antibody.

ABSTRACT The 2H2 monoclonal antibody recognizes the precursor peptide on immature dengue virus and might therefore be a useful tool for investigating the conformational change that occurs when the immature virus enters an acidic environment. During dengue virus maturation, spiky, immature, noninfectious virions change their structure to form smooth-surfaced particles in the slightly acidic environment of the trans-Golgi network, thereby allowing cellular furin to cleave the precursor-membrane proteins. The dengue virions become fully infectious when they release the cleaved precursor peptide upon reaching the neutral-pH environment of the extracellular space. Here we report on the cryo-electron microscopy structures of the immature virus complexed with the 2H2 antigen binding fragments (Fab) at different concentrations and under various pH conditions. At neutral pH and a high concentration of Fab molecules, three Fab molecules bind to three precursor-membrane proteins on each spike of the immature virus. However, at a low concentration of Fab molecules and pH 7.0, only two Fab molecules bind to each spike. Changing to a slightly acidic pH caused no detectable change of structure for the sample with a high Fab concentration but caused severe structural damage to the low-concentration sample. Therefore, the 2H2 Fab inhibits the maturation process of immature dengue virus when Fab molecules are present at a high concentration, because the three Fab molecules on each spike hold the precursor-membrane molecules together, thereby inhibiting the normal conformational change that occurs during maturation.

Sequential Assembly of the Wedge of the Baseplate of Phage T4 in the Presence and Absence of Gp11 as Monitored by Analytical Ultracentrifugation

The baseplate wedge of bacteriophage T4 consists of seven gene products, namely, gp11, gp10, gp7, gp8, gp6, gp53, and gp25, which assemble strictly in this order with an exception that gp11 can bind to gp10 at any stage of the assembly. In this study, all the seven corresponding genes are expressed as recombinant proteins and all the possible combinations of the gene products are tested for interactions by analytical ultracentrifugation. No interactions among gene products that violate the strict sequential binding are observed except that gp6, gp53, and gp25 interact with each other weakly, but significantly. However, when gp6 is previously bound to the precursor complex, only gp53 binds to gp6 strongly and then gp25 binds to complete the wedge formation. This result indicates that the strict sequential association is based on the conformational change of the complex upon addition of each gene product. The binding constant between subunits in the intermediate complexes is too high to be measured. In fact, the binding of gp11 to gp10 is so tight that the binding constant could not be determined by trace sedimentation equilibrium. Also, no indication of dissociation of the intermediate complexes is found in sedimentation velocity, which indicates that other subunit interactions in the intermediate complexes are also strong. The 43.7 S complex, which formed upon addition of gp53, is a hexamer of the wedge complex and resembles the star-shaped baseplate. The s-value of the baseplate-like complex decreased to 40.6 S upon association with gp11 in spite of the increased molecular weight, which is reflected in the sharper edges of the baseplate-like structure which would have a higher friction.

Structural studies of Chikungunya virus maturation

Significance Chikungunya virus (CHIKV) belongs to the alphavirus family, the members of which have enveloped icosahedral capsids. The maturation process of alphaviruses involves proteolysis of some of the structural proteins before assembling with nucleocapsids to produce mature virions. We mutated the proteolytic cleavage site on E2 envelope protein, which is necessary in initiating the maturation process. Noninfectious virus-like particles (VLP) equivalent to “immature” fusion incompetent particles were produced to study the immature conformation of CHIKV. We describe the 6.8-Å resolution electron microscopy structure of “immature” CHIK VLPs. Structural differences between the mature and immature VLPs show that posttranslational processing of the envelope proteins and nucleocapsid is necessary to allow exposure of the fusion loop on glycoprotein E1 to produce an infectious virus. Cleavage of the alphavirus precursor glycoprotein p62 into the E2 and E3 glycoproteins before assembly with the nucleocapsid is the key to producing fusion-competent mature spikes on alphaviruses. Here we present a cryo-EM, 6.8-Å resolution structure of an “immature” Chikungunya virus in which the cleavage site has been mutated to inhibit proteolysis. The spikes in the immature virus have a larger radius and are less compact than in the mature virus. Furthermore, domains B on the E2 glycoproteins have less freedom of movement in the immature virus, keeping the fusion loops protected under domain B. In addition, the nucleocapsid of the immature virus is more compact than in the mature virus, protecting a conserved ribosome-binding site in the capsid protein from exposure. These differences suggest that the posttranslational processing of the spikes and nucleocapsid is necessary to produce infectious virus.

Development of a Novel Virus-Like Particle Vaccine Platform That Mimics the Immature Form of Alphavirus

ABSTRACT Virus-like particles (VLPs) are noninfectious multiprotein structures that are engineered to self-assemble from viral structural proteins. Here, we developed a novel VLP-based vaccine platform utilizing VLPs from the chikungunya virus. We identified two regions within the envelope protein, a structural component of chikungunya, where foreign antigens can be inserted without compromising VLP structure. Our VLP displays 480 copious copies of an inserted antigen on the VLP surface in a highly symmetric manner and is thus capable of inducing strong immune responses against any inserted antigen. Furthermore, by mimicking the structure of the immature form of the virus, we altered our VLPs in vivo dynamics and enhanced its immunogenicity. We used the circumsporozoite protein (CSP) of the Plasmodium falciparum malaria parasite as an antigen and demonstrated that our VLP-based vaccine elicits strong immune responses against CSP in animals. The sera from immunized monkeys protected mice from malaria infection. Likewise, mice vaccinated with P. yoelii CSP-containing VLPs were protected from an infectious sporozoite challenge. Hence, our uniquely engineered VLP platform can serve as a blueprint for the development of vaccines against other pathogens and diseases.