R. Holland Cheng

Architecture of ribonucleoprotein complexes in influenza A virus particles

In viruses, as in eukaryotes, elaborate mechanisms have evolved to protect the genome and to ensure its timely replication and reliable transmission to progeny. Influenza A viruses are enveloped, spherical or filamentous structures, ranging from 80 to 120 nm in diameter. Inside each envelope is a viral genome consisting of eight single-stranded negative-sense RNA segments of 890 to 2,341 nucleotides each. These segments are associated with nucleoprotein and three polymerase subunits, designated PA, PB1 and PB2; the resultant ribonucleoprotein complexes (RNPs) resemble a twisted rod (10–15 nm in width and 30–120 nm in length) that is folded back and coiled on itself. Late in viral infection, newly synthesized RNPs are transported from the nucleus to the plasma membrane, where they are incorporated into progeny virions capable of infecting other cells. Here we show, by transmission electron microscopy of serially sectioned virions, that the RNPs of influenza A virus are organized in a distinct pattern (seven segments of different lengths surrounding a central segment). The individual RNPs are suspended from the interior of the viral envelope at the distal end of the budding virion and are oriented perpendicular to the budding tip. This finding argues against random incorporation of RNPs into virions, supporting instead a model in which each segment contains specific incorporation signals that enable the RNPs to be recruited and packaged as a complete set. A selective mechanism of RNP incorporation into virions and the unique organization of the eight RNP segments may be crucial to maintaining the integrity of the viral genome during repeated cycles of replication.

Congo red and thioflavin-T analogs detect Aβ oligomers

Several small molecule ligands for amyloid‐β (Aβ) fibrils deposited in brain have been developed to facilitate radiological diagnosis of Alzheimer’s disease (AD). Recently, the build‐up of Aβ oligomers (AβO) in brain has been recognized as an additional hallmark of AD and may play a more significant role in early stages. Evidence suggests that quantitative assessment of AβO would provide a more accurate index of therapeutic effect of drug trials. Therefore, there is an urgent need to develop methods for efficient identification as well as structural analysis of AβO. We found that some well established amyloid ligands, analogs of Congo red and thioflavin‐T (ThT), bind AβO with high affinity and detect AβO in vitro and in vivo. Binding studies revealed the presence of binding sites for Congo red‐ and thioflavin‐T‐analogs on AβO. Furthermore, these ligands can be used for imaging intracellular AβO in living cells and animals and as positive contrast agent for ultrastructural imaging of AβO, two applications useful for structural analysis of AβO in cells. We propose that by improving the binding affinity of current ligands, in vivo imaging of AβO is feasible by a ‘signal subtraction’ procedure. This approach may facilitate the identification of individuals with early AD.

Biological and immunological characteristics of hepatitis E virus-like particles based on the crystal structure

Hepatitis E virus (HEV) is a causative agent of acute hepatitis. The crystal structure of HEV-like particles (HEV-LP) consisting of capsid protein was determined at 3.5-Å resolution. The capsid protein exhibited a quite different folding at the protruding and middle domains from the members of the families of Caliciviridae and Tombusviridae, while the shell domain shared the common folding. Tyr-288 at the 5-fold axis plays key roles in the assembly of HEV-LP, and aromatic amino acid residues are well conserved among the structurally related viruses. Mutational analyses indicated that the protruding domain is involved in the binding to the cells susceptive to HEV infection and has some neutralization epitopes. These structural and biological findings are important for understanding the molecular mechanisms of assembly and entry of HEV and also provide clues in the development of preventive and prophylactic measures for hepatitis E.

Distinct cellular receptor interactions in poliovirus and rhinoviruses

Receptor binding to human poliovirus type 1 (PV1/M) and the major group of human rhinoviruses (HRV) was studied comparatively to uncover the evolution of receptor recognition in picornaviruses. Surface plasmon resonance showed receptor binding to PV1/M with faster association and dissociation rates than to HRV3 and HRV16, two serotypes that have similar binding kinetics. The faster rate for receptor association to PV1/M suggested a relatively more accessible binding site. Thermodynamics for receptor binding to the viruses and assays for receptor‐mediated virus uncoating showed a more disruptive receptor interaction with PV1/M than with HRV3 or HRV16. Cryo‐electron microscopy and image reconstruction of receptor–PV1/M complexes revealed receptor binding to the ‘wall’ of surface protrusions surrounding the ‘canyon’, a depressive surface in the capsid where the rhinovirus receptor binds. These data reveal more exposed receptor‐binding sites in poliovirus than rhinoviruses, which are less protected from immune surveillance but more suited for receptor‐mediated virus uncoating and entry at the cell surface.

Structure of Hepatitis E Virion-sized Particle Reveals an RNA-dependent Viral Assembly Pathway

Hepatitis E virus (HEV) induces acute hepatitis in humans with a high fatality rate in pregnant women. There is a need for anti-HEV research to understand the assembly process of HEV native capsid. Here, we produced a large virion-sized and a small T=1 capsid by expressing the HEV capsid protein in insect cells with and without the N-terminal 111 residues, respectively, for comparative structural analysis. The virion-sized capsid demonstrates a T=3 icosahedral lattice and contains RNA fragment in contrast to the RNA-free T=1 capsid. However, both capsids shared common decameric organization. The in vitro assembly further demonstrated that HEV capsid protein had the intrinsic ability to form decameric intermediate. Our data suggest that RNA binding is the extrinsic factor essential for the assembly of HEV native capsids.

Essential Elements of the Capsid Protein for Self-Assembly into Empty Virus-Like Particles of Hepatitis E Virus

ABSTRACT Hepatitis E virus (HEV) is a noncultivable virus that causes acute liver failure in humans. The viruss major capsid protein is encoded by an open reading frame 2 (ORF2) gene. When the recombinant protein consisting of amino acid (aa) residues 112 to 660 of ORF2 is expressed with a recombinant baculovirus, the protein self-assembles into virus-like particles (VLPs) (T.-C. Li, Y. Yamakawa, K. Suzuki, M. Tatsumi, M. A. Razak, T. Uchida, N. Takeda, and T. Miyamura, J. Virol. 71:7207-7213, 1997). VLPs can be found in the culture medium of infected Tn5 cells but not in that of Sf9 cells, and the major VLPs have lost the C-terminal 52 aa. To investigate the protein requirement for HEV VLP formation, we prepared 14 baculovirus recombinants to express the capsid proteins truncated at the N terminus, the C terminus, or both. The capsid protein consisting of aa residues 112 to 608 formed VLPs in Sf9 cells, suggesting that particle formation is dependent on the modification process of the ORF2 protein. In the present study, electron cryomicroscopy and image processing of VLPs produced in Sf9 and Tn5 cells indicated that they possess the same configurations and structures. Empty VLPs were found in both Tn5 and Sf9 cells infected with the recombinant containing an N-terminal truncation up to aa residue 125 and C-terminal to aa residue 601, demonstrating that the aa residues 126 to 601 are the essential elements required for the initiation of VLP assembly. The recombinant HEV VLPs are potential mucosal vaccine carrier vehicles for the presentation of foreign antigenic epitopes and may also serve as vectors for the delivery of genes to mucosal tissue for DNA vaccination and gene therapy. The results of the present study provide useful information for constructing recombinant HEV VLPs having novel functions.

Membrane proteins organize a symmetrical virus.

Alphaviruses are enveloped icosahedral viruses that mature by budding at the plasma membrane. According to a prevailing model maturation is driven by binding of membrane protein spikes to a preformed nucleocapsid (NC). The T = 4 geometry of the membrane is thought to be imposed by the NC through one‐to‐one interactions between spike protomers and capsid proteins (CPs). This model is challenged here by a Semliki Forest virus capsid gene mutant. Its CPs cannot assemble into NCs, or its intermediate structures, due to defective CP–CP interactions. Nevertheless, it can use its horizontal spike–spike interactions on membrane surface and vertical spike–CP interactions to make a particle with correct geometry and protein stoichiometry. Thus, our results highlight the direct role of membrane proteins in organizing the icosahedral conformation of alphaviruses.

Combining the rapid MTT formazan exocytosis assay and the MC65 protection assay led to the discovery of carbazole analogs as small molecule inhibitors of Aβ oligomer-induced cytotoxicity

The discovery of small molecule inhibitors of cytotoxicity induced by amyloid-beta (Abeta) oligomers, either applied extracellularly or accumulated intraneuronally, is an important goal of drug development for Alzheimers disease (AD), but has been limited by the lack of efficient screening methods. Here we describe our approach using two cell-based methods. The first method takes advantage of the unique ability of extracellularly applied Abeta oligomers to rapidly induce the exocytosis of formazan formed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). We employed a short protocol to quantify this toxicity, and quickly identified two novel inhibitors, code-named CP2 and A5, from two compound libraries. A second independent screen of the same libraries using our previously published MC65 protection assay, which identifies inhibitors of toxicity related to intracellular Abeta oligomers, also selected the same two leads, suggesting that both assays select for the same anti-Abeta oligomer properties displayed by these compounds. We further demonstrated that A5 attenuated the progressive aggregation of existing Abeta oligomers, reduced the level of intracellular Abeta oligomers, and prevented the Abeta oligomer-induced death of primary cortical neurons, effects similar to those demonstrated by CP2. Our results suggest that, when combined, the two methods would generate fewer false results and give a high likelihood of identifying leads that show promises in ameliorating Abeta oligomer-induced toxicities within both intraneuronal and extracellular sites. Both assays are simple, suitable for rapid screening of a large number of medicinal libraries, and amenable for automation.

Multistep Regulation of Membrane Insertion of the Fusion Peptide of Semliki Forest Virus

ABSTRACT A prevailing model for virus membrane fusion proteins has been that the hydrophobic fusion peptide is hidden in the prefusion conformation, becomes exposed once the fusion reaction is triggered, and then either inserts into target membranes or is rapidly inactivated. This model is in general agreement with the structure and mechanism of class I fusion proteins, such as the influenza virus hemagglutinin. We here describe studies of the class II fusion protein E1 from the alphavirus Semliki Forest virus (SFV). SFV fusion is triggered by low pH, which releases E1 from its heterodimeric interaction with the E2 protein and induces the formation of a stable E1 homotrimer. The exposure and target membrane interaction of the E1 fusion peptide (residues 83 to 100) were followed using a monoclonal antibody (MAb E1f) mapping to E1 residues 85 to 95. In agreement with the known structure of SFV and other alphaviruses, the fusion peptide was shielded in native SFV particles and exposed when E1-E2 dimer dissociation was triggered by acidic pH. In contrast, the fusion peptide on purified E1 ectodomains (E1*) was fully accessible at neutral pH. Functional assays showed that MAb E1f binding at neutral pH prevented subsequent low-pH-triggered E1* interaction with target membranes and trimerization. E1* was not inactivated by low pH when treated either in the absence of target membranes or in the presence of fusion-inactive cholesterol-deficient liposomes. Thus, the membrane insertion of the E1 fusion peptide is regulated by additional low-pH-dependent steps after exposure, perhaps involving an E1-cholesterol interaction.

Structural Analysis of Human Rhinovirus Complexed with ICAM-1 Reveals the Dynamics of Receptor-Mediated Virus Uncoating

ABSTRACT Intercellular adhesion molecule 1 (ICAM-1) functions as the cellular receptor for the major group of human rhinoviruses, being not only the target of viral attachment but also the mediator of viral uncoating. The configurations of HRV3-ICAM-1 complexes prepared both at 4°C and physiological temperature (37°C) were analyzed by cryoelectron microscopy and image reconstruction. The particle diameters of two complexes (with and without RNA) representing uncoating intermediates generated at 37°C were each 4% larger than that of those prepared at 4°C. The larger virus particle arose by an expansive movement of the capsid pentamers along the fivefold axis, which loosens interprotomer contacts, particularly at the canyon region where the ICAM-1 receptor bound. Particle expansion required receptor binding and preceded the egress of the viral RNA. These observations suggest that receptor-mediated uncoating could be a consequence of restrained capsid motion, where the bound receptors maintain the viral capsid in an expanded open state for subsequent genome release.