Biomedical subjects

R Holland Cheng

Publications and source records attributed to R Holland Cheng.

At least 19 recordsLinked to original sources

PubMed2006

The dynamic envelope of a fusion class II virus. Prefusion stages of semliki forest virus revealed by electron cryomicroscopy.

Semliki Forest virus is among the prototypes for Class II virus fusion and targets the endosomal membrane. Fusion protein E1 and its envelope companion E2 are both anchored in the viral membrane and form an external shell with protruding spikes. In acid environments, mimicking the early endosomal milieu, surface epitopes in the virus rearrange along with exposure of the fusion loop. To visualize this transformation into a fusogenic stage, we determined the structure of the virus at gradually lower pH values. The results show that while the fusion loop is available for external interaction and the shell and stalk domains of the spike begin to deteriorate, the E1 and E2 remain in close contact in the spike head. This unexpected observation points to E1 and E2 cooperation beyond the fusion loop exposure stage and implies a more prominent role for E2 in guiding membrane close encounter than has been earlier anticipated.

Cryoelectron Microscopy↗

PubMed2006

Combining the rapid MTT formazan exocytosis assay and the MC65 protection assay led to the discovery of carbazole analogs as small molecule inhibitors of Abeta 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 Alzheimer's 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.

Amyloid beta-Peptides↗

PubMed2006

Immunological equivalence between mouse brain-derived and Vero cell-derived Japanese encephalitis vaccines.

The persistent spread via animal reservoirs urges expanding vaccination programs against pathogens like the Japanese encephalitis virus, JEV. The JEV is spreads to new areas by domestic as well as by wild animals. Although there is a safe and efficient vaccine on the market, this is derived from infected mouse brains, why today's situation requires overcoming the potential risk caused by using animal tissues. To meet this demand we have developed a Vero cell-derived JEV vaccine, using the same virus strain as in the established one. A phase III clinical study of the new vaccine has recently been completed with positive outcome. Like the established mouse brain-derived vaccine, the Vero cell-derived one is a formalin inactivated whole virus vaccine. We here demonstrate the very good agreement in immunological tests between the two antigens. The study includes analyses with two neutralizing monoclonal antibodies that blocks cell entry at a late stage in infection, assumedly interfering with fusion-related refolding in the virus fusion protein. It is obvious that the formalin inactivation treatment, with both virus preparations, retains these essential vaccine epitopes.

Adolescent↗

PubMed2006

Multiple assembly states of lumazine synthase: a model relating catalytic function and molecular assembly.

Lumazine synthases have been observed in the form of pentamers, dimers of pentamers, icosahedral capsids consisting of 60 subunits and larger capsids with unknown molecular structure. Here we describe the analysis of the assembly of native and mutant forms of lumazine synthases from Bacillus subtilis and Aquifex aeolicus at various pH values and in the presence of different buffers using small angle X-ray scattering and electron microscopy. Both wild-type lumazine synthases are able to form capsids with a diameter of roughly 160 A and larger capsids with diameters of around 300 A. The relative abundance of smaller and larger capsids is strongly dependent on buffer and pH. Both forms can co-exist and are in some cases accompanied by other incomplete or deformed capsids. Several mutants of the B. subtilis lumazine synthase, in which residues in or close to the active site were replaced, as well as an insertion mutant of A. aeolicus lumazine synthase form partially or exclusively larger capsids with a diameter of about 300 A. The mutations also reduce or inhibit enzymatic activity, suggesting that the catalytic function of the enzyme is tightly correlated with its quaternary structure. The data show that multiple assembly forms are a general feature of lumazine synthases.

Bacillus subtilis↗

PubMed2006

Efficient implementation of a filtered back-projection algorithm using a voxel-by-voxel approach.

The large amount of image data necessary for high-resolution 3D reconstruction of macromolecular assemblies leads to significant increases in the computational time. One of the most time consuming operations is 3D density map reconstruction, and software optimization can greatly reduce the time required for any given structural study. The majority of algorithms proposed for improving the computational effectiveness of a 3D reconstruction are based on a ray-by-ray projection of each image into the reconstructed volume. In this paper, we propose a novel fast implementation of the "filtered back-projection" algorithm based on a voxel-by-voxel principle. Our version of this implementation has been exhaustively tested using both model and real data. We compared 3D reconstructions obtained by the new approach with results obtained by the filtered Back-Projections algorithm and the Fourier-Bessel algorithm commonly used for reconstructing icosahedral viruses. These computational experiments demonstrate the robustness, reliability, and efficiency of this approach.

Algorithms↗

PubMed2006

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.

Animals↗

PubMed2006

Increased solubility of integrin betaA domain using maltose-binding protein as a fusion tag.

In proteomics research, generation of recombinant proteins in their native, soluble form with large quantity is often a challenging task. To tackle the expression difficulties, different expression vectors with distinct affinity fusion tags, i.e. pET-43.1a (N-utilization substance A tag), pMAL-cRI (maltose binding protein tag) (MBP tag), pGEX-4T-2 (glutathione S-transferase tag), and pET-15b (hexahistidine tag) were compared for their effects on the productivity and solubility, which were assessed by SDS-PAGE and immunoblotting, of the integrin betaA domain. The incubation temperatures were tested for its effects on these parameters. Our data suggested that MBP tag enhanced the yield and solubility of the betaA domain protein, which can also be recognized using an anti-CD18 antibody, at room temperature incubation. Thus, the nature of fusion partner chosen for expression in bacteria and its incubation temperature would significantly affect the yield and solubility of the recombinant target protein.

Animals↗

PubMed2005

Transcapsidation and the conserved interactions of two major structural proteins of a pair of phytoreoviruses confirm the mechanism of assembly of the outer capsid layer.

The strongly conserved amino acid sequences of the P8 outer capsid proteins of Rice dwarf virus (RDV) and Rice gall dwarf virus (RGDV) and the distribution of electrostatic potential on the proteins at the interfaces between structural proteins suggested the possibility that P8-trimers of RGDV might bind to the 3-fold symmetrical axes of RDV core particles, with vertical interaction between heterologous P3 and P8 proteins and lateral binding of homologous P8 proteins, thereby allowing formation of the double-layered capsids that are characteristic of viruses that belong to the family Reoviridae. We proved this hypothesis using chimeric virus-like particles composed of the P3 core capsid protein of RDV and the P8 outer capsid protein of RGDV, which were co-expressed in a baculovirus expression system. This is the first report on the molecular biological proof of the mechanism of the assembly of the double-layered capsids with disparate icosahedral lattices.

Amino Acid Sequence↗

PubMed2005

Comparative studies of the capsid precursor polypeptide P1 and the capsid protein VP1 cDNA vectors for DNA vaccination against foot-and-mouth disease virus.

BACKGROUND: Foot-and-mouth disease virus (FMDV) causes a severe livestock disease, and the virus is an interesting target for virology and vaccine studies. MATERIALS AND METHODS: Here we evaluated comparatively three different viral antigen-encoding DNA sequences, delivered via two physical means (i.e., gene gun delivery into skin and electroporation delivery into muscle), for naked DNA-mediated vaccination in a mouse system. RESULTS: Both methods gave similar results, demonstrating commonality of the observed DNA vaccine effects. Immunization with a cDNA vector expressing the major viral antigen (VP1) alone routinely failed to induce the production of anti-VP1 or neutralizing antibodies in test mice. As a second approach, the plasmid L-VP1 that produces a transgenic membrane-anchored VP1 protein elicited a strong antibody response, but all test mice failed in the FMDV challenge experiment. In contrast, for mice immunized with the viral capsid precursor protein (P1) cDNA expression vector, both neutralizing antibodies and 80-100% protection in test mice were detected. CONCLUSIONS: This strategy of using the whole capsid precursor protein P1 cDNA for vaccination, intentionally without the use of virus-specific protease or other encoding genes for safety reasons, may thus be employed as a relevant experimental system for induction or upgrading of effective neutralizing antibody response, and as a convenient surrogate test system for DNA vaccination studies of FMDV and presumably other viral diseases.

Animals↗

PubMed2005

Essential elements of the capsid protein for self-assembly into empty virus-like particles of hepatitis E virus.

Hepatitis E virus (HEV) is a noncultivable virus that causes acute liver failure in humans. The virus's 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.

Animals↗

PubMed2005

Structure and assembly of a T=1 virus-like particle in BK polyomavirus.

In polyomaviruses the pentameric capsomers are interlinked by the long C-terminal arm of the structural protein VP1. The T=7 icosahedral structure of these viruses is possible due to an intriguing adaptability of this linker arm to the different local environments in the capsid. To explore the assembly process, we have compared the structure of two virus-like particles (VLPs) formed, as we found, in a calcium-dependent manner by the VP1 protein of human polyomavirus BK. The structures were determined using electron cryomicroscopy (cryo-EM), and the three-dimensional reconstructions were interpreted by atomic modeling. In the small VP1 particle, 26.4 nm in diameter, the pentameric capsomers form an icosahedral T=1 surface lattice with meeting densities at the threefold axes that interlinked three capsomers. In the larger particle, 50.6 nm in diameter, the capsomers form a T=7 icosahedral shell with three unique contacts. A folding model of the BKV VP1 protein was obtained by alignment with the VP1 protein of simian virus 40 (SV40). The model fitted well into the cryo-EM density of the T=7 particle. However, residues 297 to 362 of the C-terminal arm had to be remodeled to accommodate the higher curvature of the T=1 particle. The loops, before and after the C-terminal short helix, were shown to provide the hinges that allowed curvature variation in the particle shell. The meeting densities seen at the threefold axes in the T=1 particle were consistent with the triple-helix interlinking contact at the local threefold axes in the T=7 structure.

BK Virus↗

PubMed2004

Budding of alphaviruses.

Alphaviruses are small highly ordered enveloped RNA viruses, which replicate very efficiently in the infected cell. They consist of a nucleocapsid (NC) and a surrounding membrane with glycoproteins. In the NC the positive single stranded RNA genome of the virus is enclosed by a T=4 icosahedral shell of capsid (C) proteins. The glycoproteins form a second shell with corresponding symmetry on the outside of the lipid membrane. These viruses mature by budding at the plasma membrane (PM) of the infected cell and enter into new cells by acid-triggered membrane fusion in endosomes. The viral glycoprotein consists of two subunits, E1, which carries the membrane fusion function, and E2, which suppresses this function until acid activation occurs. In the infected cell the RNA replication and transcription are confined to the cytoplasmic surface of endosome-derived vesicles called cytopathic vacuoles type I (CPV I). These structures are closely associated with membranes of the endoplasmic reticulum (ER), thereby creating a microenvironment for synthesis of viral proteins, assembly of the glycoproteins and formation of genome-C complexes. The budding process of the virus is initiated by C-glycoprotein interactions, possibly already before the glycoproteins arrive at the PM. This might involve a premade, ordered NC or a less ordered form of the genome-C complex. In the latter case, the interactions in the glycoprotein shell provide the major driving force for budding. The nature of the C-glycoprotein interaction has been resolved at atomic resolution by modelling. It involves hydrophobic interactions between a Tyr-X-Leu tripeptide in the internal tail of the E2 subunit and a pocket on the surface of the C protein. When the virus enters the endosome of a new cell the acid conditions trigger rearrangements in the glycoprotein shell, which result in the dissociation of the interactions that drive budding and a concomitant activation of the membrane fusion function in the E1 subunit.

Alphavirus↗

PubMed2004

Enhanced cellular receptor usage by a bioselected variant of coxsackievirus a21.

Decay-accelerating factor (DAF) functions as cell attachment receptor for a wide range of human enteroviruses. The Kuykendall prototype strain of coxsackievirus A21 (CVA21) attaches to DAF but requires interactions with intercellular cell adhesion molecule 1 (ICAM-1) to infect cells. We show here that a bioselected variant of CVA21 (CVA21-DAFv) generated by multiple passages in DAF-expressing, ICAM-1-negative rhabdomyosarcoma (RD) cells acquired the capacity to induce rapid and complete lysis of ICAM-1-deficient cells while retaining the capacity to bind ICAM-1. CVA21-DAFv binding to DAF on RD cells mediated lytic infection and was inhibited by either antibody blockade with a specific anti-DAF SCR1 monoclonal antibody (MAb) or soluble human DAF. Despite being bioselected in RD cells, CVA21-DAFv was able to lytically infect an additional ICAM-1-negative cancer cell line via DAF interactions alone. The finding that radiolabeled CVA21-DAFv virions are less readily eluted from surface-expressed DAF than are parental CVA21 virions during a competitive epitope challenge by an anti-DAF SCR1 MAb suggests that interactions between CVA21-DAFv and DAF are of higher affinity than those of the parental strain. Nucleotide sequence analysis of the capsid-coding region of the CVA21-DAFv revealed the presence of two amino acid substitutions in capsid protein VP3 (R96H and E101A), possibly conferring the enhanced DAF-binding phenotype of CVA21-DAFv. These residues are predicted to be embedded at the interface of VP1, VP2, and VP3 and are postulated to enhance the affinity of DAF interaction occurring outside the capsid canyon. Taken together, the data clearly demonstrate an enhanced DAF-using phenotype and expanded receptor utilization of CVA21-DAFv compared to the parental strain, further highlighting that capsid interactions with DAF alone facilitate rapid multicycle lytic cell infection.

Animals↗

PubMed2004

The amino-terminal region of major capsid protein P3 is essential for self-assembly of single-shelled core-like particles of Rice dwarf virus.

The core protein P3 of Rice dwarf virus constructs asymmetric dimers, one of which is inserted by the amino-terminal region of another P3 protein. The P3 proteins with serial amino-terminal deletions, expressed in a baculovirus system, formed particles with gradually decreasing stability. The capacity for self-assembly disappeared when 52 of the amino-terminal amino acids had been deleted. These results demonstrated that insertion of the amino-terminal arm of one P3 protein into another appears to play an important role in stabilizing the core particles.

Animals↗

PubMed2004

Multistep regulation of membrane insertion of the fusion peptide of Semliki Forest virus.

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.

Animals↗

PubMed2004

Nephrin strands contribute to a porous slit diaphragm scaffold as revealed by electron tomography.

Nephrin is a key functional component of the slit diaphragm, the structurally unresolved molecular filter in renal glomerular capillaries. Abnormal nephrin or its absence results in severe proteinuria and loss of the slit diaphragm. The diaphragm is a thin extracellular membrane spanning the approximately 40-nm-wide filtration slit between podocyte foot processes covering the capillary surface. Using electron tomography, we show that the slit diaphragm comprises a network of winding molecular strands with pores the same size as or smaller than albumin molecules, as demonstrated in humans, rats, and mice. In the network, which is occasionally stratified, immunogold-nephrin antibodies labeled individually detectable globular cross strands, about 35 nm in length, lining the lateral elongated pores. The cross strands, emanating from both sides of the slit, contacted at the slit center but had free distal endings. Shorter strands associated with the cross strands were observed at their base. Immunolabeling of recombinant nephrin molecules on transfected cells and in vitrified solution corroborated the findings in kidney. Nephrin-deficient proteinuric patients with Finnish-type congenital nephrosis and nephrin-knockout mice had only narrow filtration slits that lacked the slit diaphragm network and the 35-nm-long strands but contained shorter molecular structures. The results suggest the direct involvement of nephrin molecules in constituting the macromolecule-retaining slit diaphragm and its pores.

Animals↗

PubMed2003

Structural and functional analysis of integrin alpha2I domain interaction with echovirus 1.

Integrins are cell surface receptors for several microbial pathogens including echovirus 1 (EV1), a picornavirus. Cryo-electron microscopy revealed that the functional domain (alpha(2)I) of human alpha(2)beta(1) integrin binds to a surface depression on the EV1 capsid. This three-dimensional structure of EV1 bound to alpha(2)I domain provides the first structural details of an integrin interacting with a picornavirus. The model indicates that alpha(2)beta(1) integrin cannot simultaneously bind both EV1 and the physiological ligand collagen. Compared with collagen binding to the alpha(2)I domain, the virus binds with a 10-fold higher affinity but in vitro uncoating of EV1 was not observed as a result of attachment of alpha(2)I. A molecular model, constructed on the basis of the EV1-integrin complex, shows that multiple alpha(2)beta(1) heterodimers can bind at adjacent sites around the virus 5-fold symmetry axes without steric hindrance. In agreement with this, virus attachment to alpha(2)beta(1) integrin on the cell surface was found to result in integrin clustering, which can give rise to signaling and facilitate the initiation of the viral entry process that takes place via caveolae-mediated endocytosis.

Capsid↗

PubMed2003

Characterization of self-assembled virus-like particles of human polyomavirus BK generated by recombinant baculoviruses.

The major structural protein of the human polyomavirus BK (BKV), VP1, was expressed by using recombinant baculoviruses. A large amount of protein with a molecular mass of about 42 kDa was synthesized and identified by Western blotting. The protein was detected exclusively in the nuclei by immunofluorescent analysis and it was released into culture medium. The expressed BKV VP1 protein was self-assembled into virus-like particles (BK-VLPs) with two different sizes (50 and 26 nm in diameter), which migrated into four different bands in CsCl gradient with buoyant densities of 1.29, 1.30, 1.33, and 1.35 g/cm(3). The immunological studies on the BK-VLPs suggested that they have similar antigenicity with those of authentic BKV particles. Cryoelectron microscopy and 3D image analysis further revealed that the larger BK-VLPs were composed of 72 capsomers which all were pentamers arranged in a T = 7 surface lattice. This system provides useful information for detailed studies of viral morphogenesis and the structural basis for the antigenicity of BKV.

Amino Acid Sequence↗