Biomedical subjects

C Cheng Kao

Publications and source records attributed to C Cheng Kao.

At least 19 recordsLinked to original sources

PubMed2007

Core-controlled polymorphism in virus-like particles.

This study concerns the self-assembly of virus-like particles (VLPs) composed of an icosahedral virus protein coat encapsulating a functionalized spherical nanoparticle core. The recent development of efficient methods for VLP self-assembly has opened the way to structural studies. Using electron microscopy with image reconstruction, the structures of several VLPs obtained from brome mosaic virus capsid proteins and gold nanoparticles were elucidated. Varying the gold core diameter provides control over the capsid structure. The number of subunits required for a complete capsid increases with the core diameter. The packaging efficiency is a function of the number of capsid protein subunits per gold nanoparticle. VLPs of varying diameters were found to resemble to three classes of viral particles found in cells (T=1, 2, and 3). As a consequence of their regularity, VLPs form three-dimensional crystals under the same conditions as the wild-type virus. The crystals represent a form of metallodielectric material that exhibits optical properties influenced by multipolar plasmonic coupling.

Crystallization↗

PubMed2007

Biochemical and functional analyses of the human Toll-like receptor 3 ectodomain.

The structure of the human Toll-like receptor 3 (TLR3) ectodomain (ECD) was recently solved by x-ray crystallography, leading to a number of models concerning TLR3 function (Choe, J., Kelker, M. S., and Wilson, I. A. (2005) Science 309, 581-585; Bell, J. K., Botos, I., Hall, P. R., Askins, J., Shiloach, J., Segal, D. M., and Davies, D. R. (2005) Proc. Natl. Acad. Sci. U. S. A. 102, 10976-10980) The structure revealed four pairs of cysteines that are putatively involved in disulfide bond formation, several residues that are predicted to be involved in dimerization between ECD subunits, and surfaces that could bind to poly(I:C). In addition, there are two loops that protrude from the central solenoid structure of the protein. We examined the recombinant TLR3 ECD for disulfide bond formation, poly(I:C) binding, and protein-protein interaction. We also made over 80 mutations in the residues that could affect these features in the full-length TLR3 and examined their effects in TLR3-mediated NF-kappaB activation. A number of mutations that affected TLR3 activity also affected the ability to act as dominant negative inhibitors of wild type TLR3. Loss of putative RNA binding did not necessarily affect dominant negative activity. All of the results support a model where a dimer of TLR3 is the form that binds RNA and activates signal transduction.

Amino Acid Sequence↗

PubMed2006

Selective repression of translation by the brome mosaic virus 1a RNA replication protein.

Differential expression of viral replication proteins is essential for successful infection. We report here that overexpression of the brome mosaic virus (BMV) 1a protein can repress viral RNA replication in a dosage-dependent manner. Using RNA replication-incompetent reporter constructs, repression of translation from BMV RNA1 and RNA2 was observed, suggesting that the effect on translation of the BMV RNA replication proteins is responsible for the decrease in RNA levels. Furthermore, repression of translation by 1a required the B box in the 5'-untranslated region (5' UTR); BMV RNA3 that lacks a B box in its 5' UTR is not subject to 1a-mediated translational inhibition. Mutations in either the methyltransferase or the helicase-like domains of 1a reduced the repression of replication and translation. These results suggest that in addition to its known functions in BMV RNA synthesis, 1a also regulates viral gene expression.

5' Untranslated Regions↗

PubMed2006

RNA recognition and cleavage by the SARS coronavirus endoribonuclease.

The emerging disease SARS is caused by a novel coronavirus that encodes several unusual RNA-processing enzymes, including non-structural protein 15 (Nsp15), a hexameric endoribonuclease that preferentially cleaves at uridine residues. How Nsp15 recognizes and cleaves RNA is not well understood and is the subject of this study. Based on the analysis of RNA products separated by denaturing gel electrophoresis, Nsp15 has been reported to cleave both 5' and 3' of the uridine. We used several RNAs, including some with nucleotide analogs, and mass spectrometry to determine that Nsp15 cleaves only 3' of the recognition uridylate, with some cleavage 3' of cytidylate. A highly conserved RNA structure in the 3' non-translated region of the SARS virus was cleaved preferentially at one of the unpaired uridylate bases, demonstrating that both RNA structure and base-pairing can affect cleavage by Nsp15. Several modified RNAs that are not cleaved by Nsp15 can bind Nsp15 as competitive inhibitors. The RNA binding affinity of Nsp15 increased with the content of uridylate in substrate RNA and the co-factor Mn(2+). The hexameric form of Nsp15 was found to bind RNA in solution. A two-dimensional crystal of Nsp15 in complex with RNA showed that at least two RNA molecules could be bound per hexamer. Furthermore, an 8.3 A structure of Nsp15 was developed using cyroelectron microscopy, allowing us to generate a model of the Nsp15-RNA complex.

Binding, Competitive↗

PubMed2006

Nanoparticle-templated assembly of viral protein cages.

Self-assembly of regular protein surfaces around nanoparticle templates provides a new class of hybrid biomaterials with potential applications in medical imaging and in bioanalytical sensing. We report here the first example of efficiently self-assembled virus-like particles (VLPs) having a brome mosaic virus protein coat and a functionalized gold core. The present study indicates that functionalized gold particles can initiate VLP assembly by mimicking the electrostatic behavior of the nucleic acid component of the native virus. These VLP constructs are symmetric, with the protein stoichiometry and packaging properties indicating similarity to the icosahedral packing of the capsid. Moreover, a pH-induced swelling transition of the VLPs is observed, in direct analogy to the native virus.

Biomimetic Materials↗

PubMed2006

Quantum dot encapsulation in viral capsids.

Incorporation of CdSe/ZnS semiconductor quantum dots (QDs) into viral particles provides a new paradigm for the design of intracellular microscopic probes and vectors. Several strategies for the incorporation of QDs into viral capsids were explored; those functionalized with poly(ethylene glycol) (PEG) can be self-assembled into viral particles with minimal release of photoreaction products and enhanced stability against prolonged irradiation.

Capsid↗

PubMed2005

Functional analysis of RNA binding by the hepatitis C virus RNA-dependent RNA polymerase.

Protein-RNA interaction plays a critical role in regulating RNA synthesis by the hepatitis C virus (HCV) RNA-dependent RNA polymerase (RdRp). RNAs of 7 nucleotides (nt) or longer had affinities 5-fold better than an RNA of 5 nt, suggesting a minimal length required for binding. To identify RNA contact sites on the HCV RdRp, a biotinylated 7-nt RNA capable of directing de novo initiation was used in a process that coupled reversible formaldehyde cross-linking, RNA affinity chromatography, and mass spectrometry. By this process, we identified 18 peptides cross-linked to the 7-nt RNA. When these identified peptides were overlaid on the three-dimensional structures of NS5B, most mapped to the fingers subdomain, connecting loops between fingers and thumb subdomains and in the putative RNA binding channel. Two of the identified peptides resided in the active site cavity of the RdRp. Recombinant HCV RdRp with single residue changes in likely RNA contact sites were generated and characterized for effects on HCV RdRp activity. Mutant proteins had significant effects on cross-linking to 7-nt RNA and reduced RNA synthesis in vitro by 2- to 20-fold compared with wild type protein. When the mutations were tested for the replication of HCV RNA in the context of the cells transfected with the HCV subgenomic replicon, all except one prevented colony formation, indicating a defect in HCV RNA replication. These biochemical and functional analyses identified a number of residues in the HCV RdRp that are important for HCV RNA synthesis.

Amino Acid Substitution↗

PubMed2005

Pokeweed antiviral protein inhibits brome mosaic virus replication in plant cells.

Pokeweed antiviral protein (PAP) is a ribosome-inactivating protein isolated from the pokeweed plant (Phytolacca americana) that inhibits the proliferation of several plant and animal viruses. We have shown previously that PAP and nontoxic mutants of PAP can directly depurinate brome mosaic virus (BMV) RNA in vitro, resulting in reduced viral protein translation. Here we expand on these initial studies and, using a barley protoplast system, demonstrate that recombinant PAP and nontoxic mutants isolated from E. coli are able to reduce the accumulation of BMV RNAs in vivo. Pretreatment of only BMV RNA3 with PAP prior to transfection of barley protoplasts reduced the accumulation of all BMV RNAs, with a more severe effect on subgenomic RNA4 levels. Using in vitro RNA synthesis assays, we show that a depurinated template causes the BMV replicase to stall at the template nucleotide adjacent to the missing base. These results provide new insight into the antiviral mechanism of PAP, namely that PAP depurination of BMV RNA impedes both RNA replication and subgenomic RNA transcription. These novel activities are distinct from the PAP-induced reduction of viral RNA translation and represent new targets for the inhibition of viral infection.

Base Sequence↗

PubMed2005

Diel infection of a cyanobacterium by a contractile bacteriophage.

Light was found to strongly influence the infection of a freshwater cyanobacterium (Synechococcus elongatus PCC 7942) by a contractile DNA phage named AS-1. Phage progeny production was correlated with the amount of light in the laboratory and occurred in a diel pattern under natural light. At least one effect of light on AS-1 infection is at the level of adsorption.

Adsorption↗

PubMed2005

Packaging of gold particles in viral capsids.

In-vitro self-assembly conditions known to result in generating infectious virions have been used in vitro to reassemble bromovirus capsid proteins around negatively charged gold nanoparticles cores. We discuss here the optical properties (elastic light scattering) and the influence of the core size and of the functional moiety on the resulting virus-like particles. Our results indicate that the formation of a closed shell, as opposed to an amorphous protein coat, does occur and that the shell/core interactions can be tuned using different coatings on the nanoparticle core. Such studies may lead to real-time monitoring of viral traffic on the scale of a single virus, as well as to the possibility of chemical sensing along the intracellular and intercellular viral pathways and contribute to a better understanding of the virus transport and cellular compartmentalization.

Bromovirus↗

PubMed2004

Requirements for brome mosaic virus subgenomic RNA synthesis in vivo and replicase-core promoter interactions in vitro.

Based solely on in vitro results, two contrasting models have been proposed for the recognition of the brome mosaic virus (BMV) subgenomic core promoter by the replicase. The first posits that the replicase recognizes at least four key nucleotides in the core promoter, followed by an induced fit, wherein some of the nucleotides base pair prior to the initiation of RNA synthesis (S. Adkins and C. C. Kao, Virology 252:1-8, 1998). The second model posits that a short RNA hairpin in the core promoter serves as a landing pad for the replicase and that at least some of the key nucleotides help form a stable hairpin (P. C. J. Haasnoot, F. Brederode, R. C. L. Olsthoorn, and J. Bol, RNA 6:708-716, 2000; P. C. J. Haasnoot, R. C. L. Olsthoorn, and J. Bol, RNA 8:110-122, 2002). We used transfected barley protoplasts to examine the recognition of the subgenomic core promoter by the BMV replicase. Key nucleotides required for subgenomic initiation in vitro were found to be important for RNA4 levels in protoplasts. In addition, additional residues not required in vitro and the formation of an RNA hairpin within the core promoter were correlated with wild-type RNA4 levels in cells. Using a template competition assay, the core promoter of ca. 20 nucleotides was found to be sufficient for replicase binding. Mutations of the key residues in the core promoter reduced replicase binding, but deletions that disrupt the predicted base pairing in the proposed stem retained binding at wild-type levels. Together, these results indicate that key nucleotides in the BMV subgenomic core promoter direct replicase recognition but that the formation of a stem-loop is required at a step after binding. Additional functional characterization of the subgenomic core promoter was performed. A portion of the promoter for BMV minus-strand RNA synthesis could substitute for the subgenomic core promoter in transfected cells. The comparable sequence from Cowpea Chlorotic Mottle Virus (CCMV) could also substitute for the BMV subgenomic core promoter. However, nucleotides in the CCMV core required for RNA synthesis are not identical to those in BMV, suggesting that the subgenomic core promoter can induce the BMV replicase in interactions needed for subgenomic RNA transcription in vivo.

Base Sequence↗

PubMed2004

The severe acute respiratory syndrome coronavirus Nsp15 protein is an endoribonuclease that prefers manganese as a cofactor.

Nonstructural protein 15 (Nsp15) of the severe acute respiratory syndrome coronavirus (SARS-CoV) produced in Escherichia coli has endoribonuclease activity that preferentially cleaved 5' of uridylates of RNAs. Blocking either the 5' or 3' terminus did not affect cleavage. Double- and single-stranded RNAs were both substrates for Nsp15 but with different kinetics for cleavage. Mn(2+) at 2 to 10 mM was needed for optimal endoribonuclease activity, but Mg(2+) and several other divalent metals were capable of supporting only a low level of activity. Concentrations of Mn(2+) needed for endoribonuclease activity induced significant conformation change(s) in the protein, as measured by changes in tryptophan fluorescence. A similar endoribonucleolytic activity was detected for the orthologous protein from another coronavirus, demonstrating that the endoribonuclease activity of Nsp15 may be common to coronaviruses. This work presents an initial biochemical characterization of a novel coronavirus endoribonuclease.

Amino Acid Sequence↗

PubMed2004

Template sequence near the initiation nucleotide can modulate brome mosaic virus RNA accumulation in plant protoplasts.

Bromoviral templates for plus-strand RNA synthesis are rich in A or U nucleotides in comparison to templates for minus-strand RNA synthesis. Previous studies demonstrated that plus-strand RNA synthesis by the brome mosaic virus (BMV) RNA replicase is more efficient if the template contains an A/U-rich template sequence near the initiation site (K. Sivakumaran and C. C. Kao, J. Virol. 73:6415-6423, 1999). These observations led us to examine the effects of nucleotide changes near the template's initiation site on the accumulation of BMV RNA3 genomic minus-strand, genomic plus-strand, and subgenomic RNAs in barley protoplasts transfected with wild-type and mutant BMV transcripts. Mutations in the template for minus-strand synthesis had only modest effects on BMV replication in barley protoplasts. Mutants with changes to the +3, +5, and +7 template nucleotides accumulated minus-strand RNA at levels similar to the the wild-type level. However, mutations at positions adjacent to the initiation cytidylate in the templates for genomic and subgenomic plus-strand RNA synthesis significantly decreased RNA accumulation. For example, changes at the third template nucleotide for plus-strand RNA3 synthesis resulted in RNA accumulation at between 18 and 24% of the wild-type level, and mutations in the third template nucleotide for subgenomic RNA4 resulted in accumulations at between 7 and 14% of the wild-type level. The effects of the mutations generally decreased as the mutations occurred further from the initiation nucleotide. These findings demonstrate that there are different requirements of the template sequence near the initiation nucleotide for BMV RNA accumulation in plant cells.

Base Sequence↗

PubMed2003

Multiple interactions within the hepatitis C virus RNA polymerase repress primer-dependent RNA synthesis.

The hepatitis C virus (HCV) RNA-dependent RNA polymerase (RdRp) initiates RNA synthesis in vivo by a de novo mechanism. In vitro, however, the HCV RdRp can initiate de novo or extend from a primed template. A novel beta-loop near the RdRp active site was previously found to prevent the use of primed templates. We found that, in addition to the beta-loop, the C-terminal tail of the HCV RdRp and the de novo initiation GTP are required to exclude the use of primed-templates. GTP binding to the NTPi site of the HCV RdRp orchestrates the participation of other structures. The interactions of the beta-loop, C-terminal tail, and GTP provide an elegant solution to ensure de novo initiation of HCV RNA synthesis.

Base Sequence↗

PubMed2003

Gold nanoparticles as spectroscopic enhancers for in vitro studies on single viruses.

Gold particles with diameters between 2.5 and 4.5 nm have been introduced in the inner cavity of an icosahedral brome mosaic virus. The optical properties of single gold-marked virions have been tested in-vitro with respect to the characteristic plasmon polariton resonance. The shift in the plasmon polariton resonance of a single Au particle encapsulated in a virus with respect to a free particle in solution indicates a close interaction between the basic residues on the inner wall of the capsid and the negative surface charge of the particle. Incorporation of a pair of Au particles, approximately 4 nm diameter, is shown to be a frequent event. In this case, the dependence of the two-particle surface plasmon spectrum on the interparticle distance and the strong particle/capsid wall interaction suggest that, in the future, it will be possible to use encapsulated Au particles to track changes in the viral capsid volume in real-time and in a physiological environment.

Bromovirus↗

PubMed2003

Arresting initiation of hepatitis C virus RNA synthesis using heterocyclic derivatives.

The hepatitis C virus (HCV) NS5B protein encodes an RNA-dependent RNA polymerase (RdRp), the primary catalytic enzyme of the HCV replicase complex. Recently, two benzo-1,2,4-thiadiazine compounds were shown to be potent, highly specific inhibitors of the genotype 1b HCV RdRp containing a carboxyl-terminal 21 residue truncation (delta21 HCV RdRp) (Dhanak, D., Duffy, K., Johnston, V. K., Lin-Goerke, J., Darcy, M., Shaw, A. N. G. B., Silverman, C., Gates, A. T., Earnshaw, D. L., Casper, D. J., Kaura, A., Baker, A., Greenwood, C., Gutshall, L. L., Maley, D., DelVecchio, A., Macarron, R., Hofmann, G. A., Alnoah, Z., Cheng, H.-Y., Chan, G., Khandekar, S., Keenan, R. M., and Sarisky, R. T. (2002) J. Biol. Chem. 277, 38322-38327). Compound 4 (C(21)H(21)N(3)O(4)S) reduces viral replication by virtue of its direct interaction with the viral polymerase rather than by nonspecific titration of nucleic acid template. In this study, we present several lines of evidence to demonstrate that this inhibitor interferes with the initiation step of RNA synthesis rather than acting as an elongation inhibitor. Inhibition of initial phosphodiester bond formation occurred regardless of whether replication was initiated by primer-dependent or de novo mechanisms. Filter binding studies using increasing concentrations of compound 4 did not interfere with the ability of delta21 HCV RdRp to interact with nucleic acid. Furthermore, varying the order of reagent addition in the primer extension assay showed no distinct differences in inhibition profile. Finally, surface plasmon resonance analyses provided evidence that a ternary complex is capable of forming between the RNA template, RdRp, and compound 4. Together, these data suggest that this heterocyclic agent interacts with the apoenzyme, as well as with the RNA-bound form of delta21 HCV RdRp, and therefore does not directly interfere with the RdRp-RNA interaction to mediate inhibition.

Enzyme Inhibitors↗

PubMed2003

Brome mosaic virus RNA syntheses in vitro and in barley protoplasts.

The RNA replicase extracted from Brome mosaic virus (BMV)-infected plants has been used to characterize the cis-acting elements for RNA synthesis and the mechanism of RNA synthesis. Minus-strand RNA synthesis in vitro requires a structure named stem-loop C (SLC) that contains a clamped adenine motif. In vitro, there are several specific requirements for SLC recognition. We examined whether these requirements also apply to BMV replication in barley protoplasts. BMV RNA3s with mutations in SLC were transfected into barley protoplasts, and the requirements for minus- and plus-strand replication were found to correlate well with the requirements in vitro. Furthermore, previous analysis of replicase recognition of the Cucumber mosaic virus (CMV) and BMV SLCs indicates that the requirements in the BMV SLC are highly specific. In protoplasts, we found that BMV RNA3s with their SLCs replaced with two different CMV SLCs were defective for replication. In vitro results generated with the BMV replicase and minimal-length RNAs generally agreed with those of in vivo BMV RNA replication. To extend this conclusion, we determined that, corresponding with the process of infection, the BMV replicases extracted from plants at different times after infection have different levels of recognition of the minimal promoters for plus- and minus-strand RNA syntheses.

Base Sequence↗

PubMed2003

Enhancer-like activity of a brome mosaic virus RNA promoter.

As with transcription from DNA templates, RNA synthesis from viral RNA templates must initiate accurately. RNA sequences named specificity and initiation determinants allow recognition of and coordinated interaction with the viral replication enzyme. Using enriched replicase from brome mosaic virus (BMV)-infected plants and variants of the promoter template for minus-strand and subgenomic RNA initiation, we found that a specificity determinant for minus-strand initiation could function at variable distances and positions from the 3' initiation site in a manner similar to enhancers of transcription from DNA templates. This determinant's addition could convert a cellular tRNA into a template for RNA synthesis by the BMV replicase in vitro. Furthermore, the same specificity element could direct internal initiation, which occurred at a highly preferred site in a manner distinct from initiation at the 3' terminus of the template. These results document two distinct modes of initiation site recognition by a viral RNA replicase.

Bromovirus↗