Search PubMed⌕ Search

Biomedical subjects

C M Rice

Publications and source records attributed to C M Rice.

At least 19 recordsLinked to original sources
PubMed2001

Induction of hepatitis C virus-specific cytotoxic T lymphocytes in mice by immunization with dendritic cells treated with an anthrax toxin fusion protein.

As a novel and safe vaccine strategy, the anthrax toxin-mediated antigen delivery system composed of lethal factor (LF) fusion protein and protective antigen (PA) has been studied to prime hepatitis C virus (HCV) core-specific cytotoxic T lymphocytes (CTLs) in vivo. The core epitope fused to LF (LF-core) together with PA induces a negligible core-specific CTL response in mice, whereas core-specific CTL are effectively primed in mice by injecting dendritic cells (DCs) treated in vitro with LF-core and PA. These findings imply that LF fusion protein plus PA in combination with dendritic cells may be useful for a novel T cell vaccine against HCV infection.

Animals↗
PubMed2001

Determinants for membrane association of the hepatitis C virus RNA-dependent RNA polymerase.

The hepatitis C virus (HCV) RNA-dependent RNA polymerase (RdRp), represented by nonstructural protein 5B (NS5B), is believed to form a membrane-associated RNA replication complex together with other nonstructural proteins and as yet unidentified host components. However, the determinants for membrane association of this essential viral enzyme have not been defined. By double label immunofluorescence analyses, NS5B was found in the endoplasmic reticulum (ER) or an ER-like modified compartment both when expressed alone or in the context of the entire HCV polyprotein. The carboxyl-terminal 21 amino acid residues were necessary and sufficient to target NS5B or a heterologous protein to the cytosolic side of the ER membrane. This hydrophobic domain is highly conserved among 269 HCV isolates analyzed and predicted to form a transmembrane alpha-helix. Association of NS5B with the ER membrane occurred by a posttranslational mechanism that was ATP-independent. These features define the HCV RdRp as a new member of the tail-anchored protein family, a class of integral membrane proteins that are membrane-targeted posttranslationally via a carboxyl-terminal insertion sequence. Formation of the HCV replication complex, therefore, involves specific determinants for membrane association that represent potential targets for antiviral intervention.

Amino Acid Sequence↗
PubMed2001

The hepatitis C virus nonstructural protein 4B is an integral endoplasmic reticulum membrane protein.

The hepatitis C virus (HCV) nonstructural protein 4B (NS4B) is a relatively hydrophobic 27-kDa protein of unknown function. A tetracycline-regulated gene expression system, a novel monoclonal antibody, and in vitro transcription-translation were employed to investigate the subcellular localization and to characterize the membrane association of this viral protein. When expressed individually or in the context of the entire HCV polyprotein, NS4B was localized in the endoplasmic reticulum (ER), as shown by subcellular fractionation, immunofluorescence analyses, and double-label confocal laser scanning microscopy. In this compartment NS4B colocalized with the other HCV nonstructural proteins. Association of NS4B with the ER membrane occurred cotranslationally, presumably via engagement of the signal recognition particle by an internal signal sequence. In membrane extraction and proteinase protection assays NS4B displayed properties of a cytoplasmically oriented integral membrane protein. Taken together, our findings suggest that NS4B is a component of a membrane-associated cytoplasmic HCV replication complex. An efficient replication system will be essential to further define the role of NS4B in the viral life cycle.

Animals↗
PubMed2001

Enhancement of Sindbis virus self-replicating RNA vaccine potency by linkage of Mycobacterium tuberculosis heat shock protein 70 gene to an antigen gene.

Recently, self-replicating RNA vaccines (RNA replicons) have emerged as an effective strategy for nucleic acid vaccine development. Unlike naked DNA vaccines, RNA replicons eventually cause lysis of transfected cells and therefore do not raise the concern of integration into the host genome. We evaluated the effect of linking human papillomavirus type 16 E7 as a model Ag to Mycobacterium tuberculosis heat shock protein 70 (HSP70) on the potency of Ag-specific immunity generated by a Sindbis virus self-replicating RNA vector, SINrep5. Our results indicated that this RNA replicon vaccine containing an E7/HSP70 fusion gene generated significantly higher E7-specific T cell-mediated immune responses in vaccinated mice than did vaccines containing the wild-type E7 gene. Furthermore, our in vitro studies demonstrated that E7 Ag from E7/HSP70 RNA replicon-transfected cells can be processed by bone marrow-derived dendritic cells and presented more efficiently through the MHC class I pathway than can wild-type E7 RNA replicon-transfected cells. More importantly, the fusion of HSP70 to E7 converted a less effective vaccine into one with significant potency against E7-expressing tumors. This antitumor effect was dependent on NK cells and CD8(+) T cells. These results indicated that fusion of HSP70 to an Ag gene may greatly enhance the potency of self-replicating RNA vaccines.

Adjuvants, Immunologic↗
PubMed2001

A map of human genome sequence variation containing 1.42 million single nucleotide polymorphisms.

We describe a map of 1.42 million single nucleotide polymorphisms (SNPs) distributed throughout the human genome, providing an average density on available sequence of one SNP every 1.9 kilobases. These SNPs were primarily discovered by two projects: The SNP Consortium and the analysis of clone overlaps by the International Human Genome Sequencing Consortium. The map integrates all publicly available SNPs with described genes and other genomic features. We estimate that 60,000 SNPs fall within exon (coding and untranslated regions), and 85% of exons are within 5 kb of the nearest SNP. Nucleotide diversity varies greatly across the genome, in a manner broadly consistent with a standard population genetic model of human history. This high-density SNP map provides a public resource for defining haplotype variation across the genome, and should help to identify biomedically important genes for diagnosis and therapy.

Chromosome Mapping↗
PubMed2001

The physical maps for sequencing human chromosomes 1, 6, 9, 10, 13, 20 and X.

We constructed maps for eight chromosomes (1, 6, 9, 10, 13, 20, X and (previously) 22), representing one-third of the genome, by building landmark maps, isolating bacterial clones and assembling contigs. By this approach, we could establish the long-range organization of the maps early in the project, and all contig extension, gap closure and problem-solving was simplified by containment within local regions. The maps currently represent more than 94% of the euchromatic (gene-containing) regions of these chromosomes in 176 contigs, and contain 96% of the chromosome-specific markers in the human gene map. By measuring the remaining gaps, we can assess chromosome length and coverage in sequenced clones.

Chromosomes, Human, Pair 1↗
PubMed2001

Cis-acting RNA elements at the 5' end of Sindbis virus genome RNA regulate minus- and plus-strand RNA synthesis.

Alphavirus genome replication is a multistep asymmetric process. Several lines of evidence suggest that the template preference of the RNA replicase is regulated by proteolytic cleavage of the viral nonstructural polyprotein. Cis-acting RNA elements in the viral genome also play crucial roles in regulating genome replication and subgenomic RNA transcription. In this report, a series of RNA templates were analyzed in vitro and in vivo to define functional elements in the 5' end of the genome. The 5' UTR was shown to contain distinct core promoter elements for both minus- and plus-strand synthesis. In addition, two conserved stem-loop structures within the nsP1 coding sequence enhanced RNA replication but were not required. Studies with chimeric templates and trans-competition experiments suggest that the 5' determinant for minus-strand initiation can differ among alphaviruses and binds to one or more limiting replicase components. The results provide compelling evidence that the 5' and 3' ends of alphavirus genome RNAs must interact to initiate replication and we propose one model for how this interaction might occur. In addition to providing new insight into the initiation of alphavirus genome replication, these results have implications for the development of improved alphavirus vector systems with reduced recombination potential.

3' Untranslated Regions↗
PubMed2001

Transcriptional activation of the interleukin-2 promoter by hepatitis C virus core protein.

Most patients infected with hepatitis C virus (HCV) become chronic carriers. Viruses that efficiently establish persistent infections must have effective ways of evading host defenses. In the case of HCV, little is known about how chronic infections are established or maintained. Besides hepatocytes, several reports suggest that HCV can infect T and B lymphocytes. Since T cells are essential for viral clearance, direct or indirect effects of HCV on T-cell function could influence the outcome of infection. Given that T-cell growth and differentiation require the cytokine interleukin 2 (IL-2), we asked whether HCV might modulate synthesis of IL-2. Portions of the HCV polyprotein were expressed in Jurkat cells under a variety of conditions. We found that the highly conserved HCV core protein, in combination with other stimuli, was able to dramatically activate transcription from the IL-2 promoter. The carboxy-terminal hydrophobic portion of the core protein was required for this activity. Activation was dependent on nuclear factor of activated T cells (NFAT), occurred in cells deficient in the tyrosine kinase p56(lck), and could be blocked by addition of cyclosporin A and by depletion of calcium. These results suggest that the HCV core protein can activate transcription of the IL-2 promoter through the NFAT pathway. This novel activity may have consequences for T-cell development and establishment of persistent infections.

Calcium↗
PubMed2001

Isolation and characterization of noncytopathic pestivirus mutants reveals a role for nonstructural protein NS4B in viral cytopathogenicity.

Isolates of bovine viral diarrhea virus (BVDV), the prototype pestivirus, are divided into cytopathic (cp) and noncytopathic (ncp) biotypes according to their effect on cultured cells. The cp viruses also differ from ncp viruses by the production of viral nonstructural protein NS3. However, the mechanism by which cp viruses induce cytopathic effect in cell culture remains unknown. Here we used a genetic approach to isolate ncp variants that arose from a cp virus at low frequency. A bicistronic BVDV (cp strain NADL) was created that expressed puromycin acetyltransferase as a dominant selectable marker. This bicistronic virus exhibited slightly slower growth kinetics and smaller plaques than NADL but remained cp. A number of independent ncp variants were isolated by puromycin selection. Remarkably, these ncp variants produced NS3 and viral RNA at levels comparable to those of the cp parent. Sequence analyses uncovered no change in NS3, but for all ncp variants a Y2441C substitution at residue 15 of NS4B was found. Introduction of the Y2441C substitution into the NADL or bicistronic cp viruses reconstituted the ncp phenotype. Y2441 is highly conserved among pestiviruses and is located in a region of NS4B predicted to be on the cytosolic side of the endoplasmic reticulum membrane. Other engineered substitutions for Y2441 also affected viral cytopathogenicity and viability, with Y2441V being cp, Y2441A being ncp, and Y2441D rendering the virus unable to replicate. The ncp substitutions for Y2441 resulted in slightly increased levels of NS2-3 relative to NS3. We also showed that NS3, NS4B, and NS5A could be chemically cross-linked in NADL-infected cells, indicating that they are associated as components of a multiprotein complex. Although the mechanism remains to be elucidated, these results demonstrate that mutations in NS4B can attenuate BVDV cytopathogenicity despite NS3 production.

Amino Acid Sequence↗
PubMed2001

Efficient translation initiation is required for replication of bovine viral diarrhea virus subgenomic replicons.

An internal ribosome entry site (IRES) mediates translation initiation of bovine viral diarrhea virus (BVDV) RNA. Studies have suggested that a portion of the N(pro) open reading frame (ORF) is required, although its exact function has not been defined. Here we show that a subgenomic (sg) BVDV RNA in which the NS3 ORF is preceded only by the 5' nontranslated region did not replicate to detectable levels following transfection. However, RNA synthesis and cytopathic effects were observed following serial passage in the presence of a noncytopathic helper virus. Five sg clones derived from the passaged virus contained an identical, silent substitution near the beginning of the NS3 coding sequence (G400U), as well as additional mutations. Four of the reconstructed mutant RNAs replicated in transfected cells, and in vitro translation showed increased levels of NS3 for the mutant RNAs compared to that of wild-type (wt) MetNS3. To more precisely dissect the role of these mutations, we constructed two sg derivatives: ad3.10, which contains only the G400U mutation, and ad3.7, with silent substitutions designed to minimize RNA secondary structure downstream of the initiator AUG. Both RNAs replicated and were translated in vitro to similar levels. Moreover, ad3.7 and ad3.10, but not wt MetNS3, formed toeprints downstream of the initiator AUG codon in an assay for detecting the binding of 40S ribosomal subunits and 43S ribosomal complexes to the IRES. These results suggest that a lack of stable RNA secondary structure(s), rather than a specific RNA sequence, immediately downstream of the initiator AUG is important for optimal translation initiation of pestivirus RNAs.

Amino Acid Sequence↗
PubMed2000

Efficient initiation of HCV RNA replication in cell culture.

Hepatitis C virus (HCV) infection is a global health problem affecting an estimated 170 million individuals worldwide. We report the identification of multiple independent adaptive mutations that cluster in the HCV nonstructural protein NS5A and confer increased replicative ability in vitro. Among these adaptive mutations were a single amino acid substitution that allowed HCV RNA replication in 10% of transfected hepatoma cells and a deletion of 47 amino acids encompassing the interferon (IFN) sensitivity determining region (ISDR). Independent of the ISDR, IFN-alpha rapidly inhibited HCV RNA replication in vitro. This work establishes a robust, cell-based system for genetic and functional analyses of HCV replication.

Amino Acid Substitution↗
PubMed2000

An SNP map of human chromosome 22.

The human genome sequence will provide a reference for measuring DNA sequence variation in human populations. Sequence variants are responsible for the genetic component of individuality, including complex characteristics such as disease susceptibility and drug response. Most sequence variants are single nucleotide polymorphisms (SNPs), where two alternate bases occur at one position. Comparison of any two genomes reveals around 1 SNP per kilobase. A sufficiently dense map of SNPs would allow the detection of sequence variants responsible for particular characteristics on the basis that they are associated with a specific SNP allele. Here we have evaluated large-scale sequencing approaches to obtaining SNPs, and have constructed a map of 2,730 SNPs on human chromosome 22. Most of the SNPs are within 25 kilobases of a transcribed exon, and are valuable for association studies. We have scaled up the process, detecting over 65,000 SNPs in the genome as part of The SNP Consortium programme, which is on target to build a map of 1 SNP every 5 kilobases that is integrated with the human genome sequence and that is freely available in the public domain.

Cell Line↗
PubMed2000

Mechanism of action of a pestivirus antiviral compound.

We report here the discovery of a small molecule inhibitor of pestivirus replication. The compound, designated VP32947, inhibits the replication of bovine viral diarrhea virus (BVDV) in cell culture at a 50% inhibitory concentration of approximately 20 nM. VP32947 inhibits both cytopathic and noncytopathic pestiviruses, including isolates of BVDV-1, BVDV-2, border disease virus, and classical swine fever virus. However, the compound shows no activity against viruses from unrelated virus groups. Time of drug addition studies indicated that VP32947 acts after virus adsorption and penetration and before virus assembly and release. Analysis of viral macromolecular synthesis showed VP32947 had no effect on viral protein synthesis or polyprotein processing but did inhibit viral RNA synthesis. To identify the molecular target of VP32947, we isolated drug-resistant (DR) variants of BVDV-1 in cell culture. Sequence analysis of the complete genomic RNA of two DR variants revealed a single common amino acid change located within the coding region of the NS5B protein, the viral RNA-dependent RNA polymerase. When this single amino acid change was introduced into an infectious clone of drug-sensitive wild-type (WT) BVDV-1, replication of the resulting virus was resistant to VP32947. The RNA-dependent RNA polymerase activity of the NS5B proteins derived from WT and DR viruses expressed and purified from recombinant baculovirus-infected insect cells confirmed the drug sensitivity of the WT enzyme and the drug resistance of the DR enzyme. This work formally validates NS5B as a target for antiviral drug discovery and development. The utility of VP32947 and similar compounds for the control of pestivirus diseases, and for hepatitis C virus drug discovery efforts, is discussed.

Animals↗
PubMed2000

Mutagenesis of the signal sequence of yellow fever virus prM protein: enhancement of signalase cleavage In vitro is lethal for virus production.

Proteolytic processing at the C-prM junction in the flavivirus polyprotein involves coordinated cleavages at the cytoplasmic and luminal sides of an internal signal sequence. We have introduced at the COOH terminus of the yellow fever virus (YFV) prM signal sequence amino acid substitutions (VPQAQA mutation) which uncoupled efficient signal peptidase cleavage of the prM protein from its dependence on prior cleavage in the cytoplasm of the C protein mediated by the viral NS2B-3 protease. Infectivity assays with full-length YFV RNA transcripts showed that the VPQAQA mutation, which enhanced signal peptidase cleavage in vitro, was lethal for infectious virus production. Revertants or second-site mutants were recovered from cells transfected with VPQAQA RNA. Analysis of these viruses revealed that single amino acid substitutions in different domains of the prM signal sequence could restore viability. These variants had growth properties in vertebrate cells which differed only slightly from those of the parent virus, despite efficient signal peptidase cleavage of prM in cell-free expression assays. However, the neurovirulence in mice of the VPQAQA variants was significantly attenuated. This study demonstrates that substitutions in the prM signal sequence which disrupt coordinated cleavages at the C-prM junction can impinge on the biological properties of the mutant viruses. Factors other than the rate of production of prM are vitally controlled by regulated cleavages at this site.

Amino Acid Sequence↗
PubMed2000

Hepatitis C virus-encoded enzymatic activities and conserved RNA elements in the 3' nontranslated region are essential for virus replication in vivo.

Hepatitis C virus (HCV) infection is a widespread major human health concern. Significant obstacles in the study of this virus include the absence of a reliable tissue culture system and a small-animal model. Recently, we constructed full-length HCV cDNA clones and successfully initiated HCV infection in two chimpanzees by intrahepatic injection of in vitro-transcribed RNA (A. A. Kolykhalov et al., Science 277:570-574, 1997). In order to validate potential targets for development of anti-HCV therapeutics, we constructed six mutant derivatives of this prototype infectious clone. Four clones contained point mutations ablating the activity of the NS2-3 protease, the NS3-4A serine protease, the NS3 NTPase/helicase, and the NS5B polymerase. Two additional clones contained deletions encompassing all or part of the highly conserved 98-base sequence at the 3' terminus of the HCV genome RNA. The RNA transcript from each of the six clones was injected intrahepatically into a chimpanzee. No signs of HCV infection were detected in the 8 months following the injection. Inoculation of the same animal with nonmutant RNA transcripts resulted in productive HCV infection, as evidenced by viremia, elevated serum alanine aminotransferase, and HCV-specific seroconversion. These data suggest that these four HCV-encoded enzymatic activities and the conserved 3' terminal RNA element are essential for productive replication in vivo.

3' Untranslated Regions↗
PubMed2000

Subcellular localization, stability, and trans-cleavage competence of the hepatitis C virus NS3-NS4A complex expressed in tetracycline-regulated cell lines.

A tetracycline-regulated gene expression system and a panel of novel monoclonal antibodies were used to examine the subcellular localization, stability, and trans-cleavage competence of the hepatitis C virus (HCV) NS3-NS4A complex in inducible cell lines. The NS3 serine protease domain and the full-length NS3 protein expressed in the absence of the NS4A cofactor were diffusely distributed in the cytoplasm and nucleus. Coexpression of NS4A, however, directed NS3 to the endoplasmic reticulum (ER) or an ER-like modified compartment, as demonstrated by colocalization with 3,3'-dihexyloxacarbocyanine iodide, protein disulfide isomerase, and calnexin, as well as subcellular fractionation analyses. In addition, coexpression with NS4A dramatically increased the intracellular stability of NS3 (mean protein half-life of 26 versus 3 h) and allowed for NS4A-dependent trans-cleavage at the NS4B-NS5A junction. Deletion analyses revealed that the hydrophobic amino-terminal domain of NS4A was required for ER targeting of NS3. These results demonstrate the importance of studying HCV proteins in their biological context and define a well-characterized cell culture system for further analyses of the NS3-NS4A complex and the evaluation of novel antiviral strategies against hepatitis C.

Animals↗