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C M Rice

Publications and source records attributed to C M Rice.

At least 127 records · Page 7Linked to original sources
PubMed1992

Heterogeneity in envelope protein sequence and N-linked glycosylation among yellow fever virus vaccine strains.

We have compared the deduced envelope (E) protein sequences of two biologically well-characterized yellow fever (YF) virus vaccine strains. The 17DD strain has been produced in Brazil for more than 50 years and used to successfully vaccinate millions of people worldwide. The 17D-213 is a candidate vaccine strain produced in tissue culture which has previously passed the monkey neurovirulence assay for testing human YF vaccines. Nucleotide sequence analysis of polymerase chain reaction-amplified cDNA revealed a number of mutations which were strain- and substrain-specific. A major difference of 17DD and 17D-213 as compared to 17D-204 and Asibi was the existence of a potential N-linked glycosylation site located at amino acid residues 153 and 151 of 17DD and 17D-213, respectively. These acceptor sites are apparently utilized for the addition of high-mannose carbohydrate chains as shown by endoglycosidase analyses of immunoprecipitated E proteins. Glycosylated E protein is also used to assemble YF vaccine virions. This work and eventual complete nucleotide sequence analysis of both vaccine strains should help to define possible changes involved in YF virus attenuation and allow their biological importance to be determined using a recently developed system for generating YF virus from cDNA. In addition, these data provide an estimate on the extent of genetic variability among YF 17D seeds and vaccines.

Amino Acid Sequence↗
PubMed1992

Examples of expression systems based on animal RNA viruses: alphaviruses and influenza virus.

Successful recovery of RNA viruses and functional RNA replicons from cDNA has greatly facilitated molecular genetic analyses of viral proteins and cis-regulatory elements. This technology allows the use of RNA virus replication machinery to express heterologous sequences. Both positive-strand and negative-strand animal RNA viruses have been engineered to produce chimeric viruses expressing protective epitopes from other pathogens and for transient expression of heterologous sequences.

Alphavirus↗
PubMed1992

The efficient intracellular sequestration of the insulin-regulatable glucose transporter (GLUT-4) is conferred by the NH2 terminus.

GLUT-4 is the major facilitative glucose transporter isoform in tissues that exhibit insulin-stimulated glucose transport. Insulin regulates glucose transport by the rapid translocation of GLUT-4 from an intracellular compartment to the plasma membrane. A critical feature of this process is the efficient exclusion of GLUT-4 from the plasma membrane in the absence of insulin. To identify the amino acid domains of GLUT-4 which confer intracellular sequestration, we analyzed the subcellular distribution of chimeric glucose transporters comprised of GLUT-4 and a homologous isoform, GLUT-1, which is found predominantly at the cell surface. These chimeric transporters were transiently expressed in CHO cells using a double subgenomic recombinant Sindbis virus vector. We have found that wild-type GLUT-4 is targeted to an intracellular compartment in CHO cells which is morphologically similar to that observed in adipocytes and muscle cells. Sindbis virus-produced GLUT-1 was predominantly expressed at the cell surface. Substitution of the GLUT-4 amino-terminal region with that of GLUT-1 abolished the efficient intracellular sequestration of GLUT-4. Conversely, substitution of the NH2 terminus of GLUT-1 with that of GLUT-4 resulted in marked intracellular sequestration of GLUT-1. These data indicate that the NH2-terminus of GLUT-4 is both necessary and sufficient for intracellular sequestration.

Amino Acid Sequence↗
PubMed1991

Processing of the yellow fever virus nonstructural polyprotein: a catalytically active NS3 proteinase domain and NS2B are required for cleavages at dibasic sites.

The vaccinia virus-T7 transient expression system was used to further examine the role of the NS3 proteinase in processing of the yellow fever (YF) virus nonstructural polyprotein in BHK cells. YF virus-specific polyproteins and cleavage products were identified by immunoprecipitation with region-specific antisera, by size, and by comparison with authentic YF virus polypeptides. A YF virus polyprotein initiating with a signal sequence derived from the E protein fused to the N terminus of NS2A and extending through the N-terminal 356 amino acids of NS5 exhibited processing at the 2A-2B, 2B-3, 3-4A, 4A-4B, and 4B-5 cleavage sites. Similar results were obtained with polyproteins whose N termini began within NS2A (position 110) or with NS2B. When the NS3 proteinase domain was inactivated by replacing the proposed catalytic Ser-138 with Ala, processing at all sites was abolished. The results suggest that an active NS3 proteinase domain is necessary for cleavage at the diabasic nonstructural cleavage sites and that cleavage at the proposed 4A-4B signalase site requires prior cleavage at the 4B-5 site. Cleavages were not observed with a polyprotein whose N terminus began with NS3, but cleavage at the 4B-5 site could be restored by supplying the the NS2B protein in trans. Several experimental results suggested that trans cleavage at the 4B-5 site requires association of NS2B and the NS3 proteinase domain. Coexpression of different proteinases and catalytically inactive polyprotein substrates revealed that trans cleavage at the 2B-3 and 4B-5 sites was relatively efficient when compared with trans cleavage at the 2A-2B and 3-4A sites.

Animals↗
PubMed1991

Rescue of Sindbis virus-specific RNA replication and transcription by using a vaccinia virus recombinant.

A heterologous system expressing functional Sindbis virus nonstructural proteins (nsPs) has several possible uses for studying Sindbis virus-specific RNA replication and transcription in vivo and in vitro. Of the many possible approaches, vaccinia virus offers an attractive transient expression system given that Sindbis virus replication can occur in cells which have been previously infected by vaccinia virus. In this report, a vaccinia virus recombinant (called vSINNS), which contains the cDNA encoding the Sindbis virus nsPs under the control of either the vaccinia virus 7.5K promoter or the bacteriophage T7 promoter, has been constructed and characterized. Upon infection of several cell types with vSINNS, Sindbis virus nsP precursors and processed forms, including nsP1, nsP2, and both phosphorylated and nonphosphorylated forms of nsP3, were synthesized. Proteins containing the putative RNA-dependent RNA polymerase domain (nsP4 and nsP34), which are normally produced in small amounts by readthrough of an opal termination codon, were not detected in vSINNS-infected cells. However, all nsP functions necessary for Sindbis virus-specific RNA synthesis must have been expressed, since both replication and subgenomic mRNA transcription of an engineered Sindbis virus defective interfering RNA in cells infected with vSINNS was observed. Furthermore, vSINNS could be used as a helper virus to amplify, to relatively high titers, a replication-defective Sindbis virus mutant containing an in-frame deletion in the conserved N-terminal domain of nsP3. These data, as well as the observation that normal yields of parental Sindbis virus are produced in cells which have been previously infected with vSINNS, indicate that expression of Sindbis virus nsPs, in the absence of Sindbis virus-specific RNA replication, is not sufficient to block the formation of active RNA replication complexes by superinfecting Sindbis virus.

Animals↗
PubMed1990

Sequence of the 3' half of the Murray Valley encephalitis virus genome and mapping of the nonstructural proteins NS1, NS3, and NS5.

We have determined the nucleotide sequence of the 3'-terminal half of the RNA genome of Murray Valley encephalitis virus (MVE) using seven overlapping cDNA clones; an estimated 80-90 nucleotides at the extreme 3'-end remain to be sequenced. In conjunction with previous sequence data for the 5' half (16), we can conclude that the MVE genome contains a long open reading frame of 10,302 nucleotides that encodes a polyprotein of 3434 residues. Comparison of the MVE deduced amino acid sequence with that of other flaviviruses shows that MVE is most closely related to Japanese encephalitis virus, consistent with serological studies. Using N-terminal amino acid sequence analysis, three nonstructural proteins (NS1, NS3, and NS5) have been identified and mapped on the MVE genome. MVE NS3 contains sequence motifs suggesting that its amino terminus may function as a serine protease. The central region of NS3 (in the linear amino acid sequence) has motifs that are found in NTP-binding proteins and helicases. MVE NS5 contains a conserved Gly-Asp-Asp sequence that is thought to be essential for RNA-dependent RNA polymerases.

Amino Acid Sequence↗
PubMed1990

Expression of the structural proteins of dengue 2 virus and yellow fever virus by recombinant vaccinia viruses.

Vaccinia virus recombinants were constructed which contained cDNA sequences encoding the structural region of dengue 2 virus (PR159/S1 strain) or yellow fever virus (17D strain). The flavivirus cDNA sequences were expressed under the control of the vaccinia 7.5k early/late promotor. Cultured cells infected with these recombinants expressed immunologically reactive flavivirus structural proteins, precursor prM and E. These proteins appeared to be cleaved and glycosylated properly since they comigrated with the authentic proteins from dengue 2 virus- and yellow fever virus-infected cells. Mice immunized with the dengue/vaccinia recombinant showed a dengue-specific immune response that included low levels of neutralizing antibodies. Immunization of mice with the yellow fever/vaccinia recombinant was less effective at inducing an immune response to yellow fever virus and in only some of the mice were low titers of neutralizing antibodies produced.

Amino Acid Sequence↗
PubMed1990

Temperature sensitive shut-off of alphavirus minus strand RNA synthesis maps to a nonstructural protein, nsP4.

Minus strand RNA synthesis by the positive strand alphaviruses, Sindbis and Semliki Forest viruses, normally occurs early in infection, is coupled to synthesis of viral nonstructural proteins and to formation of viral replication complexes, and terminates and does not occur late in infection. Previously, ts24 of the A complementation group of Sindbis virus RNA-negative mutants was found to possess, among its other temperature sensitive defects, a temperature sensitivity in the normal cessation of minus strand synthesis which enabled minus strands to be synthesized late in infection at 40 degrees in the absence of protein synthesis. Revertants of ts24 (ts24R1, ts24R2) retained the defect in the shutoff of minus strand synthesis, indicating the lesion was not conditionally lethal and could map outside the A cistron. The studies reported here used an infectious clone of Sindbis virus to identify the mutation responsible for this phenotype. Hybrid viruses were prepared from constructs containing restriction fragments of the cDNA of ts24R1 in place of the corresponding fragments in the infectious SIN HR clone and screened for their ability to synthesize minus strands at 40 degrees in the presence of cycloheximide. A unique base change of an A for a C residue at nt 6339, predicting a change from glutamine to lysine at amino acid 195 in nsP4, was found in genomes of ts24, ts24R1, and ts24R2. Other nucleotide changes present at the 5' and 3' termini did not affect minus strand synthesis. The substitution of the parental Sindbis virus sequence that encompassed nt 6339 in an infectious clone of the ts24R1 revertant eliminated the mutant phenotype. We conclude that the ability to continue minus strand synthesis at 40 degrees exhibited by ts24 and its revertants is caused by an alteration in nsP4, which is the alphavirus replicase or an essential component of the replicase. We hypothesize that this domain of nsP4 functions to fix the minus strand as the stable template of alphavirus replication complexes.

Amino Acid Sequence↗
PubMed1990

Phosphorylation of Sindbis virus nsP3 in vivo and in vitro.

nsP3 is one of four viral nonstructural proteins required for RNA replication of Sindbis virus. In this report, post-translational modifications of nsP3 which occur in both vertebrate and mosquito cell cultures have been examined. In pulse-chase experiments, analyzed by immunoprecipitation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, nsP3 was initially observed as a single species (termed nsP3a, approximately 76 kDa) which was gradually converted to slower mobility forms ranging from 78 kDa (termed nsP3b) to 106 kDa (termed nsP3c). The slower mobility forms, but not nsP3a or the other nonstructural proteins, could be labeled in vivo with [32P]orthophosphate. Treatment of nsP3 immunoprecipitates with calf intestinal alkaline phosphatase converted the slower mobility forms to nsP3a. Phosphoamino acid analysis of nsP3b and nsP3c demonstrated that both contained phosphoserine and phosphothreonine but not phosphotyrosine, nsP34, a polyprotein produced by readthrough of the in-frame opal codon preceding nsP4, was also phosphorylated on serine and threonine residues. nsP3 phosphorylation did not require ongoing RNA synthesis since phosphorylated forms were also observed in the absence of Sindbis-specific RNA synthesis. Furthermore, when immunoprecipitates of nsP3 were incubated with [gamma-32P]ATP in the presence of Mg2+ or Mn2+, a kinase activity which was able to phosphorylate nsP3 on serine and threonine residues in vitro was detected. This kinase activity was inhibited by heparin, was activated by spermidine, and could utilize GTP and ATP as the phosphate donor. These latter properties are similar to those of cellular casein kinase II. Although it is possible that this nsP3-associated kinase is of cellular origin, autophosphorylation of nsP3 has not been excluded.

Animals↗
PubMed1990

Production of yellow fever virus proteins in infected cells: identification of discrete polyprotein species and analysis of cleavage kinetics using region-specific polyclonal antisera.

Flavivirus proteins are produced by translation of a single long open reading frame and a complex series of cotranslational and post-translational proteolytic cleavages. To study these processing events in yellow fever virus (YF)-infected cells, polyclonal antisera recognizing C, prM, E, NS1, NS2B, NS3, NS4B, and NS5 were generated using peptide and fusion protein immunogens. Evidence suggests that production of the structural protein precursors involves rapid cotranslational processing consistent with signalase cleavages. The synthesis of the NS1 glycoprotein involves cleavage of polyprotein precursors (t1/2 approximately 10 minutes) which probably contain portions of the NS2A gene product. Endoglycosidase F treatment or labeling in the presence of tunicamycin suggests that YF prM and NS1 each have two N-linked oligosaccharides. NS2B is produced without any identifiable precursors or associated polyprotein species. Processing of the NS3-4-5 region is complex and occurs rapidly. A series of polyproteins can be detected whose molecular weights correlate with the cleavage sites defined by available N-terminal amino acid sequence data. However, convincing precursor-product relationships between these polyproteins and the mature NS3 and NS5 proteins could not be demonstrated. In contrast, NS4B appears to be produced by cleavage of a discrete precursor believed to be NS4AB. N-terminal sequence data for the putative NS4AB product has tentatively defined the NS3-4A cleavage site. A scheme for in vivo processing of the YF polyprotein is presented and discussed.

Amino Acid Sequence↗
PubMed1990

Evidence that the N-terminal domain of nonstructural protein NS3 from yellow fever virus is a serine protease responsible for site-specific cleavages in the viral polyprotein.

Sequence homology and molecular modeling studies have suggested that the N-terminal one-third of the flavirvirus nonstructural protein NS3 functions as a trypsin-like serine protease. To examine the putative proteolytic activity of NS3, segments of the yellow fever virus genome were subcloned into plasmid transcription/translation vectors and cell-free translation products were characterized. The results suggest that a protease activity encoded within NS2B and the N-terminal one-third of yellow fever virus NS3 is capable of cis-acting site-specific proteolysis at the NS2B-NS3 cleavage site and dilution-insensitive cleavage of the NS2A-NS2B site. Site-directed mutagenesis of the His-53, Asp-77, and Ser-138 residues of NS3 that compose the proposed catalytic triad implicates this domain as a serine protease. Infectious virus was not recovered from mammalian cells transfected with RNAs transcribed from full-length yellow fever virus cDNA templates containing mutations at Ser-138 (which abolish or dramatically reduce protease activity in vitro), suggesting that the protease is required for viral replication.

Capsid↗
PubMed1990

Mutations which alter the level or structure of nsP4 can affect the efficiency of Sindbis virus replication in a host-dependent manner.

Two mutants of Sindbis virus have been isolated which grow inefficiently at 34.5 degrees C in mosquito cells yet replicate normally in chicken embryo fibroblast cells at the same temperature. In addition, these mutants exhibit temperature-sensitive growth in both cell types and are RNA- at the nonpermissive temperatures (K.J. Kowal and V. Stollar, Virology 114:140-148, 1981). To clarify the basis of this host restriction, we have mapped the causal mutations for these temperature-dependent, host-restricted mutants. Functional mapping and sequence analysis of the mutant cDNAs revealed several mutations which mapped to the amino terminus of nsP4, the putative polymerase subunit of the viral RNA replicase. These mutations resulted in the following amino acid changes in nsP4: leucine to valine at residue 48, aspartate to glycine at residue 142, and proline to arginine at residue 187. Virus containing any of these mutations was restricted in its ability to replicate in mosquito but not chicken embryo fibroblast cells at 34.5 degrees C. In addition to its temperature-dependent, host-restricted phenotype, virus derived from one cDNA clone also exhibited decreased levels of nsP34 and nsP4 yet contained only a silent change in its genome. This C-to-U mutation occurred at nucleotide 5751, the first nucleotide after the opal termination codon separating nsP3 and nsP4. Our results suggest that this substitution decreases readthrough of the opal codon and diminishes production of nsP34 and nsP4. Such a decrease in synthesis rates might lead to levels of these products which are insufficient for viral RNA replication in mosquito cells at the higher temperature. This work provides the first evidence that nsP4 function can be strongly influenced by the host environment.

Aedes↗
PubMed1990

Pharmacologic treatment of noncognitive behavioral disturbances in elderly demented patients.

Fifty-nine elderly residents of long-term care facilities who had DSM-III diagnoses of dementia were studied in an 8-week randomized, double-blind comparison trial of haloperidol, oxazepam, and diphenhydramine to test the efficacy of these agents in the treatment of clinically significant behavioral disturbances in patients with dementia. All three agents demonstrated modest but significant efficacy as measured by clinician ratings of agitated behavior and activities of daily living. The absolute magnitude of improvement was greater for haloperidol and diphenhydramine than for oxazepam, but differences among groups did not approach statistical significance. Frequencies of acute adverse events during the trial were similar across the drug treatment groups. Although these drugs may differ in terms of long-term safety and efficacy, they appear to be equivalent for short-term management of agitated behavior in severely demented patients.

Activities of Daily Living↗
PubMed1990

Overview of flavivirus molecular biology and future vaccine development via recombinant DNA.

Studies in many laboratories over the last several years have elucidated the structures of several different flavivirus genomes. Conserved features include the production of at least 10 different virus encoded proteins from a single long open reading frame by a combination of host and virus-encoded proteases. The established gene order is 5'-C- prM(M)-E-NS1-NS2A-NS2B-NS3-NS4A-NS4B-NS 5-3' and these proteins exhibit varying degrees of homology in comparisons among flaviviruses. Conserved RNA sequences and structures have also been identified for the mosquito-borne flaviviruses but are absent in sequenced tick-bone viruses. Relevant to the development of efficacious flavivirus vaccines, studies aimed at defining the antigenic determinants necessary for eliciting protective immunity have focused primarily on the structural proteins, in particular the E protein, as well as the nonstructural secreted glycoprotein, NS1. Other work, which has led to the derivation of live-attenuated flavivirus strains, should eventually allow the genetic determinants of flavivirus attenuation and pathogenesis to be understood at the molecular level. The successful recovery infectious flaviviruses from cloned cDNA raises the possibility of manipulating these viral genomes as cDNA to construct or propagate candidate live-attenuated vaccine strains. Several applications of this technology are discussed.

Amino Acid Sequence↗
PubMed1989

Multiple tachykinins are produced and secreted upon post-translational processing of the three substance P precursor proteins, alpha-, beta-, and gamma-preprotachykinin. Expression of the preprotachykinins in AtT-20 cells infected with vaccinia virus recombinants.

The rat preprotachykinin I gene mRNA is alternatively spliced to yield three different mRNA species differing in their protein coding regions. We have produced recombinant vaccinia viruses expressing alpha-, beta-, and gamma-preprotachykinin to examine the tachykinin-related peptides produced upon post-translational processing of each individual precursor. Infection of BSC-40 or AtT-20 cell lines with a beta-preprotachykinin-encoding vaccinia virus recombinant results in the expression of the precursor protein. The pro-form (signal peptide removed) can be immunoprecipitated from extracts of infected cells. Infected cells of both types secrete into the culture medium a product(s) which reacts in radioimmunoassay with an antiserum shown to recognize precursor as well as mature substance P. Infected AtT-20, but not BSC-40, cells secrete into the culture medium a processed form(s) of beta-preprotachykinin which reacts in radioimmunoassay with an anti-serum which recognizes the amidated carboxyl terminus of substance P. The molecular nature of the tachykinin products produced in and secreted from AtT-20 cells infected with alpha-, beta-, and gamma-preprotachykinin-encoding recombinants was analyzed by combined high performance liquid chromatography and radioimmunoassay. Peptides were identified based on comigration with synthetic standards and antisera cross-reactivity. We determined that alpha-preprotachykinin is processed to the mature undecapeptide, substance P. beta-Preprotachykinin was processed into multiple products, including substance P, neurokinin A, neurokinin A(3-10), and neuropeptide K. gamma-Preprotachykinin was processed into substance P, neurokinin A, neurokinin A(3-10), and neuropeptide gamma. These five tachykinin peptide products were all routed through the regulated secretory pathway and were secreted into the medium in a cAMP-stimulatable fashion. Since all of these peptides have been shown to be biologically active, it is important to consider the biological consequences of their co-secretion in vivo.

Animals↗
PubMed1989

Sindbis virus: an efficient, broad host range vector for gene expression in animal cells.

Sindbis virus, an enveloped virus with a single-stranded RNA genome, was engineered to express a bacterial protein, chloramphenicol acetyltransferase (CAT), in cultured insect, avian, and mammalian cells. The vectors were self-replicating and gene expression was efficient and rapid; up to 10(8) CAT polypeptides were produced per infected cell in 16 to 20 hours. CAT expression could be made temperature-sensitive by means of a derivative that incorporated a temperature-sensitive mutation in viral RNA synthesis. Vector genomic RNAs were packaged into infectious particles when Sindbis helper virus was used to supply virion structural proteins. The vector RNAs were stable to at least seven cycles of infection. The expression of CAT increased about 10(3)-fold, despite a 10(15)-fold dilution during the passaging. Sindbis virus vectors should prove useful for expressing large quantities of gene products in a variety of animal cells.

Aedes↗