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C M Rice

Publications and source records attributed to C M Rice.

At least 163 records · Page 9Linked to original sources
PubMed1986

Partial N-terminal amino acid sequences of three nonstructural proteins of two flaviviruses.

Partial N-terminal amino acid sequences for the three largest nonstructural proteins of two flaviviruses, yellow fever virus and St. Louis encephalitis virus, have been obtained. The determined sequences of these proteins exhibit significant amino acid sequence homology, and allow the positioning of these three nonstructural proteins in the polyprotein sequence deduced from the nucleotide sequence of yellow fever virus (C. M. Rice, E. M. Lenches, S. R. Eddy, S. J. Shin, R. L. Sheets, and J. H. Strauss, 1985, Science 229, 726-733.) The deduced start points support the hypothesis that the N terminus of nonstructural glycoprotein NS1 results from cleavage by signalase, whereas the N termini of NS3 and NS5 result from cleavages following double basic residues that are flanked by amino acids with short side chains.

Amino Acid Sequence↗
PubMed1986

Sindbis virus mutant ts20 of complementation group E contains a lesion in glycoprotein E2.

A technique has been devised to readily obtain the entire structural protein region of Sindbis virus cloned into a plasmid vector. This method uses the fact that the nearest site for restriction enzyme HindIII to the 3' terminal poly(A) occurs at nucleotides 6266-6271 in the genomic RNA. Inserts extending from the poly(A) tract to this HindIII site are 5438 nucleotides long (excluding the poly A tract) and contain the entire 4106-nucleotide structural protein region. Using an oligo(dT)-tailed vector as a primer for first strand cDNA synthesis such clones could be obtained in high yield. We were interested in a precise determination of the mutation responsible for the temperature-sensitive phenotype of ts20, a mutant belonging to complementation group E which has a defect in the function of glycoprotein E2 at the nonpermissive temperature. Using this technique we have cloned and sequenced the structural protein region of ts20 and of several revertants and concluded that the mutation was a change from histidine to leucine at amino acid 291 of E2. Reversion to temperature insensitivity occurred by same site reversion to the parental nucleotide, restoring the original histidine as amino acid 291. Thus, complementation group E of Sindbis virus results from changes in glycoprotein E2 and together with previous results from our laboratory (Arias et al., 1983; Hahn et al., 1985) demonstrates that the three RNA+ complementation groups of Sindbis virus, C, D, and E, result from changes in the three structural proteins of the virus, capsid, glycoprotein E1, and glycoprotein E2, respectively.

Cloning, Molecular↗
PubMed1985

Nucleotide sequence of yellow fever virus: implications for flavivirus gene expression and evolution.

The sequence of the entire RNA genome of the type flavivirus, yellow fever virus, has been obtained. Inspection of this sequence reveals a single long open reading frame of 10,233 nucleotides, which could encode a polypeptide of 3411 amino acids. The structural proteins are found within the amino-terminal 780 residues of this polyprotein; the remainder of the open reading frame consists of nonstructural viral polypeptides. This genome organization implies that mature viral proteins are produced by posttranslational cleavage of a polyprotein precursor and has implications for flavivirus RNA replication and for the evolutionary relation of this virus family to other RNA viruses.

Base Sequence↗
PubMed1985

Sindbis virus proteins nsP1 and nsP2 contain homology to nonstructural proteins from several RNA plant viruses.

Although the genetic organization of tobacco mosaic virus (TMV) differs considerably from that of the tripartite viruses (alfalfa mosaic virus [AlMV] and brome mosaic virus [BMV]), all of these RNA plant viruses share three domains of homology among their nonstructural proteins. One such domain, common to the AlMV and BMV 2a proteins and the readthrough portion of TMV p183, is also homologous to the readthrough protein nsP4 of Sindbis virus (Haseloff et al., Proc. Natl. Acad. Sci. U.S.A. 81:4358-4362, 1984). Two more domains are conserved among the AlMV and BMV 1a proteins and TMV p126. We show here that these domains have homology with portions of the Sindbis proteins nsP1 and nsP2, respectively. These results strengthen the view that the four viruses share mechanistic similarities in their replication strategies and may be evolutionarily related. These results also suggest that either the AlMV 1a, BMV 1a, and TMV p126 proteins are multifunctional or Sindbis proteins nsP1 and nsP2 function together as subunits in a single complex.

Amino Acid Sequence↗
PubMed1985

Expression of Sindbis virus structural proteins via recombinant vaccinia virus: synthesis, processing, and incorporation into mature Sindbis virions.

We have obtained a vaccinia virus recombinant which contains a complete cDNA copy of the 26S RNA of Sindbis virus within the thymidine kinase gene of the vaccinia virus genome. This recombinant constitutively transcribed the Sindbis sequences throughout the infectious cycle, reflecting the dual early-late vaccinia promoter used in this construction. The Sindbis-derived transcripts were translationally active, giving rise to both precursor and mature structural proteins of Sindbis virus, including the capsid protein (C), the precursor of glycoprotein E2 (PE2), and the two mature envelope glycoproteins (E1 and E2). These are the same products translated from the 26S mRNA during Sindbis infection, and thus these proteins were apparently cleaved, glycosylated, and transported in a manner analogous to that seen during authentic Sindbis infections. By using epitope-specific antibodies, it was possible to demonstrate that recombinant-derived proteins were incorporated into Sindbis virions during coinfections with monoclonal antibody-resistant Sindbis variants. These results suggest that all the information necessary to specify the proper biogenesis of Sindbis virus structural proteins resides within the 26S sequences and that vaccinia may provide an appropriate system for using DNA molecular genetic manipulations to unravel a variety of questions pertinent to RNA virus replication.

Biological Transport↗
PubMed1985

Neomycin resistance as a dominant selectable marker for selection and isolation of vaccinia virus recombinants.

The antibiotic G418 was shown to be an effective inhibitor of vaccinia virus replication when an appropriate concentration of it was added to cell monolayers 48 h before infection. Genetic engineering techniques were used in concert with DNA transfection protocols to construct vaccinia virus recombinants containing the neomycin resistance gene (neo) from transposon Tn5. These recombinants contained the neo gene linked in either the correct or incorrect orientation relative to the vaccinia virus 7.5-kilodalton gene promoter which is expressed constitutively throughout the course of infection. The vaccinia virus recombinant containing the chimeric neo gene in the proper orientation was able to grow and form plaques in the presence of G418, whereas both the wild-type and the recombinant virus with the neo gene in the opposite polarity were inhibited by more than 98%. The effect of G418 on virus growth may be mediated at least in part by selective inhibition of the synthesis of a subset of late viral proteins. These results are discussed with reference to using this system, the conferral of resistance to G418 with neo as a positive selectable marker, to facilitate constructing vaccinia virus recombinants which contain foreign genes of interest.

Animals↗
PubMed1984

Biochemical studies of the maturation of the small Sindbis virus glycoprotein E3.

A small glycoprotein (E3) was purified from the culture fluid of Sindbis virus-infected primary chick embryo fibroblasts. Tryptic peptide mapping and pulse-chase studies verified that this protein was produced as a by-product of the cleavage of the precursor protein PE2 to produce the envelope glycoprotein E2. A 2600-fold purification was achieved via a procedure which used differential ethanol precipitation, gel filtration, ion-exchange chromatography, and affinity chromatography on a lentil lectin column. Amino acid composition analysis, N-terminal microsequencing, and labeling studies yielded information about the fine structure of E3 and its relationship to E2 and virion maturation. The N-terminal sequence of E3 is identical to that of PE2, including the result that 90% of the molecules appear to be blocked. The first 19 amino acids are uncharged and presumably serve as the signal sequence for the insertion of PE2 into the membrane of the endoplasmic reticulum, but this sequence is unusual in that it is not immediately cleaved from PE2 and is glycosylated at the asparagine at position 14. The two residues at the C-terminus of E3, Lys-Arg, are removed during or shortly after cleavage from PE2. Labeling studies imply that, although the PE2----E2 + E3 cleavage is necessary for virion budding, these two events are not closely coupled. E3 is cleaved and released into the culture fluid under conditions where virions do not bud, and the kinetics of the appearance of E3 in the culture fluid and E2 in virions are quite dissimilar. The maturation of E3 is discussed as it relates to the processing of cellular membrane and secretory glycoproteins.

Amino Acid Sequence↗
PubMed1984

Complete nucleotide sequence of the genomic RNA of Sindbis virus.

The entire nucleotide sequence of the genomic RNA of the type virus of the alphavirus genus, Sindbis virus, has been determined. The genome is 11,703 nucleotides in length, exclusive of the 5' cap and the 3'-terminal poly(A) tract. After the 5'-terminal cap there are 59 nucleotides of 5' nontranslated nucleic acid followed by a reading frame of 7539 nucleotides that encodes the nonstructural polypeptides and which is open except for a single opal termination codon. Following 48 untranslated bases located in the junction region which separates the nonstructural and structural protein coding sequences, there is an open reading frame 3735 nucleotides long that encodes the structural proteins. Finally, the 3' untranslated region is 322 nucleotides long. The nonstructural proteins are translated from the genomic RNA as two polyprotein precursors. The first is 1896 amino acids in length and terminates at an opal codon at position 1897. This polyprotein is processed to produce three polypeptides called nsP1, nsP2, and nsP3. Sites of post-translational cleavage to produce these three proteins have been tentatively located using available N-terminal amino acid sequence data. In both cases cleavage probably occurs between the two alanine residues in the sequence Gly-Ala-Ala. The fourth nonstructural protein, nsP4, is produced when readthrough of the opal codon produces a second polyprotein precursor of length 2513 amino acids, which is also cleaved posttranslationally. The structural proteins are translated from a subgenomic message which begins at nucleotide 7598, is 4106 nucleotides in length (exclusive of the poly(A) tract), and is coterminal with the 3' end of the genomic RNA. The structural proteins are also translated as a polyprotein precursor which is cleaved to produce a nucleocapsid protein and two integral membrane glycoproteins as well as two small peptides not present in the mature virion. A replication strategy for Sindbis virus based upon the complete nucleotide sequence, as well as prior data, is presented.

Amino Acid Sequence↗
PubMed1983

Ross River virus 26 s RNA: complete nucleotide sequence and deduced sequence of the encoded structural proteins.

The complete sequence of the 26 S RNA of Ross River virus (T48 strain) has been obtained and from this the amino acid sequences of the encoded structural proteins have been deduced. These include a basic capsid protein and two envelope glycoproteins. The nucleotide sequence was obtained by chemical sequence analysis of both single-stranded and double-stranded cDNA made to RNA and the sequence data so obtained was rapidly aligned by making use of the protein homology found among the alphaviruses. The polyprotein precursor encoded by the 26 S RNA of Ross River virus is 75% homologous to that of Semliki Forest virus and 48% homologous to that of Sindbis virus. The extent of homology is not uniform within a protein or between proteins and this is discussed with respect to the possible function of the various polypeptide domains in the virus life cycle. In each case the putative attachment site of the amino proximal carbohydrate chains of the three glycoproteins is conserved, whereas the attachment site of a second chain, if present, is not conserved. The 3'-untranslated region of Ross River virus RNA is 524 nucleotides long. It contains a sequence of about 50 nucleotides in length which is present in four copies but which is not shared with other alphaviruses examined.

Alphavirus↗
PubMed1983

Sequence coding for the alphavirus nonstructural proteins is interrupted by an opal termination codon.

We have obtained the nucleotide sequence of the genomic RNAs of two alphaviruses, Sindbis virus and Middelburg virus, over an extensive region encoding the nonstructural (replicase) proteins. In both viruses in an equivalent position an opal (UGA) termination codon punctuates a long otherwise open reading frame. The nonstructural proteins are translated as polyprotein precursors that are processed by posttranslational cleavage into four polypeptide chains; the sequence data presented here indicate that the COOH-terminal polypeptide, ns72, may be produced by read-through of this opal codon. The high degree of amino acid homology between the ns72 polypeptides of the two viruses, in contrast to the lack of conserved sequence upstream from the read-through site, suggests that ns72 plays an important role in viral replication, possibly modulating the action of other replicase components.

Amino Acid Sequence↗
PubMed1982

Sequence studies of several alphavirus genomic RNAs in the region containing the start of the subgenomic RNA.

The alphaviruses produce two mRNAs after infection: the genomic (49S) RNA which is translated into the nonstructural (replicase) proteins and the subgenomic (26S) RNA which serves as the mRNA for the virion structural proteins. The sequence of the region of the genomic RNA that contains the 5' end of the subgenomic RNA and the 5' flanking sequences in the genomic RNA were determined for several alphaviruses. A highly conserved sequence of 21 nucleotides was found which includes the first two nucleotides of the subgenomic RNA and the 19 nucleotides preceding it. We propose that the complement of this sequence in the minus strand is the recognition site used by the viral transcriptase for initiation of transcription of 26S RNA and that, in general, such short recognition sequences are commonly used among the RNA viruses. The COOH-terminal sequence of the nonstructural polyprotein precursor has been deduced for each virus. These protein sequences are highly homologous and are followed by multiple in-phase termination codons clustered in the nontranslated region of the 26S RNA in each case. In contrast to the proposed transcriptase recognition site, the particular triplets used for a given conserved amino acid have diverged markedly during evolution of these viruses. The protein homology is sufficient, however, for deduction of the correct coding phase of the RNA and allows the alignment of the corresponding nucleic acid sequence data from different alphaviruses without knowledge of the sequence of the entire genomes.

Alphavirus↗
PubMed1982

Sequence analysis of cDNA's derived from the RNA of Sindbis virions and of defective interfering particles.

Sindbis virus generates defective interfering (DI) particles during serial high-multiplicity passage in cultured cells. These DI particles inhibit the replication of infectious virus and can be an important factor in the establishment and maintenance of persistent infection in BHK cells. In an effort to understand how these DI particles are generated and how they interfere with the replication of standard virus, we performed a partial sequence analysis of the RNA obtained from two independently isolated populations of DI particles and from two Sindbis virus variants and compared these with the RNA of the parental wild-type virus. The 3'-terminal regions of the RNAs were sequenced by the dideoxy chain terminating method. Internal regions of the RNA were examined by restriction endonuclease digestion of cDNA's made to the various RNAs and by direct chemical sequencing of 5' end-labeled restriction fragments from cDNA made to the DI RNAs. One of the variant viruses examined was originally derived from cells persistently infected with Sindbis virus for 16 months and is resistant to interference by the DI strains used. In the 3'-terminal region of the RNA from this variant, only two base changes were found; one of these occurs in the 20-nucleotide 3'-terminal sequence which is highly conserved among alphaviruses. The DI RNA sequences were found to have been produced not by a single deletional event, but by multiple deletion steps combined with sequence rearrangements; all sequences examined are derived from the plus strand of Sindbis virion RNA. Both DI RNAs had at least 50 nucleotides of wild-type sequence conserved at the 3' terminus; in addition, they both contained conserved and perhaps amplified sequences derived from the non-26S region of the genome which may be of importance in their replication and interference ability.

Base Sequence↗
PubMed1981

Nucleotide sequence of the 26S mRNA of Sindbis virus and deduced sequence of the encoded virus structural proteins.

The nucleotide sequence of intracellular 26S mRNA of Sindbis virus has been determined by direct sequence analysis of the cDNA made to this RNA with reverse transcriptase. From this, the amino acid sequences of the encoded virus structural proteins, which include a basic capsid protein and two integral membrane glycoproteins, have been deduced. The features of these proteins as related to their functions are discussed. We suggest that three proteases are required to produce these proteins from their polyprotein precursor: a viral protease, which functions in the cytosol to release the capsid protein, signalase, which makes two cleavages to separate the glycoproteins; and a protease of the Golgi complex that cleaves after double basic residues, to process the precursor form of one of the glycoproteins.

Amino Acid Sequence↗
PubMedDate not supplied

Adult stem cells--reprogramming neurological repair?

Much excitement has surrounded recent breakthroughs in embryonic stem-cell research. Of lower profile, but no less exciting, are the advances in the field of adult stem-cell research, and their implications for cell therapy. Clinical experience from use of adult haemopoietic stem cells in haematology will facilitate and hasten transition from laboratory to clinic--indeed, clinical trials using adult human stem cells are already in progress in some disease states, including myocardial ischaemia. Here, with particular reference to neurology, we review processes that might underlie apparent changes in adult cell phenotype. We discuss implications these processes might have for the development of new therapeutic strategies using adult stem cells.

Adult↗