Biomedical subjects

C M Rice

Publications and source records attributed to C M Rice.

At least 145 records · Page 8Linked to original sources

PubMed1989

Yellow fever virus proteins NS2A, NS2B, and NS4B: identification and partial N-terminal amino acid sequence analysis.

A series of fusion proteins corresponding to the hydrophobic ns2 and ns4 regions of yellow fever virus (YF) were generated in Escherichia coli using trpE fusion vectors. Antisera to ns2 and ns4 region fusion proteins recognize virus-specific proteins of 15 and 27 kDa, respectively. N-terminal amino acid sequence analysis of the 27-kDa protein indicates that the N-terminus of YF NS4B immediately follows a signalase-like cleavage site. Additional sequence data generated by microsequence analysis of labeled proteins immunoprecipitated with mouse hyperimmune antisera have identified the 15-kDa protein as NS2B and an additional 20-kDa viral protein as NS2A. Comparison of the sequences adjacent to the N-termini of these viral proteins suggests that three distinct types of cleavage events are involved in processing the hydrophobic YF ns2 and ns4 regions. These include cleavage after a short side chain amino acid to generate the N-terminus of NS2A, cleavage after two arginine residues to produce the N-terminus of NS2B, and a cleavage site consistent with the specificity of signalase to generate the N-terminus of NS4B. Analysis of virus-specific protein patterns in several different mammalian cell lines and in Aedes albopictus cells suggests that the same cleavage sites are used in different hosts. These findings are discussed in relation to the processing of flavivirus polyproteins.

Adrenal Cortex Neoplasms↗

PubMed1989

Association of the Sindbis virus RNA methyltransferase activity with the nonstructural protein nsP1.

SVLM21 is a mutant of Sindbis virus, which in contrast to SVSTD, is able to replicate in Aedes albopictus mosquito cells deprived of methionine. We have obtained evidence that the basis of this low methionine-resistance (LMR) phenotype is the generation of an altered RNA methyltransferase with an increased affinity for S-adenosylmethionine (ado met). We now report that following the substitution of the nucleotide sequence, 126-504, from SVLM21 cDNA for the corresponding sequence of the Toto 1101 plasmid (infectious Sindbis viral RNA can be transcribed from this plasmid) we were able to generate recombinant Sindbis virus (SVMS-65a) with the LMR phenotype. (SVTOTO virus derived from Toto 1101, like SVSTD, lacks the LMR phenotype.) As was the case with SVLM21, SVMS-65a not only possessed the LMR phenotype but also showed an increased sensitivity to Neplanocin A, a potent inhibitor of S-adenosylhomocysteine (ado hcy) hydrolase. Sequencing of the nucleotide 126-504 region from SVLM21 revealed two mutations; these mutations occurred in adjacent codons and lead to two predicted amino acid changes in the SV nsPl protein; at residue 87, from Arg to Leu, and at residue 88 from Ser to Cys. Since the nucleotide sequence 126-504 lies entirely within the gene for nsP1, we conclude that the RNA methyltransferase activity generated by SV is associated with nsP1. We suggest that residues 87 and 88 in nsP1, where the amino acid changes in SVLM21 nsP1 have occurred, are at or near the binding site for ado met; we also suggest that these changes in nsP1 are responsible for the increased affinity of the SVLM21 RNA methyltransferase for ado met and thereby for the LMR phenotype. Alternatively, it is possible that the binding site for ado met is elsewhere on nsP1 or even on another protein, and that the changes at residues 87 and 88 lead to an alteration of the binding site.

Animals↗

PubMed1989

A cis-acting mutation in the Sindbis virus junction region which affects subgenomic RNA synthesis.

The synthesis of Sindbis virus minus-strand and genomic and subgenomic RNAs is believed to require specific cis-acting sequences or structures in the template RNAs and a combination of virus-specific proteins and host components which act in trans. A conserved sequence of about 21 nucleotides in the junction region and encompassing the start site for the subgenomic RNA has been proposed to function as the promoter on the minus-strand template for synthesis of the subgenomic RNA (J.-H. Ou, C. M. Rice, L. Dalgarno, E. G. Strauss, and J. H. Strauss, Proc. Natl. Acad. Sci. USA 79:5235-5239, 1982). We introduced a three-base insertion in this sequence, which also inserts a single amino acid near the COOH terminus of nsP4, in a cDNA clone of Sindbis virus from which infectious RNA transcripts can be generated. The phenotype of this mutant, called Toto1100CR4.1, was studied after RNA transfection of chicken embryo fibroblasts or BHK cells. The mutation leads to a drastic reduction in the level of the subgenomic RNA but does not alter the start site of the RNA. Probably as a consequence of depressed structural-protein synthesis, very few progeny virions are released and the mutant makes tiny or indistinct plaques even after prolonged incubation. The cis-acting effect of this mutation was demonstrated by incorporating either a wild-type or mutant junction region into a defective-interfering RNA and examining the relative synthesis of defective-interfering RNA-derived subgenomic RNA in vivo in the presence of wild-type helper virus. These results show that the junction region is recognized by yet unidentified viral trans-acting components for subgenomic RNA synthesis. When the Toto1100CR4.1 mutant was passaged in culture, plaque morphology variants readily arose. A total of 24 independent revertants were isolated, and 16 were characterized in detail. All revertants analyzed showed an increase in the level of subgenomic RNA synthesis. Sequence analysis of the junction region showed that all were pseudorevertants, with only two containing potentially compensating changes in the junction region. An assay was developed to identify revertants with second-site changes in trans-acting viral components involved in subgenomic RNA synthesis. At least two such revertants were identified. Mapping of these and other second-site compensating mutations may provide genetic clues as to which virus-specific protein(s) is responsible for interaction with the conserved junction region to promote subgenomic RNA synthesis.

Amino Acid Sequence↗

PubMed1989

Mapping of RNA- temperature-sensitive mutants of Sindbis virus: complementation group F mutants have lesions in nsP4.

Temperature-sensitive (ts) mutants of Sindbis virus belonging to complementation group F, ts6, ts110, and ts118, are defective in RNA synthesis at the nonpermissive temperature. cDNA clones of these group F mutants, as well as of ts+ revertants, have been constructed. To assign the ts phenotype to a specific region in the viral genome, restriction fragments from the mutant cDNA clones were used to replace the corresponding regions of the full-length clone Toto1101 of Sindbis virus. These hybrid plasmids were transcribed in vitro by SP6 RNA polymerase to produce infectious transcripts, and the virus recovered was tested for temperature sensitivity. After the ts lesion of each mutant was mapped to a specific region of 400 to 800 nucleotides by this approach, this region of the cDNA clones of both the ts mutant and ts+ revertants was sequenced in order to determine the precise nucleotide change and amino acid substitution responsible for each mutation. Rescued mutants, which have a uniform background except for one or two defined changes, were examined for viral RNA synthesis and complementation to show that the phenotypes observed were the result of the mutations mapped. ts6 and ts110 had a single base substitution in nsP4, resulting in replacement of Gly by Glu at position 153 or position 324, respectively. It is of interest that nsP4 contains the Gly-Asp-Asp motif characteristic of a number of viral replicases, and this, together with the fact that all RNA synthesis in ts6-infected cells and, to a lesser extent, in ts110-infected cells shut off when the cells were shifted from a permissive to a nonpermissive temperature, suggests that nsP4 is the virus polymerase. ts118 was a double mutant. It contained a single base substitution in nsP2, resulting in replacement of Val by Ala at position 425 that resulted in the formation of minute plaques, but not in a reduction in the plaque number at the nonpermissive condition. The second change, a substitution of Gln by Arg in ts118 at residue 93 in nsP4, had little apparent phenotype on its own, but in combination with the change in nsP2 led to a ts phenotype. Thus, in each case the mutation responsible for the temperature sensitivity of the three known complementation group F mutants lay in nsP4. In addition, the result with ts118 suggests that nsP2 and nsP4 may interact with each other in a complex.

Amino Acid Sequence↗

PubMed1989

Mutagenesis of the in-frame opal termination codon preceding nsP4 of Sindbis virus: studies of translational readthrough and its effect on virus replication.

Sindbis virus (SIN) contains an in-frame opal termination codon in the nonstructural protein-coding region separating nsP3 and nsP4 and provides a useful tool to study the readthrough phenomenon of the termination codon in host cells and its role in viral replication. We have changed the opal codon by site-directed mutagenesis of a full-length SIN cDNA clone to either sense amino acids (serine, tryptophan, or arginine) or the other two translation termination codons (amber or ochre). Transcripts from all of the mutant cDNA clones were infectious when used to transfect chicken embryo fibroblasts. The resulting progeny virus stocks were then used to study the effects of these mutations on viral protein and RNA synthesis, growth properties, host range, and fitness compared with the parental strain. None of the mutants showed temperature sensitivity in plaquing efficiency or plaque morphology on chicken embryo fibroblast monolayers. Relative to the wild-type parent, the mutants containing sense replacements overproduced nsP34 but not nsP4 and made slightly decreased levels of nsP3, with a delay in its appearance. This indicates that the cleavage separating nsP3 and nsP4 occurs in these mutants and also that the level of nsP4 is not regulated solely by readthrough of the opal codon. The amber and ochre mutants produced decreased levels of nsP34, and the ochre mutant grew significantly more slowly than the other mutants or wild-type virus. For all five mutants, RNA synthesis early in infection was inhibited compared with that of the parental virus. This effect was apparent at multiplicities of infection of 20 PFU per cell but not at 100 PFU per cell. Using in situ hybridization to distinguish between mutant and wild-type plaques, we have studied the behavior of the serine mutant in a high-multiplicity growth competition experiment with wild-type virus. The wild-type virus eventually outcompeted the mutant after several passages, and these results indicate that this mutation has resulted in effects that are at least partially cis acting. Furthermore, by studying the growth, plaque formation, and protein synthesis of the mutants in various cell types, we have observed host range effects of the mutations, especially in mosquito and human cells. In addition, we have demonstrated, at least indirectly, that opal, amber, and ochre termination codons in the SIN nucleotide context can be suppressed in cultured cells of chicken, human, hamster, and mosquito origin.

Animals↗

PubMed1989

Sindbis virus ts103 has a mutation in glycoprotein E2 that leads to defective assembly of virions.

Sindbis virus mutant ts103 is aberrant in the assembly of virus particles. During virus budding, proper nucleocapsid-glycoprotein interactions fail to occur such that particles containing many nucleocapsids are formed, and the final yield of virus is low. We have determined that a mutation in the external domain of glycoprotein E2, Ala-344----Val, is the change that leads to this phenotype. Mapping was done by making recombinant viruses between ts103 and a parental strain of the virus, using a full-length cDNA clone of Sindbis virus from which infectious RNA can be transcribed, together with sequence analysis of the region of the genome shown in this way to contain the ts103 lesion. A partial revertant of ts103, called ts103R, was also mapped and sequenced and found to be a second-site revertant in which a change in glycoprotein E1 from lysine to methionine at position 227 partially suppresses the phenotypic effects of the change at E2 position 344. An analysis of revertants from ts103 mutants in which the Ala----Val change had been transferred into a defined background showed that pseudorevertants were more likely to arise than were true revertants and that the ts103 change itself reverted very infrequently. The assembly defect in ts103 appeared to result from weakened interactions between the virus membrane glycoproteins or between these glycoproteins and the nucleocapsid during budding. Both the E2 mutation leading to the defect in virus assembly and the suppressor mutation in glycoprotein E1 are in the domains external to the lipid bilayer and thus in domains that cannot interact directly with the nucleocapsid. This suggests that in ts103, either the E1-E2 heterodimers or the trimeric spikes (consisting of three E1-E2 heterodimers) are unstable or have an aberrant configuration, and thus do not interact properly with the nucleocapsid, or cannot assembly correctly to form the proper icosahedral array on the surface of the virus.

Animals↗

PubMed1989

Transcription of infectious yellow fever RNA from full-length cDNA templates produced by in vitro ligation.

Yellow fever (YF) virus is the prototype member of the flavivirus family, a diverse group of human and animal pathogens. A live-attenuated strain of YF virus, called 17D, has been used successfully for human vaccination for more than 50 years. In this report we describe the construction of full-length YF 17D cDNA templates that can be transcribed in vitro to yield infectious YF virus RNA. Because of the instability of full-length YF cDNA clones and their toxic effects on Escherichia coli, we developed a strategy in which full-length templates for transcription were constructed by in vitro ligation of appropriate restriction fragments. The YF virus recovered from cDNA was indistinguishable from the parental virus by several criteria. This system should facilitate the molecular genetic analysis of flavivirus replication and attenuation and may allow YF 17D to be used as a carrier for immunologically important epitopes from other disease agents.

Base Sequence↗

PubMed1988

Genome sequences of a mouse-avirulent and a mouse-virulent strain of Ross River virus.

The nucleotide sequence of the genomic RNA of a mouse-avirulent strain of Ross River virus, RRV NB5092 (isolated in 1969), has been determined and the corresponding sequence for the prototype mouse-virulent strain, RRV T48 (isolated in 1959), has been completed. The RRV NB5092 genome is approximately 11,674 nucleotides in length, compared with 11,853 nucleotides for RRV T48. RRV NB5092 and RRV T48 have the same genome organization. For both viruses an untranslated region of 80 nucleotides at the 5' end of the genome is followed by a 7440-nucleotide open reading frame which is interrupted after 5586 nucleotides by a single opal termination codon. By homology with other alphaviruses, the 5586-nucleotide open reading frame encodes the nonstructural proteins nsP1, nsP2, and nsP3; a fourth nonstructural protein, nsP4, is produced by read-through of the opal codon. The RRV nonstructural proteins show strong homology with the corresponding proteins of Sindbis virus and Semliki Forest virus in terms of size, net charge, and hydropathy characteristics. However, homology is not uniform between or within the proteins; nsP1, nsP2, and nsP4 contain extended domains which are highly conserved between alphaviruses, while the C-terminal region of nsP3 shows little conservation in sequence or length between alphaviruses. An untranslated "junction" region of 44 nucleotides (for RRV NB5092) or 47 nucleotides (for RRV T48) separates the nonstructural and structural protein coding regions. The structural proteins (capsid-E3-E2-6K-E1) are translated from an open reading frame of 3762 nucleotides which is followed by a 3'-untranslated region of approximately 348 nucleotides (for RRV NB5092) or 524 nucleotides (for RRV T48). Excluding deletions and insertions, the genomes of RRV NB5092 and RRV T48 differ at 284 nucleotides, representing a sequence divergence of 2.38%. Sequence deletions or insertions were found only in the noncoding regions and include a 173-nucleotide deletion in the 3'-untranslated region of RRV NB5092, compared with RRV T48. In the coding regions, most of the nucleotide differences are silent; there are 36 amino acid differences in the nonstructural proteins and 12 in the structural proteins. The distribution of amino acid differences between the two RRV strains correlates with the location of domains which are poorly conserved in sequence between alphaviruses. The possible role of amino acid differences in envelope glycoproteins E1 and E2 in determining the different antigenic and biological properties of RRV NB5092 and RRV T48 is discussed.

Alphavirus↗

PubMed1988

Nonstructural proteins nsP3 and nsP4 of Ross River and O'Nyong-nyong viruses: sequence and comparison with those of other alphaviruses.

We have sequenced the nsP3 and nsP4 region of two alphaviruses, Ross River virus and O'Nyong-nyong virus, in order to examine these viruses for the presence or absence of an opal termination codon present between nsP3 and nsP4 in many alphaviruses. We found that Ross River virus possesses an in-phase opal termination codon between nsP3 and nsP4, whereas in O'Nyong-nyong virus this termination codon is replaced by an arginine codon. Previous studies have shown that two other alphaviruses, Sindbis virus and Middelburg virus, possess an opal termination codon separating nsP3 and nsP4 [E.G. Strauss, C.M. Rice, and J.H. Strauss (1983), Proc. Natl. Acad. Sci. USA 80, 5271-5275], whereas Semliki Forest virus possesses an arginine codon in lieu of the opal codon [K. Takkinen (1986), Nucleic Acids Res. 14, 5667-5682]. Thus, of the five alphaviruses examined to date, three possess the opal codon and two do not. Production of nsP4 requires readthrough of the opal codon in those alphaviruses that possess this termination codon and the function of the termination codon may be to regulate the amount of nsP4 produced. It is an open question then as to whether alphaviruses with no termination codon use other mechanisms to regulate the activity of this gene. The nsP4s of these five alphaviruses are highly conserved, sharing 71-76% amino acid sequence similarity, and all five contain the Gly-Asp-Asp motif found in many RNA virus replicases. The nsP3s are somewhat less conserved, sharing 52-73% amino acid sequence similarity throughout most of the protein, but each possesses a nonconserved C-terminal domain of 134 to 246 amino acids of unknown function.

Alphavirus↗

PubMed1988

Detection of yellow fever virus nucleic acid in infected mosquitoes by RNA:RNA in situ hybridization.

An in situ hybridization technique was developed for the strand-specific detection of yellow fever virus (YFV) RNA. An 35S-labeled, transcribed RNA probe was used to detect positive-sense polarity YFV genomic RNA in infected C6/36 (Aedes albopictus) cells, dissected mosquito tissues, and sections of plastic-embedded, YFV-infected Aedes aegypti mosquitoes. Mosquito tissues fixed in buffered Formalin retained morphological integrity. The low concentrations of probe used yielded high specific signal on infected specimens and low background signal on uninfected specimens.

Animals↗

PubMed1988

Molecular analysis of Sindbis virus pathogenesis in neonatal mice by using virus recombinants constructed in vitro.

Genetic loci affecting Sindbis virus pathogenesis in neonatal mice have been examined by using a full-length cDNA clone of the virus (Toto1101). The full-length cDNA is linked to a bacteriophage SP6 promoter to facilitate the synthesis of infectious RNA transcripts in vitro. Virus derived from Toto1101 showed reduced virulence (attenuation) in neonatal mice. Replacement of the E1 glycoprotein and 6K genes of Toto1101 with cloned E1 and 6K genes derived from a virulent Sindbis virus strain, AR339 (SB), resulted in a new construct, TR2000, that gave rise to virulent virus. Sequence determinations for the entire substituted regions of TR2000, Toto1101, and related virulent and attenuated strains identified three coding differences in E1 between Toto1101 and TR2000. These differences, individually or in combination, may be responsible for the attenuated phenotype. Previous studies in this laboratory identified another attenuating mutation at amino acid position 114 of the E2 glycoprotein (N.L. Davis, F.J. Fuller, W.G. Dougherty, R.A. Olmsted, and R.E. Johnston, Proc. Natl. Acad. Sci. USA 83:6771-6775, 1986). Substitution of Arg-114 in the mutant SB-RL for Ser-114 of SB appears to confer three distinguishing phenotypes: attenuation in neonatal mice, increased sensitivity to specific E2 monoclonal antibodies, and accelerated penetration of BHK cells. Replacement of TR2000 sequences containing the codon for amino acid 114 of E2 with corresponding fragments from cDNA clones of SB or SB-RL produced two strains of Sindbis virus (TR2100 and TR2200) which were isogenic except for the E2 114 codon (Ser and Arg, respectively). The three diagnostic phenotypes cosegregated according to the origin of the codon for amino acid 114 of E2, confirming the dramatic effect of this single amino acid substitution on these three phenotypes.

Amino Acid Sequence↗

PubMed1987

Conserved elements in the 3' untranslated region of flavivirus RNAs and potential cyclization sequences.

We have isolated a cDNA clone after reverse transcription of the genomic RNA of Asibi yellow fever virus whose structure suggests it was formed by self-priming from a 3'-terminal hairpin of 87 nucleotides in the genomic RNA. We have also isolated a clone from cDNA made to Murray Valley encephalitis virus RNA that also appears to have arisen by self-priming from a 3'-terminal structure very similar or identical to that of yellow fever. In addition, 3'-terminal sequencing of the S1 strain of dengue 2 RNA shows that this RNA is also capable of forming a 3'-terminal hairpin of 79 nucleotides. Furthermore, we have identified two 20-nucleotide sequence elements which are present in the 3' untranslated region of all three viruses; one of these sequence elements is repeated in Murray Valley encephalitis and dengue 2 RNA but not in yellow fever RNA. In all three viruses, which represent the three major serological subgroups of the mosquito-borne flaviviruses, the 3'-proximal conserved sequence element, which is found immediately adjacent to the potential 3'-terminal hairpin, is complementary to another conserved domain near the 5' end of the viral RNAs, suggesting that flavivirus RNAs can cyclize (calculated delta G less than -11 kcal; 1 kcal = 4.184 kJ).

Base Sequence↗

PubMed1987

Partial nucleotide sequence of St. Louis encephalitis virus RNA: structural proteins, NS1, ns2a, and ns2b.

cDNA clones of the St. Louis encephalitis (SLE) virus genome have been obtained and the nucleotide sequence of 4.7 kb corresponding to the 5' terminal half of the genome determined. The genome contains a 5' noncoding region of 98 nucleotides followed by a single continuous open reading frame that encodes three structural proteins in the order capsid (C), membrane precursor (prM)-membrane (M), and envelope (E). Immediately following the C-terminus of E are located nonstructural proteins NS1 through NS3. The SLE amino acid sequence homology with yellow fever (YF), Murray Valley encephalitis (MVE), West Nile (WN), and dengue-2 (DEN) viruses over the sequenced region is 39, 66, 64, and 43%, respectively. The start of each SLE protein has been assigned on the basis of N-terminal sequence data and potential proteolytic cleavage sites homologous with YF and MVE viruses. Flaviviruses have conserved glycosylation sites in prM and NS1 proteins, although only one of the two glycosylation sites in the SLE E protein is conserved in MVE and DEN viruses. An evolutionary tree showing relationships of SLE, MVE, WN, YF, and DEN-2 flaviviruses is proposed on the basis of the amino acid sequences of the C proteins.

Base Sequence↗

PubMed1987

Use of enzyme immunoassay and nucleic acid hybridization for detecting Sindbis virus in infected mosquitoes.

Aedes aegypti mosquitoes were inoculated intrathoracically with prototype Sindbis virus, held at 26.7 degrees C for from 0-95 h and placed at -70 degrees C. Individual mosquitoes were tested for virus by plaque assay in Vero cells, for viral RNA by nucleic acid hybridization using a cloned cDNA probe, and for viral protein by enzyme-linked immunosorbent assay. Virus was detected by plaque assay as early as 8 h after infection. Sindbis virus RNA was detected by nucleic acid hybridization 18 h after infection and by enzyme-linked immunosorbent assay 10 h after infection. The results of these comparisons suggest that both nucleic acid hybridization and enzyme-linked immunosorbent assay are applicable to direct detection of Sindbis virus in mosquitoes containing virus at levels usually found during arbovirus epidemics.

Animals↗

PubMed1987

Comparison of the virulent Asibi strain of yellow fever virus with the 17D vaccine strain derived from it.

We have sequenced the virulent Asibi strain of yellow fever virus and compared this sequence to that of the 17D vaccine strain, which was derived from it. These two strains of viruses differ by more than 240 passages. We found that the two RNAs, 10,862 nucleotides long, differ at 68 nucleotide positions; these changes result in 32 amino acid differences. Overall, this corresponds to 0.63% nucleotide sequence divergence, and the changes are scattered throughout the genome. The overall divergence at the level of amino acid substitution is 0.94%, but these changes are not randomly distributed among the virus protein. The capsid protein is unchanged, while proteins NS1, NS3, and NS5 contain 0.5% amino acid substitutions, and proteins ns4a and ns4b average 0.8% substitutions. In contrast, proteins ns2a and ns2b have 3.0 and 2.3% amino acid divergence, respectively. The envelope protein also has a relatively high rate of amino acid change of 2.4% (a total of 12 amino acid substitutions). The large number of changes in ns2a and ns2b, which are largely conservative in nature, may result from lowered selective pressure against alteration in this region; among flaviviruses, these polypeptides are much less highly conserved than NS1, NS3, and NS5. However, many of the amino acid substitutions in the E protein are not conservative. It seems likely that at least some of the difference in virulence between the two strains of yellow fever virus results from changes in the envelope protein that affect virus binding to host receptors. Such differences in receptor binding could result in the reduced neurotropism and vicerotropism exhibited by the vaccine strain.

Amino Acid Sequence↗

PubMed1987

Production of infectious RNA transcripts from Sindbis virus cDNA clones: mapping of lethal mutations, rescue of a temperature-sensitive marker, and in vitro mutagenesis to generate defined mutants.

We constructed full-length cDNA clones of Sindbis virus that can be transcribed in vitro by SP6 RNA polymerase to produce infectious genome-length transcripts. Viruses produced from in vitro transcripts are identical to Sindbis virus and show strain-specific phenotypes reflecting the source of RNA used for cDNA synthesis. The cDNA clones were used to confirm the mapping of the causal mutation of ts2 to the capsid protein. A general strategy for mapping Sindbis virus mutations is described and was used to identify two lethal mutations in an original full-length construct which did not produce infectious transcripts. An XbaI linker was inserted in the cDNA clone near the transcriptional start of the subgenomic mRNA; the resulting virus retains the XbaI recognition sequence, thus providing formal evidence that viruses are derived from in vitro transcripts of cDNA clones. The potential applications of the cDNA clones are discussed.

Animals↗

PubMed1987

Molecular biology of the flaviviruses.

An overview of the molecular biology of the flaviviruses is presented. The members of this virus family are enveloped positive-strand RNA viruses capable of causing a number of important human diseases.

Animals↗

PubMed1986

Partial nucleotide sequence of the Murray Valley encephalitis virus genome. Comparison of the encoded polypeptides with yellow fever virus structural and non-structural proteins.

The sequence of 5400 bases corresponding to the 5'-terminal half of the Murray Valley encephalitis virus genome has been determined. The genome contains a 5' non-coding region of about 97 nucleotides, followed by a single continuous open reading frame that encodes the structural proteins followed by the non-structural proteins. Amino acid sequence homology between the Murray Valley encephalitis and yellow fever (Rice et al., 1985) polyproteins is 42% over the region sequenced. The start points of the various Murray Valley encephalitis virus-coded proteins have been assigned on the basis of this homology and a consistent set of potential proteolytic cleavage sites identified, the sequences of which are similar in Murray Valley encephalitis and yellow fever. The deduced Murray Valley encephalitis gene order is 5'-C-prM (M)-E-NS1-ns2a-ns2b-NS3-3'. The genome organization of Murray Valley encephalitis and yellow fever appears to be identical and the sizes of the predicted virus-coded proteins similar between the two viruses. Both viruses encode a basic capsid protein followed by three glycoproteins; the glycoproteins appear to have the conventional topology of N terminus outside with a C-terminal membrane-spanning domain. There are conserved glycosylation sites in prM, the precursor to the M protein of the virion, and in NS1, a non-structural protein of uncertain function. The glycosylation sites in E, the major envelope protein of the virion, are not conserved as to position. We predict the existence, in flavivirus-infected cells, of two small, hydrophobic peptides, ns2a and ns2b, which show only limited amino acid sequence homology. Finally, about half of the amino acid sequence of NS3 has been obtained; NS3 is a hydrophilic non-structural protein that shows 55% amino acid sequence similarity between Murray Valley encephalitis and yellow fever over the region sequenced and is probably involved in RNA replication.

Amino Acid Sequence↗