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R M Elliott

Publications and source records attributed to R M Elliott.

At least 37 records · Page 2Linked to original sources
PubMed1996

Rescue of a segmented negative-strand RNA virus entirely from cloned complementary DNAs.

We provide the first report, to our knowledge, of a helper-independent system for rescuing a segmented, negative-strand RNA genome virus entirely from cloned cDNAs. Plasmids were constructed containing full-length cDNA copies of the three Bunyamwera bunyavirus RNA genome segments flanked by bacteriophage T7 promoter and hepatitis delta virus ribozyme sequences. When cells expressing both bacteriophage T7 RNA polymerase and recombinant Bunyamwera bunyavirus proteins were transfected with these plasmids, full-length antigenome RNAs were transcribed intracellularly, and these in turn were replicated and packaged into infectious bunyavirus particles. The resulting progeny virus contained specific genetic tags characteristic of the parental cDNA clones. Reassortant viruses containing two genome segments of Bunyamwera bunyavirus and one segment of Maguari bunyavirus were also produced following transfection of appropriate plasmids. This accomplishment will allow the full application of recombinant DNA technology to manipulate the bunyavirus genome.

Base Sequence↗
PubMed1995

Transcription of a recombinant bunyavirus RNA template by transiently expressed bunyavirus proteins.

We describe a convenient system for analyzing bunyavirus transcription using a recombinant RNA template derived from the plasmid pBUNSCAT, which comprises a negative-sense reporter gene (chloramphenicol acetyltransferase or CAT) flanked by the exact 5' and 3' untranslated regions of the Bunyamwera virus (BUN) S RNA segment. When cells which expressed bunyavirus proteins (either by recombinant vaccinia viruses or by the vaccinia virus-T7 system) were transfected with BUNSCAT RNA, CAT activity could be measured, indicating transcription of the negative-sense reporter RNA into mRNA. The system permits investigation of both the protein and RNA sequence requirements for transcription. Extensions of 2 bases at the 5' end or 11 or 35 bases at the 3' end of BUNSCAT RNA allowed transcription but a lower level than the wild-type template. Deletion of the 5 nucleotides at the 3' end of BUNSCAT RNA reduced CAT activity by > 99%. Investigation of the viral protein requirements of the system showed that only the bunyavirus L and N proteins were needed for CAT activity. The BUN L protein was also able to transcribe the reporter RNA in concert with the N proteins of closely related bunyaviruses such as Batai, Cache Valley, Maguari, Main Drain, and Northway, but only inefficiently with those of Kairi, Guaroa, or Lumbo viruses. When BUN L proteins containing specific mutations were expressed CAT activity was only observed using those mutated L proteins previously reported to be active in a nucleocapsid transfection assay (H. Jin and R. M. Elliott, 1992, J. Gen. Virol. 73, 2235-2244). These results illustrate the utility of this system for a detailed genetic analysis of the factors involved in bunyavirus transcription.

Amino Acid Sequence↗
PubMed1995

The 5' ends of Hantaan virus (Bunyaviridae) RNAs suggest a prime-and-realign mechanism for the initiation of RNA synthesis.

We examined the 5' ends of Hantaan virus (HTN) genomes and mRNAs to gain insight into the manner in which these chains were initiated. Like those of all members of the family Bunyaviridae described so far, the HTN mRNAs contained 5' terminal extensions that were heterogeneous in both length and sequence, presumably because HTN also "cap snatches" host mRNAs to initiate the viral mRNAs. Unexpectedly, however, almost all of the mRNAs contained a G residue at position -1, and a large fraction also lacked precisely one of the three UAG repeats at the termini. The genomes, on the other hand, commenced with a U residue at position +1, but only 5' monophosphates were found here, indicating that these chains may not have initiated with UTP at this position. Taken together, these unusual findings suggest a prime-and-realign mechanism of chain initiation in which mRNAs are initiated with a G-terminated host cell primer and genomes with GTP, not at the 3' end of the genome template but internally (opposite the template C at position +3), and after extension by one or a few nucleotides, the nascent chain realigns backwards by virtue of the terminal sequence repeats, before processive elongation takes place. For genome initiation, an endonuclease, perhaps that involved in cap snatching, is postulated to remove the 5' terminal extension of the genome, leaving the 5' pU at position +1.

Animals↗
PubMed1994

Localization of Bunyamwera bunyavirus G1 glycoprotein to the Golgi requires association with G2 but not with NSm.

The Bunyamwera bunyavirus (BUN) M RNA genome segment encodes three proteins, two glycoproteins termed G1 and G2 and a non-structural protein called NSm, in the form of a polyprotein precursor that is co-translationally cleaved to give the mature proteins. Indirect immunofluorescence experiments have shown that these proteins localize to the Golgi complex in BUN-infected cells. We have used a recombinant vaccinia virus (vTF7-3), which expresses bacteriophage T7 RNA polymerase, to drive the expression of plasmids containing either the entire BUN M segment cDNA or fragments that encode the G1, G2 and NSm proteins separately under control of the T7 promoter. After transfection of these plasmids into vTF7-3-infected cells, correctly sized and processed proteins were detected by immunoprecipitation with BUN-specific antibodies. Immunofluorescence experiments showed that G1, G2 and NSm localized to the Golgi when transiently expressed from the full-length cDNA. When G2 or NSm were expressed separately they also localized to the Golgi, but when G1 was expressed alone a staining pattern typical for the endoplasmic reticulum was obtained. However coexpression of G2 and G1 from independent plasmids resulted in G1 localizing to the Golgi. In contrast translocation of G1 to the Golgi was not observed when G1 was coexpressed with NSm, although NSm itself was still detected in the Golgi. Similar results were obtained when the proteins were expressed from transfected plasmids containing the G2-, NSm- or G1-coding sequences under control of the cytomegalovirus immediate-early promoter. The localization of G1 to the Golgi when coexpressed with G2 was confirmed by the loss of endoglycosidase H (endo H) sensitivity of G1 after approximately 60 min in a pulse-chase experiment; G1 remained sensitive to endo H when expressed either alone or in combination with NSm. These results suggest that G2 contains the Golgi targeting and/or retention signals and that G1 has to interact with this protein to localize to this cellular compartment.

Base Sequence↗
PubMed1994

The S RNA genome segments of Batai, Cache Valley, Guaroa, Kairi, Lumbo, Main Drain and Northway bunyaviruses: sequence determination and analysis.

Bunyaviruses have a genome comprising three segments of negative-sense RNA. The smallest RNA segment, S, encodes the nucleocapsid protein, N, and a nonstructural protein, NSs, in overlapping reading frames. The sequences of the S genome RNA segments of seven bunyaviruses (Batai, Cache Valley, Guaroa, Kairi, Main Drain, Northway and Lumbo) were determined from cloned cDNAs obtained using a one-step reverse transcription-PCR protocol. These sequences were compared to those of six viruses previously published, reinforcing earlier conclusions about relationships of the bunyaviruses. Sequence homologies between N proteins correlated with the subdivision of these viruses into three serogroups, Bunyamwera, California and Simbu. The encoded N proteins are either 233 or 235 amino acids in length, depending on the serogroup, whereas the NSs proteins are more variable (83 to 109 amino acids). Certain nucleotide sequence motifs are conserved in the S segments of the Bunyamwera and California serogroup viruses, including the spacing of the AUG initiation codons for the N and NSs proteins (except Guaroa virus), and a CA-rich motif in the virion-sense RNA just downstream of the predicted mRNA termination site. A duplicated sequence was observed in the 3' non-coding region of the Lumbo virus S segment, which accounts for the significantly longer S genome segment of this virus.

Amino Acid Sequence↗
PubMed1994

Assembly of herpes simplex virus type 1 capsids using a panel of recombinant baculoviruses.

Immature or B capsids of herpes simplex virus type 1 (HSV-1) are composed of seven proteins encoded by six viral genes. The proteins encoded by UL18 (VP23), UL19 (VP5), UL35 (VP26) and UL38 (VP19C) are components of the outer capsid shell whereas those specified by UL26 (VP21 and VP24) and UL26.5 (VP22a), are involved in scaffold formation. We have used a panel of recombinant baculoviruses, each expressing one of the capsid protein genes, to examine the requirements for capsid assembly. Coexpression of the six genes in insect cells resulted in the formation of capsids that were indistinguishable in appearance and protein composition from those made during HSV-1 infection of mammalian cells. This demonstrates that the proteins encoded by the known capsid genes contain all the structural information necessary for capsid assembly and that other virus-encoded proteins are not required for this process. Omission of single recombinant baculoviruses from this system allowed the role of individual HSV-1 proteins in capsid assembly to be determined. Thus, capsid assembly did not take place in the absence of VP23, VP5 or VP19C, whereas lack of VP26 had no discernible effect on capsid formation. Capsids assembled in the absence of the UL26 gene products had a large-cored phenotype resembling that previously described for the HSV-1 mutant ts1201 which has a lesion in this gene. Some apparently intact capsid shells were also made in the absence of the major scaffolding protein, VP22a, whereas the omission of both UL26 and UL26.5 resulted in the appearance of large numbers of partial and deformed capsid shells.

Animals↗
PubMed1993

Expression of the Bunyamwera virus M genome segment and intracellular localization of NSm.

Bunyamwera (BUN) virus is the prototype of the family Bunyaviridae and contains a trisegmented, single-stranded RNA genome of negative polarity. The medium (M) RNA segment encodes the two virion glycoproteins, G1 and G2, and a nonstructural protein, NSm, in the form of a polyprotein precursor which is cotranslationally cleaved. The gene order of the M segment is 5' G2-NSm-G1 3'. We have raised a monospecific antiserum in rabbits to a branched chain synthetic peptide to a region of the NSm protein which specifically immunoprecipitates NSm from BUN-infected cells. Indirect immunofluorescence experiments on BUN-infected cells using this antiserum gave a perinuclear staining pattern, suggesting that like the viral structural proteins, NSm localizes to the Golgi complex. An essentially full-length M segment cDNA was cloned into a recombinant vaccinia virus under control of bacteriophage T7 promoter and terminator sequences and expressed in cells co-infected with a second recombinant vaccinia virus which synthesizes T7 RNA polymerase. G1, G2, and NSm were detected in cells dually infected with the recombinant vaccina viruses, indicating that processing of the M segment-encoded precursor does not require other BUN proteins. Immunofluorescence experiments showed that the BUN glycoproteins expressed from this recombinant vaccinia virus system localized to the Golgi complex like authentic BUN proteins.

Amino Acid Sequence↗
PubMed1993

Non-viral sequences at the 5' ends of Dugbe nairovirus S mRNAs.

Analyses of the mRNA transcription processes of viruses in four genera (Bunyavirus, Hantavirus, Phlebovirus and Tospovirus) of the family Bunyaviridae have revealed a common mechanism of initiation using host-derived primers, known as cap-snatching. To provide similar information on the fifth genus in the family, the 5' ends of Dugbe nairovirus S mRNA species were specifically cloned and sequenced. This revealed the presence of non-viral heterogeneous sequences, five to 16 nucleotides in length (average of 10 nucleotides) at the 5' ends, confirming that cap-snatching to prime mRNA synthesis is a familial characteristic of the Bunyaviridae. Inspection of the sequences in the primers on nairovirus, bunyavirus and phlebovirus mRNAs suggests that in some cases polymerase slippage occurs shortly after initiation, resulting in a partial reiteration of the 5'-terminal nucleotides of the viral RNA.

Base Sequence↗
PubMed1993

Advanced use of tissue adhesive in hair transplantation.

BACKGROUND: Cobblestoning is an unsightly result of graft elevation in the recipient site after hair transplantation. OBJECTIVE: To describe the use of tissue adhesives for the avoidance of cobblestoning. RESULTS: The long-term cosmetic appearance of hair transplants may be improved through the use of cyanoacrylate adhesives is the virtual elimination of the common problem of graft elevation or cobblestoning, which produces an unsightly bumpiness in the recipient area. The choice of type of adhesive and the method of application are central to successful use of this technique. CONCLUSION: The indirect benefits of elimination of the need for postoperative bandages and restrictions in activities couple with the cosmetic results to produce a high degree of patient satisfaction.

Alopecia↗
PubMed1993

Characterization of Bunyamwera virus S RNA that is transcribed and replicated by the L protein expressed from recombinant vaccinia virus.

Analysis of the 5' termini of Bunyamwera virus S segment mRNAs by cloning and sequence analysis revealed the presence of nonviral, heterogeneous sequences 12 to 17 bases long. This is similar to reports for other members of the family Bunyaviridae and is taken to indicate that mRNA transcription is primed by a "cap-snatching" mechanism. The 3' end of the Bunyamwera virus S mRNA was mapped, by using an RNase protection assay, to 100 to 110 nucleotides upstream of the 3' end of the template. Previously we reported expression of the Bunyamwera virus L (polymerase) protein by recombinant vaccinia virus and demonstrated that the recombinant L protein was functional in terms of RNA synthesis activity in a nucleocapsid transfection assay (H. Jin and R. M. Elliott, J. Virol. 65: 4182-4189, 1991). In the present study we further analyze the RNAs made by using this system and show that positive-sense RNAs contain 5' nonviral sequences. Hence the initiation of mRNA transcription by the recombinant L protein resembles that seen during authentic bunyavirus infection and suggests that the L protein has the endonuclease activity which generates the primers. Some of these positive-sense transcripts terminated at the mRNA termination site, but the majority read through to the end of the template. No primer sequences were found at the 5' terminal of negative-sense RNAs. The recombinant L protein was able to replicate negative-sense RNA supplied by transfected virion-derived nucleocapsids, and both positive- and negative-sense RNAs were synthesized. These results indicate that the recombinant L protein has both transcriptase and replicase activities.

Base Sequence↗
PubMed1993

Glucagon-like peptide-1 (7-36)amide and glucose-dependent insulinotropic polypeptide secretion in response to nutrient ingestion in man: acute post-prandial and 24-h secretion patterns.

The acute effects of different macronutrients on the secretion of glucagon-like peptide-1(7-36)amide (GLP-1(7-36)amide) and glucose-dependent insulinotropic polypeptide (GIP) were compared in healthy human subjects. Circulating levels of the two hormones were measured over a 24-h period during which subjects consumed a mixed diet. In the first study, eight subjects consumed three equicaloric (375 kcal) test meals of carbohydrate, fat and protein. Small increases in plasma GLP-1(7-36) amide were found after all meals. Levels reached a maximum 30 min after the carbohydrate and 150 min after the fat load. Ingestion of both carbohydrate and fat induced substantial rises in GIP secretion, but the protein meal had no effect. In a second study, eight subjects consumed 75 g glucose or the equivalent portion of complex carbohydrate as boiled brown rice or barley. Plasma GIP, insulin and glucose levels increased after all three meals, the largest increase being observed following glucose and the smallest following the barley meal. Plasma GLP-1(7-36)amide levels rose only following the glucose meal. In the 24-h study, plasma GLP-1(7-36)amide and GIP concentrations were increased following every meal and remained elevated throughout the day, only falling to fasting levels at night. The increases in circulating GLP-1(7-36)amide and GIP levels following carbohydrate or a mixed meal are consistent with their role as incretins. The more sustained rises observed in the daytime during the 24-h study are consistent with an anabolic role in lipid metabolism.

Adult↗
PubMed1992

Defective RNAs in mosquito cells persistently infected with Bunyamwera virus.

Viral protein and RNA synthesis were compared in BHK and Aedes albopictus C6/36 (mosquito) cells infected with Bunyamwera virus. In BHK cells host protein synthesis was inhibited and viral proteins were detected until the cells died; in C6/36 cells there was little inhibition of host proteins and viral proteins could not be detected after 36 h post-infection. Relatively more S segment RNA than L or M segment RNA was produced in infected C6/36 cells compared to BHK cells. A persistent infection of C6/36 cells was established and the cells were passaged at weekly intervals for over a year. The titre of virus released from the cells and the level of viral RNA in the cells at different passages fluctuated markedly, but there was no simple relationship between virus titre and the amount of viral RNA. Northern blot analysis of viral RNA extracted from persistently infected cells revealed the presence of subgenomic RNAs derived from the L RNA segment. These defective RNAs were not packaged into nucleocapsids. The presence of the defective RNAs did not correlate with resistance of cells cloned from the persistently infected population to superinfection with homologous virus. Hence the role of these defective RNAs in the maintenance of the persistent state remains to be elucidated.

Animals↗
PubMed1992

Characterization of Bunyamwera virus defective interfering particles.

In an attempt to isolate conditional lethal amber nonsense mutants of Bunyamwera virus, five variants were found which produced small plaques on BHK and mouse L cells. Characterization of these variants by Northern blotting showed that they synthesized defective (subgenomic) RNAs derived from the L RNA segment. No subgenomic M or S segment RNAs were detected. The defective L RNAs were shown to be packaged into virus particles, and four of five preparations caused interference with the multiplication of standard virus. When defective-containing preparations were mixed with standard virus and grown in doubly infected cells a reduction in titre of standard virus of up to 400-fold was observed. Hence these preparations most probably contained defective interfering (DI) particles. Novel DI-specific polypeptides were synthesized in DI virus-infected cells. These novel proteins could be precipitated by antisera raised against either the N or C terminus, or both, of the L protein. Nucleotide sequence analysis of cloned cDNA to prominent DI RNAs in three different defective virus preparations revealed that the DI RNA in each case had suffered a single internal deletion of the L segment while retaining the 5'- and 3'-terminal sequences. The extent of the deletion ranged between 72% and 77% of the L RNA segment. Our results suggest that these DI particles may have arisen during the attempted isolation of Bunyamwera virus amber mutants on mouse L cells, since defective/subgenomic RNAs derived from the L and M segments were readily generated in mouse L cells but not in BHK cells, following infection with wild-type virus.

Animals↗
PubMed1992

Nucleotide sequence and coding strategy of the Uukuniemi virus L RNA segment.

The complete nucleotide sequence of the L RNA segment of Uukuniemi virus has been determined from cloned cDNA. The L RNA is 6423 nucleotides in length, and is of negative polarity. The viral-complementary RNA contains a single large open reading frame of 2104 codons which corresponds to the L protein (M(r) 241039). Comparison with the L protein sequences of other members of the Bunyaviridae showed homology with the Rift Valley fever phlebovirus L protein (38% amino acid identity), but no detectable similarity with bunyavirus, hantavirus or tospovirus L proteins. These data lend further support for the recent reclassification of uukuviruses and phleboviruses into the same genus, Phlebovirus, in the family Bunyaviridae. The L RNA sequence completes the determination of the Uukuniemi virus genome: since the M RNA segment is 3229 and the S RNA segment 1720 nucleotides, the whole genome comprises 11372 nucleotides.

Amino Acid Sequence↗
PubMed1992

Mutagenesis of the L protein encoded by Bunyamwera virus and production of monospecific antibodies.

Bacterial fusion proteins containing portions of the Bunyamwera virus L protein were used as immunogens to prepare antisera in rabbits. Of five fusion proteins injected into rabbits, three yielded sera that reacted with the Bunyamwera virus L protein, detected by Western blotting or immunoprecipitation. Two of these antisera were specific for either the amino- or carboxy-terminal regions of the L protein. The specificity of these antisera was confirmed by their pattern of reactivity with full-length and truncated forms of the L protein. Plasmids containing the L gene cDNA under control of a bacteriophage T7 promoter were transfected into CV-1 cells which had previously been infected with a recombinant vaccinia virus, vTF7-3, that expresses T7 RNA polymerase. Antigenically authentic L protein was expressed. Using a nucleocapsid transfection assay developed previously, we showed that the transiently expressed L protein had RNA synthesis activity. Site-specific mutations were made in the L cDNA-containing plasmid to change certain amino acids in the putative polymerase domain of the L protein. The effects of these amino acid substitutions on the RNA synthesis activity of the L protein were monitored using the nucleocapsid transfection assay. These experiments showed that residues strictly conserved between the L proteins of different viruses in the family Bunyaviridae were obligatorily required for activity, whereas non-conserved residues could be substituted without abolishing RNA synthesis capability. Our results provide direct evidence for the functional significance of particular amino acids in the polymerase domain of a negative-strand virus RNA polymerase.

Amino Acid Sequence↗
PubMed1991

Further characterization of conditional lethal amber nonsense mutants of vesicular stomatitis virus: nucleotide sequence analysis.

Conditional lethal amber nonsense mutants of vesicular stomatitis virus, Indiana serotype, classified in complementation group I (the L gene), synthesize truncated versions of the L protein. This paper reports further characterization of mutants AmbL1, AmbL2 and AmbL3 by nucleic acid sequence analysis, which was achieved by sequencing L mRNA directly using appropriate synthetic oligonucleotides. In each case a single point mutation altered a glutamine-specifying codon to an amber stop codon. The L mRNA from wild-type and revertant viruses was sequenced for comparison. Of the revertants sequenced, each had reverted by back mutation within the same codon as the original mutation. A revertant of AmbL2 reverted by a second site mutation, also within the same codon as the original mutation. These mutants may be useful for assigning functions to different parts of the L polypeptide chain.

Base Sequence↗