SEARCH · PubMed Health

Results for “Subgenomic RNA”

Explore indexed PubMed citations for clinical trials, systematic reviews and public health research. Read source abstracts and follow each citation to its original PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 55 records · Page 3Linked to original sources

PubMed1993

Subgenomic RNA sequence of human astrovirus supports classification of Astroviridae as a new family of RNA viruses.

We report the sequence of the subgenomic RNA of human astrovirus serotype 2. This 2,484-nucleotide RNA contains a single open reading frame, which encodes a protein with a predicted molecular mass of 88 kDa. We propose that this protein is the 90-kDa capsid precursor observed in infected cells. The deduced protein sequence does not contain conserved amino acid patterns reported for the capsid proteins of picornaviruses or caliciviruses, consistent with the classification of astroviruses as a new family of RNA viruses, designated Astroviridae.

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↗

PubMed2002

The subgenomic RNA of feline calicivirus is packaged into viral particles during infection.

During infection, feline calicivirus (FCV) produces an abundant subgenomic RNA of 2.4 kb that is the major template for translation of the single capsid protein. Feline cells infected with FCV (CFI/68 strain) at a high multiplicity of infection produced a population of lower density (ld) viral particles with a density of 1.35 g/cc as compared to the 1.39 g/cc density of the wild-type virus particle. The RNA isolated from the ld particles was 2.4 kb in size, the same as that of the intracellular subgenomic transcript that encodes the single capsid protein of the virus. Primer extension analysis revealed that the 5' end of the RNA from the ld particles mapped to the same genomic location as the intracellular 2.4 kb RNA. RNA protection of the ld RNA using a FCV 4.2 kb minus strand cDNA containing 1984 bases of capsid protein coding sequences, protected an RNA fragment of approximately 2000 bases. The data presented here demonstrates that the ld particles contains the FCV subgenomic RNA and not a genomic RNA containing rearrangements or deletions.

Animals↗

PubMed1992

Precise mapping and in vitro translation of a trifunctional subgenomic RNA of barley yellow dwarf virus.

The barley yellow dwarf virus genome consists of a single 5.7-kb RNA encoding six open reading frames (ORFs). The four ORFs in the 3' half of the genome were proposed to be expressed via subgenomic mRNAs. Here, we show that the PAV serotype of barley yellow dwarf virus generates two subgenomic RNAs of 2.9 and 0.8 kb in infected plants and protoplasts. The 5' end of the larger subgenomic RNA (sgRNA1) was precisely mapped to base 2769 by ribonuclease protection and primer extension methods. Synthetic sgRNA1, containing the exact 5' and 3' ends, was generated by in vitro transcription, translated in vitro, and shown to serve as a message for three ORFs. Translation initiated at the first two AUG codons in sgRNA1, yielding the 22-kDa viral coat protein (CP) and a 17-kDa protein encoded by an overlapping, out-of-frame ORF. In addition, readthrough of the amber stop codon of the CP ORF gave rise to a 72-kDa fusion product of the CP ORF and an in-frame 50K ORF immediately 3' of the CP termination codon. The 0.8-kb sgRNA2 was present in greater abundance than sgRNA1 and likely serves as a message for a 6.7K ORF at the 3' end of the genome. Sequences that may control subgenomic RNA synthesis and the translational events are compared with those of other plant viruses.

Base Sequence↗

PubMed1995

A subgenomic RNA associated with cherry leafroll virus infections.

Cherry leafroll nepovirus (CLRV) genomic RNA 1 (8 kb) and genomic RNA 2 (7 kb) have 3' polyadenylate tracts and, extending 5' from the polyadenylate, nearly identical sequences of 1.6 kb termed the 3' common region. We observed RNAs 1 and 2 and a third RNA of 1.5 kb in nucleic acid extracts of CLRV-infected Nicotiana tabacum suspension cell protoplasts and Chenopodium quinoa plants, using a hybridization probe complementary to 1 kb of the 3' common region. The third RNA was partially purified by preparative gel electrophoresis and chromatography on an oligodeoxythymidylate column. Analyses of transcripts primed by a complementary oligodeoxyribonucleotide and of cDNA clones revealed that the third RNA corresponds to the 3' 1500 nucleotide residues of RNA 1. Hence we designate the newly characterized RNA as RNA 1A. RNA 1A was not detected as encapsidated RNA in extracts of either protoplasts or C. quinoa plants. The amount of accumulated RNA 1A declined between 24 and 48 hr after inoculation of protoplasts with CLRV virions, although CLRV RNAs 1 and 2 continued to accumulate. Other results were not consistent with cleaved RNA 1 being the origin of RNA 1A. RNA 1A has the properties of a subgenomic RNA, presumably synthesized from negative-sense RNA 1 as template.

Base Sequence↗

PubMed2005

Stable deletions arising in the readthrough region of Soil-borne wheat mosaic virus RNA2 define the 5' limit of the functional promoter for the p19 subgenomic RNA.

The appearance of de novo deletion mutations in the readthrough (RT) region (nucleotide positions 861-2591) downstream of the capsid protein (CP) gene of a Japanese strain of Soil-borne wheat mosaic virus RNA2 was examined using infectious transcripts. Mutant RNA2s with different deletions predominated in independent serial passage experiments but all best-adapted mutants retained the 3'-terminal portion of the RT gene in frame with the CP gene. The longest best-adapted mutation deleted the 1434 nucleotides between positions 1061 and 2494. When the RT protein was truncated by insertion of a termination codon plus an additional nucleotide to give a +1 frame-shift, after serial passages the progeny viruses regained the ability to express the C-terminal region of RT by an internal deletion. The 5' terminus of the p19 subgenomic RNA was identified at position 2598 and an essential transcription signal for this mRNA mapped between positions 2534 and 2563. A mutant in which this essential promoter element has been deleted cannot transcribe the p19 subgenomic RNA and has lost infectivity in planta. These results indicate that the 3'-terminal region of the RT gene has a major function in cis for expression of p19, which is essential for infecting plants. A reason for retaining the RT C-terminal region in stable deletion mutants is still unknown.

3' Flanking Region↗

PubMed1994

Genetic analysis of the nsP3 region of Sindbis virus: evidence for roles in minus-strand and subgenomic RNA synthesis.

Sindbis virus nonstructural polyproteins and their cleavage products are believed to be essential components of viral RNA replication and transcription complexes. Although numerous studies have investigated the effect of mutations in nsP1-, nsP2-, and nsP4-coding regions on Sindbis virus-specific RNA synthesis, relatively little is known about the function of the region encoding nsP3. nsP3 is a phosphoprotein comprising two regions: an N-terminal portion which is highly conserved among alphaviruses and a C-terminal portion which is not conserved, varying both in sequence and in length. We have constructed a library of random linker insertion mutations in the nsP3-coding region and characterized selected viable mutants. Initially, 126 mutants containing insertions in the conserved region and 23 with insertions in the nonconserved region were screened for temperature-sensitive (ts) plaque formation or for significant differences in plaque morphology. All nonconserved-region mutants were similar to the parental virus, whereas 13 of those in the conserved region were either ts or exhibited altered plaque phenotypes. Ten of these 13 mutants were ts for plaque formation as well as RNA accumulation at 40 degrees C. Highly ts mutants CR3.36 and CR3.39 were defective in their ability to synthesize minus-strand RNAs at the nonpermissive temperature. The CR3.36 and CR3.39 insertion mutations localized to different regions near nsP3 residues 58 and 226, respectively. CR3.39 was able to complement ts mutants from Sindbis virus complementation groups A, B, F, and G. Another mutant isolated from the library, CR3.34, while not ts for plaque formation or RNA synthesis, formed smaller plaques and was defective in subgenomic RNA synthesis at all temperatures examined. These results suggest a role for nsP3 or nsP3-containing polyproteins in the synthesis of viral minus-strand and subgenomic RNAs.

Amino Acid Sequence↗

PubMed1997

Generation of cytopathogenic subgenomic RNA of classical swine fever virus in persistently infected porcine cell lines.

Two biological clones (A.1 and B.2) of the classical swine fever virus strain Alfort/187 and the recombinant virus vA187-1, derived from a cDNA clone of Alfort/187, were used to establish persistently infected cultures of the swine kidney cell lines SK-6 and PK-41. It was found that 100% of the cells in the passaged cultures were positive for viral antigen throughout the course of the experiment. Additionally, supernatants collected upon passaging of the cells continuously contained high titers of infectious virus. In six separate cultures persistently infected with either the biological clones or the recombinant virus, a cytopathic effect occurred spontaneously between passage 8 and 94. The cytopathogenic agent in the supernatants of these cultures could be passaged repeatedly, suggesting the generation of a mutant virus. Analysis of RNA from such cultures revealed the presence of a subgenomic viral RNA of approximately 8 kilobases (kb). In all six cases, this RNA had an identical internal deletion of 4764 nucleotides, including the region coding for all structural proteins. The subgenomic RNA replicated and was packaged in the presence of wild-type virus. Cells infected with cytopathogenic virus contained increased amounts of the viral protein NS3 thought to be involved in pestivirus cytopathogenicity.

Animals↗

PubMed2001

Characterization of classical swine fever virus associated with defective interfering particles containing a cytopathogenic subgenomic RNA isolated from wild boar.

Classical swine fever virus (CSFV) strain WB82, isolated from a wild boar in 1982, induced a distinct cytopathic effect (CPE) in primary swine testicle cell culture and in most of the porcine cell lines. This strain of CSFV was found to be composed of two biotypes. cytopathogenic (cp) CSFV, as a minor population, and noncytopathogenic (noncp) CSFV, as a major population. The noncp CSFV (designated strain WB82/E+) was obtained by biological cloning, and it showed the exaltation of Newcastle disease virus phenomenon. In Northern blot analysis and RT-PCR assay, CSFV RNA with a subgenomic (sg) length was detected in addition to full-length viral RNA only in the cells in which a CPE had been revealed. These RNAs represent the genomes of typical defective interfering (DI) particles because of the strict dependence on a complementing helper virus and interference with replication of the helper virus. The sg RNA, which exhibits the genomes of the DI particles, lacked the nucleotides of the viral genomic region from Npro to NS2 (4764 bases). When extracted sg RNA was transfected to the cells infected with the WB82/E+ strain, a distinct CPE was observed. Interestingly, the CPE was observed in cells infected with other heterologous noncp CSFV ALD and GPE- strains by sg RNA transfection. The results suggested that these noncp CSFVs act as helper viruses for the replication of sg RNA (DI particles). It was also shown that the cytopathogenicity of strain WB82 is caused by apoptosis.

Animals↗

PubMed1997

Studies on the translation mechanism of subgenomic RNA of potato leafroll virus.

The expression of open reading frames located on the subgenomic RNA (sgRNA) has been studied in an in vitro transcription and translation system. The obtained results indicate: a) translation of sgRNA occurs according to the scanning model since the insertion of palindrome (delta G0 = -61 kcal/mol) prevents the initiation of translation; b) ORF6 is translated by suppression of the stop codon separating ORF4 from ORF6 and the presence of suppressor tRNA is necessary for the readthrough; c) the presence of leader sequence of sgRNA (212 nucleotides) decreases the translation efficiency of ORFs located downstream and it affects the ratio of products of ORF4 and ORF5; d) 3'UTR does not influence on an expression of genes located on the sgRNA.

Base Sequence↗

PubMed1998

Subgenomic RNA promoters dictate the mode of recognition by bromoviral RNA-dependent RNA polymerases.

Both the brome mosaic virus (BMV) and cowpea chlorotic mottle virus (CCMV) RNA-dependent RNA polymerases (RdRps) were found to recognize the BMV core subgenomic promoter in the same manner, requiring specific functional groups at positions -17, -14, -13, and -11 relative to the subgenomic initiation site (+1). For CCMV subgenomic RNA synthesis, both RdRps required the same nucleotides and four additional nucleotides at positions -20, -16, -15, and -10. The -20 nucleotide is partially responsible for the differential mode of recognition of the two promoters. These data provide evidence that the RNA can induce RdRps to alter the mode of promoter recognition.

Bromovirus↗

PubMed1996

Mapping of the red clover necrotic mosaic virus subgenomic RNA.

The red clover necrotic mosaic dianthovirus capsid protein is expressed in vivo from a subgenomic RNA (sgRNA) identical to the 3'-terminal 1.5 kb of RNA-1. The 5' leader sequence of the capsid protein sgRNA is 62 nucleotides, contains a 14-nucleotide putative promoter sequence homologous to the RNA-1 5' terminus, and exhibits a high level of similarity with the tobacco mosaic virus 5' leader translational enhancer element omega. Analysis of the RNA-1 secondary structure, in the region adjacent to the position where the 5' end of the sgRNA was mapped, predicts a stable stem-loop which includes the putative sgRNA promoter element. It is suggested that this structure is important for recognition of the sgRNA transcriptional initiation from the full-length negative-sense RNA-1.

Base Sequence↗

PubMed1997

Analysis of the two subgenomic RNA promoters for turnip crinkle virus in vivo and in vitro.

Infection of plants or protoplasts with turnip crinkle virus (TCV), a monopartite RNA virus, results in the synthesis of the genomic RNA and two subgenomic (sg) RNAs. The transcription start site for the 1.45-kb sgRNA was previously mapped to position 2606 (J. C. Carrington, T. J. Morris, P. G. Stockley, and S. C. Harrison, (1987). J. Mol. Biol. 194, 265-276) corresponding to position 2607 in the TCVms isolate and the start site for the 1.7-kb sgRNA has now been mapped to position 2333 in TCVms. A 96-base sequence (90 bases upstream and 6 bases downstream) encompassing the transcription start site for the 1.45-kb sgRNA was sufficient for full promoter activity. Similarly, a 94-base sequence (90 bases upstream and 4 bases downstream) encompassing the start site was required for full activity of the 1.7-kb sgRNA promoter. The 1.45-kb sgRNA promoter, but not the 1.7-kb sgRNA promoter, was able to direct synthesis of a nontemplate RNA in vitro using partially purified TCV RNA-dependent RNA polymerase. Computer generated secondary structures for the two sgRNA promoters revealed an extensive hairpin just upstream from the transcription start site. Comparisons of corresponding sequences from related viruses indicates higher sequence conservation for the 1.45-kb sgRNA promoter compared with the 1.7-kb sgRNA promoter, despite the latter's location within the RNA-dependent RNA polymerase open reading frame.

Arabidopsis↗

PubMed1999

Minimal sequence and structural requirements of a subgenomic RNA promoter for turnip crinkle virus.

Infection of plants or protoplasts with turnip crinkle virus (TCV) results in the synthesis of the genomic RNA and two subgenomic (sg) RNAs of 1.7 kb and 1.45 kb, respectively. Both of the sgRNA promoters were characterized previously and their secondary structures predicted by computer analysis [J. Wang and A. E. Simon (1997). Virology 232, 174-186]. Secondary structure-sensitive chemical and enzymatic probes have now been used to determine the structure of the promoter directing synthesis of the 1.45-kb sgRNA, namely the 1.45-kb sgRNA promoter, in solution. The newly obtained structure conforms with the previously predicted hairpin structure except for the hairpin base: four CG base pairs and a CA bulge are present instead of an A bulge. Studies of deletions within the 96-nucleotide (nt) 1.45-kb sgRNA promoter defined a minimal 30-nt core sequence as essential for promoter activity: a 21-nt hairpin and a 9-nt flanking single-stranded sequence. Mutational analysis in the stem section of the core promoter supported a role for the primary sequence and secondary structure in promoter activity. Sequence alterations in the flanking single-stranded region further suggest that the sequence CCCAUUA, encompassing the transcription start site, is required for efficient transcription of the 1.45-kb sgRNA by the TCV RNA-dependent RNA polymerase in vivo.

Carmovirus↗

PubMedDate not supplied

Synthesis of brome mosaic virus subgenomic RNA in vitro by internal initiation on (-)-sense genomic RNA.

The genomes of many (+)-stranded RNA viruses, including plant viruses and alphaviruses, consist of polycistronic RNAs whose internal genes are expressed via subgenomic messenger RNAs. The mechanism(s) by which these subgenomic mRNAs arise are poorly understood. Based on indirect evidence, three models have been proposed: (1) internal initiation by the replicase on the (-)-strand of genomic RNA, (2) premature termination during (-)-strand synthesis, followed by independent replication of the subgenomic RNA and (3) processing by nuclease cleavage of genome-length RNA. Using an RNA-dependent RNA polymerase (replicase) preparation from barley leaves infected with brome mosaic virus (BMV) to synthesize the viral subgenomic RNA in vitro, we now provide evidence that subgenomic RNA arises by internal initiation on the (-)-strand of genomic RNA. We believe that this also represents the first in vitro demonstration of a replicase from a eukaryotic (+)-stranded RNA virus capable of initiating synthesis of (+)-sense RNA.

DNA, Recombinant↗

PubMed1998

A core promoter hairpin is essential for subgenomic RNA synthesis in alfalfa mosaic alfamovirus and is conserved in other Bromoviridae.

The nucleotide sequence immediately in front of the initiation site for subgenomic RNA 4 synthesis on RNA 3 minus strand, which has been proved to function as a core promoter, was inspected for secondary structure in 26 species of the plant virus family Bromoviridae. In 23 cases a stable hairpin could be predicted at a distance of 3 to 8 nucleotides from the initiation site of RNA 4. This hairpin contained several conserved nucleotides that are essential for core promoter activity in brome mosaic virus (R.W. Siegel, S. Adkins and C.C. Kao, Proc. Natl. Acad. Sci. USA 94, 11238-11243, 1997). Phylogenetic evidence and evidence from the effect of artificial mutations reported in the literature (E.A.G. van der Vossen, T. Notenboom and J.F. Bol, Virology 212, 663-672, 1995) indicate that the stem-loop structure is essential for promoter activity in alfalfa mosaic virus and probably in other Bromoviridae. Stability of the hairpin is most pronounced in the genera Alfamovirus and Ilarvirus which display genome activation by coat protein. The hypothesis is put forward that with these viruses the coat protein is needed for the viral RNA polymerase to interact with the core promoter hairpin leading to access for the enzyme to the initiation site of RNA 4.

Alfamovirus↗

PubMed2005

Rubella virus capsid protein modulation of viral genomic and subgenomic RNA synthesis.

The ratio of the subgenomic (SG) to genome RNA synthesized by rubella virus (RUB) replicons expressing the green fluorescent protein reporter gene (RUBrep/GFP) is substantially higher than the ratio of these species synthesized by RUB (4.3 for RUBrep/GFP vs. 1.3-1.4 for RUB). It was hypothesized that this modulation of the viral RNA synthesis was by one of the virus structural protein genes and it was found that introduction of the capsid (C) protein gene into the replicons as an in-frame fusion with GFP resulted in an increase of genomic RNA production (reducing the SG/genome RNA ratio), confirming the hypothesis and showing that the C gene was the moiety responsible for the modulation effect. The N-terminal one-third of the C gene was required for the effect of be exhibited. A similar phenomenon was not observed with the replicons of Sindbis virus, a related Alphavirus. Interestingly, modulation was not observed when RUBrep/GFP was co-transfected with either other RUBrep or plasmid constructs expressing the C gene, demonstrating that modulation could occur only when the C gene was provided in cis. Mutations that prevented translation of the C protein failed to modulate RNA synthesis, indicating that the C protein was the moiety responsible for modulation; consistent with this conclusion, modulation of RNA synthesis was maintained when synonymous codon mutations were introduced at the 5' end of the C gene that changed the C gene sequence without altering the amino acid sequence of the C protein. These results indicate that C protein translated in proximity of viral replication complexes, possibly from newly synthesized SG RNA, participate in regulating the replication of viral RNA.

Base Sequence↗

PubMed2004

Expression of gastrointestinal glutathione peroxidase is inversely correlated to the presence of hepatitis C virus subgenomic RNA in human liver cells.

There is great medical need to develop novel therapies for treatment of human hepatitis C virus (HCV). By gene expression analysis of three HCV-subgenomic RNA replicon cell lines, we identified cellular proteins whose expression is affected by the presence of HCV and therefore may serve as drug targets. Data from cDNA array filter hybridization, as well as from Northern and Western blotting, revealed that the gastrointestinal-glutathione peroxidase (GI-GPx) was drastically down-regulated (up to 20-fold) in all replicon cell lines tested. Concomitantly, total cellular glutathione peroxidase activity was drastically reduced, which rendered these human liver cells more susceptible toward oxidative stress. Interferon alpha caused down-regulation of the HCV-replicon followed by recovery of GI-GPx expression to nearly normal levels. Furthermore, expression of GI-GPx in replicon cells by gene transduction caused down-regulation of HCV RNA in a dose-dependent manner. Moreover, activating the endogenous gene coding for GI-GPx by all-trans-retinoic acid (RA) was sufficient to cause down-regulation of the HCV replicon. A small interfering RNA duplex abrogated GI-GPx up-regulation by RA and concomitantly suppression of HCV. The RA effect was dependent on the presence of sodium selenite, was reversible, and was independent of RNA-activated protein kinase. Taken together, these results show that HCV inhibits the expression of GI-GPx in replicon cells to promote its intracellular propagation. Modulation of GI-GPx activity may open new avenues of treatment for HCV patients.

Base Sequence↗