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At least 19 recordsLinked to original sources

PubMed1988

Characterization and engineering of sequences controlling in vivo synthesis of brome mosaic virus subgenomic RNA.

Expression of brome mosaic virus (BMV) coat protein and internal genes of many other positive-strand RNA viruses requires initiation of subgenomic mRNA synthesis from specific internal sites on minus-strand genomic RNA templates. Biologically active viral cDNA clones were used to investigate the sequences controlling production of BMV subgenomic RNA in vivo. Suitable duplications directed production of specifically initiated, capped subgenomic RNAs from new sites in the BMV genome. Previously implicated promoter sequences extending 20 bases upstream (-20) and 16 bases downstream (+16) of the subgenomic RNA initiation site directed only low-level synthesis. Subgenomic RNA production at normal levels required sequences extending to at least -74 but not beyond -95. Loss of an (rA)18 tract immediately upstream of the -20 to +16 "core promoter" particularly inhibited subgenomic RNA synthesis. The -38 to -95 region required for normal initiation levels contains repeats of sequence elements in the core promoter, and duplications creating additional upstream copies of these repeats stimulated subgenomic RNA synthesis above wild-type levels. At least four different subgenomic RNAs can be produced from a single BMV RNA3 derivative. For all derivatives producing more than one subgenomic RNA, a gradient of accumulation progressively favoring smaller subgenomic RNAs was seen.

Base Sequence↗

PubMed1991

A system for study of coronavirus mRNA synthesis: a regulated, expressed subgenomic defective interfering RNA results from intergenic site insertion.

A system that exploits defective interfering (DI) RNAs of mouse hepatitis virus (MHV) for deciphering the mechanisms of coronavirus mRNA transcription was developed. A complete cDNA clone of MHV DI RNA containing an inserted intergenic region, derived from the area of genomic RNA between genes 6 and 7, was constructed. After transfection of the in vitro-synthesized DI RNA into MHV-infected cells, replication of genomic DI RNA as well as transcription of the subgenomic DI RNA was observed. S1 nuclease protection experiments, sequence analysis, and Northern (RNA) blotting analysis revealed that the subgenomic DI RNA contained the leader sequence at its 5' end and that the body of the subgenomic DI RNA started from the inserted intergenic sequence. Two subgenomic DI RNAs were synthesized after inserting two intergenic sites into the MHV DI RNA. Metabolic labeling of virus-specific protein in DI RNA replicating cells demonstrated that a protein was translated from the subgenomic DI RNA, which can therefore be considered a functional mRNA. Transfection study of gel-purified genomic DI RNA and subgenomic DI RNA revealed that the introduction of the genomic DI RNA, but not subgenomic DI RNA, into MHV-infected cells was required for synthesis of the subgenomic DI RNA. A series of deletion mutations in the intergenic site demonstrated that the sequence flanking the consensus sequence of UCUAAAC affected the efficiency of subgenomic DI RNA transcription and that the consensus sequence was necessary but not sufficient for the synthesis of the subgenomic DI RNA.

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↗

PubMed1983

Nuclei purified from cauliflower mosaic virus-infected turnip leaves contain subgenomic, covalently closed circular cauliflower mosaic virus DNAs.

Nuclei isolated from cauliflower mosaic virus (CaMV) infected turnip leaves contain subgenomic CaMV DNA species in addition to the genome length CaMV DNA. These subgenomic CaMV DNA species are present as covalently closed circles (form I), relaxed circles (form II) and linear (form III) molecules. The subgenomic form I DNA species range in size from about 10% of genome length to nearly genome length. These subgenomic DNA species appear in tissue infected with cloned CaMV DNA, indicating that they arise rapidly and have not accumulated in the virus population from serial propagation of CaMV. No specific region of the CaMV genome appears to be preferentially deleted to form the subgenomic CaMV DNA species. At least three distinct subgenomic species appear to accumulate preferentially in nuclei isolated from infected tissue. Two of these abundant subgenomic CaMV DNA species are form I and the other one is form III. Some of the subgenomic CaMV DNA species appear to be minichromosomes.

Brassica↗

PubMed1992

Mechanism of coronavirus transcription: duration of primary transcription initiation activity and effects of subgenomic RNA transcription on RNA replication.

Previously, we established a system whereby an intergenic region from mouse hepatitis virus (MHV) inserted into an MHV defective interfering (DI) RNA led to transcription of a subgenomic DI RNA in helper virus-infected cells. By using this system, the duration of a primary transcription initiation activity which transcribes subgenomic-size RNAs from the genomic-size RNA template in MHV-infected cells was examined. Efficient DI genomic and subgenomic RNA synthesis was observed when the DI RNA was transfected at 1, 3, 3.5, 5, and 6 h postinfection, indicating that all activities which are necessary for MHV RNA synthesis are present continuously during the first 6 h of infection. The effect of subgenomic DI RNA synthesis on DI genomic RNA replication was then examined. Replication efficiency of the DI genomic RNA which synthesized the subgenomic RNA was approximately 70% lower than that of DI genomic RNA which did not synthesize the subgenomic DI RNA in MHV-infected cells. Cotransfection of two different-size DI RNAs demonstrated that replication of the larger DI RNA was strongly inhibited by replication of the smaller genomic DI RNA. Cotransfection of two DI RNA species of the same length into MHV-infected cells demonstrated that reduced replication of the genomic DI RNA which synthesizes the subgenomic RNA did not affect the replication of cotransfected DI RNA, demonstrating that the reduction in DI genomic RNA replication works only in cis, not in trans. Therefore, the previously proposed hypothesis that coronavirus, subgenomic RNA synthesis may inhibit the replication of genomic RNA by competing for a limited amount of virus-derived factors seems unlikely. Possible mechanisms of coronavirus transcription are discussed.

Animals↗

PubMed1986

Molecular characterisation of subgenomic single-stranded and double-stranded DNA forms isolated from plants infected with tomato golden mosaic virus.

A subgenomic single-stranded DNA present in particles of the geminivirus, tomato golden mosaic virus, has been shown by electron microscope heteroduplex mapping and Southern hybridisation analysis to consist of circular molecules, ca. 1.2 kb in size, derived from the smaller of the two genomic DNA components, DNA B, by deletion of open reading frame (ORF) BR1 and the C-terminal portion of ORF BL1. A covalently closed circular, supercoiled, double-stranded form of the subgenomic DNA has been isolated from virus-infected plants and cloned into pEMBL9. Analysis of the sequence of 22 clones across the deletion boundaries revealed only four different deletion boundaries, derived from four different left hand borders and three different right hand borders. Each border was within a region of 11 nucleotides and gave rise to a narrow size range (1248-1261 nucleotides) for the population of 22 subgenomic DNAs. However apparently smaller subgenomic DNAs were sometimes formed when plants were inoculated with cloned subgenomic DNA, or a construct derived from a subgenomic DNA in which a neomycin phosphotransferase gene had been inserted, together with the genomic DNA components. Mechanisms to account for the size, specificity and formation of the subgenomic DNA are discussed.

Base Sequence↗

PubMed1990

Coronavirus transcription: subgenomic mouse hepatitis virus replicative intermediates function in RNA synthesis.

Both genomic and subgenomic replicative intermediates (RIs) and replicative-form (RF) structures were found in 17CL1 mouse cells that had been infected with the A59 strain of mouse hepatitis virus (MHV), a prototypic coronavirus. Seven species of RNase-resistant RF RNAs, whose sizes were consistent with the fact that each was derived from an RI that was engaged in the synthesis of one of the seven MHV positive-strand RNAs, were produced by treatment with RNase A. Because the radiolabeling of the seven RF RNAs was proportional to that of the corresponding seven positive-strand RNAs, the relative rate of synthesis of each of the MHV positive-strand RNAs may be controlled by the relative number of each of the size classes of RIs that are produced. In contrast to alphavirus, which produced its subgenome-length RF RNAs from genome-length RIs, MHV RF RNAs were derived from genome- and subgenome-length RIs. Only the three largest MHV RF RNAs (RFI, RFII, and RFIII) were derived from the RIs that migrated slowest on agarose gels. The four smallest RF RNAs (RFIV, RFV, RFVI, and RFVII) were derived from RIs that migrated in a broad region of the gel that extended from the position of 28S rRNA to the position of the viral single-stranded MHV mRNA-3. Because all seven RIs were labeled during very short pulses with [3H]uridine, we concluded that the subgenome-length RIs are transcriptionally active. These findings, with the recent report of the presence of subgenome-length negative-strand RNAs in cells infected with porcine transmissible gastroenteritis virus (P. B. Sethna, S.-L. Hung, and D. A. Brian, Proc. Natl. Acad. Sci. USA 86: 5626-5630, 1989), strongly suggest that coronaviruses utilize a novel replication strategy that employs the synthesis of subgenomic negative strands to produce subgenomic mRNAs.

Animals↗

PubMed1990

Promoter for Sindbis virus RNA-dependent subgenomic RNA transcription.

Sindbis virus is a positive-strand RNA enveloped virus, a member of the Alphavirus genus of the Togaviridae family. Two species of mRNA are synthesized in cells infected with Sindbis virus; one, the 49S RNA, is the genomic RNA; the other, the 26S RNA, is a subgenomic RNA that is identical in sequence to the 3' one-third of the genomic RNA. Ou et al. (J.-H. Ou, C. M. Rice, L. Dalgarno, E. G. Strauss, and J. H. Strauss, Proc. Natl. Acad. Sci. USA 79:5235-5239, 1982) identified a highly conserved region 19 nucleotides upstream and 2 nucleotides downstream from the start of the 26S RNA and proposed that in the negative-strand template, these nucleotides compose the promoter for directing the synthesis of the subgenomic RNA. Defective interfering (DI) RNAs of Sindbis virus were used to test this proposal. A 227-nucleotide sequence encompassing 98 nucleotides upstream and 117 nucleotides downstream from the start site of the Sindbis virus subgenomic RNA was inserted into a DI genome. The DI RNA containing the insert was replicated and packaged in the presence of helper virus, and cells infected with these DI particles produced a subgenomic RNA of the size and sequence expected if the promoter was functional. The initiating nucleotide was identical to that used for Sindbis virus subgenomic mRNA synthesis. Deletion analysis showed that the minimal region required to detect transcription of a subgenomic RNA from the negative-strand template of a DI RNA was 18 or 19 nucleotides upstream and 5 nucleotides downstream from the start of the subgenomic RNA.

Animals↗

PubMed1989

Identification of the 5' end of the rubella virus subgenomic RNA.

The 5' end of the subgenomic RNA of rubella virus was determined by primer extension. The Maxam-Gilbert sequence ladder of the primer extension product contained a determinable sequence which was colinear with the complement of the sequence of the genomic RNA through nucleotide 3325 from the 3' end of the genomic RNA and a pair of bands in every lane above the determinable sequence. These results indicated that synthesis of the subgenomic RNA is initiated internally on the minus-polarity genome-length RNA template and that the length of subgenomic RNA is 3327 nucleotides excluding the poly(A) tail. There are thus 77 nucleotides between the 5' end of the subgenomic RNA and the first AUG, which is the initiation codon for the structural protein open reading frame. The initiation site of rubella virus subgenomic RNA synthesis is 20 nucleotides downstream from a block of 28 nucleotides which shares homology with the nucleotide sequence which is conserved at the subgenomic RNA initiation site in the Alphaviruses, the other genus of Togaviruses.

Animals↗

PubMed1979

An MSV-specific subgenomic mRNA in MSV-transformed G8-124 cells.

An intracellular subgenomic RNA species from MSV-transformed G8-124 cells was characterized by electron microscopy of RNA:cDNA heteroduplexes using long cDNAs both MSV and MuLV. This subgenomic RNA, 3.1 kb long, consisted of 5'-derived sequences of about 0.4 kb joined to 2.7 kb of RNA derived from the 3' end of the RNA genome. The 3'-derived sequences included the residual sequences from the MuLV pol region and the acquired cellular sequences of MSV. The genome of MSV was shown to retain approximately 0.13 kb from the 5' end of the MuLV env region, including sequences which span the point in the MuLV env mRNA. No subgenomic MSV RNA could be detected, however, which consisted of a 5'-derived leader sequence spliced to the retained env region sequences. Nor could a subgenomic MSV RNA be detected in which a 5'-derived leader sequence was joined directly to the acquired cellular sequences. Although its translation products are unknown, the subgenomic MSV RNA was present in preparations of poly(A)+ polysomal RNA, consistent with this RNA functioning as a messenger. The structure of this 3.1 kb MSV subgenomic RNA suggests a possible role in the expression of 3'-encoded MSV information, possibly including transformation-specific sequences.

Cell Transformation, Viral↗

PubMed1989

Intraviral homology and subgenomic RNAs of pepper ringspot virus.

The Tobraviruses constitute a group of rod-shaped, bipartite, plus-stranded RNA viruses. We report on homologies between the two viral genomic RNA molecules of pepper ringspot virus (PRV) and on the subgenomic components generated from them during infection. It has previously been shown that the 3'-terminal 459 nucleotides of PRV RNA-1 and RNA-2 are identical. Here it is shown that there is a second homology between RNA-1 and a region of RNA-2, located at least 240 nucleotides downstream from its 5' terminus. In agreement with strains in another Tobravirus group, we observe three subgenomic components in extracts of plants infected with PRV. Two of these, RNAs-1a (1.6 kb) and -1b (0.8 kb), are derived from RNA-1 (the larger genomic RNA). They are probably mRNAs for 29- and 16-kDa nonstructural virus proteins. The smaller genomic RNA, RNA-2, generates the subgenomic component RNA-2a (1.3-1.45 kb) which is probably an efficient capsid protein mRNA. Previously reported subgenomic components of 2.8 and 1.1 kb are shown to be electrophoretic artifacts. RNA-2a, but not 1a or 1b, is present in virion RNA preparations, indicating that it is the only subgenomic species to be encapsidated.

DNA↗

PubMed1989

Mechanism of synthesis of turnip yellow mosaic virus coat protein subgenomic RNA in vivo.

Turnip yellow mosaic virus (TYMV) possesses a monopartite single-stranded (+) sense RNA genome in which the coat protein (cp) gene is 3' proximal and is expressed in vivo via a subgenomic RNA. Evidence is presented here that this subgenomic RNA is synthesized in vivo by internal initiation of replication on (-) RNA strands of genomic length. The double-stranded RNAs (dsRNAs) from TYMV-infected plants have been isolated, purified, and characterized. Under native conditions, no dsRNAs (replicative intermediates and/or replicative forms) of subgenomic length corresponding to subgenomic cp RNA can be detected by ethidium bromide staining of RNA-sizing gels or by Northern blot hybridization using RNA probes. The presence of nascent subgenomic cp (+) RNA strands on the dsRNA of genomic length has been demonstrated using two different approaches: (1) Northern blot hybridization using (-) RNA probes under denaturing conditions and (2) characterization of the 5' ends of nascent (+) RNA strands upon labeling by vaccinia virus nucleoside-2'-methyltransferase.

Capsid↗

PubMed1991

Genomic and subgenomic RNAs of rabbit hemorrhagic disease virus are both protein-linked and packaged into particles.

The major subgenomic RNA of the calicivirus rabbit hemorrhagic disease virus which codes for the viral capsid protein has been cloned as cDNA. The nucleotide sequence of this mRNA was shown to be identical to the 3' terminal region of the genomic RNA. The 5' end of the mRNA corresponds to position 5296 of the genomic sequence; except for two differences the first 16 nucleotides of genomic and subgenomic RNAs are identical. After isolation from liver tissue viral genomic and subgenomic RNAs were found to be resistant to RNase degradation. This protection was due to RNA packaging into particles. Sucrose density gradient centrifugation of liver homogenates allowed separation of such particles containing either genomic RNA or subgenomic RNA. Genomic and subgenomic RNAs are protein-linked and for the genomic molecule this interaction is localized within the first 179 nucleotides. After radioactive labeling of purified RNA and subsequent RNase treatment a protein of 15 kDa was identified.

Animals↗

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↗

PubMed1989

Coronavirus subgenomic minus-strand RNAs and the potential for mRNA replicons.

The genome of the porcine transmissible gastroenteritis coronavirus is a plus-strand, polyadenylylated, infectious RNA molecule of approximately 20 kilobases. During virus replication, seven subgenomic mRNAs are generated by what is thought to be a leader-priming mechanism to form a 3'-coterminal nested set. By using radiolabeled, strand-specific, synthetic oligodeoxynucleotide probes in RNA blot hybridization analyses, we have found a minus-strand counterpart for the genome and for each subgenomic mRNA species in the cytoplasm of infected cells. Subgenomic minus strands were found to be components of double-stranded replicative forms and in numbers that surpass full-length antigenome. We propose that subgenomic mRNA replication, in addition to leader-primed transcription, is a significant mechanism of mRNA synthesis and that it functions to amplify mRNAs. It is a mechanism of amplification that has not been described for any other group of RNA viruses. Subgenomic replicons may also function in a manner similar to genomes of defective interfering viruses to lead to the establishment of persistent infections, a universal property of coronaviruses.

Animals↗

PubMed1992

A number of subgenomic DNAs are produced following agroinoculation of plants with beet curly top virus.

In addition to ss and ds genomic DNA, agroinoculation of Nicotiana benthamiana plants with the Logan strain of the geminivirus beet curly top virus (BCTV) consistently resulted in de novo production of subgenomic DNAs on initial passage. Single-stranded and dsDNA forms representing at least seven size classes (0.8 to 1.8 kb) of subgenomic DNA were observed in total DNA extracts from inoculated plants. Extracts from infected sugar beet and tomato contained variable but usually smaller amounts of subgenomic DNAs, suggesting that their production may be influenced by the host species. Restriction endonuclease mapping and partial nucleotide sequencing of three independent clones of a 1.5 kb size class indicated that this subgenomic DNA is produced from the standard viral genome by two separate deletion events. One deletion of 941 bp includes portions of the leftward open reading frames (ORFs) L1, L2 and L3, while the other deletion of 579 bp encompasses portions of the intergenic region and the rightward ORFs R1, R2 and R3. The data indicate that the 1.5 kb BCTV subgenomic DNA is a defective DNA that has retained cis-elements essential for replication.

Base Sequence↗

PubMed1991

Changes in subgenome types of adenovirus type 4 isolated from patients with ocular disease between 1985 and 1989 in Sapporo, Japan.

A total of 122 adenovirus type 4 (Ad4) strains isolated from patients with epidemic keratoconjunctivitis at an eye clinic in Sapporo, Japan, from 1985 to 1989 were identified as Ad4a. By the use of HinfI and TaqI restriction endonucleases, these Ad4a strains were classified into 12 subgenome types. During this observation period, two epidemics of Ad4 infection relating to epidemic keratoconjunctivitis occurred. During the Ad4a epidemic in 1985, three subgenome types became predominant successively. However, except for one strain, the prevalent strains isolated during the epidemic of Ad4a in 1988 all belonged to a single subgenome type. In the years when an Ad4a epidemic was not observed, such Ad4a strains were new or previous subgenome types. The prevalent subgenome types tended to change within several years.

Adenoviruses, Human↗

PubMed1992

Subgenomic RNAs mediate expression of cistrons located internally on the genomic RNA of tobacco necrosis virus strain A.

Upon infection of tobacco protoplasts, the genomic RNA of tobacco necrosis virus strain A (TNV-A) accumulates linearly in time. The accumulation patterns of the two subgenomic RNAs resemble those of endogenous mRNAs in that the peak levels are reached after several hours. The accumulation of the 1.3-kb subgenomic RNA is delayed by 1 h compared with that of the 1.6-kb subgenomic RNA, which illustrates the important role of the subgenomic RNAs in the regulation of TNV-A gene expression. The locations of the 5' nucleotides of the subgenomic RNAs reveal that the 5'-proximal cistrons of the 1.6- and 1.3-kb RNAs encode an 8-kDa protein from open reading frame (ORF) 3 and the coat protein from ORF 5, respectively. In a wheat germ translation system, a synthetic transcript resembling the 1.6-kb RNA expresses both ORFs 3 and 4. Moreover, the synthesis of the 6-kDa protein from ORF 4 depends on the translation efficiency of ORF 3, suggesting that in vivo, ORFs 3 and 4 are both expressed from the 1.6-kb RNA. The major in vitro translation product of TNV-A genomic RNA is the coat protein. We show that the region upstream of the coat protein promotes internal initiation of translation in vitro. However, this region is functionally inactive in vivo, suggesting that TNV-A genomic RNA is not important for coat protein synthesis in plants.

Base Sequence↗