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Molecular characterization of porcine reproductive and respiratory syndrome virus, a member of the arterivirus group. Virology
, 1993
"... Porcine reproductive and respiratory syndrome virus (PRRSV)-specific cDNA clones spanning the 3 ' terminal 5 kb of the genomic RNA were isolated, sequenced, and used as prebes for identification of PRRSV-specific RNAs. The PRRSV genome is a positive-stranded polyadenylated RNA of about 15 kb. I ..."
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Cited by 53 (0 self)
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Porcine reproductive and respiratory syndrome virus (PRRSV)-specific cDNA clones spanning the 3 ' terminal 5 kb of the genomic RNA were isolated, sequenced, and used as prebes for identification of PRRSV-specific RNAs. The PRRSV genome is a positive-stranded polyadenylated RNA of about 15 kb. In infected cells, a 3 ' coterminal nested set of six major subgenomic mRNAs could be demonstrated. Within the 3 ' terminal 3.5 kb of the PRRSV genome, six overlapping reading frames (ORFs) were identified, each most likely expressed by one of the subgenomic mRNAs. Amino acid sequence comparisons revealed that the most 3 ' terminal ORF (ORF7) encodes the PRRSV nucleocapsid protein with a calculated molecular weight of 14 kDa. It displays 44.8 % amino acid identity with the capsid protein of lactate dehydro-genase-elevating virus (LDV) and 23.6 % with that of equine arteritis virus (EAV). The product of ORF6, the second 3' terminal ORF, represents a putative membrane protein and exhibits 53.2 and 27.2 % amino acid identity with the corresponding LDV and EAV polypeptides, respectively. Similar to EAV, ORFs 2 through 5 might encode glycosylated viral proteins. The polypeptide deduced from the most 5 ' ORF (ORF1 b) contains two conserved domains common to EAV and coronavirus polymerases. Genome organization, strategy of gene expression, and the sequence of deduced proteins show that PRRSV belongs to the Arterivirus group of viruses. © 1993 Academic Press, Inc.
The genome organization of the Nidovirales: similarities and differences between arteri-, toro-, and coronaviruses
- Semin Virol
, 1997
"... Viruses in the families Arteriviridae and Coronaviridae have enveloped virions which contain nonseg-mented, positive-stranded RNA, but the constituent genera differ markedly in genetic complexity and virion structure. Nevertheless, there are striking resemblances among the viruses in the organizatio ..."
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Cited by 49 (11 self)
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Viruses in the families Arteriviridae and Coronaviridae have enveloped virions which contain nonseg-mented, positive-stranded RNA, but the constituent genera differ markedly in genetic complexity and virion structure. Nevertheless, there are striking resemblances among the viruses in the organization and expression of their genomes, and sequence conservation among the polymerase polyproteins strongly suggests that they have a common ancestry. On this basis, the International Committee on Taxonomy of Viruses recently established a new order, Nidovirales, to contain the two families. Here, the common traits and distinguishing features of the Nidovirales are reviewed. r 1997 Academic Press KEY WORDS: arterivirus; coronavirus; torovirus; polyprotein processing; RNA recombination.
A cis-acting function for the coronavirus leader in defective interfering RNA replication
- J
, 1994
"... To test the hypothesis that the 65-nucleotide (nt) leader on subgenomic mRNAs suffices as a 5'-terininal cis-acting signal for RNA replication, a corollary to the notion that coronavirus mRNAs behave as replicons, synthetic RNA transcripts of a cloned, reporter-containing N mRNA (mRNA 7) of the ..."
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Cited by 40 (11 self)
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To test the hypothesis that the 65-nucleotide (nt) leader on subgenomic mRNAs suffices as a 5'-terininal cis-acting signal for RNA replication, a corollary to the notion that coronavirus mRNAs behave as replicons, synthetic RNA transcripts of a cloned, reporter-containing N mRNA (mRNA 7) of the bovine coronavirus with a precise 5 ' terminus and a 3 ' poly(A) of 68 nt were tested for replication after being transfected into helper virus-infected cells. No replication was observed, but synthetic transcripts of a cloned reporter-containing defective interfering (DI) RNA differing from the N mRNA construct by 433 nt of continuous 5'-proximal genomic sequence between the leader and the N open reading frame did replicate and become packaged, indicating the insufficiency of the leader alone as a 5 ' signal for replication of transfected RNA molecules. The leader was shown to be a necessary part of the cis-acting signal for DI RNA replication, however, since removal of terminal bases that destroyed a predicted intraleader stem-loop also destroyed replicating ability. Surprisingly, when the same stem-loop was disrupted by base substitutions, replication appeared only minimally impaired and the leader was found to have rapidly reverted to wild type during DI RNA replication, a phenomenon reminiscent of high-frequency leader switching in the mouse hepatitis coronavirus. These results suggest that once a minimal structural requirement for leader is fulfilled for initiation of DI RNA replication, the wild-type leader is strongly preferred for subsequent replication. They also demonstrate that,
Optimization of targeted RNA recombination and mapping of a novel nucleocapsid gene mutation in the coronavirus mouse hepatitis virus
- J
, 1994
"... We have recently described a method of introducing site-specific mutations into the genome of the coronavirus mouse hepatitis virus (MHV) by RNA recombination between cotransfected genomic RNA and a synthetic subgenomic mRNA (C. A. Koetzner, M. M. Parker, C. S. Ricard, L. S. Sturman, and P. S. Maste ..."
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We have recently described a method of introducing site-specific mutations into the genome of the coronavirus mouse hepatitis virus (MHV) by RNA recombination between cotransfected genomic RNA and a synthetic subgenomic mRNA (C. A. Koetzner, M. M. Parker, C. S. Ricard, L. S. Sturman, and P. S. Masters, J. Virol. 66:1841-1848, 1992). By using a thermolabile N protein mutant ofMHV (Alb4) as the recipient virus and synthetic RNA7 (the mRNA for the nucleocapsid protein N) as the donor, we selected engineered recombinant viruses as heat-stable progeny resulting from cotransfection. We have now been able to greatly increase the efficiency of targeted recombination in this process by using a synthetic defective interfering (DI) RNA in place of RNA7. The frequency of recombination is sufficiently high that, with Alb4 as the recipient, recombinants can be directly identified without using thermal selection. The synthetic DI RNA has been used to demonstrate that the lesion in another temperature-sensitive and thermolabile MHV mutant, Albl, maps to the N gene. Sequencing of the Albl N gene revealed two closely linked point mutations that fall in a region of the N molecule previously noted as being the most highly conserved region among all of the coronavirus N proteins. Analysis of revertants of the Albl mutant revealed that one of the two mutations is critical for the temperature-sensitive phenotype; the second mutation is phenotypically silent. The unique genomic composition of RNA viruses puts them
Repair and mutagenesis of the genome of a deletion mutant of the coronavirus mouse hepatitis virus by targeted RNA recombination
, 1992
"... The genetic characterization of a nucleocapsid (N) protein mutant of the coronavirus mouse hepatitis virus (MHIV) is described. The mutant, Albany 4 (Alb4), is both temperature sensitive and thermolabile. Analysis of the progeny of a mixed infection showed that the defective Alb4 allele is recessive ..."
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Cited by 35 (10 self)
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The genetic characterization of a nucleocapsid (N) protein mutant of the coronavirus mouse hepatitis virus (MHIV) is described. The mutant, Albany 4 (Alb4), is both temperature sensitive and thermolabile. Analysis of the progeny of a mixed infection showed that the defective Alb4 allele is recessive to wild type, and its gene product is diffusible. The N protein of Alb4 was found to be smaller than its wild-type counterpart, and sequence analysis of the Alb4 N gene revealed that it contains an internal deletion of 87 nucleotides, producing an in-frame deletion of 29 amino acids. All of these properties of Alb4 made it ideal for use as a recipient in a targeted RNA recombination experiment in which the deletion in Alb4 was repaired by recombination with synthetic RNA7, the smallest MHV subgenomic mRNA. Progeny from a cotransfection of Alb4 genomic RNA and synthetic RNA7 were selected for thermal stability. Polymerase chain reaction analysis of candidate recombinants showed that they had regained the material that is deleted in the Alb4 mutant. They also had acquired a five-nucleotide insertion in the 3 ' untranslated region, which had been incorporated into the synthetic RNA7 as a molecular tag. The presence of the tag was directly verified, as well, by sequencing the genomic RNA of purified recombinant viruses. This provided a clear genetic proof that the Alb4 phenotype was due to the observed deletion in the N gene. In addition, these results demonstrated that it is possible to obtain stable, independently replicating progeny from recombination between coronavirus genomic RNA and a
The UCUAAAC promoter motif is not required for high-frequency leader recombination in bovine coronavirus defective interfering RNA
- J
, 1996
"... The 65-nucleotide leader on the cloned bovine coronavirus defective interfering (DI) RNA, when marked by mutations, has been shown to rapidly convert to the wild-type leader of the helper virus following DI RNA transfection into helper virus-infected cells. A model of leader-primed transcription in ..."
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Cited by 27 (8 self)
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The 65-nucleotide leader on the cloned bovine coronavirus defective interfering (DI) RNA, when marked by mutations, has been shown to rapidly convert to the wild-type leader of the helper virus following DI RNA transfection into helper virus-infected cells. A model of leader-primed transcription in which free leader supplied in trans by the helper virus interacts by way of its flanking 5*UCUAAAC3 * sequence element with the 3*-proximal 3*AGAUUUG5 * promoter on the DI RNA minus strand to prime RNA replication has been used to explain this phenomenon. To test this model, the UCUAAAC element which occurs only once in the BCV 5* untranslated region was either deleted or completely substituted in input DI RNA template, and evidence of leader conversion was sought. In both cases, leader conversion occurred rapidly, indicating that this element is not required on input RNA for the conversion event. Substitution mutations mapped the crossover region to a 24-nucleotide segment that begins within the UCUAAAC sequence and extends downstream. Although structure probing of the bovine coronavirus 5 * untranslated region indicated that the UCUAAAC element is in the loop of a prominent stem and thus theoretically available for base pair-directed priming, no evidence of an unattached leader early in infection that might have served as a primer for transcription was found by RNase protection studies. These results together suggest that leader conversion on the DI RNA 5 * terminus is not guided by the UCUAAAC element and might arise instead from a high-frequency, region-specific, homologous
Subgenomic RNA synthesis directed by a synthetic defective interfering RNA of mouse hepatitis virus: a study of coronavirus transcription initiation
- J. Virol
, 1994
"... We have used a full-length cDNA clone of a mouse hepatitis virus strain A59 defective interfering (DI) RNA, pMIDI-C, and cassette mutagenesis to study the mechanism of coronavirus subgenomic mRNA synthesis. Promoter sequences closely resembling those of subgenomic mRNAs 3 and 7 were inserted into MI ..."
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We have used a full-length cDNA clone of a mouse hepatitis virus strain A59 defective interfering (DI) RNA, pMIDI-C, and cassette mutagenesis to study the mechanism of coronavirus subgenomic mRNA synthesis. Promoter sequences closely resembling those of subgenomic mRNAs 3 and 7 were inserted into MIDI-C. Both subgenomic RNA promoters gave rise to the synthesis of a subgenomic DI RNA in virus-infected and DI RNA-transfected cells. From a mutagenic analysis of the promoters we concluded the following. (i) The extent of base pairing between the leader RNA and the intergenic promoter sequence does not control subgenomic RNA abundance. (ii) Promoter recognition does not rely on base pairing only. Presumably, transcription initiation requires recognition of the promoter sequence by the transcriptase. (iii) Fusion of leader and body sequences takes place at multiple-possibly random-sites within the intergenic promoter sequence. A model is presented in which, prior to elongation, the leader RNA is trimmed by a processive 3'->5 ' nuclease. Coronaviruses are pathogens of medical and veterinary importance, causing disease in humans, livestock, and fowl. The best studied member of this virus family is the mouse hepatitis virus (MHV). The MHV genome is a positive-stranded RNA molecule of exceptional length, 32 kb, two-
Heterologous gene expression from transmissible gastroenteritis virus replicon particles
- J
, 2002
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Regulation of coronavirus mRNA transcription
- J
, 1995
"... CONTENT ALERTS more»cite this article), Receive: RSS Feeds, eTOCs, free email alerts (when new articles ..."
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Cited by 25 (5 self)
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CONTENT ALERTS more»cite this article), Receive: RSS Feeds, eTOCs, free email alerts (when new articles
The RNA structures engaged in replication and transcription of the A59 strain of mouse hepatitis virus
, 2001
"... hepatitis virus-infected cells contained six species of RNA intermediates active in transcribing subgenomic mRNA. We have named these transcriptive intermediates (TIs) and native transcriptive forms (TFs) because they are not replicating genome-sized RNA. Based on solubility in high salt solutions, ..."
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Cited by 25 (1 self)
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hepatitis virus-infected cells contained six species of RNA intermediates active in transcribing subgenomic mRNA. We have named these transcriptive intermediates (TIs) and native transcriptive forms (TFs) because they are not replicating genome-sized RNA. Based on solubility in high salt solutions, approximately 70 % of the replicating and transcribing structures that accumulated in infected cells by 5–6 h post-infection were multi-stranded intermediates, the RI/TIs. The other 30 % were in double-stranded structures, the native RF/TFs. These replicating and transcribing structures were separated by velocity sedimentation on sucrose gradients or by gel filtration chromatography on Sepharose 2B and Sephacryl S-1000, and migrated on agarose gels during electrophoresis, according to their size. Digestion with RNase T1 at 1–10 units/lg RNA resolved RI/TIs into RF/TF cores and left native RF/TFs intact, whereas RNase A at concentrations of 0–02 lg/lg RNA or higher degraded both native RF/TFs and RI/TIs. Viral RI/TIs and native RF/TFs bound to magnetic beads containing oligo(dT)25, suggesting that the poly(A) sequence on the 3« end of the positive strands was longer than any poly(U) on the negative strands. Kinetics of incorporation of [3H]uridine showed that both the RI and TIs were transcriptionally active and the labelling of RI/TIs was not the dead-end product of aberrant negative-strand synthesis. Failure
