An in vitro system for the leader-primed transcription of coronavirus mRNAs.

Baker, Susan C.; Lai, Michael M. C.

doi:10.1002/j.1460-2075.1990.tb07641.x

Cited by 55 publications

(52 citation statements)

References 42 publications

(32 reference statements)

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“…This model proposes that the body TRS complement in the nascent (Ϫ) strand base pairs with the genomic (ϩ) leader TRS, thereby allowing the addition of the antileader sequence. In the alternative model of discontinuous (ϩ) strand synthesis (5,8,9,28), a 3Ј 3 5Ј exonuclease activity must be invoked (29) to explain why, after TRS base pairing, the leader TRS nucleotides are removed from the 3Ј end of the nascent sg transcript to be replaced by the complement of the (Ϫ) body TRS. Such a mechanism would be without precedent among (ϩ)RNA viruses.…”

Section: Discussionmentioning

confidence: 99%

Arterivirus discontinuous mRNA transcription is guided by base pairing between sense and antisense transcription-regulating sequences

Marle

Dobbe

Gultyaev

et al. 1999

Proc. Natl. Acad. Sci. U.S.A.

207

216

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To generate an extensive set of subgenomic (sg) mRNAs, nidoviruses (arteriviruses and coronaviruses) use a mechanism of discontinuous transcription. During this process, mRNAs are generated that represent the genomic 5 sequence, the so-called leader RNA, fused at specific positions to different 3 regions of the genome. The fusion of the leader to the mRNA bodies occurs at a short, conserved sequence element, the transcription-regulating sequence (TRS), which precedes every transcription unit in the genome and is also present at the 3 end of the leader sequence. Here, we have used site-directed mutagenesis of the infectious cDNA clone of the arterivirus equine arteritis virus to show that sg mRNA synthesis requires a base-pairing interaction between the leader TRS and the complement of a body TRS in the viral negative strand. Mutagenesis of the body TRS of equine arteritis virus RNA7 reduced sg RNA7 transcription severely or abolished it completely. Mutations in the leader TRS dramatically influenced the synthesis of all sg mRNAs. The construction of double mutants in which a mutant leader TRS was combined with the corresponding mutant RNA7 body TRS resulted in the specific restoration of mRNA7 synthesis. The analysis of the mRNA leader-body junctions of a number of mutants with partial transcriptional activity provided support for a mechanism of discontinuous minus-strand transcription that resembles similarity-assisted, copy-choice RNA recombination.T ranscriptional regulation is one of the key processes in the controlled expression of genetic information. In general, the transcription of eukaryotic DNA genomes is regulated in the nucleus, where it is subject to pre-, co-, and posttranscriptional control mechanisms (1, 2). In contrast, the transcription of eukaryotic positive-strand RNA [(ϩ)RNA] virus genomes occurs in the cytoplasm and relies on the process of RNAdependent RNA synthesis. In some cases, the genome is the only viral mRNA and gene expression is regulated solely at the (post-)translational level, primarily by the synthesis and controlled processing of precursor polyproteins. Alternatively, regulation may occur at the transcriptional level, either by segmentation of the genome or by the generation of one or multiple subgenomic (sg) mRNAs. The latter strategy usually involves the recognition of internal promoter sequences by the viral RNAdependent RNA polymerase (RdRp) complex, a process that resembles the recognition of DNA promoters by DNAdependent RNA polymerases (3, 4).Nidoviruses (arteriviruses and coronaviruses) are mammalian (ϩ)RNA viruses that appear to have evolved the use of sg mRNAs to regulate the expression of their polycistronic genome (5, 6). The nidovirus replicase is expressed from the genomic RNA as a polyprotein, but the structural proteins are translated from a set of six to eight sg mRNAs (Fig. 1). A key feature of these sg transcripts is the fact that their 5Ј and 3Ј terminal sequences are identical to those of the genome. This nested set structure results from a fusion of the ...

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Section: Discussionmentioning

confidence: 99%

Arterivirus discontinuous mRNA transcription is guided by base pairing between sense and antisense transcription-regulating sequences

Marle

Dobbe

Gultyaev

et al. 1999

Proc. Natl. Acad. Sci. U.S.A.

207

216

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“…The leaderprimed transcription model proposed that a free leader RNA was synthesized from the 3Ј end of the full-length minus strand. In trans, the free leader RNA binds with highly conserved internal IG elements in a full-length minus-strand template to prime transcription of each of the subgenomic mRNAs (1,3,6,19,25). More recently, it was suggested that leader body fusion might result from quasi-continuous synthesis across looped-out regions of the template which are brought together by proteinprotein interactions (22,23).…”

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confidence: 99%

Subgenomic Negative-Strand RNA Function during Mouse Hepatitis Virus Infection

Baric

Yount²

2000

J Virol

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Mouse hepatitis virus (MHV)-infected cells contain full-length and subgenomic-length positive-and negative-strand RNAs. The origin and function of the subgenomic negative-strand RNAs is controversial. In this report we demonstrate that the synthesis and molar ratios of subgenomic negative strands are similar in alternative host cells, suggesting that these RNAs function as important mediators of positive-strand synthesis. Using kinetic labeling experiments, we show that the full-length and subgenomic-length replicative form RNAs rapidly accumulate and then saturate with label, suggesting that the subgenomic-length negative strands are the principal mediators of positive-strand synthesis. Using cycloheximide, which preferentially inhibits negative-strand and to a lesser extent positive-strand synthesis, we demonstrate that cycloheximide treatment equally inhibits full-length and subgenomic-length negative-strand synthesis. Importantly, following treatment, previously transcribed negative strands remain in transcriptionally active complexes even in the absence of new negative-strand synthesis. These findings indicate that the subgenomic-length negative strands are the principal templates of positive-strand synthesis during MHV infection.Mouse hepatitis virus (MHV), a coronavirus in the Nidovirales order, contains a ϳ32-kb linear, single-stranded, positivepolarity RNA genome (8,21,28). Upon entry into the cell, the viral genome is transcribed into seven to eight subgenomic mRNAs ranging in size from ϳ1.0 to 32.0 kb (21, 32). The positive-strand mRNAs are arranged in a 3Ј coterminal nested set, and each contains a 5Ј-end ϳ72-nucleotide leader RNA sequence which is derived from the 5Ј end of the genome (20, 37). Leader RNA sequences are joined to body sequences of each subgenomic-length mRNA at highly conserved intergenic (IG) sites located just upstream from the coding sequences of each viral gene (7,14,27,32). In addition to the viral mRNAs, both full-length as well as subgenomic-length negative-strand RNAs and replicative form (RF) RNAs have been detected in porcine transmissible gastroenteritis virus-, bovine coronavirus-, and MHV-infected cells (13,30,(32)(33)(34)(35). The subgenomic-length negative-strand RNAs contain antileader RNA sequences (31, 35). While MHV negative-strand RNA synthesis is rapidly inhibited by inhibitors of protein synthesis, positive-strand synthesis is significantly more resistant, suggesting that continued protein synthesis is essential for MHV negative-strand synthesis (29).Several discontinuous transcription models have been proposed to explain the presence of leader RNA sequences on positive-strand RNAs and antileader RNA sequences on fulllength and subgenomic-length negative-strand RNAs (3,10,30,31,34,35). It is generally agreed that these smaller RNAs do not originate from larger precursors (3, 15, 31). The leaderprimed transcription model proposed that a free leader RNA was synthesized from the 3Ј end of the full-length minus strand. In trans, the free leader RNA binds with highly...

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“…The 3' region of leader RNA contains two or three repeats of a consensus sequence (UCUAA) complementary to negative-strand intergenic regions (Budzilowicz et al, 1985;Sheih et al, 1987). During transcription, leader RNA sequences are cleaved and fused to mRNA bodies at a junction site within the second or third (3') tandem repeat of the consensus sequence (Baker & Lai, 1990;Joo & Makino, 1992). The N protein forms high affinity complexes with the leader RNA through interaction at the 3' end of leader RNA which contains the consensus repeat S.…”

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confidence: 99%

Localization of the RNA-binding domain of mouse hepatitis virus nucleocapsid protein

Nelson¹,

Stohlman

1993

Journal of General Virology

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The 454-amino acid nucleocapsid (N) protein of mouse hepatitis virus (MHV) binds the leader RNA sequence located at the 5' ends of all plus-sense genomic and subgenomic viral mRNAs. Purified N protein was cleaved with formic acid to determine which domain interacts with the leader RNA sequence. Incubation at 42 °C resulted in partial cleavage into two fragments of Mrs of approximately 32K and 37K and three fragments of 17K, 16K and 14K. Incubation at 56 °C resulted in complete cleavage yielding only the three lower molecular mass products. Both the 32K and 37K partial cleavage products and one of the complete cleavage products bind MHV leader RNA, suggesting that the central region of the N protein contains the RNAbinding domain. Monoclonal antibody mapping of the cleavage products confirmed that the MHV leader RNA binding domain is contained within the central 140-amino acid fragment, comprising amino acids 169 to 308. Analysis of the amino acids within this domain indicates no similarity to any previously described RNAbinding protein, suggesting that N protein may possess a unique RNA-binding motif.

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An in vitro system for the leader-primed transcription of coronavirus mRNAs.

Cited by 55 publications

References 42 publications

Arterivirus discontinuous mRNA transcription is guided by base pairing between sense and antisense transcription-regulating sequences

Arterivirus discontinuous mRNA transcription is guided by base pairing between sense and antisense transcription-regulating sequences

Subgenomic Negative-Strand RNA Function during Mouse Hepatitis Virus Infection

Localization of the RNA-binding domain of mouse hepatitis virus nucleocapsid protein

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