Recombination and Coronavirus Defective Interfering RNAs

Brian, David A.; Spaan, Willy J. M.

doi:10.1006/smvy.1997.0109

Cited by 79 publications

(73 citation statements)

References 77 publications

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“…A number of models have been put forward to explain the discontinuous step in nidovirus sg RNA synthesis (2,31,33,38). Our recent data (46) are most consistent with the model of Sawicki and Sawicki (32), in which the discontinuous step occurs during minus-strand synthesis, producing minus-strand sg-sized templates for the synthesis of sg mRNAs in a process that resembles similarity-assisted RNA recombination (3,27,46).…”

supporting

confidence: 83%

“…EAV contains six or seven structural proteins (9,10,16,35): the nucleocapsid protein N, the nonglycosylated, triple-spanning membrane protein M, the small envelope protein E, and four glycoproteins, GP 2b , GP 3 , GP 4 , and GP 5 . It is unclear whether GP 3 is a structural component of the arterivirus particle in view of the conflicting data reported for PRRSV GP 3 (see reference 34 for a review).…”

mentioning

confidence: 99%

“…It is unclear whether GP 3 is a structural component of the arterivirus particle in view of the conflicting data reported for PRRSV GP 3 (see reference 34 for a review). The genes encoding the EAV structural proteins (ORF2a to -7; Fig.…”

mentioning

confidence: 99%

“…From 5Ј to 3Ј, the genes encode proteins in the following order: E-GP 2b -GP 3 -GP 4 -GP 5 -M-N. mRNA2 is presumed to be bicistronic and is thought to encode both E and GP 2b . The other five genes (encoding GP 3 to N) are expressed from sg mRNA3 to -7.…”

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confidence: 99%

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Genetic Manipulation of Arterivirus Alternative mRNA Leader-Body Junction Sites Reveals Tight Regulation of Structural Protein Expression

Pasternak

Gultyaev

Spaan

et al. 2000

J Virol

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To express its structural proteins, the arterivirus Equine arteritis virus (EAV) produces a nested set of six subgenomic (sg) RNA species. These RNA molecules are generated by a mechanism of discontinuous transcription, during which a common leader sequence, representing the 5 end of the genomic RNA, is attached to the bodies of the sg RNAs. The connection between the leader and body parts of an mRNA is formed by a short, conserved sequence element termed the transcription-regulating sequence (TRS), which is present at the 3 end of the leader as well as upstream of each of the structural protein genes. With the exception of RNA3, only one body TRS was previously assumed to be used to join the leader and body of each EAV sg RNA. Here we show that for the synthesis of two other sg RNAs, RNA4 and RNA5, alternative leader-body junction sites that differ substantially in transcriptional activity are used. By site-directed mutagenesis of an EAV infectious cDNA clone, the alternative TRSs used to generate RNA3, -4, and -5 were inactivated, which strongly influenced the corresponding RNA levels and the production of infectious progeny virus. The relative amounts of RNA produced from alternative TRSs differed significantly and corresponded to the relative infectivities of the virus mutants. This strongly suggested that the structural proteins that are expressed from these RNAs are limiting factors during the viral life cycle and that the discontinuous step in sg RNA synthesis is crucial for the regulation of their expression. On the basis of a theoretical analysis of the predicted RNA structure of the 3 end of the EAV genome, we propose that the local secondary RNA structure of the body TRS regions is an important factor in the regulation of the discontinuous step in EAV sg mRNA synthesis.The arterivirus Equine arteritis virus (EAV) is a plus-stranded RNA virus belonging to the order Nidovirales, which includes coronaviruses and arteriviruses (4, 11). The family Arteriviridae consists of EAV, Porcine reproductive and respiratory syndrome virus (PRRSV), murine Lactate dehydrogenaseelevating virus (LDV), and Simian hemorrhagic fever virus (SHFV) (see reference 34 for a recent review).The EAV genome is a single 12.7-kb RNA molecule (11). Two large replicase polyproteins, the ORF1a and ORF1ab proteins, are encoded in the 5Ј three-fourths of the genome. Translation of the ORF1b-encoded part of the ORF1ab protein involves a ribosomal frameshift (7). These polypeptides are proteolytically cleaved to yield 12 nonstructural proteins (36,37,44,45,48). In addition to genome replication, these proteins function in the production of a 3Ј-coterminal nested set of subgenomic (sg) RNAs, from which the viral structural proteins are translated (Fig. 1A).EAV contains six or seven structural proteins (9, 10, 16, 35): the nucleocapsid protein N, the nonglycosylated, triple-spanning membrane protein M, the small envelope protein E, and four glycoproteins, GP 2b , GP 3 , GP 4 , and GP 5 . It is unclear whether GP 3 is a structural component of...

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supporting

confidence: 83%

mentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

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Genetic Manipulation of Arterivirus Alternative mRNA Leader-Body Junction Sites Reveals Tight Regulation of Structural Protein Expression

Pasternak

Gultyaev

Spaan

et al. 2000

J Virol

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“…It was proposed that the process may mechanistically resemble similarity-assisted copy choice RNA recombination (1,2,26). As in the case of RNA recombination, the higher-order structure of the template (and possibly also of the nascent strand) may play an important role, in particular in the regions where leader TRS and body TRSs reside.…”

mentioning

confidence: 99%

Discontinuous Subgenomic RNA Synthesis in Arteriviruses Is Guided by an RNA Hairpin Structure Located in the Genomic Leader Region

Born

Posthuma

Gultyaev

et al. 2005

J Virol

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Nidoviruses produce an extensive 3-coterminal nested set of subgenomic (sg) mRNAs, which are used to express structural proteins and sometimes accessory proteins. In arteriviruses and coronaviruses, these mRNAs contain a common 5 leader sequence, derived from the genomic 5 end. The joining of the leader sequence to different segments derived from the 3-proximal part of the genome (mRNA bodies) presumably involves a unique mechanism of discontinuous minus-strand RNA synthesis in which base pairing between sense and antisense transcription-regulating sequences (TRSs) plays an essential role. The leader TRS is present in the loop of a hairpin structure that functions in sg mRNA synthesis. In this study, the minimal sequences in the 5-proximal region of the Equine arteritis virus genome that are required for sg RNA synthesis were delimited through mutagenesis. A full-length cDNA clone was engineered in which this domain was duplicated, allowing us to make mutations and monitor their effects on sg RNA synthesis without seriously affecting genome replication and translation. The leader TRS present in the duplicated sequence was used and yielded novel sg mRNAs with significantly extended leaders. Our combined findings suggest that the leader TRS hairpin (LTH) and its immediate flanking sequences are essential for efficient sg RNA synthesis and form an independent functional entity that could be moved 300 nucleotides downstream of its original position in the genome. We hypothesize that a conformational switch in the LTH region regulates the role of the 5-proximal region of the arterivirus genome in subgenomic RNA synthesis.Many eukaryotic plus-strand RNA (ϩRNA) viruses generate subgenomic (sg) mRNAs as a strategy to regulate the expression of one or several viral proteins (21). In addition to internal initiation of translation, sg mRNA synthesis provides a mechanism to express genes positioned downstream of the 5Ј-proximal gene in polycistronic genomes of ϩRNA viruses of eukaryotes. Different mechanisms are employed by ϩRNA viruses to achieve the transcription of sg mRNAs (47). The generation of an extensive 3Ј-coterminal nested set of sg mRNAs to express structural and/or accessory proteins is a common property of viruses belonging to the order Nidovirales (Coronaviridae, Arteriviridae, and Roniviridae) (6, 34). In the case of arteriviruses and coronaviruses, all transcripts contain a common 5Ј sequence element, the so-called "leader," which is colinear with the 5Ј-proximal part of the genome and is fused to different regions derived from the 3Ј-proximal third of the genome, which are termed "mRNA bodies" (Fig. 1A). For two other nidovirus subgroups, roniviruses and toroviruses (with the exception of sg mRNA2 of the latter), sg mRNAs without a common 5Ј leader sequence have been described (3,32,45).For sg RNAs of coronaviruses and arteriviruses, and also the largest sg RNA of toroviruses (45), the fusion of the sg RNA body to the common leader sequence has been postulated to involve the discontinuous extension of minu...

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