Subgenomic RNAs of Lelystad virus contain a conserved leader-body junction sequence

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...

Section: Vol 74 2000 Eav Alternative Leader-body Junction Sites 11649mentioning

confidence: 99%

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

Pasternak

Gultyaev

Spaan

et al. 2000

“…These are subsequently cleaved by virus-encoded proteases to yield at least 12 nonstructural proteins, including the viral RdRp (reviewed in reference 24). In addition, a set of subgenomic mRNAs, collectively expressing the viral structural proteins, are produced through a process of discontinuous mRNA transcription (5,8,14,16). This process has not been unraveled completely and may occur during plus-or minus-strand synthesis (reviewed in references 9, 23, and 26).…”

mentioning

confidence: 99%

Kissing Interaction between 3′ Noncoding and Coding Sequences Is Essential for Porcine Arterivirus RNA Replication

Verheije

Olsthoorn

Kroese

et al. 2002

Self Cite

We used an infectious cDNA clone of Porcine reproductive and respiratory syndrome virus (PRRSV) to investigate the presence of essential replication elements in the region of the genome encoding the structural proteins. Deletion analysis showed that a stretch of 34 nucleotides (14653 to 14686) within ORF7, which encodes the nucleocapsid protein, is essential for RNA replication. Strand-specific reverse transcription-PCR analysis of viral RNA isolated from transfected BHK-21 cells revealed that this region is required for negative-strand genomic RNA synthesis. The 34-nucleotide stretch is highly conserved among PRRSV isolates and folds into a putative hairpin. A 7-base sequence within the loop of this structure was suggested to base-pair with a sequence present in the loop of a hairpin located in the 3 noncoding region, resulting in a kissing interaction. Mutational analyses confirmed that this kissing interaction is required for RNA replication.Positive-strand RNA viruses replicate in infected cells by a process that is mediated by RNA-dependent RNA polymerase (RdRp). In this process, genomic RNA serves as a template for the production of negative-strand antigenomic RNA, which is used in turn as a template for the synthesis of new plus strands. The process of replication requires the recruitment of the RdRp to specific sequences or structures within the templates, also known as cis-acting replication elements. These elements are usually located in the noncoding regions at the termini of the viral RNA, where RdRp complexes initiate the synthesis of plus and minus strands (2). Only a few examples are known of cis-acting replication elements that reside within a coding region (7,10,11,20).Porcine reproductive and respiratory syndrome virus (PRRSV) is a positive-stranded RNA virus that belongs to the Arteriviridae family (reviewed in reference 24), together with Equine arteritis virus (EAV), Lactate dehydrogenase-elevating virus (LDV), and Simian hemorrhagic fever virus (SHFV) (17). On the basis of their similar genomic organization and replication strategy, the arteriviruses have been grouped into the order of Nidovirales together with the coronaviruses and the toroviruses (3).The genome of PRRSV is 15.1 kb (17), of which the 5Ј two-thirds is translated into two large polyproteins. These are subsequently cleaved by virus-encoded proteases to yield at least 12 nonstructural proteins, including the viral RdRp (reviewed in reference 24). In addition, a set of subgenomic mRNAs, collectively expressing the viral structural proteins, are produced through a process of discontinuous mRNA transcription (5,8,14,16). This process has not been unraveled completely and may occur during plus-or minus-strand synthesis (reviewed in references 9, 23, and 26). Besides the coding regions, the PRRSV genome contains a 5Ј untranslated region (5ЈUTR) of 221 nucleotides (nt) (24), which carries a cap at its 5Ј end (15, 21), and a 3ЈUTR of 114 nt to which the poly(A) tail is attached (17).Little is known about the requirements for arterivi...

“…PRRSV is enveloped and has a polyadenylated, singlestranded, nonsegmented, positive-sense RNA genome 15 kb in size (7,14,34,35). The genome consists of eight open reading frames (ORFs) that are expressed through the production of a nested set of subgenomic 3Ј-coterminal mRNAs (14,34,35).…”

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confidence: 99%

Evolution of Porcine Reproductive and Respiratory Syndrome Virus during Sequential Passages in Pigs

Chang¹,

Yoon²,

Zimmerman³

et al. 2002

118

Porcine reproductive and respiratory syndrome (PRRS) viruses are recognized as possessing a high degree of genetic and antigenic variability. Viral diversity has led to questions regarding the association of virus mutation and persistent infection in the host and has raised concerns vis-à-vis protective immunity, the ability of diagnostic assays to detect novel variants, and the possible emergence of virulent strains. The purpose of this study was to describe ongoing changes in PRRS virus during replication in pigs under experimental conditions. Animals were inoculated with a plaque-cloned virus derived from VR-2332, the North American PRRS virus prototype. Three independent lines of in vivo replication were maintained for 367 days by pig-to-pig passage of virus at 60-day intervals. A total of 315 plaque-cloned viruses were recovered from 21 pigs over the 367-day observation period and compared to the original plaque-cloned virus by virus neutralization assay, monoclonal antibody analysis, and sequencing of open reading frames (ORFs) 1b (replicase), 5 (major envelope protein), and 7 (nucleocapsid) of the genome. Variants were detected by day 7 postinoculation, and multiple variants were present concurrently in every pig sampled over the observation period. Sequence analysis showed ORFs 1b and 7 to be highly conserved. In contrast, sequencing of ORF 5 disclosed 48 nucleotide variants which corresponded to 22 amino acid variants. Although no epitopic changes were detected under the conditions of this experiment, PRRS virus was shown to evolve continuously in infected pigs, with different genes of the viral genome undergoing various degrees of change.