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

Abstract

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...

Binding of Cellular Proteins to the Leader RNA of Equine Arteritis Virus

“…These structures are recognized to play important roles in the replication cycle of several (+) strand RNA viruses [4,5,13]. Nevertheless, it is noteworthy that the RNA stem-loop structure containing the leader TRS element [12], and for which there is a proven significance in EAV RNA synthesis, was not covered by the RNA probes used in our study.…”

“…These results argue for the presence of multiple protein binding sites within the EAV leader genomic RNA, a characteristic already observed in other (+) strand RNA viruses [8,11]. Interestingly, the binding regions identified here in the (+) strand of the EAV leader RNA contain several RNA secondary structures [6,12]. These structures are recognized to play important roles in the replication cycle of several (+) strand RNA viruses [4,5,13].…”

Myristoylation of the arterivirus E protein: the fatty acid modification is not essential for membrane association but contributes significantly to virus infectivity

“…To generate an EAV mutant carrying the G2A mutation in the E protein, a GAC mutation was introduced at nt 9755 of the genome in the EAV full-length cDNA clone pEAV211 (van den Born et al, 2005;van Dinten et al, 1997). The mutation was first engineered in a BamHI-EcoRI fragment (nt 9150-11488), which was sequenced (GATC Biotech) and transferred to pEAV211.…”

The Intraleader AUG Nucleotide Sequence Context is Important for Equine Arteritis Virus Replication

“…Each open reading frame (ORF) in the EAV genome is preceded by the pentanucleotide sequence motif 5¢-UCAAC-3¢, also called the leader-body junction site [3,4] or the transcription regulating sequence (TRS) [5]. The TRS motifs, in conjunction with neighboring nt, are key elements involved in the synthesis of the 3¢-coterminal nested set of six subgenomic (sg) mRNAs [6,7] through their fusion, most likely by a discontinuous minus-strand transcription mechanism, with a common leader sequence of 206 nt identical to and derived from the extreme 5¢ terminus of the viral genome [8].…”