5′-Proximal Hot Spot for an Inducible Positive-to-Negative-Strand Template Switch byCoronavirus RNA-Dependent RNA Polymerase

2014

J Virol

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Genomes of positive (؉)-strand RNA viruses use cis-acting signals to direct both translation and replication. Here we examine two 5=-proximal cis-replication signals of different character in a defective interfering (DI) RNA of the bovine coronavirus (BCoV) that map within a 322-nucleotide (nt) sequence (136 nt from the genomic 5= untranslated region and 186 nt from the nonstructural protein 1 [nsp1]-coding region) not found in the otherwise-identical nonreplicating subgenomic mRNA7 (sgmRNA7). The natural DI RNA is structurally a fusion of the two ends of the BCoV genome that results in a single open reading frame between a partial nsp1-coding region and the entire N gene. (i) In the first examination, mutation analyses of a recently discovered long-range RNA-RNA base-paired structure between the 5= untranslated region and the partial nsp1-coding region showed that it, possibly in concert with adjacent stem-loops, is a cis-acting replication signal in the (؉) strand. We postulate that the higher-order structure promotes (؉)-strand synthesis. (ii) In the second examination, analyses of multiple frame shifts, truncations, and point mutations within the partial nsp1-coding region showed that synthesis of a PEFP core amino acid sequence within a group A lineage betacoronavirus-conserved NH 2 -proximal WAPEFPWM domain is required in cis for DI RNA replication. We postulate that the nascent protein, as part of an RNA-associated translating complex, acts to direct the DI RNA to a critical site, enabling RNA replication. We suggest that these results have implications for viral genome replication and explain, in part, why coronavirus sgmRNAs fail to replicate. What constitutes the cis-acting requirements for coronavirus RNA replication has remained an intriguing question since it was discovered that the subgenomic mRNAs (sgmRNAs) of coronaviruses (used primarily to synthesize viral structural proteins) are both (i) 5= and 3= coterminal with the genome for at least ϳ70 and 1,670 nucleotides (nt), respectively, lengths greater than those of many viral RNA polymerase promoters (1-3), and (ii) are present in sgmRNA-length replication-intermediate-like doublestranded RNA structures that are involved in sgmRNA synthesis (4-6) yet fail to replicate when transfected, as synthetic transcripts, into virus-infected cells (Fig. 1) (7). If replication of the coronavirus sgmRNAs normally occurs during infection, it might be expected that they would replicate following their transfection into virus-infected cells, since all trans-acting factors required for viral RNA replication are present. In coronaviruses, the 5= twothirds of the single-stranded positive (ϩ)-strand ϳ30-kb coronavirus genome is used as mRNA for synthesis of overlapping polyproteins 1a (ϳ4,000 amino acids [aa]) and 1ab (ϳ7,000 aa), which are proteolytically processed into the 16 replicase proteins that make up the replication/transcription complex, whereas the 3= one-third of the genome is transcribed into a 3= nested set of sgmRNAs that are coterminal with ...

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Dependence of Coronavirus RNA Replication on an NH ₂ -Terminal Partial Nonstructural Protein 1 in cis

Fan

2014

J Virol

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“…The leader is 65-90 nt long, depending on the species of coronavirus, and the template switch likely occurs during synthesis of (−)-strand templates for sgmRNA synthesis (2, 3) ( Fig. 1, Upper), although some switching during (+)-strand synthesis (4) may occur as well (5). With few exceptions, sgmRNAs generated from 3′-proximal template-switching donor sites on the genome are progressively more abundant [up to 70-fold more at the peak time of RNA synthesis, at 6-8 h postinfection (6)] than those generated from 5′-proximal sites.…”

mentioning

confidence: 99%

Subgenomic messenger RNA amplification in coronaviruses

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

2010

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112

146

Coronaviruses possess the largest known RNA genome, a 27-to 32-kb (+)-strand molecule that replicates in the cytoplasm. During virus replication, a 3′ coterminal nested set of five to eight subgenomic (sg) mRNAs are made that are also 5′ coterminal with the genome, because they carry the genomic leader as the result of discontinuous transcription at intergenic donor signals during (−)-strand synthesis when templates for sgmRNA synthesis are made. An unanswered question is whether the sgmRNAs, which appear rapidly and abundantly, undergo posttranscriptional amplification. Here, using RT-PCR and sequence analyses of head-totail-ligated (−) strands, we show that after transfection of an in vitro-generated marked sgmRNA into virus-infected cells, the sgmRNA, like the genome, can function as a template for (−)-strand synthesis. Furthermore, when the transfected sgmRNA contains an internally placed RNA-dependent RNA polymerase templateswitching donor signal, discontinuous transcription occurs at this site, and a shorter, 3′ terminally nested leader-containing sgmRNA is made, as evidenced by its leader-body junction and by the expression of a GFP gene. Thus, in principle, the longer-nested sgmRNAs in a natural infection, all of which contain potential internal template-switching donor signals, can function to increase the number of the shorter 3′-nested sgmRNAs. One predicted advantage of this behavior for coronavirus survivability is an increased chance of maintaining genome fitness in the 3′ one-third of the genome via a homologous recombination between the (now independently abundant) WT sgmRNA and a defective genome.bovine coronavirus | discontinuous transcription | negative-strand RNA ligation | negative-strand RNA synthesis | severe acute respiratory syndrome B y virtue of an RNA-dependent RNA polymerase (RdRp) template switch, coronaviruses, which include the severe acute respiratory syndrome (SARS) virus, generate a 3′-coterminal nested set of subgenomic mRNAs (sgmRNAs) that also contain the leader of the genome (1). The leader is 65-90 nt long, depending on the species of coronavirus, and the template switch likely occurs during synthesis of (−)-strand templates for sgmRNA synthesis (2, 3) (Fig. 1, Upper), although some switching during (+)-strand synthesis (4) may occur as well (5). With few exceptions, sgmRNAs generated from 3′-proximal template-switching donor sites on the genome are progressively more abundant [up to 70-fold more at the peak time of RNA synthesis, at 6-8 h postinfection (6)] than those generated from 5′-proximal sites. The sgmRNAs are translated to virion structural proteins or to nonstructural accessory proteins, both of which may function as virulence factors or as inhibitors of host immune responses (7,8).The function of the common leader on sgmRNAs is not known, but we postulated that it [in its (−)-strand form, called the antileader] serves as a promoter for sgmRNA replication, providing a means for sgmRNA amplification (6, 9). The replication model was deemed feasible because the ant...

“…dent RNA polymerase (RdRp) template switch that causes leader switching ( Fig. 1B) (37,39). It was shown, however, that the 5Ј-terminal 13 nt which comprise part of SL1 are required for DI RNA replication, making the formerly defined SLI a probable cis-replication element (5).…”

mentioning

confidence: 99%

An Optimal cis -Replication Stem-Loop IV in the 5′ Untranslated Region of the Mouse Coronavirus Genome Extends 16 Nucleotides into Open Reading Frame 1

Guan

2011

J Virol

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cis-acting structures in positive-strand [(ϩ)-strand] RNA virus genomes that function as signals for genome translation, transcription, and replication are potential sites for antiviral drug design. The fact that replication signals from evolutionarily divergent coronaviruses can be exchanged, for example, from the 3Ј untranslated region (UTR) of the (group 2a) bovine coronavirus (BCoV) to the (group 2a) mouse hepatitis coronavirus (MHV) (21)(22) or from the (group 2b) severe acute respiratory syndrome (SARS) coronavirus (SCoV) to MHV (15), would suggest that designed therapeutic agents against common signals might be broadly effective. Several studies have identified cis-replication structures in the 3Ј UTR of MHV, heretofore recognized as the group 2 type species, and in the 3Ј UTR of BCoV that are likely involved in the initial steps of genome translation and initiation of negativestrand [(Ϫ)-strand] synthesis. These elements, identified in either a helper virus-dependent defective interfering (DI) RNA of MHV or BCoV or by reverse genetics in the fulllength MHV genome, are the following: (i) the 3Ј poly(A) tail (26, 34), (ii) the 3Ј-terminal 55 nucleotides (nt) of the 3Ј UTR (14,23,26,42), and (iii) an upstream bulged stem-loop (SL) and associated hairpin pseudoknot (13,(21)(22)36). Curiously, an ϳ140-nt hypervariable region in the 3Ј UTR that comprises part of a 3Ј-proximal bulged stem-loop and harbors a coronavirus-universal octamer sequence (GGAAGAGC) is not required for MHV replication in cell culture but plays a role in MHV pathogenesis (14).cis-replication structures have also been identified in the 5Ј-proximal region of group 2 coronaviruses, but few studies have characterized their common signaling features. In BCoV, the very 5Ј end of the genome exhibits a puzzling hypervariability during virus replication in that 5Ј extensions of up to 9 nt and varying sequences within the first 14 nt are found, but the full significance of this in virus replication is unknown (19). The higher-order RNA structures within the BCoV 5Ј UTR were initially predicted by the Tinoco algorithm (35) and more recently by the mfold algorithm of Zuker (31, 41). They have been characterized as SLs I to IV (Fig. 1B) and were shown to be consistent with enzyme structure probing analyses (5-6, 32-33). More recent characterizations based on comparisons with MHV have shown SLI to be comprised of two smaller stem-loops, named SL1 and SL2 (Fig. 1B) (24)(25)(27)(28). In the context of the DI RNA in BCoV-infected cells, it was not feasible to determine whether SLI or SLII is a bona fide cisreplication element, since SLI resides within the 65-nt leader and SLII resides largely within the leader, and transfected DI RNAs containing an experimentally mutated leader rapidly acquired the leader sequence of the helper virus genome in a process known as "leader switching" (5-6, 29). SLII, furthermore, harbors the leader-associated transcription regulatory core sequence (UCUAAAC) responsible for the RNA-depen-* Corresponding author. Mailing addres...