Characterization of an Essential RNA Secondary Structure in the 3′ Untranslated Region of the Murine Coronavirus Genome

Abstract

We have previously identified a functionally essential bulged stem-loop in the 3 untranslated region of the positive-stranded RNA genome of mouse hepatitis virus. This 68-nucleotide structure is composed of six stem segments interrupted by five bulges, and its structure, but not its primary sequence, is entirely conserved in the related bovine coronavirus. The functional importance of individual stem segments of this stem-loop was characterized by genetic analysis using targeted RNA recombination. We also examined the effects of stem segment mutations on the replication of mouse hepatitis virus defective interfering RNAs. These studies were complemented by enzymatic and chemical probing of the stem-loop. Taken together, our results confirmed most of the previously proposed structure, but they revealed that the terminal loop and an internal loop are larger than originally thought. Three of the stem segments were found to be essential for viral replication. Further, our results suggest that the stem segment at the base of the stem-loop is an alternative base-pairing structure for part of a downstream, and partially overlapping, RNA pseudoknot that has recently been shown to be necessary for bovine coronavirus replication.Mouse hepatitis virus (MHV), one of the best-characterized members of the coronavirus family, has a single-stranded, positive-sense RNA genome some 31 kb in length. Upon infection, the first two-thirds of this exceptional molecule is translated into an RNA-dependent RNA polymerase. Coronavirus RNA synthesis then proceeds by a unique and incompletely understood mechanism described by conflicting models (1, 16, 24, 44-46, 54, 55, 60). Initially, the genomic RNA becomes the template for, at the least, a full-length (negative-sense) antigenome. Further events produce a series of smaller, subgenomic RNAs of both polarities. The positive-sense subgenomic RNAs form a 3Ј nested set, with each containing a 70-nucleotide (nt) leader that is identical to the 5Ј end of the genome and is joined at a downstream site to a stretch of sequence identical to the 3Ј end of the genome. The negativesense subgenomic RNAs form a 5Ј nested set and are roughly 1/10 to 1/100 as abundant as their positive-sense counterparts, with each possessing the complement of this arrangement, including a 5Ј oligo(U) tract and a 3Ј antileader (10, 47).Many advances in investigating the mechanism of coronavirus RNA synthesis have been enabled by the discovery of defective interfering (DI) RNAs of MHV (29, 30, 52) and of other coronaviruses (5,34,39). DI RNAs are extensively deleted genomic remnants that replicate by using the RNA synthesis machinery of a helper virus, often interfering with viral genomic RNA replication. Studies of naturally occurring and artificially constructed DI RNAs, which can be transfected into helper virus-infected cells, have mapped cis-acting sequence elements from the genome that participate in replication and transcription. These deletion analyses have demonstrated that the minimal extent of the 3Ј terminus o...

Coronavirus cis-Acting RNA Elements

“…The structures and several potentially important substructures of both the BSL and PK have been characterized in significant detail for BCoV and MHV (Goebel et al, 2004a;Hsue et al, 2000;Williams et al, 1999). As indicated earlier, the BSL and PK regions overlap by several nucleotides.…”

“…Also in this case, our knowledge of coronavirus 3 0 cis-acting elements is largely based on studies using betacoronaviruses, such as MHV. A combination of bioinformatics, biochemical analyses, and functional studies was used to identify and characterize cis-acting RNA elements in the 3 0 UTR (Goebel et al, 2004aHsue and Masters, 1997;Hsue et al, 2000;Liu et al, 2001Liu et al, , 2013Stammler et al, 2011;Williams et al, 1999;Z€ ust et al, 2008). More recently, these studies were extended to alphacoronaviruses using genus-wide bioinformatics analyses.…”

“…The 5 0 -most RNA structure in this region is a bulged stem-loop (BSL) of 68 nts. It is located immediately downstream of the N gene stop codon and was shown to be required for MHV DI RNA replication (Hsue and Masters, 1997;Hsue et al, 2000). Despite limited sequence similarity in this genome region, the BSL structure is predicted to be conserved in betacoronaviruses (Goebel et al, 2004a;Hsue and Masters, 1997).…”

RNA structure analysis of alphacoronavirus terminal genome regions

“…For example, the presence of a BSL followed by a PK structure is limited to betacoronaviruses, while other genera appear to contain only one of these elements, with the PK being conserved in alphacoronaviruses and the BSL in gammacoronaviruses (Dalton et al, 2001;Hsue and Masters, 1997;Williams et al, 1999) (see Section 2.6). The structures and functionally important substructures of both the BSL and PK have been characterized in significant detail for BCoV and MHV (Goebel et al, 2004a;Hsue et al, 2000;Williams et al, 1999). In the primary structure, the BSL and PK regions overlap by several nucleotides.…”

Two cellular proteins that interact with a stem loop in the simian hemorrhagic fever virus 3′(+)NCR RNA

“…Formation of RNA structures by the 3 sequences of coronaviruses has been previously reported. For MHV, a bulged secondary structure located within the 3 NCR adjacent to the nucleocapsid (N) gene stop codon was demonstrated to be required for viral replication (Hsue and Masters, 1997;Hsue et al, 2000). The 3 side of the bottom portion of this structure was subsequently shown to form a pseudoknot with another 3 NCR SL located downstream (Goebel et al, 2004).…”