Characterization of the RNA Components of a Putative Molecular Switch in the 3′ Untranslated Region of the Murine Coronavirus Genome

Abstract

RNA virus genomes contain cis-acting sequence and structural elements that participate in viral replication. We previously identified a bulged stem-loop secondary structure at the upstream end of the 3 untranslated region (3 UTR) of the genome of the coronavirus mouse hepatitis virus (MHV). This element, beginning immediately downstream of the nucleocapsid gene stop codon, was shown to be essential for virus replication. Other investigators discovered an adjacent downstream pseudoknot in the 3 UTR of the closely related bovine coronavirus (BCoV). This pseudoknot was also shown to be essential for replication, and it has a conserved counterpart in every group 1 and group 2 coronavirus. In MHV and BCoV, the bulged stem-loop and pseudoknot are, in part, mutually exclusive, because of the overlap of the last segment of the stem-loop and stem 1 of the pseudoknot. This led us to hypothesize that they form a molecular switch, possibly regulating a transition occurring during viral RNA synthesis. We have now performed an extensive genetic analysis of the two components of this proposed switch. Our results define essential and nonessential components of these structures and establish the limits to which essential parts of each element can be destabilized prior to loss of function. Most notably, we have confirmed the interrelationship of the two putative switch elements. Additionally, we have identified a pseudoknot loop insertion mutation that appears to point to a genetic interaction between the pseudoknot and a distant region of the genome.Embedded in the genomes of RNA viruses are cis-acting sequence and structural elements that participate in replication, transcription, translation, and packaging. Some of these signals are thought to facilitate the selective interactions of viral RNAs with the machinery of RNA synthesis, while others enable or modulate events that occur during viral protein synthesis or assembly. The coronaviruses and arteriviruses, members of the order Nidovirales, are positive-strand RNA viruses with very large genomes (13 to 15 kb for arteriviruses, 27 to 31 kb for coronaviruses). In addition to the production of progeny genome copies, nidovirus replication entails the synthesis of a 3Ј nested set of subgenomic (sg) mRNAs (15). Each sgRNA contains a 5Ј leader segment connected to a body segment that is identical to the 3Ј end of the genome, starting at a given point preceding one of the downstream open reading frames. Accumulating evidence, particularly from landmark studies using a full-length infectious cDNA of equine arterivirus, supports a model in which the discontinuous step in sgRNA formation occurs during negative-strand RNA synthesis (1,28,31,35). Thus, for nidoviruses, the earliest steps of both genome replication and sgRNA transcription initiate at the 3Ј end of the genome; therefore, it is reasonable to expect that at least part of the regulation of these processes is implicit in the sequence and structure of the 3Ј untranslated region (3Ј UTR). Indeed, studies of defective interfering...

A Pseudoknot in a Preactive Form of a Viral RNA Is Part of a Structural Switch Activating Minus-Strand Synthesis

“…This process would allow progeny to be "stamped" off of the original parental genome, reducing the number of potentially deleterious mutations (3). Recent evidence for alternative RNA structures in the 5Ј and 3Ј regions of plant, animal, and bacterial RNA viruses (5,8,13,18,24) suggests that conformational switches leading to initiation of minus-strand synthesis and possibly restricting RdRp access to de novo-synthesized plus strands may be a contributing factor to the replication of a number of plus-strand RNA viruses.…”

“…Changes in viral RNA conformation in 3Ј regions of the genome that hide or expose the 3Ј terminus (13,18,24) or permit the formation of important cis-acting structures (11) likely regulate initiation of minusstrand synthesis. Evidence for important alternative structures with no known function has also been reported (4,5). Since RNA switches usually involve long-distance tertiary interactions that stabilize one of the RNA conformations (8,11,12), removal of cis-acting elements from their natural context for biochemical and biophysical analyses may lead to an oversimplification of how diverse elements are involved in many viral processes.…”

Conformational changes involved in initiation of minus-strand synthesis of a virus-associated RNA

“…For example, Barley yellow dwarf virus is proposed to repress (À)-strand synthesis by embedding its 3¢ end in a ''pocket'' structure, thus making it unavailable to the RdRp (Koev et al 2002). A similar molecular switch in the Mouse hepatitis virus genome involves sequences within a stem-loop and pseudoknot in the 3¢ untranslated region (UTR) (Goebel et al 2004). In addition to cis-acting sequences, trans-acting cellular factors or virally encoded proteins may affect the balance between alternative structural conformations (Olsthoorn et al 1999;Schuppli et al 2000).…”

Structural Domains within the 3′ Untranslated Region of Turnip Crinkle Virus

“…Recent reports that portions of RNA viral genomes undergo conformational shifts to execute different functions (10,13,21,28) complicate efforts to assign biological roles to groups of cis-acting elements that may not structurally coexist. The ability of viral RNAs to assume multiple conformations combined with evidence that newly synthesized plus strands may assume forms that suppress transcription (43) supports the "stamping model" hypothesis for viral replication.…”