The UCUAAAC promoter motif is not required for high-frequency leader recombination in bovine coronavirus defective interfering RNA

Abstract

The 65-nucleotide leader on the cloned bovine coronavirus defective interfering (DI) RNA, when marked by mutations, has been shown to rapidly convert to the wild-type leader of the helper virus following DI RNA transfection into helper virus-infected cells. A model of leader-primed transcription in which free leader supplied in trans by the helper virus interacts by way of its flanking 5UCUAAAC3 sequence element with the 3-proximal 3AGAUUUG5 promoter on the DI RNA minus strand to prime RNA replication has been used to explain this phenomenon. To test this model, the UCUAAAC element which occurs only once in the BCV 5 untranslated region was either deleted or completely substituted in input DI RNA template, and evidence of leader conversion was sought. In both cases, leader conversion occurred rapidly, indicating that this element is not required on input RNA for the conversion event. Substitution mutations mapped the crossover region to a 24-nucleotide segment that begins within the UCUAAAC sequence and extends downstream. Although structure probing of the bovine coronavirus 5 untranslated region indicated that the UCUAAAC element is in the loop of a prominent stem and thus theoretically available for base pair-directed priming, no evidence of an unattached leader early in infection that might have served as a primer for transcription was found by RNase protection studies. These results together suggest that leader conversion on the DI RNA 5 terminus is not guided by the UCUAAAC element and might arise instead from a high-frequency, region-specific, homologous recombination event perhaps during minus-strand synthesis rather than by leader priming during plus-strand synthesis.

RNA structure analysis of alphacoronavirus terminal genome regions

“…The predicted SL3 (named SL-II in BCoV DI RNA studies) appears to be conserved only in a subset of beta-and gammacoronaviruses (Chen and Olsthoorn, 2010). For BCoV and closely related viruses, the TRS-L core sequence (CS) has been proposed to be exposed in the SL3 loop region, a structure similar to the TRS-L hairpin structure reported for the related arterivirus equine arteritis virus (EAV) (Chang et al, 1996;van den Born et al, 2004van den Born et al, , 2005. By contrast, in most other coronaviruses, the TRS-L CS and flanking regions were suggested to be located in nonstructured regions (Chen and Olsthoorn, 2010;Stirrups et al, 2000;Wang and Zhang, 2000).…”

“…However, DI RNAs also have disadvantages, a major one being homologous recombination between the RNA replicon and the helper virus genome. Thus, for example, artificial DI RNAs containing mutant 5 leader sequences rapidly acquire the leader sequence of the helper virus, a process commonly referred to as "leader switching" (Chang et al, 1994(Chang et al, , 1996. The latter occurs very often if poorly replicating mutant DI RNAs are to be characterized which generally require amplification steps by serial passaging to determine their phenotype.…”

“…Cis-acting RNA structures in the 5 -terminal region of the coronavirus genome have first been studied for BCoV using DI RNAbased systems (Brown et al, 2007;Chang et al, 1994Chang et al, , 1996Gustin et al, 2009;Raman et al, 2003;Raman and Brian, 2005). In the 5terminal 215 nts of the BCoV genome, four stem-loops (designated I [comprised of Ia and Ib], II, III, and IV) were defined.…”

Coronavirus cis-Acting RNA Elements

“…A major problem in studies using DI RNAs for defining elements required for replication was the highfrequency homologous recombination between the RNA replicon and the helper virus genome. For example, BCoV-derived artificial DI RNAs containing base substitutions within 5 0 leader sequences rapidly acquired the leader sequence of the helper virus (Chang et al, 1994(Chang et al, , 1996. This "leader switching" was regularly observed in serial passaging experiments aimed to rescue (or amplify) DI RNAs for further phenotypic characterization.…”

“…Elements The majority of the 5 0 -proximal RNA structures and sequences essential for coronavirus genome replication have first been characterized for BCoV using DI RNA-based systems (Brown et al, 2007;Chang et al, 1994Chang et al, , 1996Gustin et al, 2009;Raman and Brian, 2005;Raman et al, 2003). The 5 0 -proximal 215 nts of the BCoV genome were predicted to harbor four stem-loops (SLs) that, in the older literature, were termed SL I (comprised of Ia and Ib), II, III, and IV.…”