Enhancer-like Properties of an RNA Element That Modulates Tombusvirus RNA Accumulation

Abstract

Prototypical defective interfering (DI) RNAs of the plus-strand RNA virus tomato bushy stunt virus contain four noncontiguous segments (regions I-IV) derived from the viral genome. Region I corresponds to 5'-noncoding sequence, regions II and III are derived from internal positions, and region IV represents a 3'-terminal segment. We analyzed the internally located region III in a prototypical DI RNA to understand better its role in DI RNA accumulation. Our results indicate that (1) region III is not essential for DI RNA accumulation, but molecules that lack it accumulate at significantly reduced levels ( approximately 10-fold lower), (2) region III is able to function at different positions and in opposite orientations, (3) a single copy of region III is favored over multiple copies, (4) the stimulatory effect observed on DI RNA accumulation is not due to region III-mediated RNA stabilization, (5) DI RNAs lacking region III permit the efficient accumulation of head-to-tail dimers and are less effective at suppressing helper RNA accumulation, and (6) negative-strand accumulation is also significantly depressed for DI RNAs lacking region III. Collectively, these results support a role for region III as an enhancer-like element that facilitates DI RNA replication. A scanning-type mutagenesis strategy was used to define portions of region III important for its stimulatory effect on DI RNA accumulation. Interestingly, the results revealed several differences in the requirements for activity when region III was in the forward versus the reverse orientation. In the context of the viral genome, region III was found to be essential for biological activity. This latter finding defines a critical role for this element in the reproductive cycle of the virus.

Expression of tombusvirus open reading frames 1 and 2 is sufficient for the replication of defective interfering, but not satellite, RNA

“…Tombusviruses can support the replication of homologous or heterologous DI RNA in plant (Havelda et al, 1998) or yeast cells (Pantaleo et al, 2003(Pantaleo et al, , 2004Panavas & Nagy, 2003). Tombusvirus DI RNAs have been widely used for the analysis of cis-acting factors directing the replication of viral RNA both in vivo Chang et al, 1995;Ray & White, 1999White & Morris, 1994;Wu & White, 1998;Wu et al, 2001) and in vitro (Nagy & Pogany, 2000; Panavas et al, 2002a, b;Panavas & Nagy, 2003).…”

“…Tombusviruses can support the replication of homologous or heterologous DI RNA in plant (Havelda et al, 1998) or yeast cells (Pantaleo et al, 2003(Pantaleo et al, , 2004Panavas & Nagy, 2003). Tombusvirus DI RNAs have been widely used for the analysis of cis-acting factors directing the replication of viral RNA both in vivo Chang et al, 1995;Ray & White, 1999White & Morris, 1994;Wu & White, 1998;Wu et al, 2001) and in vitro (Nagy & Pogany, 2000; Panavas et al, 2002a, b;Panavas & Nagy, 2003).The molecular biology of tombusvirus sat RNAs has been much less studied. So far, the best characterized sat RNA is the 621 nt sat RNA of Cymbidium ringspot virus (CymRSV) whose cis-acting sequence requirements essential for replication and the significance and nature of multimeric forms found in infected tissues have been studied (Rubino et al, 1990;Dalmay & Rubino, 1994.…”

A second functional RNA domain in the 5′ UTR of the Tomato bushy stunt virus genome: Intra- and interdomain interactions mediate viral RNA replication

“…For TBSV, viral RNA replication has been studied extensively using defective interfering (DI) RNAs (White and Morris 1994a,b;Chang et al 1995;Wu and White 1998;Ray andWhite 1999, 2003;Nagy and Pogany 2000;Panavas et al 2002a,b;Panavas and Nagy 2003). DI RNAs are genomederived deletion mutants that are noncoding but maintain important RNA replication elements that allow them to be amplified when p33 and p92 are provided in trans (White 1996).…”

Análisis Funcional De Proteínas Codificadas Por El Virus De La Rotura Del Color De La Flor Del Pelargonium

“…A partir de esta última se iniciará la síntesis de nuevas cadenas positivas, tras la unión de la enzima a la región 3´ UTR en la cadena negativa (reflejada en el extremo 5´ del gRNA(+) [Panavas et al, 2002a;Wang & Wong, 2004]. Además de estos promotores, se han descrito elementos estimuladores de la replicación (replication enhancers), que actúan potenciando la síntesis de las cadenas positivas, y elementos supresores de la replicación, que provocan una disminución de la síntesis de las cadenas negativas [Ray & White, 1999;Nagy et al, 1999Nagy et al, , 2001Pogany et al, 2003;Zhang et al, 2004aZhang et al, , 2004bNa & White, 2006].…”

“…Asimismo, se han descrito elementos que regulan los niveles de síntesis de las cadenas (+) y (-), tales como estimuladores de la replicación que potencian la síntesis de cadenas positivas y represores de la replicación que disminuyen la producción de cadenas negativas [Ray & White, 1999;Nagy et al, 1999Nagy et al, , 2001Pogany et al, 2003;Zhang et al, 2004aZhang et al, , 2004bNa & White, 2006]. Además de los elementos implicados en la replicación, se ha descrito la presencia de reguladores traduccionales en la 3´ UTR de distintos miembros de la familia Tombusviridae que parecen promover una traducción eficiente de los vRNA en ausencia de estructuras cap Stupina et al, 2008;Truniger et al, 2008;Wang et al, 2011b].…”