Role of an Internal and Two 3′-Terminal RNA Elements in Assembly of Tombusvirus Replicase

Abstract

Plus-strand RNA virus replication requires the assembly of the viral replicase complexes on intracellular membranes in the host cells. The replicase of Cucumber necrosis virus (CNV), a tombusvirus, contains the viral p33 and p92 replication proteins and possible host factors. In addition, the assembly of CNV replicase is stimulated in the presence of plus-stranded viral RNA (Z. Panaviene et al., J. Virol. 78:8254-8263, 2004). To define cis-acting viral RNA sequences that stimulate replicase assembly, we performed a systematic deletion approach with a model tombusvirus replicon RNA in Saccharomyces cerevisiae, which also coexpressed p33 and p92 replication proteins. In vitro replicase assays performed with purified CNV replicase preparations from yeast revealed critical roles for three RNA elements in CNV replicase assembly: the internal p33 recognition element (p33RE), the replication silencer element (RSE), and the 3 -terminal minus-strand initiation promoter (gPR). Deletion or mutagenesis of these elements reduced the activity of the CNV replicase to a minimal level. In addition to the primary sequences of gPR, RSE, and p33RE, formation of two alternative structures among these elements may also play a role in replicase assembly. Altogether, the role of multiple RNA elements in tombusvirus replicase assembly could be an important factor to ensure fidelity of template selection during replication.The genomes of plus-stranded RNA viruses are replicated by viral replicase complexes assembled on intracellular membranes (1, 4). The replicase complex consists of virus-encoded proteins, such as the RNA-dependent RNA polymerase (RdRp), auxiliary viral proteins, and possibly host-derived proteins and the viral RNA template (2, 10). The viral RNA serves a more complex role than just being used as a template for replication. For example, the assembly of viral replicases of Brome mosaic virus (BMV) and Alfalfa mosaic virus requires the plus-stranded viral RNA in order to be functional (28, 37). In the case of Cucumber necrosis virus (CNV), the viral (ϩ)RNA, but not the (Ϫ)RNA, stimulated replicase assembly by 40-fold in yeast, a model host (22). All these works led to the model that the viral (ϩ)RNA likely serves as a platform to bring the viral replication proteins and host protein factors together, leading to efficient assembly of the viral replicase.Tombusviruses are nonsegmented plus-stranded viruses that code for five proteins. These include the p33 and p92 replication proteins (Fig. 1), a cell-to-cell movement protein (p22), a coat protein (p41), and a suppressor of gene silencing (p19). The overlapping p33 and p92 replicase proteins are essential for replication of the genomic RNA (gRNA) in plant cells (14,20,33,40). The p92 replication protein has the RdRp signature motifs in its unique C terminus, whereas the auxiliary p33 plays a role in template selection and the recruitment of the viral RNA into replication (12,26). In addition, mutagenesis of p33 within its RNA-binding site (an arginine-proline-rich motif, te...

Conformational organization of the 3′ untranslated region in the tomato bushy stunt virus genome

“…Similar RSEÀ39CSS interactions are also proposed for Turnip crinkle virus (TCV) (Zhang et al 2004) and are predicted in other genera in the family Tombusviridae (Koev et al 2002;Na and White 2006). For TBSV (genus Tombusvirus, family Tombusviridae), this interaction mediates replicase complex formation in vivo and modulates negative-strand synthesis in vitro (Panaviene et al 2005;Pogany et al 2003). The latter activity has also been demonstrated in vitro for TCV (genus Carmovirus, family Tombusviridae) and the interaction is required for optimal virus accumulation in vivo (Zhang et al 2004(Zhang et al , 2006b).…”

“…Therefore, the in vivo defects in DI RNA accumulation observed are likely due, at least in part, to reduced ability to form the RSEÀ39CSS interaction. S-Z formation could act to prevent misfolding or stabilize the RSEÀ39CSS domain once formed, which in turn would facilitate replication complex formation and/or 39 end protection (Pogany et al 2003;Panaviene et al 2005). What is interesting is that S-Z appears to be a polar dualfunction element that is increasingly important for translation in its upper region and replication in its lower portion (Fig.…”

“…Additionally, this structure was recently shown to be important for formation of tombusvirus replication complexes (Panaviene et al 2005). Thus, the closed default structure may be important for both genome protection and early replication events.…”

Identification of Essential Host Factors Affecting Tombusvirus RNA Replication Based on the Yeast Tet Promoters Hughes Collection

“…Each sample was further mixed with 600 l prechilled extraction buffer, and unbroken cells were removed by centrifugation at 100 ϫ g for 5 min at 4°C (26). The supernatant was centrifuged for 10 min at 21,000 ϫ g at 4°C, and then the pellet was resuspended and used in a standard CNV replicase assay (29,30). Because no template was added to the in vitro reaction, the replicase preparation could only use the endogenous template present within the enriched membrane fraction.…”

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

“…Estudios previos con algunos miembros de la familia Tombusviridae indicaban que una de las funciones llevadas a cabo por este tipo de proteínas es probablemente la de dirigir y/o facilitar el ensamblaje del complejo replicativo en membranas celulares específicas [Weber-Lotfi et al, 2002;Navarro et al, 2004;Turner et al, 2004;Panavas et al, 2005a;Mochizuki et al, 2009]. Por otra parte, para la proteína auxiliar de la replicación, p33, de un par de tombusvirus, el TBSV y el CNV, se habían descrito propiedades de unión a ssRNAs que eran consistentes con la posible implicación de este producto en el reclutamiento del molde hacia los sitios de síntesis del vRNA [Rajendran & Nagy, 2003;Panaviene et al, 2003Panaviene et al, , 2005Pogany et al, 2005;Panavas et al, 2005a]. Los resultados obtenidos en esta Tesis proporcionan datos en ambas direcciones para la proteína p27 del PFBV, lo que sugiere que estas funciones están conservadas en las proteínas auxiliares de la replicación de los distintos componentes de la familia.…”