cis-Acting sequences required for coat protein binding and in vitro assembly of Potato virus X

Abstract

The 5' region of Potato virus X (PVX) RNA containing an AC-rich single-stranded region and stem-loop 1 (SL1) has been shown to be important for PVX replication (Miller, E.D., Plante, C.A., Kim, K.-H., Brown, J.W., Hemenway, C., 1998. Stem-loop structure in the 5' region of potato virus X genome required for plus-strand RNA accumulation. J. Mol. Biol. 284, 591-608.). Here, we describe the involvement of SL1 for binding to the PVX coat protein (CP) using an in vitro assembly system and various deletion mutants of the 5' region of PVX RNA. Internal and 5' terminal deletions of the 5'-nontranslated region of PVX RNA were assessed for their effects on formation of assembled virus-like particles (VLPs). Mutant RNAs that contain the top region of SL1 or sequences therein bound to CP to form VLPs. In contrast, transcripts of mutants that disrupt SL1 RNA structure were unable to form VLPs. SELEX was used to further confirm the specific RNA recognition of PVX CP using RNA transcripts containing randomized sequences of the upper portion of SL1. Wild-type (wt) sequences along with many other sequences that resemble SL1 structure were selected after fourth and fifth rounds of SELEX (27.0% and 44.4%, respectively). RNA transcripts from several SELEX winners that are predicted to form stable stem-loop structures very closely resembling wt PVX SL1 VLPs. RNA transcripts not predicted to form secondary structures similar to SL1 did not form VLPs in vitro. Taken together, our results suggest that RNA secondary structural elements within SL1 and/or sequences therein are crucial for formation of VLPs and are required for the specific recognition by the CP subunit.

Identification of Tobacco Proteins Associated with the Stem-Loop 1 RNAs of Potato virus X

“…PVX CP gene was cloned and expressed in E. coli using pET vector system (Novagen). The purification of His-tagged CP fusion protein was purified as described previously (Kwon et al, 2005). The extracted protein contents were determined with a Bradford analysis and used as positive control for north-…”

“…As a positive control, we used PVX CP, which was previously demonstrated to bind to SL1(+) RNA in vitro (Kwon et al, 2005). Coomassie Brilliant Blue staining of the gel revealed numerous bands ( Fig.…”

“…1B). Previous studies demonstrated that the SL1 RNAs are involved in PVX translation, RNA replication, and initiation of PVX assembly (Kim and Hemenway, 1997;Kwon et al, 2005).…”

Cell-to-cell movement of three genera (+) ss RNA plant viruses

“…5 0 SL1 is a multifunctional element, involved in virus replication, cellto-cell transport and virions assembling. Host proteins may identify 5 0 SL1 by capsidation of viral particles, they can promote translation of replication of genome RNA (Kwon et al 2005). A series of deletion mutation was used to (Rouleau et al 1995;Oparka et al 1999).…”

Study of the Initial Steps of Potato Virus X Assembly

“…Kim and C. Hemenway (6) suggested that 5´-end of PVX RNA can be involved into coordination of viral part assembly. The region responsible for initiation of PVX assembling is not completely characterized, though in different publications there are evidences for its location at the 5´-end of RNA molecule (7)(8)(9). Recently published data suggest a key role of a cap-structure at the 5´-end of RNA in assembling vRNP of PVX through changing conformation of the 5´-end of RNA and, as a result, the CP recognition at initiation of the particle assembly (10).…”