Insights into the Single-Cell Reproduction Cycle of Members of the Family Bromoviridae : Lessons from the Use of Protoplast Systems

Abstract

2The development of single-cell protoplast systems is certainly one of the milestones in the history of plant virology, allowing for the analysis of viral molecular processes at the cellular level. The use of plant cell-based systems in the study of the Bromoviridae family of multipartite single-stranded plant RNA viruses facilitated the discovery and dissection of viral processes engaged in the single-cell reproduction cycle: replication, transcription, protein synthesis, movement, virion assembly, and RNA recombination. This review summarizes the application of protoplast systems to the analysis of consecutive steps of the bromovirus life cycle, emphasizing their temporal and spatial patterns during virus multiplication.Our knowledge of viral infection at the individual cell level determines our understanding of the infection at the entire plant body level. The idea of applying single-cell systems in virology was introduced with the first attempts to transfect Escherichia coli cells with a T4 anti-E. coli bacteriophage (27). E. C. Cocking was the first (1960) to enzymatically isolate plant cells (19), which were then infected with Tobacco mosaic tobamovirus (TMV) (20). The ensuing pioneer work showed that the infection was synchronous and that the uptake of viral particles/RNAs by protoplasts was efficient enough to support virus replication (5, 118). The first viral infection of protoplasts involved the use of poly-L-ornithine (5), but the trials that followed exploited the fusogenic polymers (24), liposomes (28), or electroporation (78,129). By 1980, these procedures had been successfully employed to transfect protoplasts from more than six species of plants, including various members of the Bromoviridae family, e.g., Brome mosaic bromovirus (BMV; 80), Cucumber mosaic cucumovirus (CMV; 69), and Cowpea chlorotic mottle virus (CCMV; 130). The first attempts were based on the transfection of plant cells with whole-virus particles (104). Later on, viral RNA and its chemical modifications were used as the inoculum (62). Further advances in nucleic acid technology, especially the accessibility of infectious transcripts, have broadened the application of protoplast systems to the study of Bromoviridae.The Bromoviridae constitute one of the most important families of plant RNA viruses. They are distributed worldwide, they infect an extensive range of hosts, and some of them (e.g., CMV and Broad bean mottle bromovirus) are responsible for major crop epidemics (111, 63). The family consists of five genera named after their most representative members: Alfamovirus, Bromovirus, Cucumovirus, Ilarvirus, and Oleavirus. All these viruses possess tripartite, single-stranded, positive-sense RNA genomes (Fig. 1). RNA1 and RNA2 encode the RNAdependent RNA polymerase (RdRp) proteins 1a and 2a, respectively. The dicistronic RNA3 encodes the movement protein (MP) and coat protein (CP). The latter is translated from subgenomic RNA 4 (sgRNA4). Many of the family members, such as CMV, BMV, and Alfalfa mosaic alfamovirus (AMV), repres...

Subcelullar localization of proteins associated with Prune dwarf virus replication

“…Nevertheless, a strong labeling of vesicles and ER was showed in case of Brome mosaic virus (BMV), another member of the Bromoviridae family (Bamunusinghe et al 2011). Sztuba-Solińska andBujarski (2008) showed that the sites of BMV replication are ER membranes, and the vesicles differentiating from the ER membrane can be a potential site of particles accumulation. BMV creates special membrane structuresspherules in which replication occurs.…”

Subgenomic messenger RNAs: Mastering regulation of (+)-strand RNA virus life cycle

“…1A) (Wierzchoslawski et al, 2006). The binding of another RDRP molecule to the near-by SGP promoter core hairpin which initiates the SG RNA4 transcription might pose an additional obstacle to the progression of the RDRP during (+) strand synthesis (Wierzchoslawski et al, 2006;Sztuba-Solińska and Bujarski, 2008).…”

Recombination of 5′ subgenomic RNA3a with genomic RNA3 of Brome mosaic bromovirus in vitro and in vivo

“…Also, in Cucumber mosaic cucumovirus (CMV), which is closely related to BMV, the RNA3 component lacks the polyA tract and it does not result in formation of homologous recombinants (de Wispelaere et al, 2005). Yet another effect might be due to the close proximity of the sgRNA4 transcription site, which may enhance RdRp recruitment (Sztuba-Solińska and Bujarski, 2008).…”