RESUMEN
RNA transport and localization are evolutionarily conserved processes that allow protein translation to occur at specific subcellular sites and thereby having fundamental roles in the determination of cell fates, embryonic patterning, asymmetric cell division, and cell polarity. In addition to localizing RNA molecules to specific subcellular sites, plants have the ability to exchange RNA molecules between cells through plasmodesmata (PD). Plant RNA viruses hijack the mechanisms of intracellular and intercellular RNA transport to establish localized replication centers within infected cells and then to disseminate their infectious genomes between cells and throughout the plant organism with the help of their movement proteins (MP). In this chapter, we describe the transient expression of the tobacco mosaic virus movement protein (TMV-MP) and the application of the MS2 system for the in vivo labeling of the MP-encoding mRNA. The MS2 method is based on the binding of the bacteriophage coat protein (CP) to its origin of assembly (OAS) in the phage RNA. Thus, to label a specific mRNA in vivo, a tandem repetition of a 19-nucleotide-long stem-loop (SL) sequence derived from the MS2 OAS sequence (MSL) is transcriptionally fused to the RNA under investigation. The RNA is detected by the co-expression of fluorescent protein-tagged MS2 CP (MCP), which binds to each of the MSL elements. In providing a detailed protocol for the in vivo visualization of TMV-MP mRNA tagged with the MS2 system in Nicotiana benthamiana epidermal cells, we describe (1) the specific DNA constructs, (2) Agrobacterium tumefaciens-mediated transfection for their transient expression in plants, and (3) imaging conditions required to obtain high-quality mRNA imaging data.
Asunto(s)
Agrobacterium tumefaciens/genética , Levivirus/metabolismo , Proteínas de Movimiento Viral en Plantas/genética , Transporte de ARN/genética , ARN Mensajero/metabolismo , ARN de Planta/metabolismo , ARN Viral/genética , Virus del Mosaico del Tabaco/metabolismo , Transporte Biológico , Proteínas de la Cápside/genética , Proteínas de la Cápside/metabolismo , Clonación Molecular , Expresión Génica , Vectores Genéticos , Levivirus/genética , Proteínas Luminiscentes , Microscopía Fluorescente , Proteínas de Movimiento Viral en Plantas/metabolismo , Plantas Modificadas Genéticamente/genética , Plasmodesmos/metabolismo , ARN Mensajero/genética , Nicotiana/genética , Nicotiana/metabolismo , Virus del Mosaico del Tabaco/genéticaRESUMEN
To investigate the role of protein-protein and protein-nucleic acid interactions in virus assembly, we compared the stabilities of native bacteriophage MS2, virus-like particles (VLPs) containing nonviral RNAs, and an assembly-defective coat protein mutant (dlFG) and its single-chain variant (sc-dlFG). Physical (high pressure) and chemical (urea and guanidine hydrochloride) agents were used to promote virus disassembly and protein denaturation, and the changes in virus and protein structure were monitored by measuring tryptophan intrinsic fluorescence, bis-ANS probe fluorescence, and light scattering. We found that VLPs dissociate into capsid proteins that remain folded and more stable than the proteins dissociated from authentic particles. The proposed model is that the capsid disassembles but the protein remains bound to the heterologous RNA encased by VLPs. The dlFG dimerizes correctly, but fails to assemble into capsids, because it lacks the 15-amino acid FG loop involved in inter-dimer interactions at the viral fivefold and quasi-sixfold axes. This protein was very unstable and, when compared with the dissociation/denaturation of the VLPs and the wild-type virus, it was much more susceptible to chemical and physical perturbation. Genetic fusion of the two subunits of the dimer in the single-chain dimer sc-dlFG stabilized the protein, as did the presence of 34-bp poly(GC) DNA. These studies reveal mechanisms by which interactions in the capsid lattice can be sufficiently stable and specific to ensure assembly, and they shed light on the processes that lead to the formation of infectious viral particles.
Asunto(s)
ADN Viral , Levivirus , Proteínas Virales , Naftalenosulfonatos de Anilina/química , Colorantes Fluorescentes/química , Guanidina/química , Calor , Levivirus/química , Levivirus/genética , Levivirus/metabolismo , Sustancias Macromoleculares , Mutación , Conformación de Ácido Nucleico , Conformación Proteica , Desnaturalización Proteica , Urea/química , Proteínas Virales/química , Proteínas Virales/genética , Proteínas Virales/metabolismoRESUMEN
The effects of intracellular levels of polyamines on both the in vivo inhibition of protein synthesis and the decrease of translation accuracy induced by streptomycin have been studied in polyamine-auxotrophic strains of Escherichia coli infected with the MS2 bacteriophage. The amount of viral coat protein formed was strongly reduced upon addition of increasing concentrations of streptomycin to polyamine-supplemented bacteria. In contrast, the antibiotic almost did not inhibit coat protein synthesis in polyamine-starved cells. The increase of mistranslation frequency elicited by streptomycin was only observed in bacteria grown with putrescine. In these cells several coat protein-satellites were detected after two-dimensional gel electrophoresis. These proteins, more basic than the normal MS2 coat protein, contain multiple substitutions of lysine for asparagine.