Live‐cell imaging of viral RNA genomes using a Pumilio‐based reporter

Abstract

SummaryWe describe a method for localizing plant viral RNAs in vivo using Pumilio, an RNA-binding protein, coupled to bimolecular fluorescence complementation (BiFC). Two Pumilio homology domain (PUMHD) polypeptides, fused to either the N-or C-terminal halves of split mCitrine, were engineered to recognize two closely adjacent eight-nucleotide sequences in the genomic RNA of tobacco mosaic virus (TMV). Binding of the PUMHDs to their target sites brought the split mCitrine halves into close proximity, allowing BiFC to occur and revealing the localization of viral RNA within infected cells. The bulk of the RNA was sequestered in characteristic inclusion bodies known as viral replication complexes (VRCs), with a second population of RNA localized in discrete particles distributed throughout the peripheral cytoplasm. Transfer of the TMV Pumilio recognition sequences into the genome of potato virus X (PVX) allowed the PVX RNA to be localized. Unlike TMV, the PVX RNA was concentrated in distinctive 'whorls' within the VRC. Optical sectioning of the PVX VRCs revealed that one of the viral movement proteins was localized to the centres of the RNA whorls, demonstrating significant partitioning of viral RNA and proteins within the VRC. The utility of Pumilio as a fluorescence-based reporter for viral RNA is discussed.

Pumilio-Based RNA In Vivo Imaging

“…Pumilio-based RNA imaging has enabled subcellular detection of the mRNAs of mitochondrial NADH dehydrogenase and -actin, as well as retroviral RNA in mammalian cultured cells (20)(21)(22)(23), and of the genomes of Tobacco mosaic virus, Potato virus X and Turnip mosaic virus in live plant tissue (24)(25)(26)(27). Of particular relevance to the topic of this volume, Pumilio-based RNA imaging has recently shown that Potato virus X is present in membrane structures at the entrances of plasmodesmata that also contain the viral replicase (27).…”

“…The advantages of using modified PUMHDs for RNA imaging are 1) untagged, native RNA species expressed from their native genomic context can be localised (20,21,24), avoiding both the need to modify the RNA and to introduce expression constructs into the genome; 2) if instead PUMHD recognition sequences are engineered into an RNA as a tag, no extensive secondary structures are introduced which might affect RNA function and localisation (though the PUMHD can actually access substantially structured target motifs (18)); 3) PUMHD variants can have 10-to 100-fold higher RNA affinities than stem-loop binding peptides used in alternative RNA imaging systems, thus PUMHD-based imaging is potentially more sensitive.…”

“…Alternatively, the two PUMHD variants can be fused separately to two halves of a split FP, so that when both fusion proteins bind to the same target RNA, the split FP halves come into close proximity and re-fold into the complete FP, resulting in bimolecular fluorescence complementation (PUM-BiFC) at the location of the RNA (20,24) (Fig 1B). This principle is the only form of Pumilio-based RNA imaging so far applied in plants ( [24][25][26][27].…”

“…This principle is the only form of Pumilio-based RNA imaging so far applied in plants ( [24][25][26][27].…”

“…An additional side-effect of RNA-independent formation of PUM-BiFC complexes is that these pre-formed complexes can be recruited to the target RNA by the interaction of only one of the two PUMHDs. Thus, RNAs can be imaged with only a single binding site (24,25) or when the orientation of PUMHD fusions would be expected to prevent proximity of split FP halves after RNA binding (see below) (22). Whilst this may sometimes be useful, it is not generally desirable, as it reduces the overall specificity of the PUM-BiFC system and eliminates the benefits of using two RNA binding domains.…”