Helper Virus-Independent Transcription and Multimerization of a Satellite RNA Associated with Cucumber Mosaic Virus

Abstract

Satellite RNAs are the smallest infectious agents whose replication is thought to be completely dependent on their helper virus (HV). Here we report that, when expressed autonomously in the absence of HV, a variant of satellite RNA (satRNA) associated with Cucumber mosaic virus strain Q (Q-satRNA) has a propensity to localize in the nucleus and be transcribed, generating genomic and antigenomic multimeric forms. The involvement of the nuclear phase of Q-satRNA was further confirmed by confocal microscopy employing in vivo RNA-tagging and double-stranded-RNA-labeling assays. Sequence analyses revealed that the Q-satRNA multimers formed in the absence of HV, compared to when HV is present, are distinguished by the addition of a template-independent heptanucleotide motif at the monomer junctions within the multimers. Collectively, the involvement of a nuclear phase in the replication cycle of Q-satRNA not only provides a valid explanation for its persistent survival in the absence of HV but also suggests a possible evolutionary relationship to viroids that replicate in the nucleus.

A Bromodomain-Containing Host Protein Mediates the Nuclear Importation of a Satellite RNA of Cucumber Mosaic Virus

“…We previously demonstrated that Q-satRNA has a propensity to enter the nucleus in the presence and absence of its HV (13). To shed more light on the subcellular compartmentalization of Q-satRNA, we wanted to analyze the distribution of Q-satRNA in the presence and absence of HV during early stages of replication.…”

“…Thus, we performed a bacteriophage MS2-coat protein (MS2-CP) RNA tagging assay that allows visualizing the subcellular location of Q-satRNA in living cells (31) in wild-type (wt) and BRP1-suppressed transgenic lines of N. benthamiana. Briefly, a 23-nt MS2-CP binding site was engineered into stem loop C of Q-satRNA by the double joint PCR method as described previously (13). This approach allows specific interaction between RNA molecules bearing binding sites and RNAbinding proteins (such as MS2-CP), permitting the visualization of subcellular locations of RNA molecules in living cells when these molecules are coexpressed with an RNA-binding protein fused with GFP (such as MS2-CP-GFP).…”

“…The construction and characteristic features of agroconstructs of genomic RNA of the Q strain of Cucumber mosaic virus, Q-satRNA, and RNA5 (Q5) have been described previously (13,23). The construction and characteristic features of agroconstructs of VIRP1-FLAG (renamed in this study as BRP1-FLAG) and VIRP1-green fluorescent protein (VIRP1-GFP; renamed in this study as BRP1-GFP) were as described previously (22).…”

“…The bacteriophage MS2-CP RNA tagging assay to localize the Q-satRNA in wild-type and ph5.2nb lines of N. benthamiana was performed as described previously (13). Prior to performing confocal microscopy using Leica TCS SP2, at 2 days postinfection (dpi), the leaves were infiltrated with 1:1,000 dilution of 4=,6-diamidino-2-phenylindole (DAPI) in phosphate-buffered saline (PBS).…”

“…Recent application of molecular and cell biology approaches showed that when expressed in the absence of Cucumber mosaic virus Q strain, Q-satRNA has the propensity to localize in the nucleus and be transcribed to generate multimers of genomic and antigenomic strands (13,14). This previously unrecognized novel feature could account for the persistent survival of Cucumber mosaic virus Q-satRNA in the absence of HV (1,15).…”