The Viroid, the Polymerase, and the Transcription Factor: Replication of a Naked, Noncoding RNA Pathogen by Host Proteins

An amazing, perhaps frightening, example of the versatility and power of RNA, viroid plant pathogens consist of a circular, noncoding RNA genome of roughly 200 to 400 nucleotides, with no protein capsule or membrane (reviewed in Ding, 2009). Viroids rely on host proteins for replication, subcellular localization, cell-to-cell movement, and systemic transmission. Viroids pass from plant to plant via mechanical means or insect vectors and cause devastating diseases in many crop plants. Moreover, the simplicity of viroids and their high mutation and recombination rates make them devilishly tricky to control (reviewed in Kovalskaya and Hammond, 2014); for example, the strong secondary structure of the viroid genome and other aspects of viroid biology can defeat RNA interference-mediated defenses in plant cells.

For replication, the viroid genome is transcribed by plant DNA-dependent RNA polymerases (DdRPs), which also have RNA-dependent RNA polymerase (RdRP) activity. Transcription of the viroid genome by a rolling-circle mechanism forms a concatenated (−) intermediate that then is transcribed and cut to form (+) copies. To understand viroid replication, Wang et al. (2016) looked for factors that interact with Potato spindle tuber viroid (PSTVd) in Nicotiana benthamiana. They first used RNA immunoprecipitation with antibodies to DdRP II (Pol II) to show that Pol II interacts with both (+)- and (−)-PSTVd. The authors also used electrophoretic mobility shift assays to show that PSTVd interacts with the eukaryotic general Transcription Factor IIIA (TFIIIA). In land plants, TFIIIA has two splice variants, one encoding a highly conserved protein with nine zinc fingers (TFIIIA-9ZF) and one with seven (TFIIIA-7ZF). Intriguingly, these splice variants differed in their binding to the (+) and (−) forms of PSTVd: TFIIIA-9ZF bound to (+)-PSTVd and TFIIIA-7ZF bound to both (+)- and (−)-PSTVd, as shown by electrophoretic mobility shift and immunoprecipitation assays. Furthermore, RNase protection assays showed that the two forms of TFIIIA bound to different parts of the PSTVd genome, with TFIIIA-7ZF binding to the left-terminal region, which is important for initiation of transcription, and TFIIIA-9ZF binding to the right region, which is important for systemic trafficking (see figure).

N. benthamiana TFIIIA-7ZF and -9ZF bind to different parts of the PSTVd genome. RNase protection assays show that TFIIIA-7ZF binds to the region critical for replication and TFIIIA-9ZF binds to a region critical for trafficking. T1, RNase T1, which cleaves single-stranded RNA to the 3′ of G residues; V1, RNase V1, which cleaves double-stranded RNA. Red circles and diamonds indicate protected sites for RNases T1 and V1, respectively. (Reprinted from Wang et al. [2016], Figure 6.)

The authors next examined the functional implications of this difference in binding by the two forms of TFIIIA. RNA interference targeting TFIIIA decreased the levels of TFIIIA-7ZF but unexpectedly increased the levels of TFIIIA-9ZF; these plants showed very low accumulation of PSTVd when infected with the viroid. Transient overexpression of TFIIIA-7ZF in infected plants caused higher accumulation of PSTVd RNA, but TFIIIA-9ZF and a green fluorescent protein control had no effect. Examination of cells transiently expressing yellow fluorescent protein-tagged TFIIIAs showed that both TFIIIA-9ZF and -7ZF localized in the nucleolus, but TFIIIA-7ZF also localized in the nucleoplasm, in speckles reminiscent of Pol II transcription sites. Indeed, TFIIIA-7ZF and -9ZF both interact with Pol II, although the subcellular localization of TFIIIA-9ZF indicates that this interaction may not occur in vivo. Finally, the authors used an in vitro reaction system to show that adding TFIIIA-7ZF increases transcription of PSTVd by Pol II. Thus, these results provide strong evidence that TFIIIA-7ZF, but not TFIIIA-9ZF, has important functions in the transcription of PSTVd. The function of TFIIIA-7ZF in plant cells remains to be identified, but decreasing the amount of the splice form that encodes TFIIIA-7ZF may hold promise for control of viroid infections. Moreover, examination of this system and the mechanism by which TFIIIA-7ZF acquired its interaction with Pol II and the ability to promote RdRP activity of a DdRP remain intriguing questions for future research.