A Dual Strategy for the Suppression of Host Antiviral Silencing: Two Distinct Suppressors for Viral Replication and Viral Movement Encoded by Potato Virus M

Abstract

Viruses encode RNA silencing suppressors to counteract host antiviral silencing. In this study, we analyzed the suppressors encoded by potato virus M (PVM), a member of the genus Carlavirus. In the conventional green fluorescent protein transient coexpression assay, the cysteine-rich protein (CRP) of PVM inhibited both local and systemic silencing, whereas the triple gene block protein 1 (TGBp1) showed suppressor activity only on systemic silencing. Furthermore, to elucidate the roles of these two suppressors during an active viral infection, we performed PVX vector-based assays and viral movement complementation assays. CRP increased the accumulation of viral RNA at the single-cell level and also enhanced viral cell-to-cell movement by inhibiting RNA silencing. However, TGBp1 facilitated viral movement but did not affect viral accumulation in protoplasts. These data suggest that CRP inhibits RNA silencing primarily at the viral replication step, whereas TGBp1 is a suppressor that acts at the viral movement step. Thus, our findings demonstrate a sophisticated viral infection strategy that suppresses host antiviral silencing at two different steps via two mechanistically distinct suppressors. This study is also the first report of the RNA silencing suppressor in the genus Carlavirus.Viruses are obligate intracellular pathogens that manipulate and exploit the molecular mechanism of the host to survive in a hostile cellular environment. The presence of viruses and their propagation induces diverse mechanisms in the host for combating viral infection at both the single-cell and the wholeorganism levels. In plants, one of the most important defense mechanisms against viruses is RNA silencing, which is a sequence-specific RNA degradation process regulated by small RNA molecules (11,12). Since RNA viruses go through singlestranded RNA or double-stranded RNA (dsRNA) stages at a given point in their replication, they are both active initiators and targets of host RNA silencing. When plant RNA viruses invade host cells, dsRNAs derived from their replication intermediates or highly structured genomic RNAs are recognized by plant dicer-like nucleases and processed into small interfering RNAs (siRNAs) of 21 to 24 nucleotides in length. The siRNAs are then recruited to a multiprotein effector complex called the RNA-induced silencing complex (RISC), which includes the slicer endonuclease Argonaute and subsequently mediates the cleavage of cognate viral RNAs. RNA silencing is a non-cell-autonomous event in higher plants and, once RNA silencing is induced in the initial cell, it spreads over the whole organism through the vasculature and from cell to cell presumably via plasmodesmata, mediated by mobile small RNA signals (3,14,22,36,48,51). The cell-to-cell and long-distance movement of virus-derived small RNA signals likely serves to immunize surrounding naive cells ahead of the infection front.

An RNA Virus-Encoded Zinc-Finger Protein Acts as a Plant Transcription Factor and Induces a Regulator of Cell Size and Proliferation in Two Tobacco Species

“…The p12-like protein of another carlavirus, potato virus M, has a different function and acts in concert with the TGB1 (for Triple Gene Block) protein as a silencing suppressor to enhance viral RNA accumulation, cell-to-cell movement, and systemic transport (Senshu et al, 2011). On the other hand, we demonstrated explicitly that CVB p12 did not suppress RNA interference and, moreover, did not suppress microRNA-mediated mRNA cleavage (Lukhovitskaya et al, 2005(Lukhovitskaya et al, , 2009).…”

In Planta Recognition of a Double-Stranded RNA Synthesis Protein Complex by a Potexviral RNA Silencing Suppressor

“…Hence, the amplification of RNA silencing by RDR6 is also important in terms of inhibiting virus movement, suggesting that inhibition of this process may have a significant beneficial effect on plant viruses. Therefore, VSRs that prevent the systemic spread of RNA silencing, such as TGBp1 of potato carlavirus M, p50 of apple chlorotic leaf spot trichovirus, and the coat protein of citrus tristeza closterovirus, may also inhibit the function of SGS3/RDR6 (Lu et al, 2004;Yaegashi et al, 2007;Senshu et al, 2011). Future studies will make it clear whether these VSRs actually suppress the activities of SGS3 and/or RDR6, thereby inhibiting the amplification of RNA silencing, to facilitate the escape of plant viruses from secondary siRNAs in both replicating and newly entered cells.…”

“…Confocal laser scanning microscopy analysis to detect CFP and YFP was performed as described previously (Senshu et al, 2011). For the BiFC assay, Agrobacterium tumefaciens culture with a binary vector expressing YFP N -tagged protein and a vector expressing YFP C -tagged protein was infiltrated into N. benthamiana leaves.…”

Fig mosaic emaravirus p4 protein is involved in cell-to-cell movement

“…In this assay, we utilized a GFP-tagged movement-defective mutant of PVX (PVXDTGBp1-GFP; Senshu et al, 2011), which lacks most of the triple gene block protein 1 gene (TGBp1). The pEarleyGate 100 was used for the expression of GUS, TGBp1 and FMV-p4.…”