One of the systems of plant defense against viral infection is RNA silencing, or RNA interference (RNAi), in which small RNAs derived from viral genomic RNAs and/or mRNAs serve as guides to target an Argonaute nuclease (AGO) to virus-specific RNAs. Complementary base pairing between the small interfering RNA incorporated into the AGO-based protein complex and viral RNA results in the target cleavage or translational repression. As a counter-defensive strategy, viruses have evolved to acquire viral silencing suppressors (VSRs) to inhibit the host plant RNAi pathway. Plant virus VSR proteins use multiple mechanisms to inhibit silencing. VSRs are often multifunctional proteins that perform additional functions in the virus infection cycle, particularly, cell-to-cell movement, genome encapsidation, or replication. This paper summarizes the available data on the proteins with dual VSR/movement protein activity used by plant viruses of nine orders to override the protective silencing response and reviews the different molecular mechanisms employed by these proteins to suppress RNAi.

Plant viruses exploit the endomembrane system of infected cells for their replication and cell-to-cell transport. The replication of viral RNA genomes occurs in the cytoplasm in association with reorganized endomembrane compartments induced by virus-encoded proteins and is coupled with the virus intercellular transport via plasmodesmata that connect neighboring cells in plant tissues. The transport of virus genomes to and through plasmodesmata requires virus-encoded movement proteins (MPs). Distantly related plant viruses encode different MP sets, or virus transport systems, which vary in the number of MPs and their properties, suggesting their functional differences. Here, we discuss two distinct virus transport pathways based on either the modification of the endoplasmic reticulum tubules or the formation of motile vesicles detached from the endoplasmic reticulum and targeted to endosomes. The viruses with the movement proteins encoded by the triple gene block exemplify the first, and the potyviral system is the example of the second type. These transport systems use unrelated mechanisms of endomembrane reorganization. We emphasize that the mode of virus interaction with cell endomembranes determines the mechanism of plant virus cell-to-cell transport.

Movement proteins (MPs) of plant viruses enable the translocation of viral genomes from infected to healthy cells through plasmodesmata (PD). The MPs functions involve the increase of the PD permeability and routing of viral genome both to the PD entrance and through the modified PD. Hibiscus green spot virus encodes two MPs, termed BMB1 and BMB2, which act in concert to accomplish virus cell-to-cell transport. BMB1, representing an NTPase/helicase domain-containing RNA-binding protein, localizes to the cytoplasm and the nucleoplasm. BMB2 is a small hydrophobic protein that interacts with the endoplasmic reticulum (ER) membranes and induces local constrictions of the ER tubules. In plant cells, BMB2 localizes to PD-associated membrane bodies (PAMBs) consisting of modified ER tubules and directs BMB1 to PAMBs. Here, we demonstrate that BMB1 and BMB2 interact in vitro and in vivo, and that their specific interaction is essential for BMB2-directed targeting of BMB1 to PAMBs. Using mutagenesis, we show that the interaction involves the C-terminal BMB1 region and the N-terminal region of BMB2.

To move from cell to cell through plasmodesmata, many plant viruses require the concerted action of two or more movement proteins (MPs) encoded by transport gene modules of virus genomes. A tetra-cistron movement block (TCMB) is a newly discovered transport module comprising four genes. TCMB encodes three proteins, which are similar to MPs of the transport module known as the “triple gene block”, and a protein unrelated to known viral MPs and containing a double-stranded RNA (dsRNA)-binding domain similar to that found in a family of cell proteins, including AtDRB4 and AtHYL1. Here, the latter TCMB protein, named vDRB for virus dsRNA-binding protein, is shown to bind both dsRNA and single-stranded RNA in vitro. In a turnip crinkle virus-based assay, vDRB exhibits the properties of a viral suppressor of RNA silencing (VSR). In the context of potato virus X infection, vDRB significantly decreases the number and size of “dark green islands”, regions of local antiviral silencing, supporting the VSR function of vDRB. Nevertheless, vDRB does not exhibit the VSR properties in non-viral transient expression assays. Taken together, the data presented here indicate that vDRB is an RNA-binding protein exhibiting VSR functions in the context of viral infection.

The genomes of viruses in the Allexivirus genus encode the p42 protein, which is considered the hallmark of the genus. The functions of p42 have not yet been studied experimentally and cannot be predicted based on sequence similarity, as p42-related proteins are not found among known cell or viral proteins. Here, p42 of Shallot virus X (ShVX), the type allexivirus, is demonstrated to be translated via a leaky scanning mechanism on a template comprising three “triple gene block” (TGB) transport genes and the p42 gene. Sequence analysis shows that this p42 expression mechanism is conserved in the vast majority of allexiviruses. p42 binds single-stranded RNA (ssRNA) but not double-stranded RNA (dsRNA) in vitro and localizes to the cytoplasm in association with microtubules and microtubule-bound bodies. In transient expression assays, p42 exhibits weak but detectable suppression of silencing induced by ssRNA but not by dsRNA. In addition, p42 suppresses silencing in the context of virus infection. Furthermore, p42 inhibits nonsense-mediated RNA decay (NMD) induced by a long 3′-terminal untranslated region of mRNA. Taken together, these findings provide initial evidence that the ShVX TGB/p42 gene module functions as a single genomic unit in terms of protein expression, that p42 acts as a suppressor of NMD and silencing, and that it may have multiple roles, while the precise biological significance of p42 in these roles remains to be experimentally confirmed.