Acquisition and transmission by an insect vector is central to the infection cycle of the majority of plant pathogenic viruses. Plant viruses can interact with their insect host in a variety of ways including both non-persistent and circulative transmission; in some cases, the latter involves virus replication in cells of the insect host. Replicating viruses can also elicit both innate and specific defense responses in the insect host. A consistent feature is that the interaction of the virus with its insect host/vector requires specific molecular interactions between virus and host, commonly via proteins. Understanding the interactions between plant viruses and their insect host can underpin approaches to protect plants from infection by interfering with virus uptake and transmission. Here, we provide a perspective focused on identifying novel approaches and research directions to facilitate control of plant viruses by better understanding and targeting virus–insect molecular interactions. We also draw parallels with molecular interactions in insect vectors of animal viruses, and consider technical advances for their control that may be more broadly applicable to plant virus vectors.

Many plant viruses express their proteins through a polyprotein strategy, requiring the acquisition of protease domains to regulate the release of functional mature proteins and/or intermediate polyproteins. Positive-strand RNA viruses constitute the vast majority of plant viruses and they are diverse in their genomic organization and protein expression strategies. Until recently, proteases encoded by positive-strand RNA viruses were described as belonging to two categories: (1) chymotrypsin-like cysteine and serine proteases and (2) papain-like cysteine protease. However, the functional characterization of plant virus cysteine and serine proteases has highlighted their diversity in terms of biological activities, cleavage site specificities, regulatory mechanisms, and three-dimensional structures. The recent discovery of a plant picorna-like virus glutamic protease with possible structural similarities with fungal and bacterial glutamic proteases also revealed new unexpected sources of protease domains. We discuss the variety of plant positive-strand RNA virus protease domains. We also highlight possible evolution scenarios of these viral proteases, including evidence for the exchange of protease domains amongst unrelated viruses.

Strawberry mottle virus (SMoV) belongs to the family Secoviridae (order Picornavirales) and has a bipartite genome with each RNA encoding one polyprotein. All characterized secovirids encode a single protease related to the picornavirus 3C protease. The SMoV 3C-like protease was previously shown to cut the RNA2 polyprotein (P2) at a single site between the predicted movement protein and coat protein (CP) domains. However, the SMoV P2 polyprotein includes an extended C-terminal region with a coding capacity of up to 70 kDa downstream of the presumed CP domain, an unusual characteristic for this family. In this study, we identified a novel cleavage event at a P↓AFP sequence immediately downstream of the CP domain. Following deletion of the PAFP sequence, the polyprotein was processed at or near a related PKFP sequence 40 kDa further downstream, defining two protein domains in the C-terminal region of the P2 polyprotein. Both processing events were dependent on a novel protease domain located between the two cleavage sites. Mutagenesis of amino acids that are conserved among isolates of SMoV and of the related Black raspberry necrosis virus did not identify essential cysteine, serine, or histidine residues, suggesting that the RNA2-encoded SMoV protease is not related to serine or cysteine proteases of other picorna-like viruses. Rather, two highly conserved glutamic acid residues spaced by 82 residues were found to be strictly required for protease activity. We conclude that the processing of SMoV polyproteins requires two viral proteases, the RNA1-encoded 3C-like protease and a novel glutamic protease encoded by RNA2. IMPORTANCE Many viruses encode proteases to release mature proteins and intermediate polyproteins from viral polyproteins. Polyprotein processing allows regulation of the accumulation and activity of viral proteins. Many viral proteases also cleave host factors to facilitate virus infection. Thus, viral proteases are key virulence factors. To date, viruses with a positive-strand RNA genome are only known to encode cysteine or serine proteases, most of which are related to the cellular papain, trypsin, or chymotrypsin proteases. Here, we characterize the first glutamic protease encoded by a plant virus or by a positive-strand RNA virus. The novel glutamic protease is unique to a few members of the family Secoviridae, suggesting that it is a recent acquisition in the evolution of this family. The protease does not resemble known cellular proteases. Rather, it is predicted to share structural similarities with a family of fungal and bacterial glutamic proteases that adopt a lectin fold.

Plant viruses encode movement proteins (MP) to facilitate cell-to-cell transport through plasmodesmata. In this study, using trans-complementation of a movement-defective turnip vein-clearing tobamovirus (TVCV) replicon, we show for the first time for cytorhabdoviruses (lettuce necrotic yellows virus (LNYV) and alfalfa dwarf virus (ADV)) that their P3 proteins function as MP similar to the TVCV P30 protein. All three MP localized to plasmodesmata when ectopically expressed. In addition, we show that these MP belong to the 30K superfamily since movement was inhibited by mutation of an aspartic acid residue in the critical 30K-specific LxD/N50-70G motif. We also report that Nicotiana benthamiana microtubule-associated VOZ1-like transcriptional activator interacts with LNYV P3 and TVCV P30 but not with ADV P3 or any of the MP point mutants. This host protein, which is known to interact with P3 of sonchus yellow net nucleorhabdovirus, may be involved in aiding the cell-to-cell movement of LNYV and TVCV.

Plant viruses have evolved to undermine the RNA silencing pathway by expressing suppressor protein(s) that interfere with one or more key components of this antiviral defense. Here we show that the recently identified RNA silencing suppressor (RSS) of lettuce necrotic yellows virus (LNYV), phosphoprotein P, binds to RNA silencing machinery proteins AGO1, AGO2, AGO4, RDR6 and SGS3 in protein-protein interaction assays when transiently expressed. In planta, we demonstrate that LNYV P inhibits miRNA-guided AGO1 cleavage and translational repression, and RDR6/SGS3-dependent amplification of silencing. Analysis of LNYV P deletion mutants identified a C-terminal protein domain essential for both local RNA silencing suppression and interaction with AGO1, AGO2, AGO4, RDR6 and SGS3. In contrast to other viral RSS known to disrupt AGO activity, LNYV P sequence does not contain any recognizable GW/WG or F-box motifs. This suggests that LNYV P may represent a new class of AGO binding proteins.

RNA silencing in plants and insects provides an antiviral defense and as a countermeasure most viruses encode RNA silencing suppressors (RSS). For the family Rhabdoviridae, no detailed functional RSS studies have been reported in plant hosts and insect vectors. In agroinfiltrated Nicotiana benthamiana leaves we show for the first time for a cytorhabdovirus, lettuce necrotic yellows virus (LNYV), that one of the nucleocapsid core proteins, phosphoprotein (P) has relatively weak local RSS activity and delays systemic silencing of a GFP reporter. Analysis of GFP small RNAs indicated that the P protein did not prevent siRNA accumulation. To explore RSS activity in insects, we used a Flock House virus replicon system in Drosophila S2 cells. In contrast to the plant host, LNYV P protein did not exhibit RSS activity in the insect cells. Taken together our results suggest that P protein may target plant-specific components of RNA silencing post siRNA biogenesis.

Strawberry mottle virus (SMoV, family Secoviridae, order Picornavirales) is one of several viruses found in association with strawberry decline disease in Eastern Canada. The SMoV genome consists of two positive-sense single-stranded RNAs, each encoding one large polyprotein. The RNA1 polyprotein (P1) includes the domains for a putative helicase, a VPg, a 3C-like cysteine protease and an RNA-dependent RNA polymerase at its C-terminus, and one or two protein domains at its N-terminus. The RNA2 polyprotein (P2) is predicted to contain the domains for a movement protein (MP) and one or several coat proteins at its N-terminus, and one or more additional domains for proteins of unknown function at its C-terminus. The RNA1-encoded 3C-like protease is presumed to cleave the two polyproteins in cis (P1) and in trans (P2). Using in vitro processing assays, we systematically scanned the two polyproteins for cleavage sites recognized by this protease. We identified five cis-cleavage sites in P1, with cleavage between the putative helicase and VPg domains being the most efficient. The presence of six protein domains in the SMoV P1, including two upstream of the putative helicase domain, is a feature shared with nepoviruses but not with comoviruses. Results from trans-cleavage assays indicate that the RNA1-encoded 3C-like protease recognized a single cleavage site, which was between the predicted MP and coat protein domains in the P2 polyprotein. The cleavage site consensus sequence for the SMoV 3C-like protease is AxE (E or Q)/(G or S).

Rhabdoviruses are taxonomically classified in the family Rhabdoviridae, order Mononegavirales. As a group, rhabdoviruses can infect plants, invertebrates and vertebrates. Plant cyto- and nucleorhabdoviruses infect a wide variety of species across both monocot and dicot families, including agriculturally important crops such as lettuce, wheat, barley, rice, maize, potato and tomato. Plant rhabdoviruses are transmitted by and replicate in hemipteran insects such as aphids (Aphididae), leafhoppers (Cicadellidae), or planthoppers (Delphacidae). These specific interactions between plants, viruses and insects offer new insights into host adaptation and molecular virus evolution. This review explores recent advances as well as knowledge gaps in understanding of replication, RNA silencing suppression and movement of plant rhabdoviruses with respect to both plant and insect hosts.