AlphaFold modeling of nepovirus 3C-like proteinases provides new insights into their diverse substrate specificities.

The majority of picornaviral 3C proteinases (3Cpro) cleavage sites possess glutamine at the P1 position. Plant nepovirus 3C-like proteinases (3CLpro) show however much broader specificity, cleaving not only after glutamine, but also after several basic and hydrophobic residues. To investigate this difference, we employed AlphaFold to generate structural models of twelve selected 3CLpro, representing six substrate specificities. Generally, we observed favorable correlations between the architecture and charge of nepovirus proteinase S1 subsites and their ability to accept or restrict larger residues. The models identified a conserved aspartate residue close to the P1 residue in the S1 subsites of all nepovirus proteinases examined, consistent with the observed strong bias against negatively-charged residues at the P1 position of nepovirus cleavage sites. Finally, a cramped S4 subsite along with the presence of two unique histidine and serine residues explains the strict requirement of the grapevine fanleaf virus proteinase for serine at the P4 position.

Re-examination of nepovirus polyprotein cleavage sites highlights the diverse specificities and evolutionary relationships of nepovirus 3C-like proteases.

Plant-infecting viruses of the genus Nepovirus (subfamily Comovirinae, family Secoviridae, order Picornavirales) are bipartite positive-strand RNA viruses with each genomic RNA encoding a single large polyprotein. The RNA1-encoded 3C-like protease cleaves the RNA1 polyprotein at five sites and the RNA2 polyprotein at two or three sites, depending on the nepovirus. The specificity of nepovirus 3C-like proteases is notoriously diverse, making the prediction of cleavage sites difficult. In this study, the position of nepovirus cleavage sites was systematically re-evaluated using alignments of the RNA1 and RNA2 polyproteins, phylogenetic relationships of the proteases, and sequence logos to examine specific preferences for the P6 to P1' positions of the cleavage sites. Based on these analyses, the positions of previously elusive cleavage sites, notably the 2a-MP cleavage sites of subgroup B nepoviruses, are now proposed. Distinct nepovirus protease clades were identified, each with different cleavage site specificities, mostly determined by the nature of the amino acid at the P1 and P1' positions of the cleavage sites, as well as the P2 and P4 positions. The results will assist the prediction of cleavage sites for new nepoviruses and help refine the taxonomy of nepoviruses. An improved understanding of the specificity of nepovirus 3C-like proteases can also be used to investigate the cleavage of plant proteins by nepovirus proteases and to understand their adaptation to a broad range of hosts.

Proteolytic Processing of Plant Proteins by Potyvirus NIa Proteases.

The NIa protease of potyviruses is a chymotrypsin-like cysteine protease related to the picornavirus 3C protease. It is also a multifunctional protein known to play multiple roles during virus infection. Picornavirus 3C proteases cleave hundreds of host proteins to facilitate virus infection. However, whether or not potyvirus NIa proteases cleave plant proteins has so far not been tested. Regular expression search using the cleavage site consensus sequence [EQN]xVxH[QE]/[SGTA] for the plum pox virus (PPV) protease identified 90 to 94 putative cleavage events in the proteomes of Prunus persica (a crop severely affected by PPV), Arabidopsis thaliana, and Nicotiana benthamiana (two experimental hosts). In vitro processing assays confirmed cleavage of six A. thaliana and five P. persica proteins by the PPV protease. These proteins were also cleaved in vitro by the protease of turnip mosaic virus (TuMV), which has a similar specificity. We confirmed in vivo cleavage of a transiently expressed tagged version of AtEML2, an EMSY-like protein belonging to a family of nuclear histone readers known to be involved in pathogen resistance. Cleavage of AtEML2 was efficient and was observed in plants that coexpressed the PPV or TuMV NIa proteases or in plants that were infected with TuMV. We also showed partial in vivo cleavage of AtDUF707, a membrane protein annotated as lysine ketoglutarate reductase trans-splicing protein. Although cleavage of the corresponding endogenous plant proteins remains to be confirmed, the results show that a plant virus protease can cleave host proteins during virus infection and highlight a new layer of plant-virus interactions. IMPORTANCE Viruses are highly adaptive and use multiple molecular mechanisms to highjack or modify the cellular resources to their advantage. They must also counteract or evade host defense responses. One well-characterized mechanism used by vertebrate viruses is the proteolytic cleavage of host proteins to inhibit the activities of these proteins and/or to produce cleaved protein fragments that are beneficial to the virus infection cycle. Even though almost half of the known plant viruses encode at least one protease, it was not known whether plant viruses employ this strategy. Using an in silico prediction approach and the well-characterized specificity of potyvirus NIa proteases, we were able to identify hundreds of putative cleavage sites in plant proteins, several of which were validated by downstream experiments. It can be anticipated that many other plant virus proteases also cleave host proteins and that the identification of these cleavage events will lead to novel antiviral strategies.

Mapping of sequences in the 5' region and 3' UTR of tomato ringspot virus RNA2 that facilitate cap-independent translation of reporter transcripts in vitro.

Tomato ringspot virus (ToRSV, genus Nepovirus, family Secoviridae, order Picornavirales) is a bipartite positive-strand RNA virus, with each RNA encoding one large polyprotein. ToRSV RNAs are linked to a 5'-viral genome-linked protein (VPg) and have a 3' polyA tail, suggesting a non-canonical cap-independent translation initiation mechanism. The 3' untranslated regions (UTRs) of RNA1 and RNA2 are unusually long (~1.5 kb) and share several large stretches of sequence identities. Several putative in-frame start codons are present in the 5' regions of the viral RNAs, which are also highly conserved between the two RNAs. Using reporter transcripts containing the 5' region and 3' UTR of the RNA2 of ToRSV Rasp1 isolate (ToRSV-Rasp1) and in vitro wheat germ extract translation assays, we provide evidence that translation initiates exclusively at the first AUG, in spite of a poor codon context. We also show that both the 5' region and 3' UTR of RNA2 are required for efficient cap-independent translation of these transcripts. We identify translation-enhancing elements in the 5' proximal coding region of the RNA2 polyprotein and in the RNA2 3' UTR. Cap-dependent translation of control reporter transcripts was inhibited when RNAs consisting of the RNA2 3' UTR were supplied in trans. Taken together, our results suggest the presence of a CITE in the ToRSV-Rasp1 RNA2 3' UTR that recruits one or several translation factors and facilitates efficient cap-independent translation together with the 5' region of the RNA. Non-overlapping deletion mutagenesis delineated the putative CITE to a 200 nts segment (nts 773-972) of the 1547 nt long 3' UTR. We conclude that the general mechanism of ToRSV RNA2 translation initiation is similar to that previously reported for the RNAs of blackcurrant reversion virus, another nepovirus. However, the position, sequence and predicted structures of the translation-enhancing elements differed between the two viruses.

Expanding Repertoire of Plant Positive-Strand RNA Virus Proteases.

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 (Family Secoviridae, Order Picornavirales) Encodes a Novel Glutamic Protease To Process the RNA2 Polyprotein at Two Cleavage Sites.

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.

Expression and antiviral function of ARGONAUTE 2 in Nicotiana benthamiana plants infected with two isolates of tomato ringspot virus with varying degrees of virulence.

ARGONAUTEs (notably AGO1 and AGO2) are effectors of plant antiviral RNA silencing. AGO1 was shown to be required for the temperature-dependent symptom recovery of Nicotiana benthamiana plants infected with tomato ringspot virus (isolate ToRSV-Rasp1) at 27 °C. In this study, we show that symptom recovery from isolate ToRSV-GYV shares similar hallmarks of antiviral RNA silencing but occurs at a wider range of temperatures (21-27 °C). At 21 °C, an early spike in AGO2 mRNAs accumulation was observed in plants infected with either ToRSV-Rasp1 or ToRSV-GYV but the AGO2 protein was only consistently detected in ToRSV-GYV infected plants. Symptom recovery from ToRSV-GYV at 21 °C was not prevented in an ago2 mutant or by silencing of AGO1 or AGO2. We conclude that other factors (possibly other AGOs) contribute to symptom recovery under these conditions. The results also highlight distinct expression patterns of AGO2 in response to ToRSV isolates and environmental conditions.

Corrigendum: Identification of Cleavage Sites Recognized by the 3C-Like Cysteine Protease within the Two Polyproteins of Strawberry Mottle Virus.

[This corrects the article on p. 745 in vol. 8, PMID: 28496438.].

Identification of Cleavage Sites Recognized by the 3C-Like Cysteine Protease within the Two Polyproteins of Strawberry Mottle Virus.

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).

Characterization of a Non-Canonical Signal Peptidase Cleavage Site in a Replication Protein from Tomato Ringspot Virus.

The NTB-VPg polyprotein from tomato ringspot virus is an integral membrane replication protein associated with endoplasmic reticulum membranes. A signal peptidase (SPase) cleavage was previously detected in the C-terminal region of NTB-VPg downstream of a 14 amino acid (aa)-long hydrophobic region (termed TM2). However, the exact location of the cleavage site was not determined. Using in vitro translation assays, we show that the SPase cleavage site is conserved in the NTB-VPg protein from various ToRSV isolates, although the rate of cleavage varies from one isolate to another. Systematic site-directed mutagenesis of the NTB-VPg SPase cleavage sites of two ToRSV isolates allowed the identification of sequences that affect cleavage efficiency. We also present evidence that SPase cleavage in the ToRSV-Rasp2 isolate occurs within a GAAGG sequence likely after the AAG (GAAG/G). Mutation of a downstream MAAV sequence to AAAV resulted in SPase cleavage at both the natural GAAG/G and the mutated AAA/V sequences. Given that there is a distance of seven aa between the two cleavage sites, this indicates that there is flexibility in the positioning of the cleavage sites relative to the inner surface of the membrane and the SPase active site. SPase cleavage sites are typically located 3-7 aa downstream of the hydrophobic region. However, the NTB-VPg GAAG/G cleavage site is located 17 aa downstream of the TM2 hydrophobic region, highlighting unusual features of the NTB-VPg SPase cleavage site. A putative 11 aa-long amphipathic helix was identified immediately downstream of the TM2 region and five aa upstream of the GAAG/G cleavage site. Based on these results, we present an updated topology model in which the hydrophobic and amphipathic domains form a long tilted helix or a bent helix in the membrane lipid bilayer, with the downstream cleavage site(s) oriented parallel to the membrane inner surface.

Genome sequence analysis of five Canadian isolates of strawberry mottle virus reveals extensive intra-species diversity and a longer RNA2 with increased coding capacity compared to a previously characterized European isolate.

In this study, we report the genome sequence of five isolates of strawberry mottle virus (family Secoviridae, order Picornavirales) from strawberry field samples with decline symptoms collected in Eastern Canada. The Canadian isolates differed from the previously characterized European isolate 1134 in that they had a longer RNA2, resulting in a 239-amino-acid extension of the C-terminal region of the polyprotein. Sequence analysis suggests that reassortment and recombination occurred among the isolates. Phylogenetic analysis revealed that the Canadian isolates are diverse, grouping in two separate branches along with isolates from Europe and the Americas.

Complete genome sequence of three tomato ringspot virus isolates: evidence for reassortment and recombination.

The genome sequence of tomato ringspot virus (ToRSV, a subgroup C nepovirus) is currently available for one raspberry isolate. In this study, we describe the complete genome sequence of three additional isolates from raspberry (Rasp1-2014), grapevine (GYV-2014) and prunus (13C280). The degree of nucleotide sequence identity shared between RNA1 and RNA2 in the 5'-terminal 900 nucleotides and 3' untranslated region varied from 98-99 % (13C280, GYV-2014) to 80 % (Rasp1-2014). Phylogenetic studies revealed distinct origins for Rasp1-2014 RNA1 and RNA2, suggesting reassortment. Two recombination events were also identified in the 3' UTR and 5'-terminal region of RNA1.

The Cucumber leaf spot virus p25 auxiliary replicase protein binds and modifies the endoplasmic reticulum via N-terminal transmembrane domains.

Cucumber leaf spot virus (CLSV) is a member of the Aureusvirus genus, family Tombusviridae. The auxiliary replicase of Tombusvirids has been found to localize to endoplasmic reticulum (ER), peroxisomes or mitochondria; however, localization of the auxiliary replicase of aureusviruses has not been determined. We have found that the auxiliary replicase of CLSV (p25) fused to GFP colocalizes with ER and that three predicted transmembrane domains (TMDs) at the N-terminus of p25 are sufficient for targeting, although the second and third TMDs play the most prominent roles. Confocal analysis of CLSV infected 16C plants shows that the ER becomes modified including the formation of punctae at connections between ER tubules and in association with the nucleus. Ultrastructural analysis shows that the cytoplasm contains numerous vesicles which are also found between the perinuclear ER and nuclear membrane. It is proposed that these vesicles correspond to modified ER used as sites for CLSV replication.

Temperature-dependent symptom recovery in Nicotiana benthamiana plants infected with tomato ringspot virus is associated with reduced translation of viral RNA2 and requires ARGONAUTE 1.

Symptom recovery in nepovirus-infected plants has been attributed to the induction of RNA silencing. However, recovery is not always accompanied with viral RNA clearance. In this study, we show that recovery of Nicotiana benthamiana plants infected with the tomato ringspot virus (ToRSV) is associated with a reduction of the steady-state levels of RNA2-encoded coat protein (CP) and movement protein but not of RNA2. In vivo labeling experiments revealed efficient synthesis of the CP early in infection, but reduced RNA2 translation later in infection. Silencing of Argonaute1-like (Ago1) genes prevented both symptom recovery and RNA2 translation repression. Similarly, growing the plants at lower temperature (21 °C rather than 27 °C) alleviated the recovery and the translation repression. Taken together, our results suggest that recovery of ToRSV-infected plants is associated with an Ago1-dependent mechanism that represses the translation of viral RNA2.

Tomato ringspot virus coat protein binds to ARGONAUTE 1 and suppresses the translation repression of a reporter gene.

RNA silencing regulates plant gene expression and antiviral defenses and functions by cleaving target RNAs or repressing translation. As a counter defense, many plant viruses encode suppressor proteins that sequester small RNAs or inactivate Argonaute (AGO) proteins. All known plant virus silencing suppressor activities eventually inhibit the degradation of target mRNAs. Using a transiently expressed green fluorescent protein (GFP) reporter gene, we show that Tomato ringspot virus (ToRSV) coat protein (CP) is a suppressor of RNA silencing that enhances GFP expression but does not prevent the degradation of the GFP mRNA or the accumulation of GFP small interfering RNAs (siRNAs). Coexpression of the CP with GFP resulted in increased association of residual GFP mRNAs with polysome fractions and reduced association of GFP siRNAs with monosome fractions. AGO1 was co-immunoprecipitated with the CP and CP expression destabilized AGO1. A WG motif within the CP was critical for the enhanced GFP expression, AGO1 interaction, and AGO1 destabilization, suggesting that the ToRSV CP acts as an AGO-hook protein and competes for AGO binding with a plant cellular GW/WG protein involved in translation repression.

In vitro and in vivo evidence for differences in the protease activity of two arabis mosaic nepovirus isolates and their impact on the infectivity of chimeric cDNA clones.

Regulated processing of nepovirus polyproteins allows the release of mature proteins and intermediate polyproteins. Infectious cDNA clones of the mild NW isolate of arabis mosaic virus (ArMV) and chimeric clones incorporating RNA1 segments of Lv, a severe isolate, were generated. Clones containing the Lv X2-NTB cleavage site were not infectious unless the Lv protease was present. The Lv and NW X2-NTB cleavage sites differ at positions P6, P4 and P2. In vitro, processing at the X2-NTB site was undetectable or reduced in chimeric polyproteins containing the Lv X2-NTB site and the NW protease but was restored when both the Lv protease and X2-NTB site were present. In contrast, cleavage at this site was increased in polyproteins that contained the NW X2-NTB site and the Lv protease. These results show that the ArMV-Lv protease has greater activity and is active on a greater range of cleavage sites than that of ArMV-NW.

The SNARE protein Syp71 is essential for turnip mosaic virus infection by mediating fusion of virus-induced vesicles with chloroplasts.

All positive-strand RNA viruses induce the biogenesis of cytoplasmic membrane-bound virus factories for viral genome multiplication. We have previously demonstrated that upon plant potyvirus infection, the potyviral 6K2 integral membrane protein induces the formation of ER-derived replication vesicles that subsequently target chloroplasts for robust genome replication. Here, we report that following the trafficking of the Turnip mosaic potyvirus (TuMV) 6K2 vesicles to chloroplasts, 6K2 vesicles accumulate at the chloroplasts to form chloroplast-bound elongated tubular structures followed by chloroplast aggregation. A functional actomyosin motility system is required for this process. As vesicle trafficking and fusion in planta are facilitated by a superfamily of proteins known as SNAREs (soluble N-ethylmaleimide-sensitive-factor attachment protein receptors), we screened ER-localized SNARES or SNARE-like proteins for their possible involvement in TuMV infection. We identified Syp71 and Vap27-1 that colocalize with the chloroplast-bound 6K2 complex. Knockdown of their expression using a Tobacco rattle virus (TRV)-based virus-induced gene silencing vector showed that Syp71 but not Vap27-1 is essential for TuMV infection. In Syp71-downregulated plant cells, the formation of 6K2-induced chloroplast-bound elongated tubular structures and chloroplast aggregates is inhibited and virus accumulation is significantly reduced, but the trafficking of the 6K2 vesicles from the ER to chloroplast is not affected. Taken together, these data suggest that Syp71 is a host factor essential for successful virus infection by mediating the fusion of the virus-induced vesicles with chloroplasts during TuMV infection.

Salicylic acid-dependent restriction of Tomato ringspot virus spread in tobacco is accompanied by a hypersensitive response, local RNA silencing, and moderate systemic resistance.

Tomato ringspot virus (ToRSV, a Nepovirus sp.) systemically infects many herbaceous plants. Viral RNA accumulates in symptomatic leaves and in young, asymptomatic leaves that emerge late in infection. Here, we show that systemic infection by ToRSV is restricted in tobacco. After an initial hypersensitive response in inoculated leaves, only a few plants showed limited systemic symptoms. Viral RNA did not usually accumulate to detectable levels in asymptomatic leaves. ToRSV-derived small-interfering RNAs and PR1a transcripts were only detected in tissues that contained viral RNA, indicating local induction of RNA silencing and salicylic acid (SA)-dependent defense responses. Lesion size and viral systemic spread were reduced with SA pretreatment but enhanced in NahG transgenic lines deficient in SA accumulation, suggesting that SA-dependent mechanisms play a key role in limiting ToRSV spread in tobacco. Restriction of virus infection was enhanced in transgenic lines expressing the P1-HC-Pro suppressor of silencing. Knocking down the SA-inducible RNA-dependent RNA polymerase 1 exacerbated the necrotic reaction but did not affect viral systemic spread. ToRSV-infected tobacco plants were susceptible to reinoculation by ToRSV or Tobacco mosaic virus, although a small reduction in lesion size was observed. This moderate systemic resistance suggests inefficient induction or spread of RNA silencing and systemic acquired resistance signal molecules.