A Newly Identified Virus in the Family Potyviridae Encodes Two Leader Cysteine Proteases in Tandem That Evolved Contrasting RNA Silencing Suppression Functions

The Potyviridae represent the largest group of known plant RNA viruses and account for more than half of the viral crop damage worldwide. The leader proteases of viruses within the family vary greatly in size and arrangement and play key roles during the infection. Here, we experimentally demonstrate the presence of a distinct pattern of leader proteases, HCPro1 and HCPro2 in tandem, in a newly identified member within the family. Moreover, HCPro1 and HCPro2, which are closely related and typically characterized with a short size, have evolved contrasting RNA silencing suppression activity and seem to function in a coordinated manner to maintain viral infectivity. Altogether, the new knowledge fills a missing piece in the evolutionary relationship history of potyvirids and improves our understanding of the diversification of potyvirid genomes.

ABSTRACT

Potyviridae is the largest family of plant-infecting RNA viruses and includes many agriculturally and economically important viral pathogens. The viruses in the family, known as potyvirids, possess single-stranded, positive-sense RNA genomes with polyprotein processing as a gene expression strategy. The N-terminal regions of potyvirid polyproteins vary greatly in sequence. Previously, we identified a novel virus species within the family, Areca palm necrotic spindle-spot virus (ANSSV), which was predicted to encode two cysteine proteases, HCPro1 and HCPro2, in tandem at the N-terminal region. Here, we present evidence showing self-cleavage activity of these two proteins and define their cis-cleavage sites. We demonstrate that HCPro2 is a viral suppressor of RNA silencing (VSR), and both the variable N-terminal and conserved C-terminal (protease domain) moieties have antisilencing activity. Intriguingly, the N-terminal region of HCPro1 also has RNA silencing suppression activity, which is, however, suppressed by its C-terminal protease domain, leading to the functional divergence of HCPro1 and HCPro2 in RNA silencing suppression. Moreover, the deletion of HCPro1 or HCPro2 in a newly created infectious clone abolishes viral infection, and the deletion mutants cannot be rescued by addition of corresponding counterparts of a potyvirus. Altogether, these data suggest that the two closely related leader proteases of ANSSV have evolved differential and essential functions to concertedly maintain viral viability.

IMPORTANCE The Potyviridae represent the largest group of known plant RNA viruses and account for more than half of the viral crop damage worldwide. The leader proteases of viruses within the family vary greatly in size and arrangement and play key roles during the infection. Here, we experimentally demonstrate the presence of a distinct pattern of leader proteases, HCPro1 and HCPro2 in tandem, in a newly identified member within the family. Moreover, HCPro1 and HCPro2, which are closely related and typically characterized with a short size, have evolved contrasting RNA silencing suppression activity and seem to function in a coordinated manner to maintain viral infectivity. Altogether, the new knowledge fills a missing piece in the evolutionary relationship history of potyvirids and improves our understanding of the diversification of potyvirid genomes.

INTRODUCTION

The Potyviridae family represents the largest group of plant-infecting RNA viruses and includes many agriculturally and economically important viral pathogens, such as potato virus Y, soybean mosaic virus, and plum pox virus (PPV) (1–4). A large majority of viruses in the family (here referred to as potyvirids) have one positive-sense single-stranded RNA genome (8.2 to 11.3 kb) and flexuous filamentous particles of 11 to 20 nm by 680 to 900 nm in size (2). Based on the genome composition and structure, sequence similarity, and transmission vector, potyvirids are currently sorted into the 10 following definitive genera: Bevemovirus, Brambyvirus, Bymovirus, Ipomovirus, Macluravirus, Poacevirus, Potyvirus, Rymovirus, Tritimovirus, and Roymovirus (2, 5). Exceptionally, bymoviruses have bipartite genomes (RNA1, 7.2 to 7.6 kb; RNA2, 2.2 to 3.6 kb) that are packaged into two modal particles of 250 to 300 nm and 500 to 600 nm in length (1, 2, 6). The genomes of all potyvirids, excluding bymoviruses, contain a single long open reading frame (ORF) and another relatively short ORF (PIPO) that results from RNA polymerase slippage in the P3-coding region (7–10). In particular, the same slippage event occurs in the P1-coding region of sweet potato-infecting potyviruses, given raise to an additional ORF (PISPO) (9, 11, 12). Upon translation, the resulting viral polyproteins are proteolytically processed by virus-encoded protease domains into 10 to 12 mature factors (5). Both the organization and sequence composition of potyvirid genomes from P3 to CP are relatively conserved (3, 5). However, the leader proteases encoded by the 5′-terminal regions vary greatly in number, arrangement, and sequence similarity (3, 5). Therefore, the polyprotein segment from P3 to CP is defined as the conserved central and carboxyl region, whereas the segment upstream of P3 is defined as the hypervariable N-terminal region (5).

The hypervariable N-terminal region contributes to the diversification of potyvirids (5). In fact, potyvirid genomes vary greatly in size, and this difference is mainly attributed to their hypervariable 5′-terminal regions (corresponding to the genomic RNA2 of bymoviruses) (5). This genomic region encodes one or two leader proteases arranged in a quite variable manner. Most potyvirids, including potyviruses, rymoviruses, brambyviruses, roymoviruses, tritimoviruses, poaceviruses, and a member of the Ipomovirus genus (sweet potato mild mottle virus [SPMMV]), share the same pattern of leader proteases, a chymotrypsin-like serine protease (P1) and a cysteine protease (HCPro) in tandem (1, 3, 5). However, P1 is missing and only HC-Pro is present in the N-terminal region of viruses from the Macluravirus and Bevenevirus genera, whereas HCPro is missing and either one or two P1s are present in ipomoviruses (except for SPMMV) (2, 3, 5). Remarkably, P1 and HCPro are the most divergent factors among all mature potyvirid proteins (3, 13–16). In the case of HCPro, for instance, its molecular mass ranges from ∼28 kDa in bymoviruses to ∼50 kDa in potyviruses and rymoviruses (5).

Besides the role as peptide cutters, proteases from plant viruses also function in different steps of the viral infection cycle (17–19). In recent years, several significant advances describing the biological roles of potyvirid leader proteases have been achieved. For instance, P1 can be sorted in two groups based on protein features and function: type A and type B (13), with those of the first group acting as important determinants in host adaption (5, 20–22). In turn, type B P1s (tested so far) display RNA silencing suppression activity (23–26). HCPro is a well-studied multifunctional protein that plays diverse roles during the viral infection cycle, including (i) plant-to-plant transmission, (ii) RNA silencing suppression, (iii) polyprotein maturation, and (iv) enhancement of viral particle yield (3, 27–32). Remarkably, whereas all these studies involved viruses from some, but not all, HCPro from different genera, the biological significance of HCPro from viruses in Macluravirus, Brambyvirus, and Bymovirus is largely unknown.

RNA silencing is a well-studied fundamental, evolutionarily conserved mechanism to regulate gene expression in eukaryotes and functions as a primary immune defense against viral infection in plants (33, 34). In turn, plant viruses have evolved to express viral suppressors of RNA silencing (VSRs) to counteract RNA silencing machinery (34). These VSRs vary highly in sequence, share no structural similarities, and interfere with specific steps of the RNA silencing pathway through a variety of mechanisms. Readers are referred to several excellent reviews for more information (18, 34–36). HCPro from viruses in Potyvirus is a well-studied VSR protein. Most potyviral HCPro(s) suppress host RNA silencing via sequestrating 21- and 22-nucleotide (nt) small RNA of viral sequences (vsiRNA). Alternatively, others might perturb different steps of the RNA silencing pathway, such as inhibition of methylation of vsiRNA, interference with effector protein (AGO1), and disturbance of RDR6-mediated amplification of viral double-stranded RNA (3, 5). Nevertheless, RNA silencing suppression and its underlying mechanism of HCPro in other genera of the family have rarely been reported.

Recently, we identified two novel virus species within the family Potyviridae, Areca palm necrotic spindle-spot virus (ANSSV) and Areca palm necrotic ringspot virus (ANRSV), which are highly epidemic in main growing regions of areca palm in China, and severely threaten its industrial development (37, 38). ANSSV and ANRSV are closely related and taxonomically classified into a putative new genus (Arepavirus) of the family. As a matter of interest, these viruses seem to have a unique pattern of leader proteases formed, apparently, by two HCPro factors (HCPro1 and HCPro2) in tandem. In this study, we experimentally demonstrate that this distinctive pattern is truly encoded by the 5′-terminal region of the ANSSV genome. Further, we found that HCPro1 and HCPro2 have evolved contrasting RNA silencing suppression activity. Eventually, infectivity tests of both wild-type and a series of ANSSV-derived cDNA clones revealed that HCPro1 and HCPro2 might function in a coordinated manner to maintain viral infectivity. The obtained results greatly advance our understanding of the evolution and functional diversification of potyvirids.

RESULTS

Both putative cysteine protease domains in the genomic 5′-terminal region of ANSSV have self-cleavage activity.

Previous bioinformatic analysis predicted the presence of two conserved cysteine protease domains of the peptidase_C6 superfamily in the N terminus of ANSSV polyprotein (Fig. 1A). The catalytic activity sites for them were “Gly159, Cys161, and His233” and “Gly461, Cys463, and His535,” respectively. Thus, it was speculated that the 5′-terminal region of the ANSSV genome encoded two cysteine proteases in tandem (HCPro1-HCPro2) resulting from cis-cleavage at their C terminus (Fig. 1A) (37, 38). To experimentally test the cis-cleavage activity of these two domains, a 2,142-nt fragment that corresponds to the 5′-terminal region of the ANSSV genome (HCPro1-HCPro2-P3N cistrons, here named tHCPro-P3N) was introduced into an expression vector, pF3K WG (BYDV) Flexi. An in vitro translation assay showed the production of the whole tHCPro-P3N as well as the five expected cis-cleavage products (I, II, III, IV, and V), which were absent in the mock control (Fig. 1B and C). Theoretically, a sixth product that represents P3N (∼16.7 kDa) has to be produced; however, it could not be discriminated in the analysis due to strong background signals generated by a wheat germ extract (WGE)-based in vitro translation system (Fig. 1C). To further demonstrate the presence of two bona fide cysteine-like proteases in tHCPro-P3N, two mutants were constructed, tHCPro-P3N(C161A) and tHCPro-P3N(C463A), in which the residues C161 and C463 in the expected catalytic sites were mutated by alanine (Fig. 1B). An in vitro translation assay showed two major products (I and II) for tHCPro-P3N(C161A) and three bands (I, III, and V) for tHCPro-P3N(C463A) (Fig. 1C), indicating that the mutation of C161 or C463 abolishes the cis-cleavage activity of the corresponding cysteine protease domains.

FIG 1.

WGE-based in vitro translation analysis of tHCPro-P3N and its derivatives, tHCPro-P3N(C161A) and tHCPro-P3N(C463A). (A) Schematic diagram of a 2,142-nt coding region of the genomic N terminus of ANSSV (tHCPro-P3N). Previous bioinformatic analysis predicted the presence of two conserved cysteine protease domains of peptidase_C6 superfamily in the genomic 5′-terminal region of ANSSV (37, 38). After cis-cleavage of the two domains, two cysteine proteases (HCPro1 and HCPro2) would be generated. Red arrows indicate the putative cleavage sites of the two cysteine protease domains (37). (B) A predicted profile of products for tHCPro-P3N, tHCPro-P3N(C161A), and HCPro-P3N(C463A) after in vitro translation followed by self-cleavage of cysteine protease domains. (C) SDS-PAGE analysis of WGE-based in vitro translation products of tHCPro-P3N, tHCPro-P3N(C161A), and tHCPro-P3N(C463A). Mock, empty vector control.

Delineation of HCPro1 and HCPro2 in the ANSSV genome.

For potyvirids, the cleavage site of leader cysteine protease is located immediately downstream from the cysteine protease domain, more precisely, at the C terminus of a conserved G residue at the “P1” position of the cleavage site (39). Following these criteria, 258G/V259 and 574G/G575 were initially believed to be the cleavage sites of the two cysteine protease domains of ANSSV, respectively (37, 38). To test this hypothesis, G258A and G574A were engineered into the plasmid that expresses tHCPro-P3N for a further in vitro translation assay (Fig. 2A). The results showed that products from tHCPro-P3N(G574A) (Fig. 2B) are equivalent to those from tHCPro-P3N(C463A) (Fig. 1C), indicating that 574G/G575 is indeed the cleavage site of HCPro2. However, products from tHCPro-P3N(G258A) resulted equivalently to those from the tHCPro-P3N (Fig. 2B), thereby suggesting that 258G/V259 is not the cleavage site of HCPro1. Another suspected cleavage site, 273G/S274, was screened by an in vitro translation assay after the introduction of a G273A mutation into the construct that expresses tHCPro1-P3N (Fig. 2A). We found that tHCPro-P3N(G273A) give rise to a profile of products (Fig. 2B) that is equivalent to that of HCPro-P3N(C161A) (Fig. 1C). Therefore, the cis-cleavage sites of HCPro1 and HCPro2 were located at position 273G/V274 and 574G/G575, respectively, so that the precise position of these two viral cistrons in the ANSSV genome is delimited (Fig. 2C).

FIG 2.

Mapping the cis-cleavage sites of the two cysteine protease domains in the 5′-terminal region of ANSSV genome. (A) Schematic diagram of tHCPro-P3N showing two suspected cleavage sites (258G/V259 and 273G/S274) and one site (574G/G575) for the two cysteine protease domains, respectively. The suspected cleavage sites are indicated by dotted arrows. (B) SDS-PAGE analysis of in vitro translation products of tHCPro-P3N, tHCPro-P3N(G258A), tHCPro-P3N(G273A), and tHCPro-P3N(G574A). The bands represented by I, II, III, IV, and V are diagrammatically shown in Fig. 1B. Mock, empty vector control. (C) The accurate schematic diagram of the genomic N terminus of ANSSV. The precise cis-cleavage sites of the two cysteine protease domains are indicated by red arrows.

Conserved and divergent features of HCPro.

For potyvirids, HCPro is one of the most variable proteins (3). In the present study, a comprehensive analysis of 44 representative HCPro sequences distributed into 10 definitive genera and one proposed genus (Arepavirus) within the family Potyviridae was performed. Multiple sequence alignment revealed that HCPro from most potyvirids (including potyviruses, rymoviruses, roymoviruses, poaceviruses, and ipomoviruses) are longer than 450 amino acids (aa) in size, whereas those of the other four genera (i.e., Arepavirus, Bymovirus, Bevemovirus, and Macluravirus) were shorter than 300 aa (Fig. 3 and 4). The significant variation in size of HCPro is mainly attributed to their N-terminal moieties (Fig. 4). Pairwise amino acid sequence comparison revealed that the intergenus similarity of HCPro ranges from 10.5% to 32.1% (data not shown). The corresponding values for cysteine protease domain and N-terminal moiety are 12.9 to ∼57.3% and 0 to ∼25.4%, respectively (data not shown). Thus, we propose that potyvirid’s HCPro factors comprise a variable N-terminal region (N) and a conserved C-terminal cysteine protease domain (D) (Fig. 4).

FIG 3.

Phylogenetic analysis of the conserved cysteine protease domain of potyvirid HCPros. A total of 44 representative species in 10 definitive and 1 recently proposed genera of the Potyviridae family were employed for the phylogenetic analysis. The amino acid sequences of cysteine protease domain of HCPro of these virus species were aligned using ClustalX, and the phylogenetic tree was constructed with MEGA 7.0 using the maximum likelihood method. The numbers at the branch nodes indicate bootstrap support (1,000 replicates). Values below 70% are not shown.

FIG 4.

Multiple alignment of amino acid sequences of the leader cysteine proteases (HCPro, P2-1, HCPro1, and HCPro2) of 14 representative viruses distributing into 11 genera of the Potyviridae family. The variable N-terminal region and conserved cysteine protease domain of the leader cysteine proteases are defined. Three residues, Gly, Cys, and His, as the putative catalytic activity sites, are indicated by asterisks.

A phylogenetic analysis of HCPro was performed. Unfortunately, a phylogenetic tree based on full-length HCPro sequences failed to accurately reflect the taxonomic relationship of these virus species (data not shown), presumably due to the high variability at the N region. Thus, phylogenetic analysis based on the D domain was carried out. The result showed that these HCPros can be classified into three groups (named I, II, and III) (Fig. 3). Group I encompasses HCPro factor from four genera (i.e., Potyvirus, Rymovirus, Brambyvirus, and Roymovirus). Except for the HCPro from blackberry virus Y (Brambyvirus genus) with 326 aa, they have a relatively big size (453 to 466 aa). Importantly, so far, all tested HCPros in group I display RNA silencing suppression activity (5). Group II comprises the HCPro products from three genera, Poacevirus, Ipomovirus, and Tritimovirus. The size of proteins in group II varies greatly, with a range that goes from 384 to 471 aa. Intriguingly, viruses for which HCPro is clustered in group II rely on P1 (type B) rather than HCPro for RNA silencing suppression activity (5). Group III comprises HCPro factors from four genera, Arepavirus, Bymovirus, Bevemovirus, and Macluravirus, and they are typically characterized by small HCPros (230 to 301 aa). To date, the functional roles of HCPro belonging to group III have not been defined.

HCPro2 is a viral suppressor of RNA silencing (VSR).

Usually, one of the leader proteases of potyvirids executes essential roles in RNA silencing suppression, such as HCPro factor from members of the Potyvirus genus (27, 28) and type B P1 protein from other potyvirids (5). Therefore, the capacity of HCPro1, HCPro2, and tHCPro from ANSSV to counteract the plant-based RNA silencing was investigated. Constructs expressing tHCPro-Myc, HCPro1-Myc, and HCPro2-Myc in plants by agro-infiltration were generated (Fig. 5A) and then introduced in Agrobacterium tumefaciens. Each of the generated A. tumefaciens strains was later coinfiltrated with another strain that carries a plasmid that expresses the green fluorescent protein (GFP) reporter into leaves of both wild-type and 16c Nicotiana benthamiana plants (Fig. 5B and C). The coinfiltration of GFP along with either empty vector or the silencing suppressor P19 from tobacco bushy stunt virus (TBSV) was included as negative and positive controls, respectively (40, 41) (Fig. 5B and C). At 3 days postinoculation (dpi), leaf patches expressing tHCPro-Myc/GFP or HCPro2-Myc/GFP displayed strong GFP fluorescence (Fig. 5C). No obvious GFP fluorescence was observed on leaf patches expressing HCPro1-Myc/GFP or negative control (Fig. 5C). Western blot analysis showed that the abundance of GFP protein in leaf patches coinfiltrated with tHCPro-Myc/GFP or HCPro2-Myc/GFP was significantly higher than that of HCPro1-Myc/GFP or the negative control (Fig. 5D). Of note, the Northern blot analysis showed that the enhancing levels of GFP expression in the presence of tHCPro-Myc and HCPro2-Myc occur at the level of RNA accumulation. Consequently, a larger amount of GFP mRNA transcripts was detected in samples from leaf patches expressing GFP along with either tHCPro-Myc or HCPro2-Myc (Fig. 5D and E). GFP-derived small interfering RNAs (siRNAs) were observed as a consequence of GFP mRNA silencing in samples from leaf patches expressing most of the constructs. Importantly, these molecules were hardly detectable in samples from leaf patches expressing tHCPro-Myc/GFP, tHCPro2-Myc/GFP, or the P19/GFP positive control (Fig. 5D and E). Remarkably, the expression levels of HCPro1, HCPro2, and tHCPro from above leaf patches were hardly detected by Western blotting assay (Fig. 5F), whereas they were rescued to a varied extent when coinfiltrated with the P19-expressing construct (Fig. 5F). Together, all these data suggest that HCPro2 has RNA silencing suppression activity, whereas HCPro1 does not.

FIG 5.

RNA silencing suppression test of tHCPro, HCPro1, and HCPro2 of ANSSV. (A) Schematic diagram of tHCPro-Myc, HCPro1-Myc, or HCPro2-Myc. Diagrams of wild-type tHCPro (I) and its derivatives, tHCPro-Myc, HCPro1-Myc, and HCPro2-Myc (II), are shown. Bent arrows indicate the self-cleavage site of HCPro1 or HCPro2. (B) Patch design used for coinfiltration in wild-type N. benthamiana or 16c leaves with a GFP-expressing construct together with a candidate protein-expressing construct (indicated by X), a P19-expressing positive control, or a negative empty vector, pCaMterX control (indicated by delta). (C) Representative photographs of coinfiltrated wild-type N. benthamiana (upper) or 16c (lower) leaves taken at 3 dpi under UV light. Bar, 25 mm. (D) Immunoblot and Northern blot analyses of GFP silencing in coinfiltrated leaf patches. Coomassie blue staining of the large subunit of rubisco was used as a loading control for the immunoblot. Ethidium bromide (EB) staining of rRNA and tRNA served as the loading controls for Northern blot assays of GFP mRNA transcripts and GFP-derived siRNAs, respectively. (E) Quantitative analysis of GFP mRNA (left) and GFP-derived siRNA (right) signals shown in panel D. The signal intensity values are presented as the mean ± standard deviation (SD) (n = 3). The average delta values were designated 100% to normalize the data. Statistically significant differences, determined by an unpaired two-tailed Student’s t test, are indicated by asterisks. *, P < 0.05; **, P < 0.01; NS, no significant difference. (F) Immunoblot analysis of the transient expression of tHCPro-Myc, HCPro1-Myc, and HCPro2-Myc in N. benthamiana; (top) Western blot detection of the expression of tHCPro-Myc, HCPro1-Myc, and HCPro2-Myc; leaf patches indicated in panel B were sampled at 3 dpi; (bottom) Western blot analysis of the expression of tHCPro-Myc, HCPro1-Myc, and HCPro2-Myc. Leaf patches of coinfiltration of TBSV P19, together with tHCPro-Myc, HCPro1-Myc, or HCPro2-Myc, were sampled at 3 dpi for the analysis.

The RNA silencing suppression activity of HCPro2 is independent of its release from the preceding HCPro1.

The leader proteases encoded by potyviruses, which are the predominant virus genus of potyvirids, are P1 and HCPro in tandem. As mentioned above, HCPro is the potyviral VSR factor, and its activity depends on its release from the preceding P1 (20, 21, 42). To investigate whether the detachment from HCPro1 is essential for RNA silencing suppression activity of HCPro2, two mutants of tHCPro, tHCPro(C161A) and tHCPro(G273A), in which the cis-cleavage activity of HCPro1 was compromised (Fig. 1 and 2), were constructed (Fig. 6A) and subjected to an RNA silencing suppression test. Coexpression with GFP showed that leaf patches expressing tHCPro(C161A)/GFP or tHCPro(G273A)/GFP displayed clear GFP fluorescence, similar to that of leaf patches expressing wild-type tHCPro/GFP (Fig. 6B). In line with this observation, a higher accumulation level of GFP protein and GFP mRNA transcript was detected in leaf patches expressing active VSRs but not in the case of the negative control (Fig. 6C and D). Consistently, GFP-derived siRNA accumulated significantly more in leaf patches corresponding to the negative control than in those of the other treatments (Fig. 6C and D). These results indicate that the self-cleavage of HCPro1 to release free HCPro2 is not essential for HCPro2-mediated antisilencing activity.

FIG 6.

The inactivation of the self-cleavage of HCPro1 has no influence on the antisilencing activity of HCPro2. (A) Schematic diagram of wild-type tHCPro and its derivatives, tHCPro(C161A) and tHCPro(G273A). The shaded regions in blue represent the cysteine protease domain of HCPro1 and HCPro2. The cis-cleavage sites of the cysteine protease domains are indicated by blue arrows. (B) Representative photographs of coinfiltrated N. benthamiana leaves taken at 3 dpi under UV light. Patch distribution for coinfiltration is exactly the same as that shown in Fig. 5B, except that tHCPro-expressing construct serves as a positive control. Bar, 25 mm. (C) Immunoblot and Northern blot analyses of GFP silencing in coinfiltrated leaf patches. Coomassie blue staining of the large subunit of rubisco was used as a loading control for the immunoblot. EB staining of rRNA and tRNA served as the loading controls for Northern blot assays of GFP mRNA transcripts and GFP-derived siRNAs, respectively. Delta, an empty vector control (pCaMterX/35S:GFP). (D) Quantitative analysis of GFP mRNA (left) and GFP-derived siRNA (right) signals shown in panel C. The signal intensity values are presented as mean ± SD (n = 3). The average delta values were designated 100% to normalize the data. Statistically significant differences, determined by an unpaired two-tailed Student’s t test, are indicated by asterisks: *, P < 0.05; **, P < 0.01.

Both N2 and D2 domains of HCPro2 confer RNA silencing suppression activity independently.

To determine the domain(s) of HCPro2 that confer(s) antisilencing activity, two constructs that transiently express N2 and D2 of HCPro2, respectively, were constructed (Fig. 7A). Transient coexpression experiments showed that leaf patches corresponding to N2/GFP and D2/GFP, similar to those of HCPro2/GFP, displayed strong GFP fluorescence (Fig. 7B). Consistently, Western blot analysis revealed that the abundance of GFP protein in leaf patches expressing N2/GFP, D2/GFP, and HCPro2/GFP was significantly higher than that of the negative control (Fig. 7C). Supporting the above-described conclusion, a higher accumulation level of GFP mRNA transcripts in leaf patches expressing N2/GFP, D2/GFP, and HCPro2/GFP than that of the negative control was observed (Fig. 7C and D).

FIG 7.

RNA silencing suppression test of the variable N-terminal region (N) and conserved C-terminal cysteine protease domain (D) of HCPro1 and HCPro2. (A) Schematic diagram of HCPro1 and HCPro2 showing the N and D domains. (B) Representative photographs of coinfiltrated N. benthamiana leaves at 3 dpi under UV light. Patch distribution for coinfiltration is exactly the same as that shown in Fig. 5B, except that the HCPro2-expressing construct served as a positive control. Bar, 25 mm. (C) Immunoblot and Northern blot analyses of GFP silencing in coinfiltrated leaf patches. Coomassie blue staining of the large subunit of rubisco was used as a loading control for the immunoblot. EB staining of rRNA served as the loading controls for Northern blot assays of GFP mRNA transcripts. Delta, an empty vector control (pCaMterX/35S:GFP). (D) Quantitative analysis of GFP mRNA signals shown in panel C. The signal intensity values are presented as the mean ± SD (n = 3). The average delta value was designated 100% to normalize the data. Statistically significant differences, determined by an unpaired two-tailed Student’s t test, are indicated by asterisks. *, P < 0.05; **, P < 0.01; NS, no significant difference.

The HCPro1 D1 domain prevents the RNA silencing suppression activity of the whole HCPro1 protein.

Given that the cysteine protease domains of HCPro1 and HCPro2 are conserved (Fig. 4) and that HCPro2 D2 has antisilencing activity, we wondered whether the conserved cysteine protease domain of HCPro1 (D1) has RNA silencing suppression activity. We constructed two plasmids to express N1 or D1 domains of HCPro1 separately (Fig. 7A). Surprisingly, a coinfiltration experiment showed that leaf patches producing N1/GFP, but not D1/GFP, displayed strong GFP fluorescence (Fig. 7B), indicating that the variable N-terminal region rather than the conserved cysteine protease domain from HCPro1 has RNA silencing suppression activity. Detection of GFP by Western blotting, as well as detection of GFP mRNA transcripts by Northern blotting confirmed the above-described result (Fig. 7C and D). Since the whole HCPro1 lacks RNA silencing suppression activity (Fig. 5), we speculated that the conserved cysteine protease domain of HCPro1 might repress the antisilencing activity of the HCPro1 N1 domain. To test this idea, we swapped the protease domain of HCPro1 and HCPro2 to generate two chimeras: N1-D2 and N2-D1 (Fig. 8A), and then carried out an RNA silencing suppression test. Similar to the positive control that expresses HCPro2/GFP, leaf patches corresponding to N1-D2 displayed strong GFP fluorescence signals (Fig. 8B). However, similar background fluorescence was observed in leaf patches of N2-D1/GFP and the negative control (Fig. 8B). As expected, Western blot analysis of GFP and Northern blot detection of GFP mRNA transcripts supported the above-described conclusion (Fig. 8C and D). These results suggest that the C-terminal cysteine protease domain of HCPro1 is a genuine regulator of the N-terminal domain-mediated silencing suppression.

FIG 8.

RNA silencing suppression test of two chimeras (N1-D2 and N2-D1). (A) Schematic diagram of two hybrids (N1-D2 and N2-D1) that were generated by swapping the D domain of HCPro1 and HCPro2. (B) Representative photographs of coinfiltrated N. benthamiana leaves at 3 dpi under UV light. The patch design for coinfiltration is exactly the same as that shown in Fig. 5B, except that the HCPro2-expressing construct is served as a positive control. Bar, 25 mm. (C) Immunoblot and Northern blot analyses of GFP gene silencing in coinfiltrated leaf patches. Coomassie blue staining of the large subunit of rubisco was used as a loading control for the immunoblot. EB staining of rRNA served as the loading controls for Northern blot assays of GFP mRNA transcripts. Delta, an empty vector control (pCaMterX/35S:GFP). (D) Quantitative analysis of GFP mRNA signals shown in panel C. The signal intensity values are presented as the mean ± SD (n = 3). The average delta value was designated 100% to normalize the data. Statistically significant differences, determined by an unpaired two-tailed Student’s t test, are indicated by asterisks. *, P < 0.05; ***, P < 0.001; NS, no significant difference.

Development of ANSSV-derived full-length infectious cDNA clones.

Conventional cloning technologies were adopted to construct the complete cDNA clone of ANSSV-HNBT with the low-copy mini-binary T-DNA vector pCB301-35S-Nos (43) as the backbone. The entire genomic cDNA of ANSSV-HNBT was placed between the 35S promoter and Nos terminator in pCB301 to generate the complete cDNA clone of ANSSV (named pSS-I) (Fig. 9A and B). For pSS-I, a 220-bp intron 2 of the NiR gene of Phaseolus vulgaris was inserted into the P3-coding region of the ANSSV genome in order to attenuate the toxicity of this region in Escherichia coli (Fig. 9A). To test the infectivity of pSS-I, 10 N. benthamiana seedlings at 6- to 7-leaf stage were infiltrated with an A. tumefaciens strain that harbors this plasmid. At 25 dpi, the newly emerging leaves of all plants infiltrated with pSS-I showed vein-clearing symptoms, which were absent in the mock control (Fig. 9C). Typical symptoms of viral infection, such as foliar chlorosis and leaf distortion, were observed in systemic leaves at 35 dpi (Fig. 9C). The presence of ANSSV was confirmed by reverse transcriptase PCR (RT-PCR) amplification of CP- and P3-derived fragments (Fig. 9C). As expected, Sanger sequencing of the PCR products demonstrated that the intron sequence in P3 was completely spliced out from the viral progeny (Fig. 9C).

FIG 9.

The infectivity test of ANSSV-derived cDNA clones. (A) T-DNA construct representing the complete cDNA clone of ANSSV (pSS-I). The entire genomic cDNA of ANSSV (from the 5′ terminus: N1, N2, M, and C) harboring an intron in the P3-coding region was placed between the 35S promoter and the Nos terminator of mini-binary plant expression vector pCB301-35S-Nos (43). The intron (indicated by a short rectangular box) and its flanking P3-coding sequences are shown. The transcription start site is indicated by a bent arrow. Nucleotides belonging to the pCB301 backbone are indicated in lowercase. Viral nucleotides are indicated in uppercase. The PstI site is shown in italicized uppercase. 35S, the cauliflower mosaic virus 35S promoter; Nos, the nopaline synthase terminator; LB, left border; RB, right border. (B) Schematic representation of pSS-I and pSS-I-G. For pSS-I-G, the GFP-coding sequence was integrated into the NIb/CP junction of ANSSV. (C) Infectivity test of pSS-I in N. benthamiana. The representative photographs of agro-infiltrated plants were taken at 25 dpi (panel I) and 35 dpi (panel II). Bar, 25 mm. The close view of leaf regions (indicated by red box) is shown. Mock, empty vector control. The presence of ANSSV was detected by RT-PCR to target its CP- (left) and P3-derived (right) fragments (III). Sanger sequencing revealed the intron sequence was completely spliced from virus progeny of the pSS-I clone (IV). The red arrow indicated the insertion site of the intron in pSS-I-G. (D) Infectivity test of pSS-I-G in N. benthamiana. The representative photograph of plants infiltrated with pSS-I-G are taken at 30 dpi under UV light (I). The close view of the leaf region (indicated by white box) is shown. Bar, 3 cm. RT-PCR with a pair of primers analyzed the presence of the GFP cistron in virus progeny derived from the pSS-I-G clone (II). The two primers were positioned upstream and downstream, respectively, of the GFP-coding region in pSS-I-G. Western blot detection of GFP in infected N. benthamiana plants at 30 dpi (III). Arrow, free GFP (∼27.8 kDa). A Coomassie brilliant blue-stained gel was used as a loading control.

To create a GFP-tagged version of ANSSV, the GFP cistron was integrated into the NIb/CP intercistronic junction in pSS-I to build the clone pSS-I-G (Fig. 9B). To examine the infectivity of pSS-I-G, eight N. benthamiana plants (6- to 7-leaf stage) were inoculated with an A. tumefaciens strain that carries pSS-I-G, and then GFP fluorescence signals were recorded at different time points. At 30 dpi, strong fluorescence signals were observed for all infiltrated plants under a UV lamp at the veins of upper noninoculated leaves (Fig. 9D). The stable presence of foreign GFP cistron in the ANSSV genome of the virus progeny was confirmed by RT-PCR (Fig. 9D). Furthermore, GFP production in systemically infected tissues was confirmed by Western blotting analysis. A major band corresponding to the predicted size for the recombinant GFP (approximately 27.8 kDa) was detected, indicating that free GFP protein was efficiently processed and released from the virus genome-encoded polyprotein (Fig. 9D).

Deletion of either HCPro1 or HCPro2 prevents systemic infection of the resulting ANSSV mutants, and neither of them can be functionally replaced, respectively, by the P1 and HCPro of a potyvirus (TuMV).

To test the importance of HCPro1 and HCPro2 during the viral infection cycle, two ANSSV-derived cDNA clones, termed pSS-I-G(Δ1) and pSS-I-G(Δ2), in which the complete HCPro1- or HCPro2-coding region was deleted, were constructed (Fig. 10A). At 30 dpi, obvious GFP fluorescence was observed in upper noninoculated leaves, flowers, and stems of all plants inoculated with wild-type pSS-I-G. In contrast, the GFP fluorescence was absent in plants inoculated with either pSS-I-G(Δ1) or pSS-I-G(Δ2) (Fig. 10B). These plants were sequentially monitored until 60 dpi with equivalent observations (data not shown). Both RT-PCR and Western blot assays confirmed the presence of the recombinant virus in upper noninoculated leaves of plants infected with pSS-I-G but not in those inoculated with pSS-I-G(Δ1) or pSS-I-G(Δ2) (Fig. 10C). Altogether, these results suggest that both the HCPro1- and HCPro2-coding regions are indispensable for viral infection.

FIG 10.

The infectivity test of two ANSSV deletion mutants, as well as four hybrid viruses between ANSSV and TuMV. (A) Schematic diagram of T-DNA constructs representing different viral clones. (B) Infectivity test of pSS-I-G(Δ1), pSS-I-G(Δ2), pSS-I-G(P1), and pSS-I-G(HCPro) in N. benthamiana. Ten seedlings at the 6- to 8- leaf stage were agroinfiltrated with each construct. The representative photographs of agro-infiltrated plants were taken at 30 dpi under UV light. Bar, 5 cm. (C) RT-PCR detection and/or Western blot assay of viral infection in N. benthamiana plants inoculated with the indicated constructs. The newly expanded leaves of inoculated plants were sampled at 30 dpi. (D) Infectivity test of pSS-I-G(P1HCPro) and pCBTuMV(HCPro1/2) in N. benthamiana. Ten seedlings at the 6- to 8- leaf stage were agroinfiltrated with each construct. For pSS-I-G and pSS-I-G(P1HCPro), the representative photographs of inoculated plants were taken at 30 dpi (under UV lamp), and for pCBTuMV and pCBTuMV(HCPro1/2), at 14 dpi. (E) RT-PCR detection of viral infection in N. benthamiana plants agroinfiltrated with the indicated constructs. For pSS-I-G and pSS-I-G(P1HCPro), the newly expanded leaves of inoculated seedlings were assayed at 30 dpi, and for pCBTuMV and pCBTuMV(HCPro1/2), at 14 dpi. Arrow, free GFP (∼27.8 kDa). A Coomassie brilliant blue-stained gel was used as a loading control.

Potyviruses (members of the genus Potyvirus), the predominant and well-studied virus species within the family Potyviridae, encode leader serine and cysteine proteases (P1-HCPro) in tandem, such as a 362-aa P1 and 458-aa HCPro for turnip mosaic virus (TuMV). Potyviral P1 plays multiple accessory roles during the viral infection cycle (44–47), but it seems to be unnecessary for viral systemic infection (20, 42). Potyviral HCPro is the VSR factor, which could be replaced by other heterologous VSRs to support viral systemic infection (48, 49). Unlike the leader proteases P1 and HCPro that potyviruses possess, ANSSV belonging to another genus (Arepavirus) has, instead, an HCPro1 and HCPro2 of 273 and 301 aa in length, respectively (Fig. 3 and 4). To further dissect additional functions of HCPro1 or HCPro2 during infection, we built two chimeric clones, pSS-I-G(P1) and pSS-I-G(HCPro), in which either HCPro1 or HCPro2 of ANSSV was substituted by the corresponding counterparts, P1 or HCPro from TuMV, respectively (Fig. 10A). N. benthamiana plants inoculated with pSS-I-G(P1) or pSS-I-G(HCPro) did not display GFP fluorescence in upper noninoculated leaves at 30 dpi (Fig. 10B). Further, two more hybrid viruses, pSS-I-G(P1HCPro) and pCBTuMV(HCPro1/2), were created via swapping the complete HCPro1-HCPro2 of ANSSV and P1-HCPro of TuMV (Fig. 10A). Similar to pSS-I-G(P1) and pSS-I-G(HCPro), pSS-I-G(P1HCPro) compromised viral infectivity (Fig. 10D). Surprisingly, the HCPro1-HCPro2 of ANSSV that has RNA silencing suppression activity was unable to replace the P1-HCPro of TuMV to maintain viral infectivity for pCBTuMV(HCPro1/2) (Fig. 10D), indicating that the unique TuMV P1-HCPro might function in other essential step(s) in viral infection beyond RNA silencing suppression. The above-described results were confirmed by RT-PCR and/or Western blot assays (Fig. 10C and E). All in all, replacement of HCPro1 or/and HCPro2 in ANSSV by the corresponding counterparts of a heterologous potyvirus has a deleterious effect, suggesting that HCPro1 and HCPro2 function in a coordinated manner to maintain viral infection.

DISCUSSION

In summary, this study with ANSSV experimentally demonstrates for the first time the presence of a unique pattern of leader proteases formed by HCPro1 and HCPro2 in tandem in a member of the family Potyviridae, which is the most important group of viruses infecting plants with RNA genomes. Interestingly, whereas HCPro1 and HCPro2 are closely related (Fig. 4), they greatly differ from their counterparts in other potyvirids when comparing sizes and amino acid composition (Fig. 4; data not shown). Despite their similarity, these two leader cysteine proteases are divergent in terms of their capacity to block RNA silencing, an evolutionarily conserved and sequence-specific immune response against viral infections in plants and other eukaryotes (33, 34, 50, 51). Our results also suggest that (i) HCPro1 and HCPro2 are indispensable for viral compatible infection and (ii) HCPro2 has, apart from RNA silencing suppression activity, additional essential functions that cannot be replaced by a heterologous VSR.

The potyviral HCPro is a well-studied VSR. It works mainly by direct interaction with, and sequestration of, vsiRNAs as well as, apparently, via interference with diverse steps of the RNA silencing machinery (3, 5, 52–56). Although both HCPro1 and HCPro2 from ANSSV do not have the well-conserved, antisilencing-related FRNK motif at their central regions (3), HCPro2 is a potent VSR, and HCPro1 does the same when its C-terminal part is deleted. Therefore, we envisage a scenario in which HCPro2 (and the N terminus of HCPro1) uses a different strategy than potyviral HCPro to block RNA silencing. Indeed, HCPro2 behaves differently than its potyviral counterpart, as both its variable N-terminal region and conserved C-terminal cysteine protease work as VSRs independently (Fig. 7). Moreover, whereas the potyviral HCPro becomes active after its release from the upstream protease P1 (20, 21, 57), the ANSSV HCPro2 silencing suppression activity is independent of the release from its upstream protease HCPro1. Intriguingly, potyviral P1 is dispensable for viral viability (20, 42), while HCPro1, the counterpart in ANSSV, is indispensable (Fig. 10). From now on, our further efforts will be focused on unraveling (i) the meaning of these differences between arepaviruses and potyviruses and, of course, (ii) the molecular mechanism by which HCPro1 and HCPro2 from ANSSV act coordinately to interfere with antiviral silencing.

Several Western blot assays failed to detect the expression level of HCPro1, HCPro2, and tHCPro from the corresponding coinfiltrated leaf patches (Fig. 5F). Therefore, we performed a coinfiltration assay of HCPro1, HCPro2, or tHCPro, together with a P19-expressing construct. It was found that the accumulation of HCPro2 was significantly increased (Fig. 5F), possibly implying a certain counter-counter-defense response deployed by host cells. Still, the expression of HCPro1 and the HCPro2 released from the precursor tHCPro was largely unrescued (Fig. 5F). Based on this, we propose that some cellular degradation machinery, such as selective autophagy and/or ubiquitin-proteasome (34, 41, 58–60), might be involved in the degradation of HCPro1 as well as HCPro2 when the last is expressed attached to the first. Therefore, further dissection on the molecular mechanism underlying the degradation of HCPro1 and its complex relationship with HCPro2 remains to be investigated.

For potyviruses, deletion of the whole HCPro or the introduction of point mutations abolishing HCPro-mediated silencing suppression generates a defective virus that is no longer able to initiate an infection (61). However, as in the case of PPV, the replacement of HCPro (or P1-HCPro) by other heterologous VSRs generates viable viruses able to reach upper noninoculated leaves with different efficiencies (48, 49, 62). Unlike PPV, the chimeric ANSSV clone, which was generated via replacement of HCPro2 (or HCPro1-HCPro2) by the potent VSR HCPro (or P1-HCPro) from TuMV, is unable to even initiate infection in N. benthamiana (Fig. 10), suggesting that HCPro2 plays other essential function(s), apart from RNA silencing suppression, during the viral infection cycle. It has to be mentioned that the complete P1-HCPro of TuMV is unable to be substituted by HCPro1-HCPro2 of ANSSV that has RNA silencing suppression activity to support viral infection (Fig. 10). This seems to be contradictory to previous observations of the well-studied PPV (48, 49, 62). It is noted that the hypervariable leader proteases of different potyvirids (even among viruses in the same genus) (5) might play multiple (but not exactly the same) essential roles in successful viral infection. For instance, apart from RNA silencing suppression activity, potato virus A (a member of the Potyvirus genus) HCPro promotes viral genome translation via inducing the formation of RNA granules (63, 64). This will be a fascinating and promising research direction in the future. Nevertheless, it remains to be determined whether all these mutant viruses generated in this study that fail to be systemic infections have defects in the infection of the inoculated leaves.

Based on the results presented here, we built a simplified model that might reflect the evolution of potyvirids (Fig. 11). It is logical to think that the conserved region from P3 to CP, which is proteolytically processed by the viral-encoded NIa-Pro, is the most ancient and basic viral module (3). Two separate lineages would have evolved from that (Fig. 11). One lineage corresponds to some ipomoviruses which harbor a single leader P1, possibly by acquiring P1 into the basic module. Although the genesis of the P1 element has not been determined, we attempt to speculate that an undiscovered virus that has a bi-segmented genome similar to that of bymoviruses might serve as a donor of P1 (Fig. 11). Later, with a putative duplication of P1, other ipomoviruses evolved two P1s in tandem (P1a and P1b). The other lineage might have evolved bevemoviruses and macluraviruses, in which the basic module incorporated a single leader HCPro of small size likely from RNA-2 of bymoviruses. A duplication of HCPro would have generated ANSSV and ANRSV arepaviruses with a distinct pattern of leader proteases: HCPro1 and HCPro2 in tandem. Finally, a recombination event might have occurred between the single P1-typed ipomovirus and HCPro-typed bevemovirus/macluravirus, giving rise to the most widespread potyvirid species, which have P1 and HCPro as leader proteases (Fig. 11). All in all, the study presented here with an arepavirus fills a missing piece in the evolutionary path of viruses in the Potyviridae family.

FIG 11.

A proposed model that reflects an evolutionary relationship of viruses in the Potyviridae family. The conserved region starting from P3 through CP is proposed to be a basic module for two separate evolutionary lineages. Two separate evolutionary lineages would be expanded along the basic module. A recombination event might occur between the single P1-typed ipomovirus and HCPro-typed bevemovirus/macluravirus for the origin of virus species with leader P1 and HCPro. SqVYV, Squash vein yellowing virus; CVYV, Cucumber vein yellowing virus; CBSV, Cassava brown streak virus; UCBSV, Uganda cassava brown streak virus.

MATERIALS AND METHODS

Viral isolate and plant materials.

In this study, an ANSSV isolate HNBT, whose genome was completely determined (GenBank accession no. MH330686) (37), was subjected to construction of the indicated plasmids as well as generation of ANSSV-derived infectious cDNA clones. The seedlings of wild-type N. benthamiana or the green fluorescent protein gene (GFP)-transgenic line (16c) were maintained in a growth chamber with 16 h of light at 25°C and 8 h of darkness at 23°C.

Plasmid construction.

To detect two putative cysteine protease domains in the presence of the N terminus of ANSSV polyprotein, a 2,142-nt coding region that corresponds to the genomic 5′-terminal region of ANSSV (tHCPro-P3N) was amplified by RT-PCR. The resulting product was integrated into pF3K WG (BYDV) Flexi (Promega) by AsiSI/PmeI sites to generate plasmid pF3-tHCPro-P3N. Two derivatives of pF3-tHCPro-P3N, pF3-tHCPro-P3N(C161A) and pF3-tHCPro-P3N(C463A), in which the residues C161 and C463 in catalytic activity sites were, respectively, mutated into alanine, were constructed using the overlapping PCR method. To map the cis-cleavage sites of HCPro1 and HCPro2, the other three mutants, pF3-tHCPro(G258A), pF3-tHCPro(G273A), and pF3-tHCPro(G574A), in which the residues G258 and G273 at the “P1” position of two suspected cis-cleavage sites of HCPro1 and the residue G574 of HCPro2, were, respectively, mutated into alanine, were created.

For RNA silencing suppression assays, we constructed three plasmids (pCaM-tHCPro-Myc, pCaM-HCPro1-Myc, and pCaM-HCPro2-Myc) that transiently express tHCPro-Myc, HCPro1-Myc, and HCPro2-Myc in planta. Briefly, the tHCPro, HCPro1, and HCPro2 coding regions from pF3-tHCPro-P3N were amplified, followed by integration into a binary plant expression vector, pCaMterX (65). Similarly, four additional plasmids, pCaM-N1, pCaM-N2, pCaM-D1, and pCaM-D2, which express the N and D domains of HCPro1 or HCPro2, were constructed.

To determine the repression of the D1 domain on antisilencing activity of HCPro1 N1, the overlapping PCR method was adopted to produce two plasmids, pCaM-N1D2 and pCaM-N2D1, for which the D domains of HCPro1 and HCPro2 were swapped. To investigate whether the detachment of HCPro1 affects antisilencing activity of HCPro2, two derivatives of pCaM-tHCPro-Myc, pCaM-tHCPro(C161A), and pCaM-tHCPro(G273A), for which the cis-cleavage of HCPro1 was abolished, were constructed. In brief, two plasmids, pF3-tHCPro-P3N(C161A) and pF3-tHCPro(G273A), were subjected to PCR amplification of tHCPro-P3N(C161A) and tHCPro(G273A), respectively, and the resulting PCR products were cloned into pCaMterX by XhoI/KpnI sites.

For generation of digoxin-labeled GFP probe, total RNA was extracted from 16c leaf tissues, followed by RT-PCR amplification of the complete GFP-coding region. The resulting PCR product was ligated into pGEM-T easy vector (Promega) to produce the plasmid pGEM-GFP.

All constructs above were verified by DNA sequencing.

In vitro translation.

A WGE-based in vitro translation assay was employed to assess the cis-cleavage activity of the indicated cysteine proteases or their mutants. For the assay, transcription and translation reactions were coupled using the T_NT SP6 high-yield wheat germ protein expression system and the FluoroTect Green_Lys in vitro translation labeling system (Promega), essentially following the manufacturer’s protocols. Subsequently, an aliquot of 4 μl of translation product for each sample was mixed with 10% SDS protein loading buffer, denatured at 60°C for 10 min, and separated by 12% SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Protein bands were visualized under a fluorescence scanning imager (Typhoon FLA; GE Healthcare).

Sequence analysis.

Nucleotide sequence analysis, such as protein sequence deduction, was performed using the Lasergene software package version 7.1. The HCPro sequences (or their truncated versions) of 44 representative species in 10 definitive genera and our recently proposed genus (Arepavirus) within the Potyviridae family, as previously reported (37, 38), were subjected to pairwise calculation of amino acid sequence identities, multiple alignment, and phylogenetic analysis. The pairwise calculation was carried out via ClustalW2 (https://www.ebi.ac.uk/Tools/msa/clustalw2/). Multiple alignment was performed using ClustalX 1.8.1 (http://www.clustal.org/), followed by the description with the Espript 3.0 program (http://espript.ibcp.fr/ESPript/ESPript/) (66). A phylogenetic tree was constructed with the maximum-likelihood method using MEGA 7.0.20. The robustness of the inferred evolutionary relationships was assessed by 1,000 bootstrap replicates.

Agroinfiltration assays.

Fully expanded leaves from wild-type N. benthamiana and 16c line seedlings at the 6- to 8-leaf stage were used for infiltration of A. tumefaciens cultures of the strain GV3101 harboring the relevant plasmids by following a previously described protocol (43, 67). For the single-stranded RNA-mediated silencing suppression assay, the classic two-component transient coexpression was performed by agroinfiltration of GFP-expressing construct pCHF3-35S-GFP (35S:GFP) (40) together with any of the tested plasmids. Briefly, two agrobacterial cultures, harboring the 35S:GFP and a candidate protein-expressing construct were adjusted into OD₆₀₀ of 1.0, mixed with equal volume, and coinfiltrated into leaves. GFP fluorescence was monitored with a high-intensity UV lamp (Black-Ray B-100AP, UVP), and the infiltrated leaves were photographed at the indicated dpi. For an infectivity test of ANSSV-derived cDNA clones, agrobacterial culture harboring pSS-I, pSS-I-G, pSS-I-G(Δ1), pSS-I-G(Δ2), pSS-I-G(P1), or pSS-I-G(HCPro) (these constructs are described below) was adjusted to an optical density at 600 nm (OD₆₀₀) of 1.0 and infiltrated into the third newly emerging leaves of N. benthamiana seedlings.

Northern blot and siRNA blot analyses.

In an RNA silencing suppression assay, Northern blotting was employed to detect the expression of the GFP gene. Briefly, total RNA was extracted from fresh leaf tissues using TRIzol (Invitrogen), separated in 1.2% agarose gel containing 2% formaldehyde, and transferred to Hybond-N⁺ membranes (GE Healthcare) by upward capillary transfer in 20× SSC buffer (1× SSC is 0.15 M NaCl plus 0.015 M sodium citrate). The membranes were hybridized to GFP-specific probes labeled with digoxigenin, which was prepared using a DIG Northern starter kit (Roche) with the linearized plasmid pGEM-GFP as the template. The detection of DIG signals was performed using CDP-star reagent in the kit. RNA bands were visualized using an ImageQuant LAS 4000 mini biomolecular imager (GE Healthcare) and analyzed with ImageJ software (68) for signal quantification.

The accumulation level of GFP-derived siRNAs was detected essentially as previously described (40). In brief, an aliquot of 5 μg of low-molecular-mass RNAs per sample was fractionated on a 15% PAGE containing 7 M urea and transferred to a Hybond-N⁺ membrane (GE Healthcare) by a semidry transfer instrument (LIUYI). After UV cross-linking and prehybridization, the blots were hybridized with a digoxigenin-labeled GFP-specific small RNA probe with an average length of approximately 50 nt, which was prepared by hydrolysis of antisense GFP RNA probe with carbonate buffer (23, 69). Detection and imaging of hybridization signals were the same as described above.

Immunoblotting and antibodies.

Fresh leaf tissues of N. benthamiana were collected at the indicated days and subjected to extraction of total proteins, essentially as described previously (43). Total proteins were separated by 12% SDS-PAGE, electroblotted onto a polyvinylidene difluoride membrane, and detected by immunoblotting using an anti-GFP or anti-Myc polyclonal antibody (Abcam). Goat anti-rabbit immunoglobulin antibody (Abcam) conjugated to horseradish peroxidase was used as the secondary antibody. The blots were treated with enhanced chemiluminescence detection reagents (Thermo Fisher Scientific) and visualized using an ImageQuant LAS 4000 mini biomolecular imager (GE Healthcare).

Construction of ANSSV-derived cDNA clones and infectivity test.

Standard DNA manipulation technologies were used to construct a complete cDNA clone of ANSSV-HNBT with a low-copy mini-binary T-DNA vector pCB301 as the backbone (70), essentially as described previously (71). Briefly, a modified pCB301 vector with our desired multiple cloning sites was amplified from pVPH (43). The entire ANSSV genome might be split into four portions (from the 5′ terminus: N1, N2, M, and C) that are delimited with unique MluI, BamHI, and AatII sites (Fig. 9A). Among them, the MluI and AatII sites were artificially created by mutating A (nucleotide position 2566) and T (position 5677) into corresponding G and C during primer synthesis; however, these mutations did not alter the encoded amino acids. The fragments M, C, and N1 were amplified by RT-PCR and integrated, step-by-step, into the modified pCB301 vector with the multiple cloning sites (Fig. 9A) to generate an intermediate vector. To compromise the toxicity of the P3 coding region to E. coli, a 220-bp intron 2 of the NiR gene of P. vulgaris was amplified from pT7-PLDMV-In2 (72) and integrated into the N2 fragment by the overlapping PCR method. The resulting N2 fragment containing the intron was cloned into the above-described intermediate vector by the MluI/BamHI sites. Finally, the entire genomic cDNA of ANSSV-HNBT was placed between the 35S promoter and Nos terminator in the pCB301 backbone to generate the complete cDNA clone of ANSSV (pSS-I) (Fig. 9A and B).

To develop a recombinant infectious ANSSV clone tagged by GFP, a GFP-coding region was inserted into the NIb/CP intercistronic junction. The original cleavage site “ANKEFQ/MD” at the NIb/CP junction, which is proteolytically recognized by NIa-Pro, was engineered into the NIb/GFP and GFP/CP junctions. The complete GFP-coding region was amplified from pVPH-GFP (43), and an overlapping PCR strategy was adopted to construct this clone. Using pSS-I-G as a parental clone, we constructed HCPro1- and HCPro2-deleted ANSSV mutants pSS-I-G(Δ1) and pSS-I-G(Δ2) using the overlapping PCR method. To evaluate whether the HCPro1, HCPro2, and HCPro1-HCPro2 of ANSSV could be replaced by the corresponding counterparts of a heterologous potyvirus and retain viral viability, the P1, HCPro, and P1-HCPro cistrons of TuMV (accession no. EF028235) were amplified from pCBTuMV-GFP/mCherry (73) to create three chimeric ANSSV clones using the overlapping PCR method, pSS-I-G(P1), pSS-I-G(HCPro), and pSS-I-G(P1HCPro), in which HCPro1, HCPro2, and HCPro1-HCPro2 of ANSSV were substituted with the corresponding counterparts P1, HCPro, and P1-HCPro of TuMV, respectively. In addition, we created another chimeric viral clone, pCBTuMV(HCPro1/2), in which the complete P1-HCPro of TuMV was substituted with the HCPro1-HCPro2 of ANSSV. All constructs were confirmed by DNA sequencing.

For plants inoculated with pSS-I or its derivatives, the presence of virus was detected by RT-PCR at the days indicated. Total RNAs were extracted from newly emerging leaves using TRIzol and treated with DNase I. The first-strand cDNAs were generated using the RevertAid First Strand cDNA synthesis kit (Thermo Fisher Scientific) with random hexamer primers, followed by PCR with Phusion high-fidelity DNA polymerase (Thermo Fisher Scientific) to target the ANSSV CP region. To assess whether the intron sequence was spliced in virus progenies of pSS-I, systemic leaves of plants inoculated with pSS-I were subjected to total RNA extraction, followed by RT-PCR detection of the ANSSV P3 region covering the intron sequence. The PCR product was sequenced for confirmation. For plants inoculated with GFP-tagged ANSSV clones, viral infection accompanying the expression of GFP protein was monitored by the UV lamp and Western blot analysis using an anti-GFP polyclonal antibody as described above (Abcam). For plants inoculated with pCBTuMV and pCBTuMV(HCPro1/2), viral infection was detected by RT-PCR to target the TuMV CP region.