Identification of a conserved binding site for WD40-domain proteins in PVA HCPro

The C-terminal region of PVA HCPro contains a five amino acid motif ‘AELPR’. This sequence is highly conserved in the genus Potyvirus (Fig 1A and S1A Fig). The percentage of conservation of the individual residues in the AELPR motif ranges from 86.6 to 100% in the HCPro sequences of the 119 potyvirus species we compared. Alanine, glutamic acid and proline were the most conserved residues (Fig 1A). This motif falls within the known crystal structure of the C-terminal cysteine proteinase domain of HCPro of turnip mosaic virus (TuMV; genus Potyvirus) [28] (Fig 1B). The catalytically active residues surround the motif as visible from the sequence comparison presented in (S1A Fig; marked by arrows). It is an inherently linear region between an α-coil and a β-sheet (highlighted in red) on the HCPro surface (Fig 1C) thus enabling direct interactions with possible binding partners. Based on predictions (with P < 0.05) by the Eukaryotic Linear Motif (ELM) Resource for Functional Sites in Proteins (http://elm.eu.org/), there are three motifs overlapping with ‘AELPR’ between amino acids 398–406 ‘TSAAEL’, ‘SAAELPRI’ and ‘ELPRI’, which have the potential to act as a short linear motif capable of interacting with WD40 domain-containing proteins. In the stretch 5 in the alignment of S1A Fig at least one of these motifs is retained leading to 100% conservation in the 119 potyvirus sequences. A molecular docking model (created using ClusPro online server: https://cluspro.bu.edu/) between TuMV HCPro and VCS suggested the presence of the ‘AELPR’ motif at the site of interaction (S1B–S1F Fig). Because of the properties described above, we hypothesized that this sequence could be the binding site to host proteins containing WD40 domains. The AELPR motif was mutated by replacing the charged residues glutamic acid and arginine with alanines. These mutations disrupted all the three possible forms, ‘TSAAEL’, ‘SAAELPRI’ and ‘ELPRI’, of WD40 domain- interacting motifs. The mutated virus and protein were named PVA^WD and HCPro^WD, respectively. Fig 1D and 1E depicts the PVA and HCPro constructs that were prepared for this study. All constructs used in this study are described in the S1 Table (see also S1 Reference).

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Fig 1. AELRP sequence in HCPro is predicted to bind to proteins containing a WD40-domain.

(A) Percentage of amino acid conservation within the AELPR motif in 119 potyviral HCPro sequences. This motif was identified by scanning the PVA HCPro sequence for short linear motifs (SLiMs) via ELM server (http://elm.eu.org/). The AELPR motif was predicted to be a putative WD40 -domain-interacting motif. (B) Ribbon diagram of X-ray crystallographic structure of the cysteine protease domain of TuMV HCPro (PDB ID: 3RNV). Secondary structure of the ‘AELPR’ motif has been highlighted in red. (C) Surface diagram of 3RNV showing the accessibility of the WD40-domain -interacting motif in HCPro (highlighted in red). (D—F) Schematic representation of the constructs prepared for this study (not in scale): (D) HCPro overexpression constructs. (E) icDNAs of the full length PVA constructs. (F) VCS overexpression constructs.

https://doi.org/10.1371/journal.ppat.1008956.g001

Infection profile of PVA^WD

In order to investigate the effects of the mutation in HCPro^WD on PVA infection we measured viral expression levels from local and systemic leaves. Expression of the viral polyprotein was assessed from the activity levels of the RLUC reporter gene incorporated between the NIb and CP cistrons [29]. In local leaves, PVA^WD accumulated with approximately five-fold lower efficiency than PVA^WT (Fig 2A). As the mutated motif lies in close vicinity to the cysteine protease domain of HCPro, it was necessary to confirm that this mutation did not hamper its autocatalytic activity. A western blot analysis of the same samples was carried out with an α-HCPro antibody (lower panel Fig 2A). HCPro^WD accumulation level was lower than that of wild-type HCPro (HCPro^WT) in the blot, which was in line with the difference in the polyprotein expression levels of PVA^WD and PVA^WT. The presence of monomeric HCPro^WD derived from PVA^WD infection (Fig 2A) confirmed that HCPro^WD had retained its polyprotein processing ability.

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Fig 2. HCPro^WD mutation reduces local and prevents systemic PVA infection without affecting its autocatalytic proteinase activity.

(A) Virus-derived RLUC activity levels of PVA^WT and PVA^WD to evaluate the effect of the mutation in WD40-domain -interacting motif of HCPro on PVA gene expression. PVA^WD exhibits a ~5 fold lower expression level than PVA^WT. All the constructs were infiltrated at OD₆₀₀ = 0.01, and the samples were collected at 5 dpi. A western blot analysis with α-HCPro antibody of the same samples is presented in the lower panel. The presence of monomeric HCPro^WD derived from PVA^WD confirms the preservation of the autocatalytic activity of HCPro^WD’s cysteine ​​protease domain. Statistically significant difference between the samples is denoted by an asterisk (*P < 0.05; n = 3). (B) Complementation of PVA^ΔHCPro infectivity by various HCPro mutants. Agrobacterium carrying different HCPro variants were infiltrated as follows: silencing-deficient HCPro^SDM and eIF4E binding-deficient HCPro^4EBM mutants at OD₆₀₀ = 1, HCPro^WD and HCPro^WT at OD₆₀₀ = 0.3. PVA^ΔHCPro was infiltrated at OD₆₀₀ = 0.1. GUS was used as the control, as well as to equalize infiltrated Agrobacterium cell numbers among all the sets. Firefly luciferase (FLUC) at OD₆₀₀ = 0.01 was added as internal control for normalization of RLUC activity. Samples were collected at 3 dpi. A western blot analysis (lower panel) with α-HCPro antibodies demonstrates comparative expression levels of all HCPro variants from their corresponding expression constructs. Different letters above the bars indicate a statistically significant difference (student’s t-test P < 0.05; n = 4). (C) A time course experiment of systemic infection by PVA^WD. N. benthamiana leaves were infiltrated with Agrobacterium carrying PVA^WD / PVA^WT constructs (OD₆₀₀ = 0.01). Samples were collected from newly emerging systemic leaves at 5, 6, 7 dpi, respectively and RLUC activity was measured. Statistical significance was assessed using student’s t-test (*P < 0.05; n = 3).

https://doi.org/10.1371/journal.ppat.1008956.g002

Additionally, we compared the complementation efficiency of HCPro^WD with respect to HCPro^WT and two other HCPro mutants, the RNA silencing suppression-deficient HCPro (HCPro^SDM) and eIF(iso)4E binding-deficient HCPro (HCPro^4EBM) [10]. For this, we infiltrated the plants with PVA^ΔHCPro and complemented the infection with GUS (OD₆₀₀ = 1), HCPro^SDM (OD₆₀₀ = 1), HCPro^4EBM (OD₆₀₀ = 1), HCPro^WD (OD₆₀₀ = 0.3), or HCPro^WT (OD₆₀₀ = 0.3). Neither HCPro^SDM nor HCPro^4EBM could elevate RLUC expression from PVA^ΔHCPro showing no sign of complementation. HCPro^WD on the other hand promoted PVA^ΔHCPro gene expression significantly (Fig 2B) and HCPro^WT complemented PVA^ΔHCPro most efficiently. Interestingly, the complementation efficiencies of HCPro^WD and HCPro^WT also exhibited approximately a five-fold difference, which was similar to differences in the gene expression efficiency of PVA^WD and PVA^WT (Fig 2A). Infiltration ODs of all the HCPros were adjusted to achieve equal expression (lower panel of Fig 2B). Notably, same infiltration ODs of HCPro^WD/WT resulted in similar level of HCPro expression (lower panel of Fig 2B; also see S2 Fig for the expression levels of their Strep-RFP tagged versions). Therefore, the defective infection in PVA^WD is not because of the lower expression level of HCPro^WD, rather it is something related to its functional aspects.

An interesting difference in the viral gene expression was noticed while monitoring the infection level of PVA^WD and PVA^WT in systemically infected leaves. There was more than a 1000-fold reduction in the RLUC activity in PVA^WD compared to PVA^WT (Fig 2C) indicating that the systemic spread of PVA^WD was severely compromised. Despite the difference, the low level of PVA^WD expression was detectable in the systemic leaves already at 5 dpi. We followed the development of RLUC activity at 6 and 7 dpi, but did not see any signs that the mutant would reach wild-type expression levels. Since HCPro can affect the potyviral infection cycle at multiple levels we next assessed the effect of the HCPro^WD mutation on further known properties of HCPro.

The silencing suppression capacity of the HCPro^WD mutant is compromised

Ability to suppress RNA silencing is undoubtedly one of the most studied properties of HCPro. To assess whether the HCPro^WD mutation affected the silencing suppression capacity of HCPro we used a dsRNA-triggered silencing assay based on the simultaneous transient expression of a transgene and a hairpin targeting it (Fig 3A). We tested the ability of HCPro to rescue the expression of the silenced transgene. The study was carried out in three different contexts. In the first case, RLUC was expressed along with a hairpin-RLUC (pHG-RLUC) construct and either HCPro^WD or HCPro^WT was expressed to rescue RLUC expression (Fig 3B). Secondly, YFP and a hairpin targeting it (pHG-GFP) were expressed in the presence of HCPro^WD / HCPro^WT and PVA^ΔHCPro. PVA^ΔHCPro was included to see if other PVA proteins could contribute to the silencing suppression process (Fig 3C). Finally, pHG-RLUC was employed to target RLUC expressed from PVA^ΔHCPro RNA and either HCPro^WD or HCPro^WT was expressed to rescue viral gene expression (Fig 3D). In all cases, HCPro^WT successfully recovered the transgene and viral expression while HCPro^WD did not. These experiments clearly demonstrated that HCPro^WD is deficient in dsRNA-triggered RNA silencing suppression.

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Fig 3. HCPro^WD is defective in suppressing hairpin-triggered RNA silencing, inducing PGs and forming infection associated HMW complexes.

(A) A schematic representation of the constructs used to study the suppression of hairpin-triggered RNA silencing. The hairpin RNAs (pHG-RLUC and pHG-GFP) targeting monocistronic RLUC, YFP, and RLUC-expressing PVA RNA were expressed to trigger RNA silencing. HCPro^WD and HCPro^WT were expressed to test their capacity to suppress RNA silencing. (B) The capacity of HCPro^WD to suppress RNA silencing. N. benthamiana plants were agroinfiltrated with the RLUC expression construct (OD₆₀₀ = 0.01), pHG-RLUC construct (OD₆₀₀ = 0.4) and HCPro^WD / HCPro^WT constructs (OD₆₀₀ = 0.3) as indicated. Empty pHG-CTRL and GUS were expressed as the controls. All constructs were co-infiltrated and the samples were analyzed for RLUC activity at 3 dpi. (C) The effect of other PVA proteins on HCPro^WD’s capacity to suppress RNA silencing. Agrobacterium carrying YFP expression construct (OD₆₀₀ = 0.01) and pHG-GFP (OD₆₀₀ = 0.4) were co-infiltrated as in (B) with HCPro^WD / HCPro^WT and PVA^ΔHCPro (OD₆₀₀ = 0.1). YFP fluorescence was quantitated from leaf discs collected at 4 dpi using a 96-well plate reader at Ex/Em 500/530 nm [51]. (D) Suppression of RNA silencing targeting PVA RNA-derived RLUC expression. The experimental setup was similar to that of (B). Agrobacterium carrying PVA^ΔHCPro was infiltrated at OD₆₀₀ = 0.05. RLUC activities were measured from samples collected at 3dpi. The number of plants equals 4 (n = 4) in (B), n = 9 in (C) and n = 6 in (D). Different letters above the bars indicate a statistically significant difference (student’s t-test *P < 0.05). (E, F) Variation in the amount of PGs during transient expression of HCPro^WD (E) and HCPro^WT (F) infiltrated at OD₆₀₀ = 0.1. P0^YFP was co-expressed at OD₆₀₀ = 0.1 to visualize PGs. Samples collected at 3 dpi were examined with an epifluorescence microscope under an FITC filter. (G) Amount of PGs induced by HCPro^WD / HCPro^WT overexpression. HCPro^WD / HCPro^WT were agroinfiltrated at OD₆₀₀ = 0.1 and 0.3 as indicated, while P0^YFP was agroinfiltrated at OD₆₀₀ = 0.1. The number of PGs per mm² was calculated from nine independent areas. Asterisks denote statistical significance between the sample sets (student’s t-test *P < 0.05, ***P < 0.001). (H) TwinStrep tag-affinity purifications were carried out from leaf samples collected at 3 dpi from plants infiltrated with either PVA^WD-Strep-RFP or PVA^WT-Strep-RFP (OD₆₀₀ = 0.1). The purification resulted in the pulldown of HCPro along with its in vivo binding partners. Purified proteins were analyzed by western blotting using α-HCPro, α-CI, α-SAMS and α-VPg antibodies, respectively. All the blots were made from the same gel by cutting the PVDF membrane into strips after the transfer. In addition, two lanes of the same gel were silver-stained. The low molecular weight bands marked with asterisk demonstrates equal loading of the HCPro^WD-Strep-RFP and HCPro^WT-Strep-RFP samples.

https://doi.org/10.1371/journal.ppat.1008956.g003

In our earlier work, we proposed a role for HCPro in formation of PVA induced granules (PGs), and we suggested PGs have a role in suppression of RNA silencing [10]. Since HCPro^WD was unable to suppress RNA silencing, we next compared its PG formation efficiency to that of HCPro^WT. For this we co-infiltrated leaves with either HCPro^WD or HCPro^WT and P0^YFP, which is a visual marker for PGs [10], all at OD₆₀₀ = 0.1. The extent of PG induction by the HCPro variants was calculated at 3 dpi. The amount of PGs was reduced by almost five-fold in the presence of HCPro^WD as compared to HCPro^WT (Fig 3E–3G), which showed that the impaired ability of HCPro^WD to act as a suppressor of RNA silencing correlated with its capacity to induce PG formation. Increasing the OD of infiltration (to OD₆₀₀ = 0.3) did not increase granule count either for HCPro^WD or HCPro^WT. Rather, a reduction in the granule count was noticed in the case of HCPro^WT, indicating that the potency of the PG-inducing property of HCPro does not necessarily correlate with the increased protein amount (Fig 3G). No granules were detected in the control images where P0^YFP was expressed alone with 35S-GUS (S3 Fig).

HCPro^WD is defective in forming PVA infection-associated high molecular weight (HMW) complexes

Previously we reported the formation of highly stable HMW complexes during PVA infection [12]. These complexes need HCPro to form and they contain several infection-associated host and viral proteins. We also suggested that these complexes play a role in PVA translation [12]. In this study, we tested if the HCPro^WD mutation changed HCPro’s ability to form these HMW complexes. For this, we adopted a similar strategy reported in Ivanov et al. [12] with minor modifications. N. benthamiana plants were infected with PVA^WD-Strep-RFP or PVA^WT-Strep-RFP at OD₆₀₀ = 0.1. Since PVA^WD gene expression in systemic leaves was low (Fig 2C), samples were collected from the local leaves at 3 dpi. The affinity purification of HCPro^{WD/WT-Strep-RFP} was performed following the method described in [30]. The purified samples were analysed by probing the protein gel blots with antibodies recognizing some of the previously identified HCPro^WT interactors including S–adenosyl-L–methionine synthase (SAMS), VPg and CI [12]. The HMW bands detected in the immunoblots had a similar electrophoretic mobility as those in the silver stained gel (Fig 3H). Based on visual inspection the amounts of SAMS, CI, and VPg were reduced in the HCPro^WD samples compared to HCPro^WT (Fig 3H). LC-MS/MS analysis of the same samples confirmed the western blot results (S2 Table). Interestingly, the silver stained gel showed an overall enrichment of complexes in the HMW region in HCPro^WT samples compared to HCPro^WD. Equivalent loading of the samples is demonstrated by the equal intensity of a 55 kDa band in the silver stained gel (left panel Fig 3H).

HCPro^WD is unable to sequester WD40 domain-containing host protein VCS into PGs

Earlier, Hafrén et al. [10] established VCS as a component of HCPro-induced PGs and an important factor in PVA infection. Furthermore, systemic infection, PVA translation, and coat protein (CP) accumulation were all affected by VCS knockdown [10]. Since VCS is a WD40 repeat-containing protein and the effects of low VCS levels on infection were parallel with the defects observed in PVA^WD infection, we decided to assess whether the HCPro^WD -binding site is needed to connect it to VCS. To this end, we tested whether VCS localized to PG structures in the presence of HCPro^WD. For this experiment, we first generated VCS expression constructs. N. benthamiana annotated transcript data available on the Sol Genomics Network (http://solgenomics.net) document different variants of VCS. We used BLAST searches to identify three genes in the N. benthamiana genome with circa 50% identity to the Arabidopsis thaliana VARICOSE (Q9LTT8): VCS-A (Niben101Scf00654g02003.1), VCS-B (Niben101Scf39216g00007.1) and VCS-C (Niben101Scf21107g00015.1). We generated expression constructs of these three N. benthamiana genes along with their tagged versions (see Fig 1F).

Next we infiltrated N. benthamiana leaves with either RFP-tagged HCPro or the mutated version (HCPro^WT-Strep-RFP / HCPro^WD-Strep-RFP at OD₆₀₀ = 0.1) to induce PGs, YFP-tagged VCS (VCS-A^YFP, VCS-B^YFP and VCS-C^YFP each at OD₆₀₀ = 0.1 cumulatively denoted by VCS^YFP), and another PG marker P0^CFP (OD₆₀₀ = 0.1). Their localization was monitored via confocal microscopy at 3 dpi. The results reveal that HCPro^WT was able to sequester VCS into the PGs. Co-localization of the PG marker P0^CFP [10] in the same spot validated them as bona fide PGs. Unless the threshold effect prevented the detection of an overlap, HCPro^WD-Strep-RFP -induced cytoplasmic foci did not co-localize with either VCS^YFP or P0^CFP (Fig 4A–4F). Pairwise overlaps of the fluorescence signals corresponding to Fig 4A–4F are shown in S4 Fig, and the accumulations of HCPro^{WD/WT-Strep-RFP} and VCS^YFP related to the Fig 3A–3F is shown in the S5 Fig. Although a small difference could be detected in the accumulation levels of HCPro^WT and HCPro^WD, they were still comparable.

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Fig 4. HCPro^WD is defective in recruiting VCS to PGs.

(A-F) Localization of Strep-RFP-tagged -HCPro, YFP -tagged-VCS and CFP tagged -P0 in N. benthamiana epidermal cells. Agrobacterium carrying either HCPro^WT-Strep-RFP or HCPro^WD-Strep-RFP was infiltrated at OD₆₀₀ = 0.1, while Agrobacteria harboring VCS-A^YFP, VCS-B^YFP and VCS-C^YFP were each infiltrated at OD₆₀₀ = 0.1 (together denoted as VCS^YFP). Agrobacterium harboring P0^CFP, infiltrated at OD₆₀₀ = 0.1, was expressed as another marker for PGs. Samples were analyzed at 3 dpi by confocal laser scanning microscopy in sequential scanning mode. (A) P0^CFP, (B) VCS^YFP, (C) HCPro^WT-Strep-RFP, (D) P0^CFP, (E) VCS-^YFP and (F) HCPro^WD-Strep-RFP. (G-L) Localization of HCPro^{WT/WD-Strep-RFP} and VCS^YFP and their overlays in N. benthamiana epidermal cells. Experimental details are otherwise the same as in Fig 4A–4F, except for the absence of P0^CFP. (G) HCPro^WT-Strep-RFP, (H) VCS-^YFP and (I) Overlay of HCPro^WT-Strep-RFP and VCS^YFP. (J) HCPro^WD-Strep-RFP, (K) VCS^YFP and (L) Overlay of HCPro^WD-Strep-RFP and VCS^YFP. Degree of co-localization between HCPro^WT-Strep-RFP / HCPro^WD-Strep-RFP and VCS^YFP is calculated from representative images and presented in S6 Fig. (M-R) Epifluorescence microscopy showing the distribution of HCPro^{WT/WD-Strep-RFP} and P0^YFP within a representative area of examination. HCPro and P0^YFP, which both typically co-localize in PGs, were co-infiltrated to visualize the PGs. The experiment was conducted similarly as Fig 3E and 3F. The only exception was that HCPro^{WT/WD-Strep-RFP} was used instead of HCPro^WT/WD. Images were acquired at 3 dpi with a 10X objective. HCPro^{WT/WD-Strep-RFP} was visualized under an RFP filter (M, P respectively). P0^YFP from the corresponding sample sets was visualized under the FITC filter (N, Q respectively), and their overlays are presented in (O, R), respectively.

https://doi.org/10.1371/journal.ppat.1008956.g004

HCPro^WD mutation impairs HCPro-VCS co-localization

Next, we compared the extent of VCS co-localization with HCPro^WD and HCPro^WT. For this we used similar experimental setup as Fig 4A–4F, except that P0^CFP was omitted. Confocal microscopy images from 3 dpi samples clearly show that HCPro^WT-Strep-RFP and VCS^YFP accumulated together to distinct foci with near perfect co-localization between these proteins (Fig 4G–4I). On the other hand, while HCPro^WD-Strep-RFP accumulated in distinct foci (Fig 4J), VCS^YFP remained diffuse in the cytoplasm (Fig 4K). The overlay image (Fig 4L) shows reduced co-localization between VCS^YFP and HCPro^WD-Strep-RFP. The amount of co-localization with VCS^YFP was calculated from the confocal image data for both HCPro^WT-Strep-RFP and HCPro^WD-Strep-RFP (S6 Fig). Approximately a 48% reduction was observed with the HCPro^WD mutant. The relative expression levels corresponding to Fig 4G–4L of HCPro^WD-Strep-RFP and HCPro^WT-Strep-RFP and that of VSC^YFP in the presence of both HCPro versions were comparable (S7 Fig). These data along with Fig 4A–4F advocate the importance of the WD40 domain-binding motif in HCPro-VCS co-localization in planta.

HCPro^WD mutation impairs HCPro’s ability to induce functional PGs

The low level of co-localization between VCS and HCPro^WD was an interesting finding especially since we still observed plenty of HCPro^WD-Strep-RFP in PG-like foci (Fig 4F–4J). Epifluorescence microscopy revealed a more than 75% coincidence of HCPro^WT with PG marker P0^YFP (S8A Fig, Fig 4M–4O). The amount of co-localization between HCPro^WD and P0^YFP was ten-fold lower (S8A Fig). This shows that only a marginal portion of HCPro^WD-Strep-RFP containing foci were PGs, which typically contain P0^YFPas found in Hafrén et al.[10]. To show the lack of co-localization of HCPro^WD and P0^YFP in detail, an image of a single intact cell is provided in (S8B–S8D Fig). A reference PG induced by HCPro^WT is shown in (S8E–S8G Fig). Cumulatively the confocal and epifluorescence microscopy results indicate that the WD domain-interacting motif in HCPro is required for HCPro’s ability to recruit vital granule components to form functional PGs.

HCPro-VCS association in HMW complexes is affected by the HCPro^WD mutation

In order to demonstrate the association of HCPro and VCS in HMW complexes formed during PVA infection in N. benthamiana, we affinity purified Strep-tagged HCPro and tested the samples for the co-immunoprecipitation of VCS. In the affinity purification experiment, the plants were infected with PVA^WD-Strep-RFP or PVA^WT-Strep-RFP at OD₆₀₀ = 0.1. Samples were collected from local leaves at 3 dpi and the affinity purification of HCPro^{WD/WT-Strep-RFP} was performed following the method described in [30]. The presence of HCPro and VCS in the purified samples was investigated by immunoblotting with α-HCPro and α-VCS antibodies (Fig 5A). Equivalent loading of the purified HCPro^WD-Strep-RFP and HCPro^WT-Strep-RFP samples is demonstrated by the equal intensities of low molecular weight bands, the major one marked with an arrow at the ~70 kDa region in the silver stained gel. The lack of any prominent bands in the GUS control indicated a negligible amount of non-specific interactions with Strep-Tactin matrix during purification. Both HCPro and VCS bands were detected in the HMW- region (>250 kDa). In contrast to the HCPro^WT-Strep-RFP, top-most band containing VCS (marked with an asterisk) was missing in HCPro^WD-Strep-RFP samples. Interestingly, the top-most band was also absent in the silver stained gel and drastically reduced in the α-HCPro immunoblot in the HCPro^WD-Strep-RFP pull-down samples. A comparison between the input and the eluted samples indicated specific enrichment of HMW complexes upon purification (S9 Fig). Additionally, it further validated the specificity of disappearance of the top-most VCS band from HCPro^WD-Strep-RFP purified samples. Nevertheless, HMW complexes approximately 250 kDa in size were still present in HCPro^WD-Strep-RFP samples. This suggests that the mutation in the WD40 domain-interacting motif of HCPro affected but did not completely abolish the formation of HCPro- and VCS-containing HMW-complexes during PVA infection.

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Fig 5. The WD mutation weakens VCS association with HCPro in HMW RNP complexes and the stability of the complexes.

(A) HCPro and VCS assemble into HMW complexes during infection. For affinity purification, PVA^{WD- Strep-RFP} / PVA^WT-Strep-RFP constructs were agroinfiltrated at OD₆₀₀ = 0.1 and the infected local leaves were sampled at 3 dpi. Both HCPro and VCS antibodies recognized HMW bands with a similar electrophoretic mobility in the purified HCPro^WT-Strep-RFP samples. The uppermost HCPro and VCS containing band was missing in HCPro^WD-Strep-RFP samples (marked with asterisks) both in the silver-stained gel and in the western blots. Equal loading is demonstrated in the silver-stained gel by the low molecular weight bands present in the samples (marked with an arrow). All the samples were run in the same gel. (B) Validation of the HCPro^WD-Strep-RFP / HCPro^WT-Strep-RFP affinity purification procedure by SDS PAGE and silver staining. HMW complexes are visible in the HCPro^{WD/WT-Strep-RFP} eluates. The purified products were treated with DNase, Proteinase K and RNase A and subsequently subjected to SDS-PAGE. The silver-stained gels demonstrate the stability of the purified HMW complexes. (C) RNase degradation of HCPro-associated HMW RNP complexes. To achieve maximum separation of the HMW RNP complexes, samples similar to those in A) were loaded in varying quantities and run for a longer time in a 10% SDS-PAGE gel. In all the cases, PVA^WD-Strep-RFP and PVA^WT-Strep-RFP samples were loaded equally similarly as in A). Samples presented in the right panel are similar to those shown in the adjacent left panel except that three folds more was loaded to visualize the effect of RNase A treatment on complexes in PVA^WD-Strep-RFP samples. All the samples in C) were part of the same gel but the right panel was developed for a longer time than the left panel during silver staining.

https://doi.org/10.1371/journal.ppat.1008956.g005

HMW complexes formed during PVA^{WD Strep-RFP} infection are sensitive to RNase treatment

Results presented in Fig 3H and Fig 5A indicated compositional differences between HCPro^WD and HCPro^WT -containing HMW complexes. Several HMW components such as CI, VPg, SAMS and VCS were specifically reduced in HCPro^WD samples. Next, we probed into the stability of the HMW complexes. For this, we treated the affinity-purified samples with DNase, proteinase K or RNase A followed by SDS-PAGE analysis. Both HCPro^WD and HCPro^WT–containing HMW complexes resisted DNase treatment whereas proteinase K degraded them completely (Fig 5B). Intriguingly, RNase A affected HCPro^WD-HMW complexes, while it did not affect the HCPro^WT ones (Fig 5B). A bulk reduction of the HCPro^WD-HMW complexes upon RNase A treatment suggested that they are ribonucleoprotein (RNP) complexes. To probe into this phenomenon further, we ran the RNase A degraded complexes longer in the gel and optimized the loading quantity to get a better look on the degraded complexes (Fig 5C). Smearing of the bands rather than the presence of individual low-molecular weight proteins suggests the existence of loosely bound multiprotein complexes migrating at the HMW region even when the RNA fraction exposed to RNase treatment had been degraded (Fig 5C). The data suggest that the impaired assembly of HCPro^WD and the other HMW components compromised the stability of these complexes rendering the RNA prone to degradation by RNase A.

VCS knockdown reduces PVA^WT gene expression and accumulation to the level of PVA^WD

We considered the WD-mutation -mediated defect in HCPro-VCS association could be a possible reason behind the aberrant behaviour of HCPro^WD and sub-optimal performance of PVA^WD. To test this idea we characterized the role of HCPro-VCS interaction in PVA infection. We studied how limited VCS availability affected PVA^ΔHCPro, PVA^WD and PVA ^WT infection. For this, we transiently knocked down VCS by infiltrating leaves with a hairpin construct targeting VCS (pHG-VCS). PVA^ΔHCPro, PVA^WD and PVA ^WT expression levels were then measured both in the control and VCS-silenced background. VCS silencing was validated via reverse transcription-polymerase chain reaction (RT-PCR) (S10 Fig). Following a pattern similar to Fig 2A, PVA^WD gene expression level was approximately 5-fold lower than that of PVA^WT (Fig 6A) and approximately 20-fold higher than that of PVA^ΔHCPro in the control plants. On the other hand, in VCS knock-down experiments PVA^WT gene expression decreased to the level of PVA^WD, while that of PVA^WD remained unaffected. Interestingly, gene expression of PVA^ΔHCPro increased upon VCS downregulation (Fig 6A), showing that in the absence of HCPro, VCS is an antiviral protein. vRNA levels followed the same pattern as RLUC expression throughout this experiment (Fig 6B). In contrast to the replicating viruses, RLUC and vRNA were expressed at similar levels from the replication-deficient variants of PVA (PVA^{ΔGDD-ΔHCPro}, PVA^{ΔGDD-HCProWD} and PVA^{ΔGDD- HCProWT}) and did not change upon VCS silencing (Fig 6C and 6D). Thus, the VCS-HCPro interaction is especially important for the functions of the replicating PVA RNA. Interestingly, VCS seemed to promote PVA^WT infection in the presence and restrict it in the absence of HCPro. PVA^WD was insensitive to VCS knockdown while PVA^WT decreased to the level of PVA^WD supporting the role of impaired HCPro-VCS interaction in the reduced PVA^WD infection. Our interpretation of this result is that HCPro has the capacity to convert VCS from an antiviral protein to the support of the PVA infection.

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Fig 6. VCS knockdown brings down PVA^WT expression level to that of PVA^WD.

(A, B) N. benthamiana leaves were infiltrated with Agrobacterium carrying a silencing vector (pHG-VCS) expressing a hairpin RNA targeting VCS (OD₆₀₀ = 0.4). An empty Hellsgate plasmid (pHG-CTRL) was used as a control. Agrobacterium carrying PVA^ΔHCPro, PVA^WD and PVA^WT constructs were co-infiltrated (OD₆₀₀ = 0.01) with Agrobacterium carrying 35S-FLUC (OD₆₀₀ = 0.01) for normalization of the virus-derived RLUC values. Normalized RLUC (A) and PVA RNA (B) levels were quantitated at 4 dpi. (C) Normalized RLUC and (D) RNA values derived from non-replicating PVA RNAs: PVA^{ΔGDD-ΔHCPro}, PVA ^{ΔGDD-HCProWD} and PVA ^{ΔGDD-HCProWT}. VCS silencing was done similarly as in (A). Agrobacterium carrying PVA^{ΔGDD-ΔHCPro}, PVA ^{ΔGDD-HCProWD} and PVA ^{ΔGDD-HCProWT} constructs were co-infiltrated (OD₆₀₀ = 0.05) with Agrobacterium carrying 35S-FLUC (OD₆₀₀ = 0.01) for normalization of the virus-derived RLUC values. Samples were collected at 3 dpi. The number of plants per experiment was 6 in (A, B) and 3 in (C). Statistical significance was assessed using student’s t-test. Different letters above the bars indicate a statistically significant difference (P < 0.05).

https://doi.org/10.1371/journal.ppat.1008956.g006

VPg, HCPro and VCS together promote PVA translation

We proposed in our previous paper [12] that the presence of HCPro and AGO1 on polysomes may indicate that RNA silencing-related translational repression is involved in PVA infection and HCPro is required to relieve it. Also, we reported a role for VPg and HCPro in PVA translational regulation, and proposed the possible involvement of VCS in VPg-mediated translational enhancement [10, 31, 32]. In this study, we probed into the role of VCS in PVA translation by overexpressing either VPg, VCS or both together with PVA^{ΔGDD-ΔHCPro}, PVA^{ΔGDD-HCProWD} and PVA^{ΔGDD-HCProWT}. The replication-deficient PVAs were used in this experiment to narrow down the effects of VCS, VPg and HCPro to aspects pertaining to PVA translation. More vigorous protein production from an unaltered RNA concentration, which affects the ratio of the protein and its mRNA, has been regarded as an indicator of release from translational repression [33]. Therefore, we calculated the RLUC/vRNA ratios from PVA^{ΔGDD-ΔHCPro}, PVA^{ΔGDD-HCProWD} and PVA^{ΔGDD-HCProWT} upon VPg and VCS overexpression. In this experiment, all the constructs were co-infiltrated and samples were collected at 3 dpi. The absolute values for RLUC activities and RNA levels were quantitated and are presented in S11A and S11B Fig. Overexpression of VCS was validated via qPCR (S11C and S11D Fig).

Irrespective of which HCPro variant was present, VPg enhanced the RLUC/vRNA ratio similarly for all of the replication-deficient PVAs (Fig 7A). The absolute RLUC activity measurements show that both PVA^{ΔGDD-HCProWD} and PVA^{ΔGDD-HCProWT} are equally enhanced by VPg (S11A Fig). This suggests that VPg-mediated enhancement of translation does not require an intactWD40 domain-binding motif in HCPro. We further validated this by supplementing non-replicating PVA^{ΔGDD-ΔHCPro} with VPg together with either GUS (OD₆₀₀ = 1), HCPro^SDM (OD₆₀₀ = 1), HCPro^4EBM (OD₆₀₀ = 1), HCPro^WD (OD₆₀₀ = 0.3), or HCPro^WT (OD₆₀₀ = 0.3). VPg did not enhance translation in the presence of HCPro^SDM and HCPro^4EBM. However, in the presence of both HCPro^WD and HCPro^WT, VPg caused a similar extent of RLUC enhancement (S12 Fig), which further confirmed that the HCPro^WD mutation does not prevent the effect of VPg on PVA translation, whereas HCPro^SDM and HCPro^4EBM mutants do. Overexpression of VCS alone did not have a significant effect on the RLUC / vRNA ratio of any replication-deficient PVA. Finally, co-expression of VPg and VCS increased the RLUC/vRNA ratio of PVA^{ΔGDD-HCProWT} significantly more than that of PVA^{ΔGDD-ΔHCPro} and PVA^{ΔGDD-HCProWD}. Both PVA^{ΔGDD-ΔHCPro} and PVA^{ΔGDD-HCProWD} performed similarly in the simultaneous overexpression of VPg and VCS. In fact, here the addition of VCS did not change the situation where VPg alone was overexpressed. Taken together, HCPro and VCS need to interact in order to achieve full translational benefit generated by the combined effect of VPg and VCS (Fig 7A).

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Fig 7. Both VPg and VCS-HCPro interaction promote PVA translation.

(A) RLUC/vRNA ratio of non-replicating variants of PVA RNA (PVA^{ΔGDD-ΔHCPro}, PVA^{ΔGDD-HCProWD} and PVA^{ΔGDD-HCProWT}) was quantitated during VPg, VCS and VPg + VCS overexpression. All virus constructs were infiltrated at OD₆₀₀ = 0.05 and all overexpression constructs at OD₆₀₀ = 0.25. VCS overexpression was initiated with a mix of Agrobacterium carrying VCS-A, VCS-B and VCS-C constructs each at OD₆₀₀ = 0.25. Samples for the dual luciferase assay and RNA quantification via qPCR were collected at 3 dpi. Statistical significance was assessed using student’s t-test (*P < 0.05). Different letters above the bars indicate a statistically significant difference. (B—D) Fold change in the RLUC/vRNA ratio in replicating variants of PVA- (PVA^ΔHCPro, PVA^WD and PVA^WT) upon VCS-silencing (B, C) and overexpression (D, E). The ratios were determined both without (B, D) and with (C, E) overexpressed VPg. All PVA constructs were infiltrated at OD₆₀₀ = 0.01. The silencing constructs were infiltrated one day before virus infiltration at OD₆₀₀ = 0.4, whereas the overexpression constructs were co-infiltrated with PVA constructs. VPg was infiltrated at OD₆₀₀ = 0.4. The VCS overexpression set contained a mix of VCS-A, VCS-B and VCS-C each infiltrated at OD₆₀₀ = 0.1. All samples were collected at 4 dpi. The concentration of infiltrated Agrobacterium between different samples in (A-E) was equalized with Agrobacterium carrying either pHG-CTRL or GUS. The number of plants per experiment was 4 in (A), 6 in (B, C) and 5 in (D, E). Statistical significance between the sets is denoted by asterisk (student’s t-test *P < 0.05, **P < 0.01).

https://doi.org/10.1371/journal.ppat.1008956.g007

Next we studied the effect of VPg and VCS on the translation of replicating PVAs. The RLUC/vRNA ratios of PVA^ΔHCPro, PVA^WD and PVA^WT were calculated from VCS knock-down and overexpression experiments. In addition, we included transiently overexpressed VPg in the experiment to study its contribution to the RLUC/vRNA ratio with and without VCS. Samples were collected from the infiltrated leaves at 4 dpi. Silencing and overexpression of VCS were validated via qPCR (S13 Fig). After measuring RLUC activity and vRNA levels (S14 Fig) we calculated the RLUC/vRNA ratio for each sample. VCS knock-down either in the presence or in the absence of VPg did not significantly alter the RLUC/vRNA ratio of any PVA variant (Fig 7B and 7C). A small increase in the RLUC/vRNA ratio was detected upon VCS overexpression in plants infected with PVA^WT (Fig 7D). This ratio increased further when both VCS and VPg were co-expressed in PVA^WT infected plants (Fig 7E). Similar to the replication-deficient PVAs (S11A and S11B Fig), the change in the ratios caused by simultaneous overexpression of VPg and VCS were due to reduced vRNA accumulation and unaffected RLUC. While active PVA translation benefits from the collaboration of VPg, VCS and HCPro, the reduced RNA amount during VCS overexpression may indicate that other viral processes become unbalanced.

VCS-HCPro interaction is required for particle formation

The importance of VCS for systemic infection and CP accumulation was proposed in Hafren et al. [10]. Here we show that in PVA^WD viral gene expression remained low in systemic leaves suggesting that very little of vRNA reached the upper leaves (see Fig 2C). Since systemic movement is often associated with the formation of stable virions [34–38], we next tested the ability of PVA^WD to form particles or virus-like particles (VLPs). For this, we sampled locally infected leaves at 3 dpi. In order to obtain equivalent infection PVA^WD was infiltrated in a higher amount (OD₆₀₀ = 1) compared to PVA^WT (OD₆₀₀ = 0.1). Immunocapture (IC)-qRT-PCR was performed to measure the abundance of vRNA assembled into VLPs or virions. Although RLUC activities were broadly similar, a drastic reduction in the amount of particles was noticed in PVA^WD-infected plants as compared to PVA^WT-infected plants (Fig 8A). CP accumulation was validated via western blotting. A slight reduction in the intensity of the CP band was observed in PVA^WD (Fig 8A). To visually inspect the particles, we extracted the particles in phosphate buffer and subjected them to transmission electron microscopy. Virus particles were abundantly detected in PVA^WT-infected samples but none were observed in PVA^WD-infected samples. A few short and loose particle-like structures were occasionally detected; however, they were virtually indistinguishable from imaging artefacts (Fig 8B). Taken together, IC-qRT-PCR results coupled with the electron microscopy study provided solid evidence that either PVA^WD does not form particles or the particles formed are fundamentally defective.

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Fig 8. PVA^WD is defective in particle formation.

(A) PVA particle accumulation in PVA^WD and PVA^WT infected local leaves at 3 dpi, as measured by immunocapture qRT-PCR. N. benthamiana plants were agroinfiltrated with PVA^WD (OD₆₀₀ = 1) and PVA^WT (OD₆₀₀ = 0.1) to ensure comparable RLUC expression level between PVA^WD and PVA^WT (upper panel). The corresponding particle numbers and CP accumulation visualized by western blotting with anti-CP antibodies are presented in the middle and lower panels, respectively. (B) Electron microscopy images from the same samples as in A) showing normal virus particles formed by PVA^WT. No particles were found in PVA^WD, only few deformed particle-like structures were observed (marked by an arrow in the lower electron micrograph). The number of plants per experiment was 3. Statistical significance was assessed using student’s t-test (*P < 0.05). (C) Both PVA^WT and PVA^WD infections were allowed to spread in the local leaves until 9 dpi, and EM images of the infected tissues were taken.Pinwheel inclusions (marked as ‘CI’) were observed. However, the pinwheels from PVA^WT were associated with stacks of particles (marked as ‘PS’) in vicinity while those from PVA^WD were devoid of them.

https://doi.org/10.1371/journal.ppat.1008956.g008

Finally, in order to probe into the nature of infection in planta we allowed the infection to spread in local leaves for 9 days, and visualized the leaves under transmission electron microscopy. Pinwheel structures characteristic for potyvirus infection were detected in both PVA^WT and PVA^WD samples (Fig 8C right panel). PVA^WD infected cells were, however, devoid of particle stacks observed in abundance in PVA^WT infected samples (Fig 8C left panel) [39]. While PVA^ΔHCPro failed to form pinwheel structures (Fig 9A) typically seen in PVA^WT infected cells (Fig 9B), PVA^WD infected cells showed dense aggregations of cylindrical inclusion protein (CI)-induced pinwheel structures (Fig 9C). Although it is not clear what the abundance of these pinwheel structures means, it could indicate that the HCPro^WD mutation disrupts the homeostasis of CI and pinwheel structures. It also shows that although the expression level of PVA^WD was high the infection was not able to launch robust particle formation.

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Fig 9. Homeostasis of pinwheel inclusions is disrupted in PVA^WD infection.

(A) PVA^ΔHCPro infected leaf tissues did not show any pinwheel inclusions or any other visible symptoms of infection at 9 dpi. (B) PVA^WT infected cell showing characteristic pinwheel inclusions (CI) and associated particle stacks (marked as PS). (C) PVA^WD infected cells showing in several instances dense patches of pinwheel inclusions (marked as CIA). All the images were taken at 8000X magnification.

https://doi.org/10.1371/journal.ppat.1008956.g009

Plants

All experiments were carried out using Nicotiana benthamiana plants as the host. The plants were grown in an environmentally controlled greenhouse at 18 h light and 6 h dark photoperiod at 22 ^oC and 18 ^oC respectively. Relative humidity was maintained at 50%.

Recombinant viruses and HCPro overexpression constructs

Mutation in the WD40 domain-interacting motif of HCPro was introduced in the wild type P1-C-terminus-HCPro (or HCPro^Strep-RFP) P3-N-terminus fragment (P1-HCPro-P3) in the pGEM-T-Easy vector (Promega) using a two-step site directed mutagenesis described by Edelheit et al. [52]. The primer sequences are as follows: forward primer- 5’-CAA GTG CCG CCG CGC TTC CAG CAA TTT TGG TTG-3’; reverse primer- 5’-CAA CCA AAA TTG CTG GAA GCG CGG CGG CAC TTG-3’ (changed nucleotides are marked in bold). The mutated fragments (P1-HCPro^WD-P3 or P1-HCPro^WD-Strep-RFP-P3) were then used to replace the P1-HCPro-P3 fragment in PVA or PVA^WT-Strep-RFP icDNAs in the pUC18 vector using SexAI and NruI restriction sites. A non-replicating variant PVA^{ΔGDD-HCProWD} was prepared by replacing the wild-type NIb gene with the mutated ΔGDD version as described in Eskelin et al. [29]. Finally, to enable Agrobacterium mediated transformation the mutated PVA icDNAs were transferred into the binary vector pRD400 using SalI and KpnI restriction sites.

For HCPro expression constructs, the mutation was introduced similarly in an interim vector containing 35S promoter-HCPro-nos terminator (pHTT690). The mutated 35S-HCPro^WD-nos fragment was cloned into pRD400 using HindIII restriction sites. Expression constructs for TwinStrep-tagged RFP fusions of HCPro^WT and HCPro^WD were generated from P1-HCPro^WT-Strep-RFP-P3 or P1-HCPro^WD-Strep-RFP-P3 fragments by PCR using the following primers: forward- 5’-TCC GCT CGA GAT GAA ATG GTC TCA TCC ACA A-3’, reverse- 5’-CGC GGA TCC TCA CGA CTC TTT TTC GAA CTG-3’. The primers were designed to add an Xho1 restriction site at the 5’ end and both a stop codon and a BamH1 restriction site to the 3’ end of the amplified fragment. XhoI-BamHI digested PCR products were ligated into similarly double-digested pHTT690H to generate 35S-TwinStrep-RFP-HCPro-nos and 35S-TwinStrep-RFP-HCPro^WD-nos. After sequence verification the expression cassettes were transferred into the pRD400 binary vector using HindIII restriction.

N. benthamiana VARICOSE expression constructs

Genes homologous to the Arabidopsis thaliana VARICOSE (VCS, accession Q9LTT8) were identified with a BLAST search of the N. benthamiana genome v1.0.1 predicted cDNAs (https://solgenomics.net/). Three N. benthamiana VCS-like genes: Niben101Scf00654g02003.1 (VCS-A), Niben101Scf39216g00007.1 (VCS-B) and VCSC Niben101Scf21107g00015.1 (VCS-C) were Gateway-cloned into expression vectors. VCS-A and VCS-C were amplified with gateway-compatible primers from a N. benthamiana cDNA library generated from total RNA using Superscript III (Invitrogen). VCS-B was synthesized and cloned into the pUC57 vector by BaseClear, Netherlands. The forward primer for VCS-A and -C was 5’-GGG GAC AAG TTT GTA CAA AAA AGC AGG CTT CAT GGC TTT CTT TGC AGA G-3´ and the reverse primers were- 5’-GGG GAC CAC TTT GTA CAA GAA AGC TGG GTT TCA TTT ACT CAT CAA CAT CGA-3’ and 5’-GGG GAC CAC TTT GTA CAA GAA AGC TGG GTT TCA TTT ACT ACA GGT CAT CAG-3’, respectively. VCS-B was amplified with the forward primer 5’-GGG GAC AAG TTT GTA CAA AAA AGC AGG CTT CAT GGC TTC TTC TCC TGG C-3’ and the reverse primer 5’-GGG GAC CAC TTT GTA CAA GAA AGC TGG GTT TCA TTT ACT CAT CAA CAT CGA AT-3’. The PCR products were cloned into pDONR-Zeo via a BP-reaction with a BP ClonaseII enzyme mix (Invitrogen). Positive entry clones were verified by sequencing and LR-cloned into the KpnI-linearized destination vector pMDC32 to generate native expression constructs. XhoI-linearised pGWB42 [53] was used as the destination vector to construct N-terminal YFP-fusions.

Agroinfiltration

Preparation of the infiltration mix as well as agroinfiltration was carried out following Ivanov et al. [12].

Luciferase assay

Samples at designated dpi were collected using a cork borer of internal diameter 5-mm. Leaf discs were either directly used for luciferase assays using a Dual luciferase kit (Promega) as in Eskelin et al. [29] or stored at -80 ^oC for later analysis.

YFP Fluorescence quantification

YFP fluorescence intensity was quantified directly from 5-mm leaf discs using a Tecan Infinite M200 monochromator-based pate reader following De et al. [54]. Ex/Em wavelength used for the detection of YFP fluorescence was 500/530 nm.

Epifluorescence and confocal microscopy

The expression and localization of HCPro^WD-Strep-RFP, HCPro^WT-Strep-RFP and VCS^YFP in N. benthamiana epidermal cells was examined with confocal laser scanning microscopy using a Leica TCS SP5II instrument (Leica Microsystems). For imaging, leaf samples were mounted upside down on an objective slide with a drop of water and placed under a cover glass. All images were acquired with a 63x water immersion objective and in sequential scanning mode in order to avoid fluorophore bleed-through. YFP was excited with a 488 nm argon laser and RFP with a DPSS 561 nm laser. Emissions were detected at 525–555 nm and at 570–630 nm for YFP and RFP, respectively. CFP was excited with an argon laser at 458 nm and emission was recorded at 470–500 nm. Line-averaging was adjusted to 4 to improve image quality. Co-localization analysis of VCS^YFP with HCPro^WT-Strep-RFP and HCPro^WD-Strep-RFP was performed with the Fiji (ImageJ) image analysis software package using the co-localization threshold-function. HCPro-containing granules were selected as ROIs for the co-localization and results are given as % intensity of VCS^YFP co-localizing with HCPro^WT-Strep-RFP or HCPro^WD-Strep-RFP.

PG visualization and quantification was carried out using epifluorescence microscopy following the method described in Hafrén et al. [10].

Strep-Tag affinity purification of HCPro and identification of co-purified proteins

HCPro-associated protein complexes were isolated from leaves infected with PVA^WT-Strep-RFP or PVA^WD-Strep-RFP infiltrated at OD₆₀₀ = 0.1. GUS-infiltrated plants were used as negative controls. Leaves were harvested at 3 dpi and immediately frozen in liquid nitrogen. Subsequently, Strep-tag mediated purification of HCPro^WT-Strep-RFP or HCPro^WD-Strep-RFP was carried out following Ivanov et al. [30] with minor modifications. 1 g frozen leaf material was homogenized in 3 mL binding buffer. The homogenate was subsequently filtered through double layered miracloth and centrifuged at 5000 ×g for 5 min at 4 ^oC to remove insoluble debris. Approximately 2 mL of cleared lysate was mixed with 50 μL resin suspension and incubated overnight with gentle rotation at 4 ^oC. The rest of the protocol is same as Ivanov et al. [30]. Final elution volume was 100 μL. Identification of proteins present in the purified fractions was carried out by western blotting and LC-MS/MS following Ivanov et al. [12].

DNase, RNase and Proteinase K stability assay

Purified products obtained after elution from Strep-Tactin resin were subjected to DNase (RQ1 RNase-Free DNase, Promega), Proteinase K (Product No. 59895, Invitrogen, Thermo Fisher Scientific) and RNase A (Fermentas) treatment. Each reaction mix comprised 3 μL eluate and 1 μL DNase (1u/μL), Proteinase K (20 μg/μL) or RNase A (10 μg/μL). Final reaction volume was made up to 10 μL with recommended buffer / water. Reaction mixes were incubated for 1 h at 37 ^oC and thereafter subjected to standard SDS-PAGE followed by silver staining.

Western blotting

Standard western blotting procedures were followed to detect eluted proteins from Strep-affinity purification experiments and from all other overexpression experiments. Western blot for HCPro was carried out following De et al. [54], while SAMS, AGO1, CI and VPg were detected similarly to Ivanov et al. [12]. A customised anti-VCS antibody recognizing all three N. benthamiana VCS proteins used in this study was produced in rabbit (Biomatik) and used at 1 μg/mL dilution. The antibody recognizes peptide sequence ‘TEGPDEEDKPQITGK’ located near the N-terminal region of all three forms of VCS (S17 Fig).

LC-MS/MS

LC-MS/MS analysis of the purified products were carried out following Ivanov et al. [12].

RT and ic-qRT-PCR

Omega E.Z.N.A. Plant RNA-purification kit (Product No. R6827-02) along with RNase-free DNase (Product No. E1091-02) was used to purify RNA from 100 mg of leaf tissues. Standard protocol [55] for ic-qRT-PCR was followed to quantify PVA particles from 100 mg of leaf tissue. Primary antibody against CP (SASA, UK) at 1:1000 dilution was used for this purpose. All cDNAs were prepared using the H Minus First Strand cDNA synthesis kit (Product No. K1632, Thermo Fisher Scientific) with random hexamer primers. qPCR was carried out using Maxima SYBR Green qPCR Master mix (Thermo Fisher Scientific) and a CFX96 Touch^TM Real-Time PCR Detection System. Amplification of PVA RNA was carried out using primers targeting the CP region: forward- 5’-CAT GCC CAG GTA TGG TCT TC-3’, reverse- 5’-ATC GGA GTG GTT GCA GTG AT-3’. The protein phosphatase 2A (PP2A) gene was used as the reference gene. PP2A was amplified with the following primers: forward- 5’-GAC CCT GAT GTT GAT GTT CG-3’, reverse- 5’-GAG GGA TTT GAA GAG AGA TTT C-3’.

Electron microscopy

PVA particles were visualized with a Jeol JEM-1400 transmission electron microscope (Jeol Ltd., Tokyo, Japan). 100 mg leaf tissue was ground in 100 μL 0.06 M phosphate buffer and incubated on ice for 1 h to let the cell debris settle. Carbon-coated EM-grids were incubated with the anti-CP antibody (1:100 dilution) for 1 h at room temperature. Excess antibody was washed with phosphate buffer. Subsequently, the antibody-coated grids were incubated for 5 min with supernatant collected from the leaf extracts after settling down of the debris. Grids were further washed with 20 drops of phosphate buffer and immediately stained with 2% Uranyl acetate for 15 sec. Excess stain was drained from the grids using filter paper and dried grids were used for visualization of the particles. EM images of the infected leaf tissues were visualized following Lõhmus et al. [56].