pva infection  (DSMZ)

Journal: PLoS Pathogens

Article Title: Association of host protein VARICOSE with HCPro within a multiprotein complex is crucial for RNA silencing suppression, translation, encapsidation and systemic spread of potato virus A infection

doi: 10.1371/journal.ppat.1008956

Figure Lengend Snippet: (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.

Article Snippet: 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.

Techniques: Sequencing, Construct, Over Expression

Journal: PLoS Pathogens

Article Title: Association of host protein VARICOSE with HCPro within a multiprotein complex is crucial for RNA silencing suppression, translation, encapsidation and systemic spread of potato virus A infection

doi: 10.1371/journal.ppat.1008956

Figure Lengend Snippet: (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 OD 600 = 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 OD 600 = 1, HCPro WD and HCPro WT at OD 600 = 0.3. PVA ΔHCPro was infiltrated at OD 600 = 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 OD 600 = 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 (OD 600 = 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).

Article Snippet: 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.

Techniques: Virus, Derivative Assay, Activity Assay, Mutagenesis, Gene Expression, Expressing, Construct, Western Blot, Preserving, Infection, Binding Assay, Control, Luciferase

Journal: PLoS Pathogens

Article Title: Association of host protein VARICOSE with HCPro within a multiprotein complex is crucial for RNA silencing suppression, translation, encapsidation and systemic spread of potato virus A infection

doi: 10.1371/journal.ppat.1008956

Figure Lengend Snippet: (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 (OD 600 = 0.01), pHG-RLUC construct (OD 600 = 0.4) and HCPro WD / HCPro WT constructs (OD 600 = 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 (OD 600 = 0.01) and pHG-GFP (OD 600 = 0.4) were co-infiltrated as in (B) with HCPro WD / HCPro WT and PVA ΔHCPro (OD 600 = 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 . (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 OD 600 = 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 OD 600 = 0.1. P0 YFP was co-expressed at OD 600 = 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 OD 600 = 0.1 and 0.3 as indicated, while P0 YFP was agroinfiltrated at OD 600 = 0.1. The number of PGs per mm 2 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 (OD 600 = 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.

Article Snippet: 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.

Techniques: Construct, Expressing, Activity Assay, Fluorescence, Derivative Assay, Microscopy, Over Expression, Purification, In Vivo, Binding Assay, Western Blot, Membrane, Staining, Molecular Weight

Journal: PLoS Pathogens

Article Title: Association of host protein VARICOSE with HCPro within a multiprotein complex is crucial for RNA silencing suppression, translation, encapsidation and systemic spread of potato virus A infection

doi: 10.1371/journal.ppat.1008956

Figure Lengend Snippet: (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 OD 600 = 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.

Article Snippet: 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.

Techniques: Infection, Affinity Purification, Construct, Purification, Staining, Western Blot, Molecular Weight, Biomarker Discovery, SDS Page, Silver Staining

Journal: PLoS Pathogens

Article Title: Association of host protein VARICOSE with HCPro within a multiprotein complex is crucial for RNA silencing suppression, translation, encapsidation and systemic spread of potato virus A infection

doi: 10.1371/journal.ppat.1008956

Figure Lengend Snippet: We present here a hypothetical model of a core complex, which contains both HCPro and VCS and guides PVA RNA through the different stages of infection. We propose that after PVA RNA is released from the viral replication complex it connects with HCPro. HCPro then recruits VCS to assist in the formation of multiprotein complexes. The composition of these complexes probably varies depending on the infection stage. Interaction between HCPro and VCS is required for the formation of PGs, which form to suppress RNA silencing and to enable active PVA translation. Finally, the multiprotein complex formed around HCPro-VCS seals the PVA particles. This is essential for both the stability of PVA RNA within particles and the systemic spread of PVA infection.

Article Snippet: 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.

Techniques: Infection