Nuclear import of CaMV P6 is required for infection and suppression of the RNA silencing factor DRB4

Abstract

Replication of Cauliflower mosaic virus (CaMV), a plant double-stranded DNA virus, requires the viral translational transactivator protein P6. Although P6 is known to form cytoplasmic inclusion bodies (viroplasms) so far considered essential for virus biology, a fraction of the protein is also present in the nucleus. Here, we report that monomeric P6 is imported into the nucleus through two importin-alpha-dependent nuclear localization signals, and show that this process is mandatory for CaMV infectivity and is independent of translational transactivation and viroplasm formation. One nuclear function of P6 is to suppress RNA silencing, a gene regulation mechanism with antiviral roles, commonly counteracted by dedicated viral suppressor proteins (viral silencing suppressors; VSRs). Transgenic P6 expression in Arabidopsis is genetically equivalent to inactivating the nuclear protein DRB4 that facilitates the activity of the major plant antiviral silencing factor DCL4. We further show that a fraction of P6 immunoprecipitates with DRB4 in CaMV-infected cells. This study identifies both genetic and physical interactions between a VSR to a host RNA silencing component, and highlights the importance of subcellular compartmentalization in VSR function.

Figure 1

(A) Schematic of P6 and its functional domains (coloured boxes). NES: nuclear export signal; domain A: P6–P6 interaction; mini-TAV: minimal domain for translational transactivation; ssRNA: single-stranded RNA binding; dsRNA: double-stranded RNA binding. The various GFP–P6 alleles were transiently expressed into BY-2 tobacco cells and observed under confocal microscope 18 h post-bombardment. Images on the left show GFP detection and those on the right are composite images of green fluorescence and DIC. Position and amino-acid sequence of the putative basic bipartite NLS are indicated. Similarities with the NLS of human ribosomal protein L22 are shown in yellow. (B) P6 regions (a–c) showing homology with non-conventional, viral NLSs (red: influenza virus 1 NP protein; yellow and green: Borna disease virus P10 protein). The effects of their deletion (singly or in combination) on P6ΔA distribution are depicted on the right. (C) Cellular localization of GFP–P6_m1 (leucine substitution in red) and P6_m1Δ_NLSΔa. (D) Cellular localization of GFP–GUS in the absence/presence of individual P6 NLS, or combination thereof. (E) GST pull down of P6 and rice importins. Radiolabelled P6 was incubated either with importin α (GST-Imp α), β (GST-Imp β) or GST alone (GST). Following SDS–PAGE, P6 was detected by autoradiography (upper panel) and total proteins by Coomassie blue staining (lower panel).

Figure 2

(A) Effect of point mutations in NLS-a (red) on GFP–P6ΔA localization. (B). N. plumbaginifolia protoplasts were co-transfected with constructs expressing P6, CAT and GUS, under the control of the CaMV 35S promoter. The GUS ORF cloned downstream of the CaMV ORF VII is used to monitor translational transactivation. CAT expression is an internal control for translation and transfection efficacy. The effects of P6, or mutated version thereof, on GUS expression are represented by histograms. GUS activity in the presence of WT P6 was arbitrarily set to 100%. The data are from two independent experiments. (C) Schematic of the pGH recombinant viral vector. ORF VI variants can be inserted owing to the SacI/KpnI restriction sites. The indicated P6 variants were cloned in pGH and inoculated to Arabidopsis upon linearization with SalI. Plants were monitored for symptom formation over an 18-day period, upon which tissues were collected for analysis of the CaMV coat protein P4. The ratio of infected to inoculated plants is shown. Coomassie blue staining shows equal protein loading. (D) Schematic of the GFP–P6 and GFP–P6_m3 proteins and analysis of their localization in BY-2 cells 18 h post-bombardment. Both alleles were introduced into pGH and infections were monitored as in (C).

Figure 3

(A) Schematic of the PVX–GFP transgene in the Arabidopsis AMP line. (B). Restauration of GFP accumulation upon transgenic expression of P6 and P38 in the AMP line (left panel). The red colour is from chlorophyll autofluorescence under UV and signifies GFP silencing. High molecular weight (HMW) RNA and siRNA were extracted and detected using a GFP-specific probe (right panel). rRNA: ethidium bromide staining of ribosomal RNA; gRNA and sgRNA: genomic and subgenomic PVX–GFP RNA respectively. (C) Leaves of AMP plants expressing P6, P6Δa or P6ΔaΔNLS, under UV light. (D) Accumulation of P6 or its variants was detected by western blot analysis using a P6 antiserum (P6, upper panel). Prot: Coomassie blue staining of total protein. PVX–GFP-derived siRNA were detected as in (B) (lower panel). (E) HMW RNA analysis of PVX gRNA and sgRNA in AMP lines expressing P6 and its variants, as in (B).

Figure 4

(A, B) P6 transgenic Arabidopsis ecotype Col-0 (B) are stunted, chlorotic and exhibit leaf serration compared with their non-transgenic counterparts (A). (C) Magnified view of (B) showing the ‘silvery' chlorosis developing on older leaves. (D) None of the P6 mutants induce developmental anomalies when expressed transgenically in the Col-0 ecotype. (E) Upper panel: western blot analysis of P6 accumulation in plants depicted in (A–D), as in Figure 3D. Lower panel: LMW RNA analysis in the corresponding genotypes using probes for tasiRNA255, miR173 and miR156.

Figure 5

(A) Schematic of the SUC-SUL transgene and phenotype of progenies expressing the P6 alleles used in Figure 4. (B) Upper panel: accumulation of P6 and its variants, as in Figures 3D and 4E. Lower panel: LMW RNA analysis in the corresponding genotypes, using a DNA probe specific for the SUL region. SUL siRNAs accumulate as 21 and 24 nt species. (C) Detection of CaMV-derived HMW (upper panel) and LMW (lower panel) RNA species in WT, dcl2 dcl4 and drb4 mutant plants. The DNA probe covers the CaMV genome. (D) Western blot analysis of DRB4 accumulation from WT and drb4 mutant plants. The arrow indicates the expected migration of DRB4 (predicted molecular mass: 43 kDa). (E) Western blot analysis of DRB4 accumulation in Arabidopsis plants infected by CaMV or expressing P6. drb4 and dcl4 mutant plants were used as controls. (F) DRB4 immunoprecipitation in mock-inoculated (left) and CaMV-infected plants (right). Immunoprecipitates were subjected to western blot analysis using a P6 antibody (upper panel). The filter was then re-hybridized with the DRB4 antibody (lower panel). The ∼50 kDa nonspecific signal is from the immunoglobulin heavy chain (Ig HC). (G) Same as in (F) but in the drb4 mutant background. DRB4 immunoprecipitates from drb4 plants are devoid of P6.