A unique N-terminal sequence in the Carnation Italian ringspot virus p36 replicase-associated protein interacts with the host cell ESCRT-I component Vps23

Abstract

Like most positive-strand RNA viruses, infection by plant tombusviruses results in extensive rearrangement of specific host cell organelle membranes that serve as the sites of viral replication. The tombusvirus Tomato bushy stunt virus (TBSV) replicates within spherules derived from the peroxisomal boundary membrane, a process that involves the coordinated action of various viral and cellular factors, including constituents of the endosomal sorting complex required for transport (ESCRT). ESCRT is comprised of a series of protein subcomplexes (i.e., ESCRT-0 -I, -II, and -III) that normally participate in late endosome biogenesis and some of which are also hijacked by certain enveloped retroviruses (e.g., HIV) for viral budding from the plasma membrane. Here we show that the replication of Carnation Italian ringspot virus (CIRV), a tombusvirus that replicates at mitochondrial membranes also relies on ESCRT. In plant cells, CIRV recruits the ESCRT-I protein, Vps23, to mitochondria through an interaction that involves a unique region in the N terminus of the p36 replicase-associated protein that is not conserved in TBSV or other peroxisome-targeted tombusviruses. The interaction between p36 and Vps23 also involves the Vps23 C-terminal steadiness box domain and not its N-terminal ubiquitin E2 variant domain, which in the case of TBSV (and enveloped retroviruses) mediates the interaction with ESCRT. Overall, these results provide evidence that CIRV uses a unique N-terminal sequence for the recruitment of Vps23 that is distinct from those used by TBSV and certain mammalian viruses for ESCRT recruitment. Characterization of this novel interaction with Vps23 contributes to our understanding of how CIRV may have evolved to exploit key differences in the plant ESCRT machinery.

Importance: Positive-strand RNA viruses replicate their genomes in association with specific host cell membranes. To accomplish this, cellular components responsible for membrane biogenesis and modeling are appropriated by viral proteins and redirected to assemble membrane-bound viral replicase complexes. The diverse pathways leading to the formation of these replication structures are poorly understood. We have determined that the cellular ESCRT system that is normally responsible for mediating late endosome biogenesis is also involved in the replication of the tombusvirus Carnation Italian ringspot virus (CIRV) at mitochondria. Notably, CIRV recruits ESCRT to the mitochondrial outer membrane via an interaction between a unique motif in the viral protein p36 and the ESCRT component Vps23. Our findings provide new insights into tombusvirus replication and the virus-induced remodeling of plant intracellular membranes, as well as normal ESCRT assembly in plants.

FIG 1

Alignment of the deduced amino acid sequences of CIRV p36 and TBSV p33. Sequences were obtained from GenBank (accession numbers CAA59477.2 and NP_062898.1) and aligned using ClustalW. Identical and similar amino acids in each protein are colored red and green or blue, respectively, and indicated also with asterisks and colons or periods, respectively. The numbers represent specific amino acid residues in full-length p36 (330 residues). Putative TMDs (shown on a gray background) were determined using TOPCONS and visual inspection, and the late-domain-like motif identified in the intervening loop sequence of p33 (23) but absent in p36 is shown on a bright blue background.

FIG 2

Inhibition of CIRV and TBSV replication in N. benthamiana by Vps4^E232Q. (A) RNA blot analysis of N. benthamiana leaves infiltrated with Agrobacterium harboring either an empty vector (35S) or a vector encoding Arabidopsis Vps4^E232Q, followed by rub inoculation with full-length infectious CIRV or TBSV cDNA. The relative positions of the CIRV and TBSV genomic (gRNA) and the two subgenomic mRNAs (sg1 and sg2) are shown to the left of the gel. The numbers of days after infiltration or inoculation are indicated above the lanes. (B) RNA blot analysis of N. benthamiana leaves agro-infiltrated with either Vps4^E232Q alone, empty vector (35S), and two other vectors (TRV1 and TRV2) encoding the full-length infectious TRV genome (TRV), or Vps4^E232Q, TRV1, and TRV2. In panels A and B, the 28S and 18S rRNAs in the corresponding ethidium bromide-stained agarose gels are shown as a loading control.

FIG 3

Relocalization of Vps23 from the cytosol and late endosomes to mitochondria in BY-2 cells coexpressing CIRV p95 and/or p36. (A to C) Representative epifluorescence micrographs of BY-2 cells (co)transformed (as indicated by panel labels) with either full-length CIRV, Rep (both p95 and p36 together), p95, p36, Myc-Vps23, or GFP-Syp21, or mock transformed with empty plasmid DNA and then immunostained for the endogenous mitochondrial protein β-ATPase (top right-hand photo in panel A). All cells shown in Fig. 3 were formaldehyde fixed and processed for immunofluorescence microscopy as described in Materials and Methods. Note that p95 and p36 were immunodetected using primary antibodies raised against a synthetic peptide that corresponds to an amino acid sequence in both proteins. p95 is produced by the translational read-through of the p36 amber stop codon (8). Selected transformed cells were also immunostained (as indicated) for endogenous mitochondrial β-ATPase or CoxII. Also shown are the corresponding merge and differential interference contrast (DIC) images. The yellow color in the merged images indicates colocalization, and the white arrowheads in panel A indicate obvious examples of Myc-Vp23 and GFP-Syp21 colocalization at late endosomes. Bar = 10 μm. (D) Representative confocal micrographs of BY-2 cells cotransformed with either p36 and Myc-Vps23 (top and middle rows), or GFP-Syp21 and Myc-Vps23 (bottom row), which were immunostained for Myc-Vps23 and endogenous CoxII (GFP fluorescence is not shown). All images are medial (midcell) optical sections of cells, and boxes represent the portions of the cells shown at higher magnification in the panels on the right. The Pearson's correlation coefficient r values were as follows: r = 0.81 for p36 and Myc-Vps23, r = 0.71 for Myc-Vps23 and CoxII, and r = 0.21 for Myc-Vps23 and CoxII. (E to H) Representative epifluorescence micrographs of BY-2 cells cotransformed (as indicated) with either p36 and either N-terminal HA epitope- or GFP-tagged Vps23 (E); p36 and a Myc-tagged N. tabacum Vps23 (F), p36 and Myc-tagged Vps28 or Vps25 (G), or p36 and various organelle marker fusion proteins, including Cherry-PTS1 (peroxisome), GFP-Syp52 (early endosome/trans-Golgi network), GFP-Syp21 (late endosome), TIC40-RFP (plastid), PAP26-Cherry (lytic vacuole) (H). Bar = 10 μm.

FIG 4

The cytosol-facing N-terminal region of p36 is both necessary and sufficient for relocalizing Vps23 to mitochondria in BY-2 cells. (A) Topology models of p36 and TraB in the mitochondrial outer membrane based on previously published results (11, 26, 50) and those presented in panel D. The numbers show the numbers of specific amino acid residues in full-length p36 (330 residues) and TraB (371 residues), including those in the names of the modified versions of p36 and p36-CAT (and p36-TraB) shown in panels B to E. The asterisk represents the relative position of the peptide sequence in the intervening loop of p36 used to generate the antiloop antibodies used in panel D. The N and C termini of the proteins are shown. Cyt, cytosol; IMS, intermembrane space. (B and C) Representative epifluorescence micrographs of BY-2 cells cotransformed with proteins as indicated by panel labels. Cells were formaldehyde fixed and processed for immunofluorescence epimicroscopy as described in the legend to Fig. 3. The Pearson's correlation coefficient r values based on confocal microscopy of cells coexpressing p36^91-330 and Myc-Vps23 or Myc-TraB and HA-Vps23 were r = 0.31 and r = 0.25, respectively. Bar = 10 μm. (D) Representative epifluorescence micrographs of BY-2 cells either nontransformed (top row) or cotransformed with the indicated proteins, fixed with formaldehyde, and then permeabilized with either Triton X-100 (which permeabilizes both the plasma membrane and all organellar membranes) or digitonin (which permeabilizes only the plasma membrane) (16). The cells were then processed for immunoepifluorescence microscopy using primary antibodies raised against endogenous cytosolic α-tubulin, endogenous mitochondrial matrix protein CoxII (anti-CoxII [α-CoxII]), the Myc epitope tag (anti-Myc [α-Myc]), an amino acid sequence in the intervening loop of p36 (antiloop [α-Loop]), or the bacterial passenger protein CAT (anti-CAT [α-CAT]), as indicated by panel labels. Note that the presence of immunostaining in digitonin-permeabilized cells indicates that endogenous α-tubulin and the expressed protein's appended Myc epitope tag(s) or CAT moiety are exposed to the cytosol. Conversely, endogenous CoxII or the intervening loop sequence of p36 is not immunodetectable in the same corresponding digitonin-permeabilized cells. (E) Representative epifluorescence micrographs of BY-2 cells transformed with p36^91-330, Myc-p36^1-90-TraB, or Myc-TraB and immunostained for endogenous mitochondrial β-ATPase or CoxII. Note that the mitochondria in the cells expressing p36^1-90-TraB, but not Myc-TraB, were aggregated, similar to the mitochondrial aggregation observed in cells expressing full-length p36 (Fig. 3).

FIG 5

The N-terminal region of p36 interacts with Vps23. (A) p36^1-90 interacts with Vps23 in the yeast two-hybrid assay. Yeast strains were cotransformed with the indicated pairs of GAL4-binding domain (BD) and GAL4-activiating domain (AD) fusion proteins or the corresponding empty BD or AD control plasmids, denoted by a hyphen in the BD or AD column. Serial (1:5) dilutions of cells were spotted onto agar plates containing either low-stringency media (SD-Leu,Trp) or high-stringency media (SD-Leu,Trp,His,Ade), where cell growth is dependent on two-hybrid protein interactions. (B) In vitro coimmunoprecipitation of p36 or p36^1-90 and Vps23. Whole-cell lysates of E. coli transformed with S-Vps23 or the corresponding empty vector were incubated with S-protein agarose and in vitro-translated (IVT) full-length Myc-p36, Myc-p36^1-90, or Myc-Vps28, and washed. Eluted proteins (and 10% of the total IVT reaction mixture) were subjected to SDS-PAGE and protein blotting and then probed with either anti-Myc or anti-S-tag antibodies. The solid (black) arrowheads indicate the relative positions of specific immunodetected proteins; the open (white) arrowheads indicate nonspecific, immunodetected proteins. Note that the immunodetected proteins indicated by the solid arrowheads are not present in the coimmunoprecipitation reactions with lysates of E. coli containing empty vector. Shown to the right is a Coomassie blue-stained gel of total soluble protein from lysates containing S-Vps23 or empty vector prior to incubation with S-protein agarose, confirming equivalent input. The numbers to the left of the gels are the molecular masses (in kilodaltons) of protein standards. (C) Interaction of p36 and Vps23 in the BiFC assay. Representative epifluorescence micrographs of BY-2 cells triple transformed (as indicated) with RFP, nYFP-Vps23, and either p36-cYFP or p36^91-330-cYFP. Relative RFP and YFP fluorescence values of transformed cells, which are delineated by the solid lines in the panels on the right were quantified using ImageJ (refer to Materials and Methods for details). Note the relatively low YFP fluorescence in the representative cell coexpressing the negative control, p36^91-330-cYFP. At least 25 transformed cells were analyzed from at least three independent experiments, and the mean YFP-to-RFP ratios (plus standard deviation [SD]) are plotted in the bar graph on the right. The two asterisks denote a statistically significant difference between the two samples (P ≤ 0.001).

FIG 6

A unique 16-amino-acid-long sequence at the N terminus of p36 is both necessary and sufficient for relocalizing Vps23 to mitochondria in BY-2 cells. (A) Representative epifluorescence micrographs of BY-2 cells cotransformed with proteins as indicated by panel labels and immunostained (as indicated) for endogenous mitochondrial β-ATPase. Numbers in the name of the construct denote the specific amino acid residues derived from full-length p36 (330 residues) or specific residues deleted from p36. Bar = 10 μm. (B) Amino acid sequence alignment of the N termini of various tombusvirus p36 and p33 proteins and cartoon illustrations of p33-36 and p36-TraB hybrid proteins. Sequences were obtained from GenBank and aligned using ClustalW. Identical and similar amino acids in each protein are indicated with asterisks and colons or periods, respectively. The unique amino acid sequence present in CIRV p36 and PeNSV p33 (residues 7 to 22) but absent in TBSV, CNV, and CyRSV p33 are shaded gray. Cartoons depict the structure and topology of p33–p36 and p36-TraB hybrid proteins in the mitochondrial outer membrane. Lines representing amino acid sequences from p33, p36, and TraB are colored red, black, and green, respectively. Numbers represent specific amino acid residues derived from either full-length p33 (296 residues), p36 (330 residues), or TraB (371 residues) and correspond to the numbers in the names of the p33–p36 and p36-TraB hybrid proteins described in panels C and D. (C and D) Representative epifluorescence micrographs of BY-2 cells cotransformed with proteins as indicated by panel labels and immunostained (as indicated) for endogenous mitochondrial β-ATPase. Numbers in the name of the construct denote the specific amino acid residues derived from full-length p36 or p33 and are as illustrated in panel B.

FIG 7

The C-terminal StBox of Vps23 is necessary and sufficient for its recruitment to mitochondria by p36 in BY-2 cells. (A) Schematic diagram illustrating the domain organization of Arabidopsis Vps23 based on Spitzer et al. (63). Numbers denote the specific amino acid residues derived from full-length Vps23 (398 residues) that delineate the protein's UEV, CC, and StBox domains, which are colored red, blue, and yellow, respectively, and which are depicted below as lines representing the various Vps23 truncation mutants described in panels B and C. Numbers denote the specific amino acid residues at the N and C termini of each Vps23 mutant. (B and C) Representative epifluorescence micrographs of BY-2 cells (co)transformed with proteins as indicated by panel labels. Names of mutants represent the specific domain(s) derived from Vps23, as illustrated in panel A. The corresponding merged images (B) and the corresponding DIC images of the UEV-GFP-transformed BY-2 cells (C) are also shown.

FIG 8

The N-terminal region of p36, like Vps28, interacts with the C-terminal StBox of Vps23 in the yeast two-hybrid assay. (A to D) Yeast strains were cotransformed with the indicated pairs of GAL4-binding domain (BD) and GAL4-activating domain (AD) fusion proteins or the corresponding empty BD or AD control plasmids (indicated by a hyphen in the BD or AD column). Serial (1:5) dilutions of cells were spotted onto agar plates containing either low-stringency medium (SD-Leu,Trp) or high-stringency medium (SD-Leu,Trp,His,Ade), where cell growth is dependent on two-hybrid protein interactions. Note that neither full-length Vps23, nor any of the truncation mutants of Vps23, Vps28, and Vps37 autoactivated the two-hybrid reporter gene system on their own.