Fig 2: Loss of VPS39 affects the synthesis of ASFV proteins during the early phase of infection. (A) Overview of identified ASFV proteins 8 h after infection in CTRL-KO and VPS39-KO cells. The number of proteins exhibiting reduced or increased amounts in VPS39-KO cells was determined. (B) Heat map of viral proteins identified at 8 h depicts changes in protein expression levels between CTRL-KO and VPS39-KO cells. (C) Line plots showing fold changes in ASFV proteins expressed in VPS39-KO cells compared to controls. Three clusters with similar profiles of protein were distinguished: downregulated (or unchanged) at 8 h and increasing over time (blue); upregulated (or unchanged) at 8 h and decreasing over time (red); and continuously downregulated during infection in VPS39-KO cells (yellow). Selected ASFV protein profiles are depicted with solid black lines. Mass-spectrometric data can be found in Data Set S1D.

Fig 3: CP204L and VPS39 accumulate at the virus factory early during infection. (A) Virus factories in WSL cells infected with ASFV were monitored by immunofluorescence at 8, 12, and 24 h postinfection. Representative images show the accumulation of CP204L (red) in early VFs and dispersion from late VFs, which are marked by the presence of late virus protein E183L (P54) (yellow). Virus factories (white arrows) are labeled by DNA staining (blue) and staining of vimentin cages (green). Bars, 10 µm. (B) Colocalization of CP204L and VPS39 in ASFV-infected WSL-VPS39-GFP cells was monitored at 4, 8, 12, and 24 h postinfection. The aggregation of VPS39-GFP near the nucleus was observed 4 h postinfection (red arrow).

Fig 4: Interaction between ASFV CP204L and porcine VPS39. (A) Coimmunoprecipitation of GFP-tagged CP204L with endogenous VPS39. GFP was immunoprecipitated in lysates from mock-infected and virus-infected cells stably expressing CP204L-GFP or GFP alone. Representative immunoblots of whole-cell lysates (WCLs) and GFP immunoprecipitates (IPs) are shown. a-Tubulin was used as a loading control in WCLs. (B) Reverse coimmunoprecipitation of VPS39-GFP with CP204L in virus-infected cells. (C) WSL cells were infected with ASFV at an MOI of 2 for 24 h. Lysates were prepared and immunoprecipitated with rabbit anti-VPS39 antibody, a nonspecific rabbit IgG control, or beads only, followed by immunoblotting. (D) Interaction between CP204L and VPS39 was tested in a yeast two-hybrid assay. CP204L and VPS39 were expressed as fusion proteins with the DBD or AD domain of Gal4. The empty vector is shown as a negative control. (E) Yeast two-hybrid mapping of the CP204L-binding region in VPS39 and the VPS39-binding region in CP204L. Various combinations of VPS39 truncations and CP204L were cotransformed into the pPC86 vector. The transformants were spotted on control plates (-Leu -Trp) and selective plates (-Leu -Trp -His +3-AT [5 mM]). Cotransfection with interacting protein fragments was indicated by growth on the selective medium. Vertical and horizontal axes indicate the first and the last amino acid residues of each tested fragment, respectively. (F) (Top) Predicted domain organization of porcine VPS39. CNH, citron homology domain; CHCR, clathrin heavy-chain repeat; TGFB1 and TGFB2, transforming growth factor beta receptor-associated domains 1 and 2. (Middle) CP204L-binding region between amino acids 271 and 541 of VPS39. (Bottom) Degree of order predicted by IUPred2A. The cutoff was set to a 0.5 probability score. (G) HEK-293T cells were transfected with expression vectors encoding GST alone or fused to CP204L and tested for interaction with 3×FLAG-tagged VPS39271–541. WCLs were prepared at 48 h posttransfection (top), and copurifications of VPS39271–541 were assayed by pulldown using glutathione-Sepharose beads (middle and bottom).

Fig 5: During infection, CP204L and VPS39 form aggregates and localize to the virus factory. Colocalization and aggregate formation between CP204L and VPS39 in WSL-VPS39-GFP cells (A) 24 h after ASFV infection and (B) CP204L plasmid transfection. Empty vector and mock-infected cells were used as negative controls. (C) Subcellular localization of CP204L in infected WSL cells (12 h after infection). Indirect immunofluorescence shows localization of CP204L (red) to virus replication sites (arrows) surrounded by a vimentin cage (green). (D) (Top) Representative image of a WSL cell infected with ASFV (24 h postinfection). The image shows a Z-stack projection (64 slices across 12.8 µm) of the cell nucleus and virus factories (blue) and the CP204L protein (red). The crosshairs were positioned to indicate virus DNA. (Bottom) Cross-section through the replication compartment and the CP204L accumulation at its periphery. (E) Protein VPS39 (green) and CP204L (red) colocalization at the periphery of the virus replication site in infected WSL-VPS39-GFP cells. Hoechst dye was used to stain cellular and virus DNA (blue). Arrows indicate virus factories. Bars, 10 µm.