Fig 2: CSDE1 regulates viral M RNA levels.Cells (HepB3 or HepB3-CSDE1C-T; B16 or B16-CSDE1C-T) were infected with VSV viruses (VSV-IFNβ or VSV-IFNβ-IGR P/MC-U) at an MOI of 3. Lanes: 1, Hep3B/VSV-hIFNβ; 2, Hep3B/VSV-hIFNβ-IGR P/MC-U; 3, Hep3B-CSDE1C-T/VSV-hIFNβ; 4, Hep3B-CSDE1C-T/VSV-hIFNβ-IGR P/MC-U; 5, B16/VSV-mIFNβ; 6, B16/VSV-mIFNβ-IGR P/MC-U; 7, B16-CSDE1C-T/VSV-mIFNβ; and 8, B16-CSDE1C-T/ VSV-mIFNβ-IGR P/MC-U. Six hours later, RNA was isolated and converted into cDNA. qrtPCR was used to assess levels of A viral P RNA (P-specific primers P1 and P2), B viral M RNA (M-specific primers M1 and M2), or C viral P–M RNA (P/M IGR-M-specific primers IGR1 and M2). Representative of three separate experiments. D–F Levels of G, L, and G–L RNA were measured as in A–C using primers G1 and 2, L1 and 2, and G1–L2, respectively. Representative of two separate experiments. Levels of each RNA species were normalized to expression in Hep3B infected with VSV-hIFNβ or to B16 infected with VSV-mIFNβ. G Hep3B parental cells (Hep3B) (Lanes 1–3) or Hep3B cells engineered to overexpress CSDE1wt (Hep3B-CSDE1wt) (lanes 4 and 5) or mutated CSDE1P5S (Hep3B-CSDE1C-T) (lanes 6 and 8) proteins, or Hep3B that had been selected in vitro for resistance to VSV-IFNβ oncolysis for 21 days (Hep3B-VSV-IFNβ-ESC) (lanes 7 and 9), were infected with wild-type VSV-IFNβ (lanes 2 and 5–7) or VSV-IFNβ-IGR P/MC-U (lanes 3, 4, 8, and 9) at an MOI of 3. Cells were collected at either 6 or 18 h post infection and levels of viral M protein were measured by western blotting. Representative of two separate experiments means ± SD of three biological replicates are shown (A–F). P-values were determined using a one-way ANOVA (A–F) on the raw data. Statistical significance was set at p < 0.05, ns > 0.05. *p < 0.05, **p < 0.1, ***p < 0.001. Source data are provided as a Source Data file.

Fig 3: Escape from VSV-hIFNβ is immunogenic.A Human CD3+ T cells activated with αCD3 and αCD28 were co-cultured with autologous CD14+-matured DC and different Hep3B cell lysates on d1, 3, and 5. On d7, CD8+ T cells were isolated and co-cultured with autologous DC and the same Hep3B cell lysates (E : T 10 : 1). B Seventy-two hours later, supernatants were assayed for IFN-γ. Means ± SD of three technical replicates from three donors. C 104 target cells (Hep3BP or Hep3B-VSV-hIFN-β 21d ESC) were treated for 24 h with hIFN-γ before being co-cultured with 105 T cells primed/expanded on Hep3BP or Hep3B-VSV-hIFNβ-21d ESC cells as in A. 105 T cells were added after 48 h. At 120 h, surviving adherent cells were counted. D NetMHC4.0 %rank of the predicted affinity of the unmutated CSDE1WT 9mer, MSFDPNLLH, and its CSDE1C-T-mutated counterpart 9mer, MSFDSNLLH, compared to 400,000 random peptides for HLA subtypes. Strong binders, %rank < 0.5, weak binders, %rank < 2. http://www.cbs.dtu.dk/services/NetMHC/. E Human CD3+ T cells activated in vitro were co-cultured with autologous DC and Hep3BP or Hep3B-VSV-hIFNβ-ESC lysates, or with DC transfected 48 h previously with 10 µg pcDNA3.1-CSDE1WT or pcDNA3.1-CSDE1C-T plasmids. Lysates, or DC, were re-added on d5. On d7, isolated CD8+ T cells were co-cultured with DC/Hep3B cell lysates or with transfected DC (E : T 10 : 1). Seventy-two hours later, supernatants were assayed for IFN-γ. Means ± SD of three technical replicates from two donors. ND, below limit of detection. F Activated human CD3+ T cells were co-cultured with autologous DC pulsed 48 h previously with 5 µg/ml peptide on d1 and 5. On d7, isolated CD8+ T cells were co-cultured with similarly pulsed DC. Seventy-two hours later, supernatants were assayed for IFN-γ. Peptide pulsing: Lane 1: SIINFEKL peptide from Ovalbumin; 2: WT CSDE1 9mer MSFDPNLLH; 3: CSDE1 20mer, MSFDPNLLHNNGHNGYPNGT, which could be processed into 9-11mer with Pro (position 5) in different positions; 4: mutated CSDE1P5S MSFDSNLLH; or 5: CSDE1P5S 20mer, MSFDSNLLHNNGHNGYPNGT, in which Ser (position 5) could be at different positions. Means ± SD of three technical replicates from three donors. P-values determined using a one-way (B) or two- way (C, F) ANOVA with a Tukey’s multiple comparisons post test. Statistical testing was on log-transformed data in C. Statistical significance, p < 0.05, ns > 0.05. Source data are provided as a Source Data file.

Fig 4: CSDE1 expressed from the virus enhances replication.A Viruses expressing CSDE1WT and CSDE1C-T were constructed and validated by western blotting for the expression of CSDE1. Stars indicate clones that were used in subsequent experiments. Representative of three separate experiments. B BHK (hamster), B16 (mouse), Hep3B, and Mel888 (human) cell lines were infected with VSV-GFP, VSV-hIFN-β, VSV-CSDE1WT, or VSV-CSDE1C-T at an MOI of 3 (triplicate wells per cell line). Forty-eight hours later, virus was titered on BHK cell by plaque assay. Representative of three separate experiments. C Murine B16 or human Hep3B cells were infected (MOI 0.01) with VSV-IFNβ, VSV-IFN-β-CSDE1WT, or with VSV-IFN-β-CSDE1C-T viruses (using species-matched IFNβ genes) for 21d as in “Methods” and ref. 10. Surviving cells were pooled and counted. D Hep3B cells were infected (MOI 0.01) with the VSV-hIFNβ-CSDE1WT virus for 21d as in “Methods” and ref. 10. Surviving cells were pooled and genomic DNA prepared. Sanger sequencing of CSDE1 is shown for two independent experiments. In these experiments, as well as in two other experiments, a mixed population of mutated and unmutated cells were selected. Means ± SD of three technical replicates are shown. P-values were determined using a two-way (B) or one-way (C) ANOVA with a Tukey’s multiple comparisons post test on log-transformed data. Statistical significance was set at p < 0.05, ns > 0.05. Source data are provided as a Source Data file.