Fig 2: Cells high in Syncytin-1 more readily support the EBV lytic phase. (A) HH514-16, LCL, or Akata cells were treated with lytic inducing agents for 24 h and assayed for ERVW-1 transcript levels using RT-qPCR. (B) HH514-16 cells were induced for 24 h with NaB, followed by FACS sorting to separate lytic from refractory cell populations and assayed for ERVW-1 transcript via RT-qPCR. (C,D) Cells were induced as in (A) followed by flow cytometry staining for Syncytin-1 and ZEBRA protein. Syncytin-1 positive cells were sub-gated roughly equally into Syncytin-1hi and Syncytin-1lo sub-populations, which were then further examined for expression of ZEBRA. Data represent averages of three independent experiments; error bars, SEM; ***p = 0.001.

Fig 3: Syncytin-1 supports the KSHV lytic cascade. BCBL-1 cells were nucleofected with scrambled control or ERVW-1 specific siRNAs for 18 h and harvested for western blot analysis for Syncytin-1 (A) or treated with TPA or VPA to induce lytic activation and harvested at 24 h (B) or 36 h (C,D) to analyze lytic readouts in each siRNA treatment group. Lytic proteins RTA and bZIP were probed using western blot in (B). Lytic transcript levels of representative genes of each KSHV kinetic class were assayed using RT-qPCR after induction with TPA (C) or VPA (D). (E–H) BCBL-1 cells were nucleofected with pSpCas9 BB-2A-puro or pSpCas9 BB-2A-puro ERVW-1 guide RNA for 24 h prior to harvest for western blot analysis (E) or treated with TPA or VPA to induce lytic activation. Cells were harvested at 24 h (F) or 36 h (G,H) to analyze lytic readouts as in (B–D), respectively. Data represent averages of three independent experiments; error bars, SEM; *p = 0.05, **p = 0.01, ***p = 0.001.

Fig 4: Depletion of Syncytin-1 impairs EBV lytic activation. Akata cells were nucleofected with scrambled control or ERVW-1 specific siRNAs for 18 h and harvested for western blot analysis for Syncytin-1 (A) or treated with rabbit anti-human IgG to induce lytic activation and harvested at 24 h (B), 36 h (C,D), or 72 h (E) to analyze lytic readouts in each siRNA treatment group. Lytic proteins EA-D and ZEBRA were probed using western blot in B. Lytic transcript levels of representative genes of each EBV kinetic class were assayed using RT-qPCR in (C). (D) EBV BamW qPCR was performed on DNA isolated from cell pellets. (E) EBV BamW qPCR was performed on DNase-treated, filtered cell culture medium. (F–I) Akata cells were nucleofected with pSpCas9 BB-2A-puro or pSpCas9 BB-2A-puro ERVW-1 guide RNA for 24 h prior to harvest for western blot analysis (F) or treated with rabbit anti-human IgG to induce lytic activation. Cells were harvested at 24 h (G) or 36 h (H,I) to analyze lytic readouts as in (B–D), respectively. Data represent averages of three independent experiments; error bars, SEM; **p = 0.01.

Fig 5: CD8+ and CD4+ T-cell responses in BALB/c mice vaccinated with the two HERV-W vaccines. (A) Schematic representation of the immunisation study with the HERV-WWT and HERV-WLQMV vaccines. BALB/c mice were immunised subcutaneously (s.c.) in the lower limb on day zero, and splenocytes were harvested either 14 days or 28 days later. For each of the two experiments, each vaccine group included ten mice. (B) Illustration of the vaccine-encoded antigens, visualising the four different 16-mer peptide pools spanning HERV-W Gag, HERV-W Env SU, or the two HERV-W TU regions: the ectodomain (Ecto) or the TM-CT. The figure also illustrates the origin of the two 9-mer peptides: peptide 28 (p28, TM-CT) and peptide 34 (p34, Ecto). Graphs show the percentages of IFN?- and TNFa-expressing CD8+ T-cells 14 (C) and 28 (D) days after immunisation or CD4+ T-cells 14 (E) and 28 (F) days after immunisation. Geometric mean fluorescent intensity (MFI) of IFN?-expressing CD8+ T-cells on day 14 (G) and day 28 (H), as well as CD4+ T-cells on day 14 (I) and day 28 (J) after vaccination. The gating strategy is illustrated in supplementary Figure S3A. The individual bullet depicts the response from one mouse, and the horizontal line indicates the mean of the group. Statistically significant differences (non-parametric, two-tailed Mann–Whitney test) between T-cell responses are marked by * = p < 0.05 and ** = p < 0.01. The dotted lines indicate a lower limit for a positive response, and they are based on the mean plus two times the standard deviation of the mean of the responses for T-cells without peptide stimulation (Neg. ctrl).