Figure S6.

Evolution of resistance to existing antivirals; FD duplexes do not induce cell death in uninfected bystander cells, related to Figures 4 and 5

(A) Outgrowth of fomivirsen-resistant virus in continuous culture. Viral titers from rounds 3–5 of continuous cultures ±25 μM fomivirsen (no dose escalation was used). Positive slope in titers in presence of fomivirsen is observed beginning at round 3 of infection.

(B) Verification that FD⁸⁶ decreases CMV titers below detection; subculturing of supernatants from continuous culture. We used the established TCID50 calculation described previously (Reed, 1938). This calculation computes both the TCID50/ml and a single-well detection limit from the initial dilution and titration factor. Because there are multiple replicates (in this case eight replicate wells) and the statistics are Boolean, it is possible to reliably calculate TCID50/ml values below the single-well detection limit for the entire row of eight wells.

(C) Fitness recovery assay: analysis of FD⁸⁶-treated continuous cultures upon removal of FD⁸⁶. Supernatants from continuous cultures on indicated days were cultured on ARPE-19 cells ± FD⁸⁶. Titers recover 1.5–2 logs upon removal of FD⁸⁶.

(D) HSV-1 resistance to acyclovir (ACV). Viral titers of ARPE-19 cells infected with HSV-1 (strain 17syn+ IE175-YFP; MOI = 1) ±100μM ACV (no dose escalation used) and supernatant transferred every 2 days over three consecutive rounds of infection. Virus titers were assayed by TCID-50 every 2 days post transfer; a positive slope in the titers (i.e., outgrowth of ACV-resistant virus) is evident despite 100 μM ACV.

(E and F) Continuous cultures of AD169 either without FD⁸⁶, with 25 μM FD⁸⁶, or with FD⁸⁶ dose escalation.

(G and H) Continuous cultures of Δcrs AD169 either without FD⁸⁶, with 25 μM FD⁸⁶, or with FD⁸⁶ dose escalation.

(I) No evidence of escape through increasing expression of UL38 or vMIA. qRT-PCR of UL38 and vMIA (normalized to beta-actin) from the AD169 continuous cultures with FD⁸⁶ dose escalation. Significance tests were carried out using a one-way ANOVA with Dunnet’s test for multiple comparisons.

(J) The AD169 strain sustains greater fitness than the Δcrs AD169 strain does over multiple weeks of FD⁸⁶ treatment in continuous culture. Data points from day 8 to 36 in (E) and (G) were compared. Significance was determined by Student’s t test, ^∗∗ < 0.01.

(K) UL38 overexpression does not rescue CMV from FD-induced open-loop lethality. MRC5 cells were nucleofected with a UL38 expression vector (and either mock/FD^Scram/FD⁸⁶), then 24 h later, infected with CMV and titered at 4 dpi.

(L) Flow cytometry viability analysis of uninfected neighboring “bystander” cells. ARPE-19 cells were nucleofected with 25 μM FD⁸⁶, and 24 h post-nucleofection, cells were infected with CMV TB40E-IE86-YFP (MOI = 0.5). At 2 hpi, cells were washed twice in PBS to remove any attached virus and co-cultured with naive mCherry-expressing ARPE-19 cells (“bystanders”). Cells were analyzed by flow cytometry at 36 hpi (i.e., once FD-mediated cytotoxicity was present but prior to virus release).