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Data Collection for:
Langelier et al. (2002). Journal of General Virology, 83, part 11, pp. 2779–2789.
The following material is available only in JGV Online:
Methods
Transfection experiments. HeLa-tTA cells were seeded in six-well plates (1.5x105 cells per well) 24 h before transfection with different plasmids (2 µg) in the presence of 6 µl of FuGENE 6 following the provider's protocol (Roche Diagnostics). After 36 h, the cells received new medium containing either CHX or CHX+TNF as indicated in the published paper or no additives for the control cultures; apoptosis was scored 14 h later. Percentage transfection (% GFP-positive cells in control cultures that were co-transfected with 2 µg of the tested plasmid and 0.5 µg of pAdCMV5-GFPQ) was determined with an EPICS XL-MCL flow cytofluorometer.
Results
Physiological amount of R1 is sufficient for protection
To determine the minimal amount of R1 necessary for protection, A549-tTA cells were infected at 5 p.f.u. per cell in the presence of increasing concentrations of doxycycline (Fig. s1A). Quantification of the R1 concentration by immunoblotting using purified R1 as a standard revealed that a doxycycline concentration of 0.1 ng/ml, which reduced the level of protection only slightly (Fig. s1A), decreased the R1 concentration from 0.4 % total cell protein in the control without doxycycline (not shown) to 0.06 % (Fig. s1B). During the course of an HSV-2 infection of BHK 21/C13 cells, a similar R1 level is attained at approximately 3 h post-infection; at 12 h, when the R1 accumulation reaches its maximal value, it represents 1 % total cell protein (data not shown). Together, these results indicated that R1 expressed at physiological levels could effectively protect cells against the death-inducing cytokine TNF.
Fig. s1. Doxycycline decreases R1 accumulation and R1 protection against TNF. (A) A549-tTA cells were infected with Ad5TR5-R1 at an m.o.i. of 5 in the presence of increasing concentrations of doxycycline (filled triangle) or mock-infected (filled square); after 7 h CHX+TNF were added. Percentage apoptosis was evaluated at 24 h post-infection. (B) Increasing amounts of proteins extracted from harvested cells were immunoblotted with an anti-R1 serum (representative experiment shown). Quantification was by densitometric comparison with a purified R1 standard. From three independent blots, the calculated mean values, expressed as a percentage of total cell protein for the 0.1, 0.3 and 1.0 ng/ml doxycycline samples, were 0.06, 0.01 and <0.002 %, respectively.
Full-length R1 inhibits Fas receptor-induced apoptosis
To further characterize the anti-apoptotic potential of R1 against death receptor activation, its action against Fas triggering was studied. The anti-Fas antibody CH11 in the presence of CHX was as effective as TNF in mediating apoptosis with HeLa-rtTA or parental HeLa cells but was less potent with A549-tTA cells, only 35 % of the cells being scored apoptotic at 48 h. Ad5TR5-R1 infection of HeLa-rtTA and A549-tTA cells blocked Fas receptor-induced apoptosis as efficiently as it did in TNF-treated cells (data not shown). To rule out the possible involvement of the tTA protein in the protective effect of R1, HeLa cells were infected with the recombinant Ad5CMV5-R1, a constitutive Ad with an improved CMV-based expression cassette. The results shown in Fig. s2(A) indicated that full protection occurred in these cells against both TNF and anti-Fas antibody. Protection against TNF was also observed in MDA-MB-231 breast cancer cells infected with the recombinant Ad5CMV5-R1 (data not shown). These observations demonstrate that the R1 anti-apoptotic activity does not require the presence of the tTA or rtTA protein and is functional in several types of cell.
Fig. s2. Protection against TNF and Fas is independent of the tTA or rtTA proteins and of the presence of CHX. (A) HeLa cells were infected with increasing m.o.i.s of Ad5CMV5-R1; after 7 h control medium (open circle) or medium containing CHX (open triangle), or CHX+TNF (filled square) or anti-Fas antiserum (filled diamond) was added and apoptosis was scored from 24 to 26 h post-infection. (B) A549-tTA cells were first infected with Ad5TR5-R1 at an m.o.i. of 10 in the absence (open triangle, cross) or presence (filled circle) of doxycycline or were mock-infected (open square, filled square). They were reinfected (open triangle, filled circle, filled square) or not (open square, cross) 7 h later with Ad5CMV-Fas-L at an m.o.i. of 10. Apoptosis was scored at the indicated times after Ad5TR5-R1 infection.
To determine whether protection could be observed when apoptosis was induced by more physiological conditions (without CHX), Ad5CMV-Fas-L, an Ad recombinant that expresses Fas-L under the control of the standard CMV promoter, was used. At 72 h post-infection with this recombinant, apoptosis occurred in more than 90 % of A549-tTA cells. In sharp contrast, when they had been pre-infected for 7 h with Ad5TR5-R1, they were efficiently protected, the level of apoptosis being similar to that seen in a mock-infected control (Fig. s2B). Adding doxycycline throughout the infection considerably reduced the protection. Together, these results demonstrate that HSV-2 R1 is able to protect cells against apoptosis induced by activation of death receptors.
Ad proteins are not involved in the anti-apoptotic action of HSV-2 R1
At the low m.o.i. of Ad recombinants used in the present study the level of expression of Ad genes should be extremely low due to the absence of Ad replication. However, the involvement of Ad protein(s) in the protective effect, even if highly unlikely, could not be completely ruled out. To study this possibility, HeLa-tTA cells were transfected with different transfer vectors expressing either the R1 protein or, for comparison, two well-known anti-apoptotic proteins, Ad E1B 19K or baculovirus p35. These cells were chosen as they could be transfected approximately 10-fold more efficiently than A549 cells. However, on infection with the Ad5TR5-GFPQ recombinant, they express the recombinant protein at a lower level, probably due to a lower level of tTA protein expression (data not shown). In a first series of experiments, expression of the recombinant proteins was driven by the regulatable TR5 promoter present in double cassette plasmids. As can be seen in Fig. s3(A), the R1 plasmid protected cells against apoptosis induced by CHX+TNF more efficiently (66 % of transfected cells) than the p35 plasmid (42 % of transfected cells) but less efficiently than the E1B 19K plasmid (87 % of transfected cells). For the three plasmids, transfection in the presence of doxycycline to repress recombinant protein expression decreased protection. Quantification of the R1 concentration in pAd5TR5-R1-transfected cells (0.05 %) suggested that some of the transfected cells could not have been protected due to limiting R1 expression. This possibility was confirmed in transfection experiments where R1 expression was driven by the constitutive CMV5 promoter. As shown in Fig. s3(B), cells transfected with pAdCMV5-R1 expressed R1 at a higher level and most of them (93 % of transfected cells) were protected. The control plasmid pAdCMV5-GFPQ did not afford any protection. From these results, we could conclude that the protective activity of R1 does not require co-expression of any Ad protein.
Fig. s3. Ad proteins are not involved in the anti-apoptotic action of HSV-2 R1. HeLa-tTA cells were transfected with different plasmids (2 µg) in the presence of FuGENE 6. After 36 h, CHX (grey bars) or CHX+TNF (black bars) were added and 14 h later apoptosis was scored under microscopic observation. The histogram values, expressed as a percentage of non-apoptotic cells, are means of two independent experiments. Cell extracts were prepared and analysed by immunoblotting for R1 quantification or PARP (116 kDa) cleavage. In panel (A), the plasmids used were pAdTR5-R1-K7-GFPQ (R1), pAdTR5-p35-K7-GFPQ (p35) or pAdTR5-19K-K7-GFPQ (19K); some dishes (+) contained doxycycline throughout the experiment. In panel (B), the plasmids used were pAdCMV5-GFPQ (GFPQ) or pAdCMV5-R1 (R1). The percentage transfection was determined as described in the published paper.
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