VIROLOGY 180, 822-825 (1991)

Mechanism of Inhibition of HSV-1 Replication by Tumor Necrosis Factor and Interferon y ELENA FEDUCHI AND Luis CARRASCO I Centro de Siologia Molecular (CSIC-UAM), Universidad Autdnoma Canto Blanco, 28049 Madrid, Spain Received May 28, 1990 ; accepted October 15, 1990

Tumor necrosis factor (TNF) synergizes with interferon (IFN-y) in the blockade of HSV-1 replication . Antibodies against ENO block this synergism, implying a role of IFNf in the antiviral activity of TNF plus IFNy . IFNJ3, added exogenously to Hep-2 cells shows antiviral activity against HSV-1 only at high concentrations, whereas IFN3z (also known as 11- 6) alone has no effect on the replication of VSV or HSV-1 even when 1,000 U/ml are present . Our results are in accordance with the idea that TNF induces IFN/i, and that both cytokines must be present in the culture medium to synergize with IFN7 in order to inhibit HSV-1 replication . co 1991 Academic Press, Inc . The search for different cytokine combinations more effective in the blockade of HSV-1 replication in human cells may lead to an improvement of the available chemotherapy against this human pathogen . In this regard, tumour necrosis factor (TNF), a cytokine with pleiotropic effects, has shown synergy with the antiviral activity of several interferons (IFNs) (1-4) . It reamains controversial whether this antiviral effect is mediated by the induction of another cytokine, i .e ., IFN/3 2 , also known as IL-6 (5) . Other results do not confirm this idea, suggesting a direct antiviral effect of TNF (1), or the induction of IFNO, as the mediator of the TNF antiviral activity (6, 7) . Using a sensitive method to detect low levels of mRNA, induction of IFN(3, mRNA by TNF in Hep-2 cells was found (7, 8) . The low levels of IFNR, mRNA detected suggest that this cytokine is made in low amounts in TNF-treated cells . However, antibodies against IFNf, partially reversed the antiviral effects of TNF on VSV growth, suggesting that both TNF itself and the induced IFNf, play a part in the antiviral response in that system (7) . In order to elucidate the role of IFNf3 in the synergistic action of TNF plus IFN-y against HSV-1 (3), we assayed its reversion by antibodies against the different human interferons . Hep-2 and Vero cells were grown on petri dishes (Nunc, Denmark) containing 10 rnl of Dulbecco's modified Eagle's medium (E4D) supplemented with 10% calf serum (GIBCO Laboratories, Grand Island, NY) and incubated at 37° in a 5% CO 2 atmosphere . HSV-1 was grown on Vero cells in E4D medium supplemented with 2% calf serum (E4D2) . I he fraction obtained after removal of cell debris by low-speed centrifugation was used as the source of virus . The virus thus obtained was titrated by plaque assay . Human lymphoblastoid

interferon, Hu IFN-(Ly) a (1 .7 x 10b U/ml), was a goner ous gift from Dr . N . Finter(Wellcome Research Laboratories, Beckenham, England) . Recombinant human y interferon, rHu IFN-y (2 x 10' U/mg), recombinant hu man tumor necrosis factor, rHu/TNF-a (6 X 10' U/mg), and antibody against TNF were generously provided by Dr . Adolf (Ernst-Boehringer Institute fOr AizneimittelForschung, Austria) . Human fibroblast interferon, I-lu IFN-/3 (5 X 10 4 U/ml) was from the Biomolecular Research Laboratories (CA) . Antibodies against IFNa, -0, and--y were from Boehringer-Mannheim . Recombinant human IFN/32 (IL-6) and antibodies against IFN/3 2 were generously provided by Dr . W . Fiers (Ghent, Belgium) and Dr . J . Content (Brussels, Belgium) . Hep-2 cells, grown in 96-well dishes, were infected with HSV-1 at a multiplicity of infection (m .o .i .) indicated in each experiment in the figure legends . Hep-2 cells were incubated with 50 ul of methionine-free medium and 1 .25 gCi of [36 S]methionine (1 .45 Ci/rnmol ; Amersham) for 1 hr, at the time after infection indicated in each experiment . Cell monolayers were dissolved in 50 Al of sample buffer, heated at 90 0 for 5 min, and applied to a 15% polyacrylamide gel and run overnight at 100 V/20 cm (3) . o test whether TNI - synergizes with IFNy by inducing a species of IFN, we treated Hep-2 cells with TNF and IFNy in the presence of antibodies against IFNa, (3, or y . Anti-IFNy antibodies failed to block the inhibition of HSV-1 protein synthesis in Hep-2 cells treated with TNF and IFNy (Fig . 1) . However, anti-IFNs antibodies reversed [he synergy such that the HSV-1 proteins were synthesized as in control cells . As a control, addition of anti IFNy antibodies did not allow the synergism, further suggesting that TNF alone cannot inhibit viral protein synthesis . We also assayed the specificity of the antibodies used to make sure that the anti IFN-/3 antibodies did not cross-react with IFN-y (results not shown) . These results indicate that IFNy plus

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gesting that not only IFN/3 and IFN-y, but TNF as well, must be present in the culture medium . In conclusion, to produce the blockade of HSV-1 replication, TNF must induce IFN/3 and both cytokines must synergize with IFNy . IFN02 (IL-6) is also induced by TNF, so we tested the antiviral effect of this cytokine against VSV, which is much more sensitive to the action of IFN than HSV-1 . Figure 3 shows that IFN/3 2 cannot inhibit VSV protein synthesis in Hep-2 cells . IFN3, not only is devoid of antiviral activity by itself (Fig . 3A), but cannot synergize with either TNF or IFNy in preventing VSV replication (Fig . 3A) . IFNa, -(3„ or -y drastically inhibit viral protein synthesis at the same concentrations (or even lower) employed with IFN,d2 , and TNF can synergize with IFNy although TNF alone has no effect on VSV replication (Fig . 3) .

FIG . 1 . Effect of IFN antibodies on the TNF clue IFNy synergism . Hep-2 cells were treated with TNF (100 ny/rnl), IFNy (10 U/ml), antiIFNrr (500 U/ml), anti-IEN,A (500 U/ml), and anti IFN-( (50 Writ as indicated in the figure . After 24 hr of treatment cells were infected with HSV (10 PFU/cell(, and 16 hr p .i . proteins were labeled with 25 pCi/ml of [° 6 Sjmethionine during 1 hr. Proteins were analyzed by PAGE as described in the text .

TNF synergize through a component(s) that is neutralized by anti-IFN3 antibodies . It must be kept in mind that antibodies against IFN,Q cross-react with FNB 2 (11) . We reasoned that if TNF induces an IFNf or an IFNflike component, supernatants of TNF-treated cells would show antiviral effects against HSV-1 . These supernatants alone cannot inhibit HSV-1 protein synthesis, but are able to synergize with IFNy (Fig . 2), This synergistic effect could be blocked by antibodies specific for IFN/3, suggesting that TNF has induced the secretion of an N3-like species into the supernatants . The results indicate that the TNF present in the supernatants and the IFNI3 induced are not enough to exert the antiviral state against HSV-1 . Antibodies against TNF or IFNy inhibit the antiviral effect of those supernatants observed in the presence of IFN-y, sug-

FIG, 2 . Effect of antibodies against different cytokines on the synergism established between IFNy arid TNF . Before infection with HSV-1 (m .o .t . 5) Help-2 cells were treated for 24 hrwith the indicated cytokines, supernatants, and antibodies (Ab) as indicated . SPa, control supernatantfrorn Hep 2 cells ; SPTNF, supernatant of Hep 2 cells treated with 100 ng/ml of TNF for 24 hr . anti-IFNA antibodies (500 U/ml), anti-IFNy antibodies (50 U/ml), anti-FNFantibodies (1 mg/ml) . Proteins were labeled and analyzed as described in the text .



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FIG . 3 . (A) Effect of IFNpz in combination with several cytokines on Hep-2 cells infected with VSV . All cytokines were added 24 hr before infection with VSV (m .o .i . 20) . Proteins were labeled and analyzed as described . (B) Effect of IFN9z and several ct~'s kines on cells infected with HSV-1 . Hep-2 cells treated for 24 hr with the indicated cytokines (lFNa, 400 U/ml ; IFNf, 400 U/ml;TNF, 100 ng/ml ; iFN-y, 10 U/ml) were infected with 5 PFU/cell of HSV-1 . Proteins were labeled during 1 hr at 16 hr p .i, and analyzed by PAGE .

Interferons have different antiviral activities depending on the virus-cell system analyzed . Although IFN82 has no antiviral activity against VSV, we tested the inhibition of HSV-1 replication by this cytokine . Figure 3B shows that 1000 U/ml IFNfz added exogenously cannot inhibit HSV-1 protein synthesis . Similarly, with the VSV system IFN02 cannot synergize with TNF or IFNy . These results clearly indicate that IFN/3, added exogenously has much more antiviral action against HSV-1 than IFN/iz, but neither of them can replace TNF to achieve the same inhibition observed with TNF plus IFNy . The mechanism underlying the antiviral activity of interferons against herpesviruses has been controversial (9, 10) . We reported that IFNa does not act at the same step as IFNf3 or IFNy in the replication of HSV-1

on HeLa cells (3) . Depending on the multiplicity of infection employed, the synthesis of HSV-1 proteins could be sensitive or resistant to IFN .TNF and IFNy can exert a synergistic blockade on the replication of HSV-1 in Hep-2 cells . The step inhibited by these two cytokines is located after virus entry into the cell, but before immediate early gene transcription (3) . The finding that antibodies against IFN/ can suppress the inhibitory act on of TNF and IFNy suggests a role of IFNR in such blockade . Most likely this IFN/-like component is not the only one that exerts the antiviral effect, since antibodies against TNF added to supernatants of TNF-treated cells also block the synergism . Therefore, the IFN/3 induced in this supernatant is not enough to exert a synergism with IFNy, although IFN/3 is necessary to establish the antiviral state induced by



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TNF and IFNy . These results indicate that the presence of all three cytokines is necessary to block efficiently HSV1 growth . The suggestion that the synergy ofTNF and IFNy in the antiviral activity is a multicomponent process occurring on one hand between TNF and FNy and on the other between the induced IFNf (4) is in accordance with our findings . ACKNOWLEDGMENTS The excellent technical assistance of Ms . M . Creme and Ms, M . J . Arnela are acknowledged . E .F. is the holder of a "PI fellowship . Fundaci6n Ram6n Areces and CICYT (81088-0233) are acknowledged for financial support .

REFERENCES 1 . FEDUCHI, E ., ALONSO, M . A., and CARRASCO, L ., (1989) .1 Vlrol . 63,1354-1359 .

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2. HUGHES, T . K ., KASPART, A . T., and COPPENHAUER, D . H ., (1988). Antiviral Res. 10, 1 9 . 3. JACOBSEN . H . . MESTAN, J ., 0.'11TTNACNr, S ., and DIEFFFNBACH, C . W ., (1989) . Mot. Cell Biol. 9, 3037-3042 . 4 . KOHASE, M ., HENRIKSEN-DESTEFANO, D ., MAY . L. T„ VILCEK, J ., are SEHGAL, P . B_ ;1986) . cell 45, 659-666 . 5. MESTAN, J ., DiuEL, W ., MITTNACHT, S ., HILLEN, H ., BLOHM, D ., MOLLER, A ., JACOBSEN, H ., and KIRSCHNER, H ., (1986) . Nature (London) 323, 816-819 . 6. MuEoz, A., and CARRASCO, L ., (1984) . J. Gen . Virot 65, 10691078 . 7. MuEoz, A ., and CARRASCO, L., (1987) . In "Mechanisms of Viral Toxicity in Animal Cells'' (L Carrasco, Ed .) . CRC Press, Boca Baton, FL . 8. REIS . L . F. L ., LOU, T . H ., and VILCEK. J ., (1989) .1 Biel. Chem. 264, 16,351 15,354 .

9. Van DAMMEN, J ., DELEY, M ., VAN SNICK, J ., DINARELLO . C . A ., and RILUAu, A ., (1987) . J. Immune,'. 139, 1867 1872 . 10. WONG, G . H ., and GOEOOEL, D . V ., (1986) . Nature (London) 323, 819-822 . 11, ZILBERSTEIN, C ., (1986) . EMBO J. 5, 2529-2537 .