The OTU enzymatic activity is required for inhibition of IFN induction and action.

(a) 293FT cells were transfected with 400 ng pHA-Ub and with 500 ng empty vector (Lane 2) or with 500 ng of plasmid expressing GV L1-169-V5 wildtype (Lane 3) or with the mutants C40A (Lane 4), H151A (Lane 5) or Q16R (Lane 6) or left untransfected (Lane 1). Thirty hours post-transfection, cells were lysed and total protein ubiquitination was determined by Western blot using anti-HA. (b) 293FT cells were transfected with 250 ng each pHis-mISG15, HA-mHerc6, mUBE1L-HA, UbcM8 alone (Lane 3) or together with 500 ng of plasmid expressing GV L1-169-V5 (Lane 4) or with one of the following mutants C40A (Lane5), H151A (Lane 6) or L16R (Lane 7), or left untransfected (Lane 1). As an extra negative control, cells were transfected with 250 ng pHis-mISG15 and 1250 ng empty plasmid (Lane 2). After 30 h of transfection cells were lysed and total ISG15ylation was analyzed by Western blot using anti-6His. The expression level of GV L1-169 and its mutants were assayed by using anti-V5. PCNA levels served as loading control in all experiments. (c) Vero cells were transfected with 350 ng of the reporter plasmid pIFNβ-luc plus 200 ng pJATLacZ combined with 700 ng of pcDNA6-GV-L1-169 or of one of the mutants C40A, H151A, or Q16R, or empty vector. After 24 hours of transfection cells were infected with Sendai virus (SV) or left uninfected. Eight hours post-infection the cells were lysed with NP40 lysis buffer and the luciferase and β-galactosidase activities determined as described in material and methods. (d) Vero cells were transfected essentially as in (c) except that 100 ng of the reporter plasmid pGL3Mx-1-luc was used. After 24 hours of transfection cells were incubated with or without IFNα. After a further 8 hours the cells were lysed and the luciferase and β-galactosidase activities determined as described in material and methods. (e) Vero cells were transfected essentially as in (c) except that 400 ng of pGAS-luc was used as the reporter plasmid. After 24 hours of transfection cells were incubated with or without IFNγ. After a further 8 hours the cells were lysed and the luciferase and β-galactosidase activities determined as described in material and methods. Error bars in (c-e) show standard error of the mean of normalised data. Results from three separate experiments were combined by setting the RLUs induced by SV or IFN in cells transfected with empty vector to 100%.