Different functions of the common P/V/W and V-specific domains of rinderpest virus V protein in blocking IFN signalling

Abstract

The V proteins of paramyxoviruses are composed of two evolutionarily distinct domains, the N-terminal 75 % being common to the viral P, V and W proteins, and not highly conserved between viruses, whilst the remaining 25 % consists of a cysteine-rich V-specific domain, which is conserved across almost all paramyxoviruses. There is evidence supporting a number of different functions of the V proteins of morbilliviruses in blocking the signalling pathways of type I and II IFNs, but it is not clear which domains of V are responsible for which activities and whether all these activities are required for effective blockade of IFN signalling. We have shown here that the two domains of rinderpest virus V protein have distinct functions: the N-terminal domain acted to bind STAT1, whilst the C-terminal V-specific domain interacted with the IFN receptor-associated kinases Jak1 and Tyk2. Effective blockade of IFN signalling required the intact V protein.

Fig. 1.

Effects of W and Vs domains on IFN signalling. (a) Protein constructs used in this study and their derivation from the RPV P gene; W*, W_Y110H. (b, c) Plasmids expressing RPV V, RPV W, GFP or GFP–Vs were transfected into Vero cells together with pJAT-lacZ and either pGL3-MX1P-luc (b) or pGAS-luc (c) as described in Methods. At 24 h post-transfection, the cells were treated with 1000 IU IFN-α ml⁻¹ for 8 h (b) or 1000 IU IFN-γ ml⁻¹ for 6 h (c). Untreated controls were used in each case. Cells were then lysed, and luciferase and β-galactosidase measured as described in Methods. The results from multiple experiments were normalized to the induction seen in cells transfected with the appropriate control, empty vector (pcDNA) or GFP. Letters above the bars for the IFN-treated samples in (b) and (c) indicate the results of statistical analysis (Tukey analysis for multiple comparisons with maximum probability for significance of P = 0.05): results that were not statistically different from one another have the same letter. (d, e) Vero-SLAM cells were transfected with plasmids encoding the indicated constructs or empty vector. The cells were lysed 48 h post-transfection; V5-tagged proteins were immunoprecipitated and STAT1 or STAT2 in the immunoprecipitates (IP) detected by Western blotting. Equal amounts of the total lysates were analysed by Western blotting to detect the expressed proteins and the levels of STAT1 and STAT2 in the cells. (f) 293FT cells were transfected with pCMYC-MDA5 together with plasmids expressing PIV5 V, RPV V, RPV W, GFP or GFP–Vs, or empty vector. Cells were lysed and immunoprecipitation with anti-V5 carried out as in (d). Immunoprecipitates were probed for MDA-5 with anti-c-myc antibody. Equal amounts of the total lysates were analysed by Western blotting to demonstrate the expression of the c-myc-tagged and V5-tagged proteins.

Fig. 2.

Interaction of components of RPV V with Jak1 and Tyk2. (a, b) 293FT cells were transfected with plasmids encoding FLAG-tagged Tyk2 (a) or FLAG-tagged Jak1 (b) together with plasmids encoding RPV V, RPV W, GFP or GFP–Vs, or empty vector. Cells were lysed 2 days post-transfection and immunoextracted with anti-V5 tag antibody. Immunoprecipitates (IP) were analysed by Western blotting with anti-FLAG or anti-V5 antibodies. Equal amounts of the total lysates were analysed also by Western blotting using anti-FLAG and anti-V5 antibodies. (c, d) Vero-SLAM cells were transfected essentially as in (a) and (b), respectively. At 30 h post-transfection, the cells were lysed in SDS-sample buffer containing phosphatase inhibitors and the levels of phosphoTyk2 (Tyk2-P) (c) or phosphoJak1 (Jak1-P) (d) in the total lysate determined by Western blotting with specific anti-phosphoTyk2 or anti-phosphoJak1 antibodies. Lysates were also analysed to determine the expression levels of individual plasmid-encoded proteins by Western blotting with anti-V5 or anti-FLAG antibodies. (e) Vero-SLAM cells were transfected with plasmids encoding RPV V, W, W_Y110H or empty vector. The cells were lysed 48 h post-transfection; V5-tagged proteins were immunoprecipitated and STAT1 in the immunoprecipitates detected by Western blotting. Equal amounts of the total lysates were analysed by Western blotting to detect the expressed proteins and the levels of STAT1 in the cells. (f) Vero-SLAM cells were transfected with FLAG-Tyk2 together with plasmids expressing RPV V, RPV W, RPV W_Y110H or empty vector. The cells were lysed and analysed as described (c). Proliferating cell nuclear antigen (PCNA) was used as a loading control throughout.

Fig. 3.

Inhibition of type I IFN action in stable cell lines. Stable cell lines transduced with lentivirus-based vectors encoding the RPV P, V, C or W proteins, or GFP or GFP–Vs were constructed as described in Methods; a cell line transduced with the base vector without an additional ORF (A549-blank) was used as a control. (a) The expression levels of the expressed viral proteins (or derivatives) were analysed by Western blotting using anti-V5 tag antibody; PCNA was used as the loading control. (b, c) Cell lines were plated on 24-well plates and treated for 24 h with the indicated concentration of IFN-α. The cells were then infected with VSV at an m.o.i. of 0.1. Cells were fixed 24 h post-infection and stained with crystal violet.