A single amino acid substitution in the V protein of Nipah virus alters its ability to block interferon signalling in cells from different species

Abstract

The V protein of the paramyxovirus Nipah virus (NiV) has been shown to antagonize the interferon (IFN) response in human cells via sequestration of STAT1 and STAT2. This study describes a mutant of the NiV V protein, referred to as V(AAHL), that is unable to antagonize IFN signalling and demonstrates that a single amino acid substitution is responsible for its inactivity. The molecular basis for this was identified as a failure to interact with STAT1 and STAT2. It was also shown that NiV V, but not V(AAHL), was functional as an IFN antagonist in human, monkey, rabbit, dog, horse, pig and bat cells, which suggests that the ability of NiV to block IFN signalling is not a major constraint that prevents this virus from crossing species barriers.

Fig. 1.

NiV V(AAHL) inhibitory activity is disrupted by a point mutation. (a) Vero cells were transfected with expression vectors for myc-tagged NiV V variants (as indicated) or empty pEF.plink2 expression vector (Ctrl). Cells were also transfected with an IFN-α/β-responsive luciferase reporter plasmid [p(9–27ISRE)4tkΔ(−39)lucter] and a housekeeping-controlled β-galactosidase reporter plasmid (pJATlacZ) (Didcock et al., 1999). Cells were stimulated with 1.8×10⁴ IU IFN-α ml⁻¹ (Roferon-A; Roche Diagnostics) (+) or left untreated (−), and 4–6 h later were lysed and assayed for luciferase and β-galactosidase activity. Luciferase values were normalized to β-galactosidase values and the results are shown as means±sd obtained from five independent transfections. Means of induction factors (induced/uninduced activity) are indicated below the graph. (b) Schematic representation of V(CDC) with STAT1- and STAT2-binding regions. Shaded areas indicate (from left to right) the STAT1-binding region, the nuclear export signal (NES), essential residues interacting with STAT2 (*) and the conserved C-terminal domain (C-term). Numbers above the bar represent amino acid positions. Nucleotide and amino acid differences in V(AAHL) are indicated in open boxes. The P206L mutation (see Fig. 3) is indicated in the filled box. Upper case is used for amino acid residues and lower case for nucleotides.

Fig. 2.

NiV V(AAHL) does not interact with STAT1 or STAT2. (a) Immunofluorescence. 2fTGH cells were transfected with myc-tagged V(AAHL), V*(CDC) or V(AAHL)-E125G expression constructs and stimulated for 70 min with 1.8×10⁴ IU IFN-α ml⁻¹ (Roferon-A; Roche Diagnostics). Cells were fixed and stained with antibodies against the myc tag (green fluorescence) and against either STAT1 (red fluorescence, left panels) or STAT2 (red fluorescence, right panels) as indicated. (b) Co-immunoprecipitation. 293 cells were transfected with expression constructs encoding STAT1 and STAT2 and either myc-tagged V(AAHL) or V*(CDC). Cells were lysed at 48 h post-transfection and complexes containing the V and STAT proteins were precipitated from the lysates using antibodies against either STAT2 or the myc tag, as indicated above each panel. The precipitates were analysed by Western blotting with antibodies detecting either STAT1 and the myc tag or STAT2, as indicated below the panels. The lower right panel confirms efficient precipitation with the anti-STAT2 antibody in all three lysates. HC, Antibody heavy chain; LC, antibody light chain.

Fig. 3.

Inhibition of IFN signalling in cells of different species. (a) Summary of signalling results. IFN-α/β signalling assays were carried out as described. +, Inhibition of signalling; −, failure to inhibit signalling. Results were classified as positive (+) when the induction factor was reduced to 30 % or lower compared with the negative control and the value of the stimulated sample was reduced to 25 % or lower compared with the negative control. (b) Details of the results for Tb1 Lu cells given in (a). Instead of commercial IFN-α, these cells were stimulated with purified and UV-inactivated supernatant from Tb1 Lu cells infected with rSV5VΔC (He et al., 2002), a strong inducer of IFN production. Results are shown as means±sd from six independent transfections. Means of induction factors (induced/uninduced activity) are indicated below the graph. (c) Comparison of inhibition of IFN signalling by NiV V(CDC)-P206L in Hep2 and Tb1 Lu cells. Results are shown as means±sd from six (Hep2) or three (Tb1 Lu) independent transfections. Means of induction factors (induced/uninduced activity) are indicated below the graph.