Interaction of mumps virus V protein variants with STAT1-STAT2 heterodimer: experimental and theoretical studies

Abstract

Background: Mumps virus V protein has the ability to inhibit the interferon-mediated antiviral response by inducing degradation of STAT proteins. Two virus variants purified from Urabe AM9 mumps virus vaccine differ in their replication and transcription efficiency in cells primed with interferon. Virus susceptibility to IFN was associated with insertion of a non-coded glycine at position 156 in the V protein (VGly) of one virus variant, whereas resistance to IFN was associated with preservation of wild-type phenotype in the V protein (VWT) of the other variant.

Results: VWT and VGly variants of mumps virus were cloned and sequenced from Urabe AM9 vaccine strain. VGly differs from VWT protein because it possesses an amino acid change Gln103Pro (Pro103) and the Gly156 insertion. The effect of V protein variants on components of the interferon-stimulated gene factor 3 (ISGF3), STAT1 and STAT2 proteins were experimentally tested in cervical carcinoma cell lines. Expression of VWT protein decreased STAT1 phosphorylation, whereas VGly had no inhibitory effect on either STAT1 or STAT2 phosphorylation. For theoretical analysis of the interaction between V proteins and STAT proteins, 3D structural models of VWT and VGly were predicted by comparing with simian virus 5 (SV5) V protein structure in complex with STAT1-STAT2 heterodimer. In silico analysis showed that VWT-STAT1-STAT2 complex occurs through the V protein Trp-motif (W174, W178, W189) and Glu95 residue close to the Arg409 and Lys415 of the nuclear localization signal (NLS) of STAT2, leaving exposed STAT1 Lys residues (K85, K87, K296, K413, K525, K679, K685), which are susceptible to proteasome degradation. In contrast, the interaction between VGly and STAT1-STAT2 heterodimer occurs in a region far from the NLS of STAT2 without blocking of Lys residues in both STAT1 and STAT2.

Conclusions: Our results suggest that VWT protein of Urabe AM9 strain of mumps virus may be more efficient than VGly to inactivate both the IFN signaling pathway and antiviral response due to differences in their finest molecular interaction with STAT proteins.

Figure 1

Decrease in pY⁷⁰¹-STAT-1 level protein by VWT of Urabe AM9 vaccine strain. (A) Detection of ISGF3 complex activated by IFN-α in human cervical carcinoma cell line. The complex was determined 48 h after transfection and 6 h after stimulation with IFN proteins separated by 7% PAGE under native conditions, semidry transfer to PVDF membrane and immunodetection with specific antibodies for pTyr⁷⁰¹-STAT1 proteins, pTyr⁶⁹⁰-STAT2 and IRF9 (ISGF-γ3). The molecular weight of the complex is 250 kDa. (B) Effect of VWT and VGly proteins on STAT1 and STAT2 phosphorylated proteins by Western blot in human cervical carcinoma cell line. Proteins were separated in 10% SDSPAGE with semidry transfer to PVDF membrane and immunodetection with antibodies against His-tag for V proteins, pTyr⁷⁰¹-STAT1, pTyr⁶⁹⁰-STAT2 and β-actin. (C) Detection of STAT1 inactive protein in cell expressing VWT and VGly proteins and 10% SDS-PAGE transfer to PVDF membrane and immunodetection with antibodies against STAT1 inactive protein and β-actin to normalize the level protein.

Figure 2

Homologous modeling and differences of theoretical 3D structure of VWT and VGly. (A) Models of V proteins, VWT and (B) VGly built with the PDB: 2B5Lc as template. Both cases show residues 155 and 103. Additionally, residue 156 is shown in B. (C) Superimposition of the 3D models of VWT (gray) and VGly (blue), Gly¹⁵⁵ (green), Gly¹⁵⁶ of VGly (red), Pro¹⁰³ of VGly (orange), Gln¹⁰³ of VWT (yellow), Trp-motif residues of binding to STAT1-STAT2 (purple), Cys-rich motif C4HC3 (residues in pink). Display in Web Lab Viewer.

Figure 3

Interaction model V-STAT1-STAT2. (A) Heterodimer model of STAT1-STAT2 by the SH2-domain (STAT1 PDB: 1YVL and 3D model of STAT2 from 1YVL and 1BF5). Heterodimer model shows the following residues: Tyr⁶⁹⁰ and Tyr⁷⁰¹ (orange), nuclear localization signal residues (pink), lysine residues to ubiquitylation (Ub) (blue). (B) Interaction of STATs heterodimer with VWT by Trp-motif and STAT2. (C) Interaction of heterodimer with VGly by STAT2, lysine residues (Ub) (blue). In B and C, the boxes at right show the interaction site.