The Measles Virus V Protein Binding Site to STAT2 Overlaps That of IRF9

Abstract

Measles virus (MeV) is a highly immunotropic and contagious pathogen that can even diminish preexisting antibodies, and remains a major cause of childhood morbidity and mortality worldwide despite the availability of effective vaccines. MeV is one of the most extensively studied viruses with respect to mechanisms of JAK-STAT antagonism. Of the three proteins translated from the MeV P gene, P and V are essential for inactivation of this pathway. However, the lack of data from direct analyses of the underlying interactions means that the detailed molecular mechanism of antagonism remains unresolved. Here we prepared recombinant MeV V protein, which is responsible for human JAK-STAT antagonism, and a panel of variants, enabling the biophysical characterization of V protein including direct V/STAT1 and V/STAT2 interaction assays. Unambiguous direct interaction between the host and viral factors, in the absence of other factors such as Jak1 or Tyk2, were observed and the dissociation constants were quantified for the first time. Our data indicate that interactions between the C-terminal region of V and STAT2 is one order of magnitude stronger than that of the N-terminal region of V and STAT1. We also clarified that these interactions are completely independent of each other. Moreover, results of size-exclusion chromatography demonstrated that addition of MeV-V displaces STAT2-core, a rigid region of STAT2 lacking the N and C-terminal domains, from pre-formed complexes of STAT2-core/IRF-associated-domain (IRF9). These results provide a novel model whereby MeV-V can not only inhibit the STAT2/IRF9 interaction but also disrupt pre-assembled interferon-stimulated gene factor 3. IMPORTANCE To evade host immunity, many pathogenic viruses inactivate host Janus kinase-signal transducer and activator of transcription (STAT) signaling pathways using diverse strategies. Measles virus utilizes P and V proteins to counteract this signaling pathway. Data derived largely from cell-based assays have indicated several amino acid residues of P and V proteins as important. However, biophysical properties of V protein or its direct interaction with STAT molecules using purified proteins have not been studied. We have developed novel molecular tools enabling us to identify a novel molecular mechanism for immune evasion whereby V protein disrupts critical immune complexes, providing a clear strategy by which measles virus can suppress interferon–mediated antiviral gene expression.

Viral pathogen-induced mechanisms to antagonize mammalian interferon (IFN) signaling pathway

“…Many viruses target STAT2 to antagonize IFN signaling. Thus, MeV V protein binds to STAT2 [ 177 , 178 ]. Yellow fever virus NS5 protein also binds STAT2 but this interaction requires STAT2 activation by IFN [ 179 ].…”

Mechanisms of Viral Degradation of Cellular Signal Transducer and Activator of Transcription 2

“…Structure–function analysis has suggested that the Cys-rich C-terminal domain (CTD), particularly the zinc fingers ( Figure 3 and Figure 4 ), of the V protein is required to stabilize the interactions of the V protein with DDB1, whereas direct binding occurs with the other regions (also see Figure 6 ) [ 105 , 106 , 107 , 108 , 109 ]. This is also supported by phylogenetic evidence; specifically, the three jeilongviruses that express V proteins incapable of zinc finger formation ( Figure 4 and Figure 5 ) also fail to affect STAT1 or STAT2 [ 110 ].…”

Evasion of Host Antiviral Innate Immunity by Paramyxovirus Accessory Proteins

“…Ultimately, V protein was proved to inhibit lysine 63 (K63)-linked polyubiquitination of IRF7 mediated by TRAF6, consequently preventing TLR7/9-dependent IFN production (Kitagawa et al, 2013). Apart from V protein discussed above, the C protein of SeV can bind to IKKα and inhibit the phosphorylation of Andrejeva et al, 2004;Komatsu et al, 2007;Sakaguchi et al, 2011;Schaap-Nutt et al, 2011;Svitek et al, 2014;Audsley et al, 2016;Sanz Bernardo et al Garcin et al, 2002;Nishio et al, 2002;Takeuchi et al, 2003;Shaw et al, 2004;Nishio et al, 2005;Nanda and Baron, 2006;Caignard et al, 2007;Devaux et al, 2007Devaux et al, , 2013Svitek et al, 2014;Ma et al, 2015;Oda et al, 2015;Li P. et al, 2019;Keiffer et al, 2020;Nagano et al, 2020 STAT2 V (CDV, HPIV4, MeV, MuV, NiV, PIV5, and PPRV) Nishio et al, 2002Nishio et al, , 2005Rodriguez et al, 2004;Ramachandran et al, 2008;Svitek et al, 2014;Nagano et Takeuchi et al, 2003Hela Caignard et al, 2007HEK293T Ramachandran et al, 2008HEK293T and 2fTGH Nagano et al, 2020None Devaux et al, 2007 IRF7, leading to the inhibition of TLR7/9 signaling. However, compared to full size C protein (aa 1-204), Y1 (aa 24-204), and Y2 (aa 30-204) could bind to IKKα and exhibit less ability to suppress TLR7/9 signaling, indicating that N-terminal of C protein is not required for interaction with IKKα.…”

“…STAT2 is a primary target for MeV V protein (Takeuchi et al, 2003;Caignard et al, 2007;Ramachandran et al, 2008). However, Nagano et al (2020) provided controversial evidence recently. They found that the different regions of V protein mediated the interactions with STAT1 or STAT2, and these interactions are independent of each other.…”

“…Mechanistic studies clarified that the C-terminal region of V interacts with the STAT2-core region. This interaction competes with IRF9 for STAT2, leading to the disruption of ISGF3 formation and suppression of antiviral genes expression (Nagano et al, 2020). In addition to the V proteins discussed above, MeV P protein also interacts with the linker domain of STAT1 and suppresses the phosphorylation of STAT1.…”