Zika Virus Targets Human STAT2 to Inhibit Type I Interferon Signaling

Abstract

The ongoing epidemic of Zika virus (ZIKV) illustrates the importance of flaviviruses as emerging human pathogens. All vector-borne flaviviruses studied thus far have to overcome type I interferon (IFN) to replicate and cause disease in vertebrates. The mechanism(s) by which ZIKV antagonizes IFN signaling is unknown. Here, we report that the nonstructural protein NS5 of ZIKV and other flaviviruses examined could suppress IFN signaling, but through different mechanisms. ZIKV NS5 expression resulted in proteasomal degradation of the IFN-regulated transcriptional activator STAT2 from humans, but not mice, which may explain the requirement for IFN deficiency to observe ZIKV-induced disease in mice. The mechanism of ZIKV NS5 resembles dengue virus (DENV) NS5 and not its closer relative, Spondweni virus (SPOV). However, unlike DENV, ZIKV did not require the E3 ubiquitin ligase UBR4 to induce STAT2 degradation. Hence, flavivirus NS5 proteins exhibit a remarkable functional convergence in IFN antagonism, albeit by virus-specific mechanisms.

Figure 1. Phylogenetically diverse NS5 proteins antagonize Type I IFN signaling

(A) Phylogenetic tree of NS5 proteins; red indicates proteins chosen for characterization. (B) Amino acid percent identity matrix of NS5 proteins. (C) ISG54 reporter assay performed in triplicate in 293T cells transfected with increasing amounts of NS5 plasmids. (D) Renilla luciferase values for (C). (E) Immunoblot of 293T cells transfected with GFP and flavivirus NS5 plasmids. GFP positive cells of similar intensity were sorted by flow cytometry. (F) HA-tag IP assay and immunoblot of 293T cells transfected with indicated HA-tagged plasmids. (G) Vero cell IFA for endogenous STAT2 levels post IFN treatment for 30 minutes. (H) Same as (G), except staining for endogenous pSTAT1. Error bars represent mean ±SD. Results are representative of three or more independent experiments.

Figure 2. ZIKV NS5 proteins antagonize Type I IFN signaling by targeting STAT2

(A) ISG54 reporter assay performed in triplicate in 293T cells transfected with increasing amounts of NS5 expressing plasmids. (B) HA-tag IP assay and immunoblot of 293T cells transfected with indicated HA-tagged plasmids. (C) Immunoblot of 293T cells transfected with increasing amounts of DENV2, ZIKV UG and FP NS5 plasmids. (D) Vero cell IFA for colocalization of overexpressed human STAT2-FLAG and ZIKV NS5-HA tagged proteins. (E) Vero cell IFA of endogenous STAT2 levels post IFN treatment for 30 minutes. Error bars represent mean ±SD. Results are representative of three or more independent experiments.

Figure 3. ZIKV degrades STAT2 in a proteasome dependent manner

(A) Vero cell IFA of endogenous STAT2 upon ZIKV UG infection and IFN treatment for 30 minutes. (B) qRT-PCR analysis of ISGs and ZIKV UG RNA upon infection in Vero cells for 24h at indicated MOI, following mock treatment or treatment with IFN for 14h. nd=not detected, error bars represent mean ±SD of triplicates. (C) Immunoblot of Vero cells infected with ZIKV UG or ZIKV PR for 24h at indicated MOI. (D) Immunoblot of 293T cells infected with DENV2 and ZIKV UG followed by mock, MG132 (MG) or Lactacystin (LC) proteasome inhibitor treatment. (E) Immunoblot of DENV2 and ZIKV UG infected WT 293T or Ubr4 deficient 293T cells, 24h post infection at MOI 5. (F) HA-tag IP assay and immunoblot of 293T cells transfected with indicated HA-tagged plasmids. Results are representative of three or more independent experiments.

Figure 4. ZIKV exhibits species-specific antagonism of STAT2

(A) Vero cell IFA of viral NS5 and E proteins upon ZIKV Fortaleza infection at MOI 5 for 24h (B) Vero cell IFA for colocalization between overexpressed mouse STAT2-FLAG and ZIKV UG NS5-HA tagged proteins. (C) HA-tag IP assay and immunoblot analysis of 293T cells transfected with ZIKV UG NS5-HA, human STAT1-FLAG, human STAT2-FLAG and mouse STAT2-FLAG plasmids. (D) Immunoblot of primary human and mouse fibroblasts infected with ZIKV Fortaleza strain at MOI 5 for indicated duration. (E) Measurement of ZIKV Fortaleza viral replication by plaque assay in primary human and mouse fibroblasts infected at MOI 0.1 for indicated duration. PFU (plaque forming units). (F) Immunoblot of wild type (WT) and IFNAR −/− MEFs infected with ZIKV Fortaleza at MOI 1, probed with anti-NS5 sera. Results are representative of three or more independent experiments.