NSs Protein of Sandfly Fever Sicilian Phlebovirus Counteracts Interferon (IFN) Induction by Masking the DNA-Binding Domain of IFN Regulatory Factor 3

Abstract

Sandfly fever Sicilian virus (SFSV) is one of the most widespread and frequently identified members of the genus Phlebovirus (order Bunyavirales, family Phenuiviridae) infecting humans. Being transmitted by Phlebotomus sandflies, SFSV causes a self-limiting, acute, often incapacitating febrile disease ("sandfly fever," "Pappataci fever," or "dog disease") that has been known since at least the beginning of the 20th century. We show that, similarly to other pathogenic phleboviruses, SFSV suppresses the induction of the antiviral type I interferon (IFN) system in an NSs-dependent manner. SFSV NSs interfered with the TBK1-interferon regulatory factor 3 (IRF3) branch of the RIG-I signaling pathway but not with NF-κB activation. Consistently, we identified IRF3 as a host interactor of SFSV NSs. In contrast to IRF3, neither the IFN master regulator IRF7 nor any of the related transcription factors IRF2, IRF5, and IRF9 were bound by SFSV NSs. In spite of this specificity for IRF3, NSs did not inhibit its phosphorylation, dimerization, or nuclear accumulation, and the interaction was independent of the IRF3 activation or multimerization state. In further studies, we identified the DNA-binding domain of IRF3 (amino acids 1 to 113) as sufficient for NSs binding and found that SFSV NSs prevented the association of activated IRF3 with the IFN-β promoter. Thus, unlike highly virulent phleboviruses, which either destroy antiviral host factors or sequester whole signaling chains into inactive aggregates, SFSV modulates type I IFN induction by directly masking the DNA-binding domain of IRF3.IMPORTANCE Phleboviruses are receiving increased attention due to the constant discovery of new species and the ongoing spread of long-known members of the genus. Outbreaks of sandfly fever were reported in the 19th century, during World War I, and during World War II. Currently, SFSV is recognized as one of the most widespread phleboviruses, exhibiting high seroprevalence rates in humans and domestic animals and causing a self-limiting but incapacitating disease predominantly in immunologically naive troops and travelers. We show how the nonstructural NSs protein of SFSV counteracts the upregulation of the antiviral interferon (IFN) system. SFSV NSs specifically inhibits promoter binding by IFN transcription factor 3 (IRF3), a molecular strategy which is unique among phleboviruses and, to our knowledge, among human pathogenic RNA viruses in general. This IRF3-specific and stoichiometric mechanism, greatly distinct from the ones exhibited by the highly virulent phleboviruses, correlates with the intermediate level of pathogenicity of SFSV.

Keywords: DNA-binding domain; IRF3; NSs; interferon beta promoter; interferon induction; sandfly fever Sicilian virus.

FIG 1

SFSV NSs and IFNB induction. (A) A549 cells were infected with SFSV, RVFV MP12, or clone 13 (Cl13) at an MOI of 1, harvested 12 hpi, and analyzed by RT-qPCR analysis for IFNB (n = 4; mean ± SD). (B) A549 cells were cotransfected with expression constructs for 3×FLAG-tagged SFSV or PTV-A NSs and nontargeting control siRNA or SFSV NSs-specific siRNA. Samples were subjected to RT-PCR analysis (upper panels) and immunoblotting using anti-FLAG and anti-tubulin antibodies (lower panel) 24 h after transfection. To exclude amplification of NSs sequences from plasmid DNA, a duplicate set of reactions was performed without the reverse transcription step (no RT). (C to F) A549 cells were pretransfected with control or SFSV NSs-targeting siRNA and infected with SFSV or RVFV MP12 at an MOI of 1. RNA was isolated 12 hpi for RT-qPCR analysis for NSs-containing RNA (C), IFNB (D), the L segment of SFSV (E), and the S segment of RVFV MP12 (n = 3; means ± SD) (F). (G) Summary of the relative fold induction data depicted in panels C to F, normalized to the mock sample pretreated with control siRNA as well as the fold induction of IFNB in siNSs-treated cells over siCTRL-treated cells that occurred in a manner independent of the viral burden (means ± SD). n.a., not applicable.

FIG 2

Effect of selected NSs proteins on phlebovirus host targets and central RIG-I signaling components. (A) A549 cells were infected at an MOI of 1 with recombinant RVFV expressing the NSs of RVFV, TOSV, SFSV, PTV-A, or PTV-B or entirely lacking an NSs-coding sequence. Cells were harvested 8 hpi for immunoblot analysis. (B) A549 cells were infected with SFSV or RVFV MP12 at an MOI of 1 and harvested 12 hpi for SDS-PAGE and Western blot analysis.

FIG 3

Influence of phlebovirus NSs proteins on IFNB promoter elements. HEK293 cells were transfected with expression plasmids for MAVS; NSs of RVFV, SFSV, PTV-A, or PTV-B; or inactive control ΔMx (0.1 ng, 1 ng, or 10 ng), as well as stimulation-dependent firefly luciferase (FF-Luc) and constitutively active Renilla luciferase reporters. Firefly luciferase was under the control of (A) the entire IFN-β promoter (n = 3; means ± SD), (B) IRF-driven PRDI (n = 3; means ±SD), or (C) NF-κB-driven PRDII (n = 3; means ± SD). Cell lysates were harvested 24 h after transfection for dual-luciferase assays. Firefly reporter activities were normalized to the Renilla reporter activities, and the positive controls were set to 100% prior to calculating means and SD across biological replicates.

FIG 4

Coimmunoprecipitation of NSs proteins with eGFP-IRF3. Selected 3×FLAG-tagged NSs proteins were coexpressed with eGFP-IRF3 in HEK293 cells. eGFP and ΔMx served as negative controls for eGFP-IRF3 and the NSs proteins, respectively. Cell lysates were subjected to immunoprecipitation via a GFP-binding nanobody immobilized on the bottom of a 96-well plate. Input samples and bound proteins were analyzed via immunoblotting (n = 3).

FIG 5

Coimmunoprecipitation of SFSV NSs with IRF proteins. 3×FLAG-tagged SFSV NSs plasmids were transfected into HEK293 cells together with eGFP-fused IRF7 (A) or IRF2, IRF5, or IRF9 (B). eGFP-IRF3 and eGFP were included as positive and negative controls, respectively. Cells were lysed, immunoprecipitation (IP) was performed via the use of GFP, and input lysates and immunoprecipitates were subjected to immunoblotting.

FIG 6

Markers of IRF3 activation under conditions of SFSV infection. (A to C) IRF3 phosphorylation and dimerization. A549 cells were infected with the indicated viruses at an MOI of 1, harvested 12 hpi (A) and 8 hpi (B), and analyzed by immunoblotting for IRF3 phosphorylation and viral nucleocapsid proteins. (C) Samples from the experiment described for panel A were additionally subjected to native PAGE, followed by immunoblotting. (D) Nuclear importation of IRF3. A549 cells seeded onto glass coverslips were infected at an MOI of 1, fixed 12 hpi with paraformaldehyde, and subsequently stained for IRF3 and the SFSV nucleocapsid protein N.

FIG 7

Phosphomimetic and dimerization-deficient IRF3 mutants. (A and B) Promoter reporter assays were performed under conditions of stimulation with a phosphomimetic, constitutively active IRF3(5D). (A) HEK293 cells were transfected with expression plasmids for NSs of RVFV, SFSV, PTV-A, or PTV-B or inactive control ΔMx, as well as firefly and Renilla luciferase reporters, under the control of the IFN-β and constitutively active simian virus 40 (SV40) promoters, respectively. IFN-β induction was stimulated by overexpression of IRF3(5D) and total plasmid adjusted to equal levels with empty vector. Firefly activities were normalized to those of Renilla, and the stimulation control was set to 100% (n = 3; means ± SD). (B) A dual-luciferase assay was performed in parallel with a PRDI-responsive firefly luciferase reporter (n = 3; means ± SD). (C and D) Interaction with IRF3 mutants. (C) 3×FLAG-tagged SFSV NSs or PTV-A NSs was coexpressed with IRF3(5D) in HEK293 cells. Cell lysates were then subjected to immunoprecipitation using an antibody against FLAG that was covalently coupled to magnetic beads beforehand. (D) GFP-IRF3(S385/386A), eGFP-IRF3, or eGFP, as well as 3×FLAG-tagged SFSV NSs, was obtained by transient transfection of HEK293 cells. Immunoprecipitation was performed via the use of GFP.

FIG 8

FIG 8 Domain mapping of binding region within IRF3. (A) Schematic representation of the IRF3 domain structure. IRF3 contains a DNA-binding domain (DBD, aa 1 to 113) with an embedded bipartite nuclear localization signal(s) (NLS; K77/R78 and R86/K87), a nuclear export signal (NES; aa 139 to 150), and a proline-rich region (Pro;aa 150 to 190) directly followed by the IRF association domain (IAD; aa 190 to 384) and a serine-rich region (SR; aa 384 to 427) at the C terminus. (B) eGFP-fused full-length IRF3, its N-terminal portion (1–190) or C-terminal portion (190–427), or eGFP alone was expressed together with 3×FLAG-tagged SFSV NSs in HEK293 cells, followed by immunoprecipitation via the use of GFP and immunoblotting. (C) A series of successively truncated eGFP-IRF3 mutants were produced from DNA templates by coupled in vitro transcription-translation and added to lysates of HEK293 cells expressing 3×FLAG-tagged SFSV NSs for subsequent immunoprecipitation via the use of GFP.

FIG 9

IFN-β promoter binding assay. HEK293 cells were cotransfected with plasmids encoding eGFP-IRF3 or eGFP or MAVS, as well as with increasing amounts of plasmids encoding 3×FLAG-tagged SFSV NSs, or the 3×FLAG-tagged control protein ΔMx, as indicated. Cell lysates were then incubated with an unlabeled, double-stranded DNA oligonucleotide comprising the IFN-β promoter or with a scrambled control oligonucleotide or were left untreated. Next, streptavidin-coated magnetic beads covered with biotinylated IFN-β promoter oligonucleotide were used to pull down activated IRF3. Bound proteins were eluted by boiling in Laemmli buffer and analyzed by immunoblotting.