Two Conserved Amino Acids within the NSs of Severe Fever with Thrombocytopenia Syndrome Phlebovirus Are Essential for Anti-interferon Activity

Recognition of viruses by host innate immune systems plays a critical role not only in providing resistance to viral infection but also in the initiation of antigen-specific adaptive immune responses against viruses. Severe fever with thrombocytopenia syndrome (SFTS) is a newly emerging infectious disease caused by the SFTS phlebovirus (SFTSV), a highly pathogenic tick-borne phlebovirus. The 294-amino-acid nonstructural protein (NSs) of SFTSV associates with TANK-binding kinase 1 (TBK1), a key regulator of host innate antiviral immunity, to inhibit interferon beta (IFN-β) production and enhance viral replication. Here, we demonstrate that two conserved amino acids at positions 21 and 23 in the NSs of SFTSV and heartland virus, another tick-borne phlebovirus, are essential for association with TBK1 and suppression of IFN-β production. Our results provide important insight into the molecular mechanisms by which SFTSV NSs helps to counteract host antiviral strategies.

ABSTRACT

The nonstructural protein (NSs) of severe fever with thrombocytopenia syndrome phlebovirus (SFTSV) sequesters TANK-binding kinase 1 (TBK1) into NSs-induced cytoplasmic structures to inhibit the phosphorylation and nuclear translocation of interferon (IFN) regulatory factor 3 (IRF3) and subsequent interferon beta (IFN-β) production. Although the C-terminal region of SFTSV NSs (NSs_66–249) has been linked to the formation of NSs-induced cytoplasmic structures and inhibition of host IFN-β responses, the role of the N-terminal region in antagonizing host antiviral responses remains to be defined. Here, we demonstrate that two conserved amino acids at positions 21 and 23 in the SFTSV and heartland virus (HRTV) NSs are essential for suppression of IRF3 phosphorylation and IFN-β mRNA expression following infection with SFTSV or recombinant influenza virus lacking the NS1 gene. Surprisingly, formation of SFTSV/HRTV NSs-induced cytoplasmic structures is not essential for inhibition of host antiviral responses. Rather, an association between SFTSV/HRTV NSs and TBK1 is required for suppression of mitochondrial antiviral signaling protein (MAVS)-mediated activation of IFN-β promoter activity. Although SFTSV NSs did not prevent the ubiquitination of TBK1, it associates with TBK1 through its N-terminal kinase domain (residues 1 to 307) to block the autophosphorylation of TBK1. Furthermore, we found that both wild-type NSs and the 21/23A mutant (NSs in which residues at positions 21 and 23 were replaced with alanine) of SFTSV suppressed NLRP3 inflammasome-dependent interleukin-1β (IL-1β) secretion, suggesting that the importance of these residues is restricted to TBK1-dependent IFN signaling. Together, our findings strongly implicate the two conserved amino acids at positions 21 and 23 of SFTSV/HRTV NSs in the inhibition of host interferon responses.

IMPORTANCE Recognition of viruses by host innate immune systems plays a critical role not only in providing resistance to viral infection but also in the initiation of antigen-specific adaptive immune responses against viruses. Severe fever with thrombocytopenia syndrome (SFTS) is a newly emerging infectious disease caused by the SFTS phlebovirus (SFTSV), a highly pathogenic tick-borne phlebovirus. The 294-amino-acid nonstructural protein (NSs) of SFTSV associates with TANK-binding kinase 1 (TBK1), a key regulator of host innate antiviral immunity, to inhibit interferon beta (IFN-β) production and enhance viral replication. Here, we demonstrate that two conserved amino acids at positions 21 and 23 in the NSs of SFTSV and heartland virus, another tick-borne phlebovirus, are essential for association with TBK1 and suppression of IFN-β production. Our results provide important insight into the molecular mechanisms by which SFTSV NSs helps to counteract host antiviral strategies.

INTRODUCTION

Mammalian innate immune systems utilize pattern recognition receptors (PRRs) to detect invading pathogens such as bacteria and viruses (1–3). Thus far, three classes of PRRs are known to be involved in the induction of type I interferons (IFNs) following the recognition of RNA viruses. Toll-like receptor 3 (TLR3) detects double-stranded RNA (dsRNA) (4), which accumulates during viral replication (5), while TLR7/8 recognizes viral genomic RNA in the endosomal compartment (6–8). Retinoic acid-inducible gene I (RIG-I)-like helicases (RLHs), including RIG-I and melanoma differentiation-associated gene 5 (MDA5), detect viral genomic RNA and dsRNA in the cytosol (9, 10). In addition, recent studies indicate that infection by certain RNA viruses, including vesicular stomatitis virus (VSV), Sendai virus, and influenza virus, stimulates IFN production in a stimulator of interferon genes (STING)-dependent manner (11, 12). These signals activate TANK-binding kinase 1 (TBK1), which phosphorylates the transcription factor interferon regulatory factor 3 (IRF3) to stimulate type I IFN production (13). Type I IFNs play a critical role not only in providing resistance to viral infection but also in initiating antigen-specific adaptive immune responses against viruses (14). Unsurprisingly, viruses have evolved strategies to evade host innate antiviral immunity (15).

Severe fever with thrombocytopenia syndrome (SFTS) is a newly emerging infectious disease caused by the SFTS phlebovirus (SFTSV), a novel phlebovirus within the family Phenuiviridae of the order Bunyavirales (16). The disease mostly affects elderly people, with a mortality rate estimated to be as high as 30% (16). Recently, IFN-α/β receptor (IFNAR) knockout mice were shown to be susceptible to SFTSV infection (17, 18), suggesting that host type I IFNs play an important role in host defenses against SFTSV infection. To evade host antiviral immunity, the 294-amino-acid nonstructural protein (NSs), encoded by the S segment of the SFTSV genome by an ambisense strategy, sequesters TBK1 into NSs-induced cytoplasmic structures, thereby inhibiting host IFN-β and NF-κB responses induced by virus infection and dsRNA treatment (19). The sequestration of RIG-I signaling molecules, including TBK1, into NSs-induced cytoplasmic structures correlates with inhibition of host antiviral responses (20, 21). In addition, the PXXP motif (P and X refer to proline and any amino acid, respectively) at residues 66 to 69 of SFTSV NSs is important for the formation of NSs-induced cytoplasmic structures and suppression of IFN-β promoter activity (21). Although the C-terminal region (residues 66 to 249) is important for these functions (22, 23), the role of the N-terminal region (residues 1 to 65) of NSs in the suppression of IFN-β promoter activity remains unclear.

Here, we demonstrated that two conserved amino acids at positions 21 and 23 in the SFTSV and heartland virus (HRTV) NSs are essential for suppression of IRF3 phosphorylation and activation of IFN-β promoter activity. Surprisingly, the formation of SFTSV/HRTV NSs-induced cytoplasmic structures is not essential for inhibition of host antiviral responses. Rather, an association between SFTSV/HRTV NSs and TBK1 is required for suppression of mitochondrial antiviral signaling protein (MAVS)-mediated activation of IFN-β promoter activity. Our findings strongly implicate the two conserved amino acids at positions 21 and 23 of SFTSV and HRTV NSs in the inhibition of host interferon responses and will aid in the development of novel therapeutic strategies to treat SFTSV or HRTV infection and associated diseases.

RESULTS

The N-terminal 30 amino acids of SFTSV NSs are required to inhibit activation of the IFN-β promoter.

Previous studies indicated that the C-terminal region (residues 66 to 249) of SFTSV NSs is important for suppression of IFN-β promoter activity (22, 23). To examine the role of the N-terminal region of NSs in antagonizing host antiviral responses, we first constructed two NSs deletion mutants lacking the N- or C-terminal 50 amino acids (designated ΔN50 and ΔC50, respectively) (Fig. 1A). We next examined whether the deletion mutants could inhibit MAVS-mediated activation of an IFN-β reporter construct (p125-luc) (24). Consistent with data from previous reports (20, 23), overexpression of full-length NSs inhibited MAVS-mediated activation of the IFN-β promoter (Fig. 1B), even with equal amounts of MAVS expression (Fig. 1C). Notably, ΔN50 lost the ability to suppress IFN-β promoter activity (Fig. 1B), whereas ΔC50 had no effect on inhibition of MAVS-mediated activation of the IFN-β promoter (Fig. 1B). In addition, while the full-length NSs relocalized TBK1 into NSs-induced cytoplasmic structures (Fig. 1D and E), ΔN50 failed to perform this function (Fig. 1D and E). Intriguingly, the ΔC50 mutant, which suppressed IFN-β promoter activity, failed to form these cytoplasmic structures (Fig. 1D and E). These results suggest that the N-terminal 50 amino acids of NSs are required to suppress activation of the IFN-β promoter.

FIG 1.

The N-terminal 50 amino acids of NSs are required to suppress activation of the IFN-β promoter. (A) Schematic diagram of wild-type (WT) SFTSV NSs and its deletion mutants. ΔN50 and ΔC50 NSs lack the N- and C-terminal 50 amino acids, respectively. (B and C) HEK293FT cells were transfected with expression plasmids encoding HA-tagged MAVS and EGFP, Flag-tagged WT SFTSV NSs, or its deletion mutants, together with IFN-β reporter plasmids. Twenty-four hours after transfection, cell lysates were collected and analyzed for luciferase activity (B) and expression (C) of HA-tagged MAVS, α-tubulin, and Flag-tagged NSs. (D) HeLa cells were cotransfected with expression plasmids encoding myc-tagged TBK1 together with Flag-tagged WT SFTSV NSs or its mutants. At 24 h posttransfection, cells were stained with anti-myc and anti-Flag antibodies and analyzed by confocal microscopy. (E) Ratios of the cellular localization patterns of myc-tagged TBK1 in the NSs-induced cytoplasmic structures (filled bar) and others (open bars) shown in panel D. The numbers of analyzed cells are shown above the columns. Data are representative of results from at least three independent experiments and are expressed as the means ± standard deviations (SD). ***, P < 0.001; N.S. not significant (B).

To map the regions of NSs that are important for suppression of IFN-β promoter activity, we generated a series of Flag-tagged NSs deletion mutants lacking the N-terminal 10 to 40 amino acids (Fig. 2A) and confirmed their expression in HEK293FT cells by immunoblot analysis (Fig. 2B). Deletion of the N-terminal 10 or 20 amino acids (designated ΔN10 and ΔN20, respectively) had no effect on inhibition of MAVS-mediated activation of the IFN-β promoter (Fig. 2C). In contrast, NSs deletion mutants lacking the N-terminal 30 or 40 amino acids (designated ΔN30 and ΔN40, respectively) completely lost the ability to suppress IFN-β promoter activity (Fig. 2C). Importantly, ΔN20 but not ΔN30 coimmunoprecipitated with TBK1 in HEK293FT cells (Fig. 2D), suggesting that interaction of NSs with TBK1 is required for suppression of IFN-β promoter activity. In addition, we found that both the ΔN10 and ΔN20 mutants relocalized TBK1 into the NSs-induced cytoplasmic structures (Fig. 2E and F), whereas deletion of the N-terminal 30 amino acids abrogated the relocalization of TBK1 (Fig. 2E and F). Together, these results suggest that amino acids 21 to 30 of NSs are important for suppression of IFN-β promoter activity.

FIG 2.

The N-terminal 30 amino acids of NSs are essential for inhibition of IFN-β promoter activity and interaction with TBK1. (A and B) Schematic diagram (A) and expression of WT SFTSV NSs and its deletion mutants (B). The ΔN10, ΔN20, ΔN30, and ΔN40 NSs mutants lack the N-terminal 10 to 40 amino acids, respectively. (C) HEK293FT cells were transfected with expression plasmids encoding HA-tagged MAVS and EGFP, Flag-tagged WT SFTSV NSs, or its deletion mutants, together with IFN-β reporter plasmids. Twenty-four hours after transfection, cell lysates were collected and analyzed for luciferase activity. (D) HEK293FT cells were cotransfected with expression plasmids encoding myc-tagged TBK1 together with EGFP and Flag-tagged WT or mutant SFTSV NSs. Twenty-four hours after transfection, cell lysates were immunoprecipitated with an anti-Flag antibody, followed by immunoblotting of total lysates (Input) and immunoprecipitates (IP) with anti-Flag or anti-myc antibodies. (E) HeLa cells were cotransfected with expression plasmids encoding myc-tagged TBK1 together with Flag-tagged WT or mutant SFTSV NSs. At 24 h posttransfection, cells were stained with anti-myc and anti-Flag antibodies and analyzed by confocal microscopy. (F) Ratios of the cellular localization patterns of myc-tagged TBK1 in the NSs-induced cytoplasmic structures (filled bars) and others (open bars) shown in panel E. The numbers of analyzed cells are shown above the columns. Data are representative of results from at least three similar experiments and are expressed as the means ± SD. ***, P < 0.001 (C).

Residues 21 to 26 of SFTSV NSs are important for suppression of IFN-β promoter activity.

To further refine the residues of the N-terminal domain of NSs that are essential for suppression of IFN-β promoter activity, we prepared a series of plasmids expressing Flag-tagged NSs with multiple alanine substitutions (Fig. 3A). Since the PXXP motif of SFTSV NSs was found to be important for suppression of IFN-β promoter activity (21), we generated a plasmid expressing Flag-tagged mutant NSs in which two prolines at positions 66 and 69 were replaced with alanine (designated SFTSV NSs 66/69A) as a positive control (Fig. 3A). The alanine mutant at positions 18 to 20 (designated SFTSV NSs 18-20A) of NSs had no effect on inhibition of MAVS-mediated activation of the IFN-β promoter compared to wild-type (WT) NSs (Fig. 3B). The SFTSV NSs 66/69A mutant displayed a slight but significant attenuation of the inhibitory effect (Fig. 3B). In contrast, inhibition of MAVS-mediated activation of the IFN-β promoter was severely impaired by alanine substitution at positions 21 to 23, 24 to 26, and 27 to 29 (designated SFTSV NSs 21-23A, SFTSV NSs 24-26A, and SFTSV NSs 27-29A, respectively) (Fig. 3B). Consistent with this observation, WT SFTSV NSs and SFTSV NSs 18-20A but not the SFTSV NSs 21-23A, 24-26A, and 27-29A mutants coimmunoprecipitated with TBK1 in HEK293FT cells (Fig. 3C) and relocalized TBK1 into NSs-induced cytoplasmic structures (Fig. 3D and E). Although the SFTSV NSs 66/69A mutant failed to sequester TBK1 into the unique cytoplasmic structures (Fig. 3D and E) (21), the 66/69A mutant still retained the ability to associate with TBK1 (Fig. 3F). Together, these results suggest that the region spanning amino acids 21 to 26 of NSs is important for suppression of IFN-β promoter activity.

FIG 3.

The N-terminal 21 to 29 amino acids of NSs are essential for inhibition of IFN-β promoter activity and interaction with TBK1. (A) Schematic diagram of WT SFTSV NSs and its alanine mutants. (B) HEK293FT cells were transfected with expression plasmids encoding HA-tagged MAVS and EGFP, Flag-tagged WT SFTSV NSs, or its alanine mutants, together with IFN-β reporter plasmids. Twenty-four hours after transfection, cell lysates were collected and analyzed for luciferase activity. (C) HEK293FT cells were cotransfected with expression plasmids encoding myc-tagged TBK1 together with EGFP or Flag-tagged WT or mutant SFTSV NSs. Twenty-four hours after transfection, cell lysates were immunoprecipitated with an anti-Flag antibody, followed by immunoblotting (IB) of total lysates (Input) and immunoprecipitates (IP) with anti-Flag or anti-myc antibodies. (D) HeLa cells were cotransfected with expression plasmids encoding myc-tagged TBK1 together with Flag-tagged WT or mutant SFTSV NSs. At 24 h posttransfection, cells were stained with anti-myc and anti-Flag antibodies and analyzed by confocal microscopy. (E) Ratios of the cellular localization patterns of myc-tagged TBK1 in the NSs-induced cytoplasmic structures (filled bars) and others (open bars) shown in panel D. The numbers of analyzed cells are shown above the columns. (F) HEK293FT cells were cotransfected with expression plasmids encoding myc-tagged TBK1 and Flag-tagged SFTSV 66/69A NSs. Twenty-four hours after transfection, cell lysates were immunoprecipitated with an anti-Flag antibody, followed by immunoblotting of total lysates (Input) and immunoprecipitates with anti-Flag or anti-myc antibodies. Data are representative of results from at least three similar experiments and are expressed as the means ± SD. ***, P < 0.001 versus WT NSs-transfected cells (B).

Residues 21 to 26 of HRTV NSs are also important for suppression of IFN-β promoter activity.

Phylogenetic analysis of the aligned amino acid sequence of NSs suggested that HRTV is most closely related to SFTSV (16, 25, 26). Sequence analysis revealed that HRTV NSs also contains conserved amino acids between positions 21 and 29 (Fig. 4A). Thus, we next examined the role of these residues in HRTV NSs in the suppression of IFN-β promoter activity. To this end, we prepared a series of plasmids expressing Flag-tagged HRTV NSs with multiple alanine substitutions. The alanine mutant at positions 18 to 20 of HRTV NSs (designated HRTV NSs 18-20A) had no effect on the inhibition of MAVS-mediated activation of the IFN-β promoter compared with WT HRTV NSs (Fig. 4B). In contrast, the 66/69A mutant (designated HRTV NSs 66/69A) severely attenuated the inhibitory effect on IFN-β promoter activity (Fig. 4B). Similarly, the inhibitory effect on MAVS-mediated activation of the IFN-β promoter was severely impaired by alanine substitution at positions 21 to 23 or 24 to 26 but not at positions 27 to 29 (designated HRTV NSs 21-23A, HRTV NSs 24-26A, and HRTV NSs 27-29A, respectively) (Fig. 4B). Consistent with this observation, WT HRTV NSs and HRTV NSs 18-20A and 27-29A, but not HRTV NSs 21-23A or 24-26A, coimmunoprecipitated with TBK1 in HEK293FT cells (Fig. 4C). In addition, both WT and alanine mutant HRTV NSs failed to form the unique cytoplasmic structures (Fig. 4D). Although the HRTV NSs 66/69A mutant failed to sequester TBK1 into the unique cytoplasmic structures (Fig. 4D), the 66/69A mutant still retained the ability to associate with TBK1 (Fig. 4E). Together, these results suggest that, similar to SFTSV NSs, residues 21 to 26 of HRTV NSs are also required for suppression of IFN-β promoter activity. In addition, formation of the unique cytoplasmic structures is unnecessary to suppress IFN-β promoter activity by HRTV NSs.

FIG 4.

The N-terminal 21 to 26 amino acids of heartland virus (HRTV) NSs are essential for inhibition of IFN-β promoter activity and interaction with TBK1. (A) Pairwise sequence alignment of the NSs derived from SFTSV strain YG1 (GenBank accession number AB817995.1) and HRTV isolate patient1 (GenBank accession number NC_024496.1) performed by using ClustalW. Alignment of the N-terminal 18 to 30 amino acids and 66 to 69 amino acids is depicted. (B) HEK293FT cells were transfected with expression plasmids encoding HA-tagged MAVS and EGFP, Flag-tagged WT HRTV NSs, or its alanine mutants, together with IFN-β reporter plasmids. Twenty-four hours after transfection, cell lysates were collected and analyzed for luciferase activity. (C) HEK293FT cells were cotransfected with expression plasmids encoding myc-tagged TBK1 together with EGFP and Flag-tagged WT or mutant HRTV NSs. Twenty-four hours after transfection, cell lysates were immunoprecipitated with an anti-Flag antibody, followed by immunoblotting of total lysates (Input) and immunoprecipitates (IP) with anti-Flag or anti-myc antibodies. (D) HeLa cells were cotransfected with expression plasmids encoding myc-tagged TBK1 together with Flag-tagged WT or mutant HRTV NSs. At 24 h posttransfection, cells were stained with anti-myc and anti-Flag antibodies and analyzed by confocal microscopy. (E) HEK293FT cells were cotransfected with expression plasmids encoding myc-tagged TBK1 and Flag-tagged HRTV 66/69A NSs. Twenty-four hours after transfection, cell lysates were immunoprecipitated with an anti-Flag antibody, followed by immunoblotting of total lysates (Input) and immunoprecipitates with anti-Flag or anti-myc antibodies. Data are representative of results from at least three similar experiments and are expressed as the means ± SD. ***, P < 0.001 versus WT NSs-transfected cells (C).

Residues 21 and 23 of SFTSV and HRTV NSs are essential for suppression of host interferon responses.

To pinpoint the N-terminal residues within positions 21 to 26 that are critical for suppression of IFN-β promoter activity, we first performed secondary structure prediction for SFTSV and HRTV NSs using the I-TASSER server (27). Both SFTSV and HRTV NSs were predicted to include a shorter β-sheet element at positions 20 to 23 (Fig. 5A). Thus, we generated plasmids expressing Flag-tagged mutant SFTSV and HRTV NSs in which two residues from positions 20 to 23 were replaced with alanine (designated SFTSV NSs 20/22A or 21/23A and HRTV NSs 20/22A or 21/23A, respectively) (Fig. 5B). We first examined the effects of these mutations on protein stability. We found that the 20/22A, 21/23A, and 66/69A NSs mutants of SFTSV (Fig. 5C) and HRTV (Fig. 5D) showed reduced protein stability compared with their WT NSs. However, SFTSV NSs 20/22A and HRTV NSs 20/22A had no effect on inhibition of MAVS-mediated activation of the IFN-β promoter compared with WT NSs (Fig. 5E). In contrast, strikingly, inhibition of MAVS-mediated activation of the IFN-β promoter was severely impaired by alanine substitution at positions 21 and 23 of SFTSV and HRTV NSs (Fig. 5E). In addition, WT NSs, SFTSV NSs 20/22A, and HRTV NSs 20/22A but not SFTSV NSs 21/23A or HRTV NSs 21/23A suppressed phosphorylation of IRF3 (Fig. 5F) and induction of IFN-β mRNA following infection with recombinant influenza virus lacking the NS1 gene (ΔNS1 virus) or SFTSV (Fig. 5G and H). Consistent with this observation, we found that WT NSs, SFTSV NSs 20/22A, and HRTV NSs 20/22A but not SFTSV NSs 21/23A or HRTV NSs 21/23A coimmunoprecipitated with TBK1 in HEK293FT cells (Fig. 5I). To rule out the possibility that the 21/23A mutants fail to associate with TBK1 due to their lower protein expression levels or inefficiency of the immunoprecipitation, we transfected HEK293FT cells with plasmids encoding myc-tagged TBK1 (500 ng) together with small amounts of Flag-tagged wild-type SFTSV NSs (2 or 20 ng), HRTV NSs (1, 10, or 100 ng), or the 21/23A mutant (500 ng). We found that wild-type SFTSV/HRTV NSs efficiently associated with TBK1 compared to the 21/23A mutant (Fig. 6A and B). In addition, small amounts of Flag-tagged wild-type SFTSV/HRTV NSs significantly inhibited MAVS-mediated activation of the IFN-β promoter compared with the 21/23A mutants (Fig. 6C). In addition, we found that both the SFTSV NSs 20/22A and 21/23A mutants reduced the number of NSs-induced cytoplasmic structures sequestrating TBK1 (Fig. 7A and B). These observation strongly suggested that interaction of SFTSV NSs with TBK1 is important for suppressing host interferon responses rather than formation of the unique cytoplasmic structures.

FIG 5.

Val21 and Leu23 of SFTSV and HRTV NSs are essential for interaction with TBK1 and subsequent inhibition of IFN-β signaling. (A) Secondary structures of the N-terminal 30 amino acids of NSs derived from SFTSV strain YG1 (GenBank accession number AB817995.1) and HRTV isolate patient1 (GenBank accession number NC_024496.1) predicted by the I-TASSER server. (B) Schematic diagram of WT SFTSV/HRTV NSs and alanine mutants. (C and D) HEK293FT cells were transfected with an expression plasmid encoding Flag-tagged WT or mutant NSs of SFTSV (C), HRTV (D), or influenza virus NS1. Twenty-four hours after transfection, cells were treated with 100 μM cycloheximide (CHX), and cell lysates were analyzed by immunoblotting with anti-Flag or antitubulin antibodies. Relative expression levels of WT or mutant NSs normalized to values for tubulin are shown. (E) HEK293FT cells were transfected with expression plasmids encoding HA-tagged MAVS and EGFP, Flag-tagged WT SFTSV/HRTV NSs, or its alanine mutants, together with IFN-β reporter plasmids. Twenty-four hours after transfection, cell lysates were collected and analyzed for luciferase activity. (F) HEK293FT cells were transfected with plasmids encoding HA-tagged IRF3 together with EGFP and Flag-tagged WT or mutant SFTSV/HRTV NSs. Twenty-four hours after transfection, cells were infected with ΔNS1 influenza virus for 11 h, and cell lysates were analyzed by immunoblotting with the indicated antibodies. (G and H) HEK293FT cells were transfected with plasmids encoding EGFP and Flag-tagged WT or mutant SFTSV/HRTV NSs. Twenty-four hours after transfection, cells were infected with ΔNS1 influenza virus (G) or SFTSV (H) for 24 h. Total RNA was extracted from virus-infected and mock-infected cells. IFN-β mRNA levels were assessed by quantitative PCR with β-actin as an internal control. (I) HEK293FT cells were cotransfected with expression plasmids encoding myc-tagged TBK1 together with EGFP and Flag-tagged WT or mutant SFTSV/HRTV NSs. Twenty-four hours after transfection, cell lysates were immunoprecipitated with an anti-Flag antibody, followed by immunoblotting of total lysates (Input) and immunoprecipitates (IP) with anti-Flag or anti-myc antibodies. Data are representative of results from at least three similar experiments and are expressed as the means ± SD. ***, P < 0.001.

FIG 6.

Effect of alanine substitution at positions 21 and 23 of SFTSV NSs on interaction with TBK1 and subsequent inhibition of IFN-β signaling. (A and B) HEK293FT cells were cotransfected with expression plasmids encoding myc-tagged TBK1 (500 ng) and the indicated amounts of Flag-tagged wild-type SFTSV NSs (2 or 20 ng), HRTV NSs (1, 10, or 100 ng), or the 21/23A mutant (500 ng). Twenty-four hours after transfection, cell lysates were immunoprecipitated with an anti-Flag antibody, followed by immunoblotting of total lysates (Input) and immunoprecipitates (IP) with anti-Flag or anti-myc antibodies. (C) HEK293FT cells were transfected with expression plasmids encoding HA-tagged MAVS and EGFP, Flag-tagged WT, or 21/23A mutant SFTSV/HRTV NSs, together with IFN-β reporter plasmids. Twenty-four hours after transfection, cell lysates were collected and analyzed for luciferase activity. Data are representative of results from at least three similar experiments and are expressed as the means ± SD. ***, P < 0.001.

FIG 7.

SFTSV/HRTV alanine mutant NSs-induced cytoplasmic structures. (A) HeLa cells were cotransfected with expression plasmids encoding myc-tagged TBK1 together with Flag-tagged WT or mutant SFTSV/HRTV NSs. At 24 h posttransfection, cells were stained with anti-myc and anti-Flag antibodies and analyzed by confocal microscopy. (B) Ratio of the cellular localization patterns of myc-tagged TBK1 in the NSs-induced cytoplasmic structures (filled bars) and others (open bars) shown in panel A. The numbers of analyzed cells are shown above the columns.

Previous studies have demonstrated that SFTSV NSs inhibits the exogenous type I IFN-induced Jak/STAT signaling (28–30). Thus, we next examined the inhibitory effects of wild-type, 20/22A mutant, and 21/23A mutant NSs on IFN-α-triggered interferon-stimulated response element (ISRE) activation. Consistent with data from previous reports (28–30), overexpression of SFTSV NSs suppressed IFN-α-triggered ISRE activation (Fig. 8). Notably, SFTSV NSs 21/23A but not 20/22A lost the ability to suppress IFN-α-triggered ISRE activation (Fig. 8). We next used an NLRP3 reconstitution assay in HEK293FT cells to test whether SFTSV NSs inhibits NLRP3 inflammasome-mediated interleukin-1β (IL-1β) secretion. Reconstitution of the NLRP3 inflammasome resulted in IL-1β secretion by HEK293FT cells, for which NLRP3 was absolutely required (Fig. 9). Consistent with data from a previous report (31), transfection of HEK293FT cells with the influenza virus NS1 protein suppressed IL-1β secretion (Fig. 9). Similarly, wild-type SFTSV/HRTV NSs significantly inhibited IL-1β secretion (Fig. 9). Alanine substitution at positions 20 to 23 of SFTSV/HRTV NSs did not change the inhibitory effect of NSs on NLRP3 inflammasome-dependent IL-1β secretion (Fig. 9), suggesting that the importance of the two conserved amino acids at positions 21 and 23 of SFTSV NSs is restricted to IFN signaling. We finally examined inhibitory effects of wild-type, 20/22A, and 21/23A NSs on SFTSV replication. Although transfection of cells with an enhanced green fluorescent protein (EGFP)-expressing plasmid, which may stimulate cytosolic DNA sensor-dependent interferon responses, suppressed SFTSV replication, the WT and the 20/22A mutant but not the 21/23A mutant of SFTSV NSs increased SFTSV replication in the human lung carcinoma cell line A549 (Fig. 10).

FIG 8.

Val21 and Leu23 of SFTSV NSs are essential for inhibition of IFN-α signaling. HEK293FT cells were transfected with expression plasmids encoding EGFP, Flag-tagged WT, or mutant SFTSV NSs, together with ISRE reporter plasmids. Six hours after transfection, cells were treated with human IFN-α for 18 h and analyzed for luciferase activity.

FIG 9.

SFTSV NSs inhibits NLRP3 inflammasome-mediated IL-1β secretion. HEK293T cells were transfected with expression plasmids encoding NLRP3, ASC, procaspase-1, pro-IL-1β, and either Flag-tagged WT, 20/22A, or 21/23A SFTSV/HRTV NSs. pCA7-EGFP and pCA7-Flag-influenza virus NS1 were used as controls. Cell-free supernatants were collected at 24 h posttransfection, and IL-1β was analyzed by an enzyme-linked immunosorbent assay (ELISA). Data are representative of results from at least three similar experiments and are expressed as the means ± SD. **, P < 0.01; ***, P < 0.001 (versus empty-plasmid-transfected cells).

FIG 10.

Effect of alanine substitution at positions 21 and 23 of SFTSV NSs on SFTSV replication. (A) A549 cells were transfected with expression plasmids encoding EGFP or Flag-tagged WT or mutant SFTSV NSs. Twenty-four hours after transfection, cells were infected with SFTSV for 72 h and analyzed for luciferase activity. Total RNA was extracted from virus-infected and mock-infected cells. SFTSV NP mRNA levels were assessed by quantitative PCR with β-actin as an internal control. Data are representative of results from at least two similar experiments and are expressed as the means ± SD. **, P < 0.01; ***, P < 0.001.

SFTSV NSs associates with TBK1 through its N-terminal domain to suppress autophosphorylation of the kinase.

To gain insight into the mechanism by which the NSs protein of SFTSV inhibits TBK1 function and phosphorylation of IRF3, we first examined the inhibitory effect of SFTSV NSs on K63-linked ubiquitination of TBK1, which triggers higher-order oligomerization of TBK1-adaptor complexes, resulting in trans-autophosphorylation (32). However, overexpression of SFTSV NSs had no effect on the inhibition of K63-linked ubiquitination of TBK1 (Fig. 11A). We next examined the inhibitory effect of SFTSV NSs on the phosphorylation of TBK1. Overexpression of wild-type TBK1 but not the kinase-inactive mutant (TBK1-K38A) induced its autophosphorylation (Fig. 11B). Strikingly, WT but not 21/23A SFTSV NSs was found to inhibit autophosphorylation of TBK1 (Fig. 11B). In addition, WT but not 21/23A SFTSV NSs associated with TBK1 through its N-terminal kinase domain (residues 1 to 307) (Fig. 11C). These results suggested that SFTSV NSs associates with TBK1 through its N-terminal domain to suppress autophosphorylation of the kinase.

FIG 11.

SFTSV NSs inhibits autophosphorylation of TBK1. (A) HEK293FT cells were cotransfected with expression plasmids encoding myc-tagged TBK1 and HA-tagged ubiquitin-K63 together with EGFP or Flag-tagged SFTSV NSs. Twenty-four hours after transfection, cell lysates were immunoprecipitated with an anti-myc antibody, followed by immunoblotting of total lysates (Input) and immunoprecipitates (IP) with anti-HA, anti-myc, or anti-Flag antibodies. (B) HEK293FT cells were transfected with plasmids encoding myc-tagged WT TBK1 or the kinase-inactive mutant (TBK K38A) together with EGFP or Flag-tagged SFTSV WT or 21/23A NSs. Twenty-four hours after transfection, cell lysates were analyzed by immunoblotting with the indicated antibodies. (C) HEK293FT cells were cotransfected with expression plasmids encoding the myc-tagged kinase domain of TBK1 (residues 1 to 307) together with EGFP or Flag-tagged SFTSV WT or 21/23A NSs. Twenty-four hours after transfection, cell lysates were immunoprecipitated with an anti-myc antibody, followed by immunoblotting of total lysates (Input) and immunoprecipitates with anti-Flag and anti-myc antibodies. Data are representative of results from at least three similar experiments.

Collectively, these results demonstrate that the two conserved amino acids at positions 21 and 23 in SFTSV and HRTV NSs are essential for their association with TBK1 and suppression of host interferon responses.

DISCUSSION

NSs of SFTSV associates with TBK1 to sequester it into NSs-induced cytoplasmic structures in what is believed to be the crucial step for evasion of host antiviral responses (20–23). Consistent with data from previous studies (20–23), we also found that TBK1 was redistributed into SFTSV NSs-induced cytoplasmic structures when HeLa cells were cotransfected with myc-tagged TBK1 and WT SFTSV NSs or deletion mutants of SFTSV NSs lacking the N-terminal 10 or 20 amino acids. Unexpectedly, we found that the SFTSV ΔC50 mutant, which failed to form unique cytoplasmic structures, completely suppressed MAVS-mediated activation of the IFN-β promoter. Consistent with data from a previous report (33), HRTV NSs that suppressed MAVS-mediated activation of the IFN-β promoter did not form the unique cytoplasmic structures. WT or deletion mutants of SFTSV or HRTV NSs that suppressed IFN-β promoter activity were associated with TBK1. Together, these results suggest that the interaction of SFTSV NSs with TBK1 may be important for suppressing IFN-β promoter activity rather than for the formation of the unique cytoplasmic structures.

Consistent with data from a previous report (21), we found that the proline residues at positions 66 and 69 in SFTSV NSs are required for the formation of the NSs-induced cytoplasmic structures. Importantly, the SFTSV 66/69A mutant still retained the ability to associate with TBK1. In addition, overexpression of the SFTSV NSs 66/69A mutant sufficiently suppressed MAVS-mediated activation of the IFN-β promoter. In contrast, the SFTSV NSs 66/69A mutant abrogated the ability to suppress IFN-β promoter activity after infection with Sendai virus (SeV) (21), suggesting that these proline residues appear to be more important for the inhibitory effects of NSs on RIG-I-like receptor signaling upstream of MAVS or TBK1-independent RNA virus recognition pathways (34).

Upon the recognition of DNA or RNA viruses by cytosolic DNA or RNA sensors such as cyclic GMP-AMP synthase (cGAS) or RIG-I, TBK1 phosphorylates their adaptor protein STING or MAVS, which in turn recruits IRF3 to activate type I IFN production (35). Infection with herpes simplex virus 1 (HSV-1) or vesicular stomatitis virus (VSV) triggers the release of mitochondrial DNA into the cytosol, which in turn stimulates the STING pathway (36). In addition, recent studies have demonstrated that TBK1 phosphorylates autophagic adaptor proteins to stimulate autophagosome maturation and elimination of bacteria by lysosomes (37, 38). Furthermore, it has been demonstrated that tripartite motif-containing 23 (TRIM23) facilitates the dimerization and trans-autophosphorylation of TBK1 to induce selective autophagy and suppress the replication of mutant HSV-1 lacking the ICP34.5 protein required for autophagy antagonism (39). These observations suggest that TBK1 plays a central role in innate antiviral immunity in response to viral infection (40, 41). Therefore, several viruses have evolved a variety of ways to counteract IFN-related responses by inhibiting phosphorylation, ubiquitination, kinase activity, or complex formation of TBK1 (41–43). Ding et al. showed that the porcine epidemic diarrhea virus nucleocapsid protein can interact with TBK1, thereby inhibiting phosphorylation and nuclear translocation of IRF3 and IFN-β production (44). Kaukinen et al. demonstrated that the hepatitis C virus protease NS2 interacts directly with TBK1 and suppresses IRF3 phosphorylation (45). In the present study, we found that SFTSV NSs associates with TBK1 through its N-terminal kinase domain to block the autophosphorylation of TBK1. Ning et al. showed that SFTSV NSs associates with TBK1 and IκB kinase ε (IKKε) (21). Since the amino acid sequence homologies of the kinase domain and ubiquitin-like domain of TBK1 with those of IKKε are 73.0% and 48.7%, respectively, the kinase domain of TBK1 seems to be important for the association with SFTSV NSs. Although the reason why SFTSV NSs forms the unique cytoplasmic structures remains elusive, our results suggest that the interaction of SFTSV NSs with TBK1 is important for suppressing host interferon responses rather than the formation of the unique cytoplasmic structures.

In summary, we show that the conserved valine and leucine residues at positions 21 and 23 in SFTSV and HRTV NSs are essential for suppression of MAVS-mediated activation of the IFN-β promoter. Both SFTSV and HRTV NSs are predicted to include a shorter β-sheet element at amino acid residues 20 to 23. Although alanine mutants at positions 20 and 22 had no effect on the inhibition of MAVS-mediated activation of the IFN-β promoter, alanine mutants at positions 21 and 23 completely abrogated the anti-interferon activity. Our findings strongly implicate the N-terminal region of SFTSV and HRTV NSs in the inhibition of host antiviral immunity and will aid in the development of novel therapeutic strategies to treat SFTSV or HRTV infection and associated diseases.

MATERIALS AND METHODS

Cells and viruses.

The human embryonic kidney cell line HEK293FT (Invitrogen) and A549 and HeLa cells were maintained in Dulbecco's modified Eagle's medium (DMEM; Nacalai Tesque) supplemented with 10% fetal bovine serum (FBS), penicillin (100 U/ml), and streptomycin (100 μg/ml) (all from Nacalai Tesque).

The recombinant influenza virus A/Puerto Rico/8/34 (H1N1) strain lacking the NS1 gene was propagated in Madin-Darby canine kidney (MDCK) cells stably expressing the influenza virus NS1 protein at 37°C for 2 days (31, 46). SFTSV strain YG1 was amplified on Vero cells and stored at −80°C until use. The infectious titer was determined by using a focus-forming assay, as described previously (47).

Antibodies.

Monoclonal antibodies against tubulin (DM1A), hemagglutinin (HA) (F-7), and normal mouse IgG1 (catalog number sc-3877) were obtained from Santa Cruz Biotechnology. Monoclonal antibody against myc (9E10) was purchased from Covance. Monoclonal antibody against Flag (M2) and rabbit polyclonal antibodies against Flag were purchased from Sigma-Aldrich. Monoclonal antibodies against phosphorylated IRF3 (p-IRF3) (Ser396; 4D4G) and p-TBK1 (Ser172; D52C2) were purchased from Cell Signaling Technology. Horseradish peroxidase-conjugated anti-mouse IgG was obtained from Jackson Immuno Research Laboratories. Horseradish peroxidase-conjugated anti-rabbit IgG was obtained from Invitrogen. Alexa Fluor 488-conjugated goat anti-mouse IgG(H+L) and Alexa Fluor 568-conjugated goat anti-rabbit IgG(H+L) antibodies were purchased from Life Technologies.

Plasmids.

A cDNA encoding SFTSV NSs was obtained by reverse transcription-PCR (RT-PCR) using total RNA extracted from SFTSV (YG1; GenBank accession number AB817995.1)-infected Vero cells (48), followed by PCR using specific primers. Oligonucleotides corresponding to both strands of the full-length sequence of NSs from HRTV isolate patient1 (GenBank accession number NC_024496.1) containing EcoRI and NotI sites at the 5′ and 3′ ends, respectively, were synthesized (Eurofins Genomics) and used to amplify and clone the gene into the eukaryotic expression vectors pCA7 (49) (a derivative of pCAGGS [50]) and pCA7-Flag to produce Flag-tagged proteins. Mutant NSs were constructed by standard PCR-based methods. The integrity of the inserts was verified by sequencing. Plasmids encoding HA-tagged human MAVS, myc-tagged human TBK1, untagged human NLRP3, ASC (apoptosis-associated speck-like protein containing a caspase recruitment domain), procaspase-1, pro-IL-1β, and Flag-tagged influenza virus NS1 proteins were described previously (31, 51). pRK5-HA-Ubiquitin-K63 was purchased from Addgene.

IFN-β and ISRE reporter assay.

HEK293FT cells seeded on 24-well cluster plates were cotransfected with 100 ng of p125-luc (a gift from T. Taniguchi, University of Tokyo), 2.5 ng of phRL-TK, and 100 ng of pcDNA3.1-HA-MAVS together with 100 ng of pCA7-EGFP or pCA7-Flag-NSs. After 24 h of transfection, cells were lysed in passive lysis buffer (Promega) and examined for luciferase activity using the dual-luciferase reporter assay system (Promega). Data were normalized for transfection efficiency against Renilla luciferase activity.

The ISRE reporter assay was performed as described previously (28). Briefly, HEK293FT cells seeded on 24-well cluster plates were cotransfected with 250 ng of pISRE-luc (Agilent Technology) and 2.5 ng of phRL-TK together with 250 ng of pCA7-EGFP or pCA7-Flag-NSs. At 6 h posttransfection, cells were treated with human IFN-α (catalog number ab48750; Abcam) for 18 h. Luciferase activities were measured with the dual-luciferase reporter assay system (Promega). Data were normalized for transfection efficiency against Renilla luciferase activity.

Coimmunoprecipitation and Western blot analysis.

Subconfluent monolayers of HEK293FT cells in 24-well cluster plates were cotransfected with 0.5 μg of pCA7-EGFP, pCA7-Flag-SFTSV NSs, or pCA7-Flag-HRTV NSs together with 0.5 μg of pcDNA3.1-myc-TBK1. At 24 h posttransfection, cells were washed with phosphate-buffered saline (PBS) and lysed in 500 μl of radioimmunoprecipitation assay (RIPA) buffer (10 mM Tris [pH 7.4], 150 mM NaCl, 1% NP-40, 1 mM EDTA, 0.1% sodium dodecyl sulfate [SDS], 0.1% sodium deoxycholate) or 1× TNT buffer (50 mM Tris [pH 7.5], 150 mM NaCl, 1% Triton X-100, 1 mM EDTA, 10% glycerol) (31). Lysates were homogenized by repetitive pipetting (30 times) through 1-ml syringes and 21-gauge needles. A small amount (27 μl) of each homogenate was mixed with SDS loading buffer (50 mM Tris [pH 6.8], 100 mM dithiothreitol [DTT], 2% SDS, 0.1% bromophenol blue, 10% glycerol) and boiled for 5 min. Cell lysates for Western blotting of NSs mutants were directly lysed in SDS loading buffer (Fig. 1C and 2B). The rest of the homogenate was centrifuged at 20,630 × g for 10 min at 4°C, and the supernatant was incubated for 60 min at 4°C with protein G-Sepharose (GE Healthcare AB), which had been pretreated with anti-Flag (M2; Sigma) or normal mouse IgG1 (catalog number sc-3877; Santa Cruz) antibodies overnight at 4°C. Complexes were obtained by centrifugation and washed four times with coimmunoprecipitation buffer (50 mM Tris [pH 7.5], 150 mM NaCl, 1% Triton X-100, 1 mM EDTA). Polypeptides within precipitated complexes were fractionated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) using 10% gels and electroblotted onto polyvinylidene difluoride (PVDF) membranes (Immobilon-P; Millipore). Membranes were incubated with mouse antitubulin (DM1A; Santa Cruz), mouse anti-myc (9E10; Covance), mouse anti-HA (F-7; Santa Cruz), mouse anti-Flag (M2; Sigma), rabbit anti-p-IRF3 (Ser396) (4D4G; Cell Signaling Technology), or rabbit anti-p-TBK1 (Ser172) (D52C2; Cell Signaling Technology) antibodies, followed by incubation with horseradish peroxidase-conjugated anti-mouse IgG (Jackson Immuno Research Laboratories) or anti-rabbit IgG (Invitrogen). The PVDF membranes were then treated with Chemi-Lumi One super (Nacalai Tesque) to elicit chemiluminescent signals, which were detected and visualized using an LAS-4000 miniapparatus (GE Healthcare).

Confocal microscopy.

HeLa cells were seeded onto coverslips in 24-well cluster plates and transfected with 0.5 μg of pCA7-Flag-NSs together with 0.5 μg of pcDNA3.1-myc-TBK1. At 24 h posttransfection, cells were fixed and permeabilized with PBS containing 4% formaldehyde and 1% Triton X-100. Cells were then washed with PBS and incubated with rabbit anti-Flag (Sigma) and anti-myc (9E10; Covance) antibodies, followed by incubation with Alexa Fluor 488-conjugated goat anti-mouse IgG(H+L) and Alexa Fluor 568-conjugated goat anti-rabbit IgG(H+L) antibodies (Life Technologies). Stained cells were observed under a confocal microscope (LSM5; Zeiss).

RT-quantitative PCR.

Total RNA was extracted from cells using TRIzol reagent (Invitrogen) and reverse transcribed into cDNA using SuperScript III reverse transcriptase (Invitrogen) with an oligo (dT)primer. SYBR Premix Ex Taq II (TaKaRa) and a LightCycler instrument (Roche Diagnostics) were used for quantitative PCR with the following primers: human IFN-β forward primer 5′-CTCCTGGCTAATGTCTATCA-3′ and reverse primer 5′-GCAGAATCCTCCCATAATAT-3′, human β-actin forward primer 5′-CTGGAACGGTGAAGGTGACA-3′ and reverse primer 5′-AAGGGACTTCCTGTAACAATGCA-3′, and SFTSV NP forward primer 5′-TGTCAGAGTGGTCCAGGATT-3′ and reverse primer 5′-ACCTGTCTCCTTCAGCTTCT-3′ (52, 53).

Pairwise sequence alignment.

Amino acid sequences of NSs from SFTSV (YG1; accession number AB817995.1) and HRTV isolate patient1 (accession number NC_024496.1) were obtained from the GenBank database at the National Center for Biotechnology Information, U.S. National Library of Medicine. Sequence alignment was performed using the ClustalW algorithm (54).

Protein structure prediction.

Protein structures of SFTSV and HRTV NSs were predicted by using the I-TASSER (http://zhanglab.ccmb.med.umich.edu/I-TASSER/) ab initio protein structure modeling tool based on amino acid sequences of NSs. The results of protein secondary structure prediction were also confirmed by using the Jpred4 modeling tool (http://www.compbio.dundee.ac.uk/jpred/) (data not shown).

Statistical analysis.

Statistical significance was tested using nonparametric one-way analysis of variance (ANOVA), using PRISM software (version 5; GraphPad software). P values of <0.05 were considered statistically significant.