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. 2020 Jan 6;94(2):e01454-19.
doi: 10.1128/JVI.01454-19. Print 2020 Jan 6.

VAMP8 Contributes to the TRIM6-Mediated Type I Interferon Antiviral Response during West Nile Virus Infection

Sarah van Tol #  1 , Colm Atkins #  2 , Preeti Bharaj  1 , Kendra N Johnson  1 , Adam Hage  1 , Alexander N Freiberg  3   4   5 , Ricardo Rajsbaum  6   4
Affiliations

VAMP8 Contributes to the TRIM6-Mediated Type I Interferon Antiviral Response during West Nile Virus Infection

Sarah van Tol et al. J Virol. .

Abstract

Several members of the tripartite motif (TRIM) family of E3 ubiquitin ligases regulate immune pathways, including the antiviral type I interferon (IFN-I) system. Previously, we demonstrated that TRIM6 is involved in IFN-I induction and signaling. In the absence of TRIM6, optimal IFN-I signaling is reduced, allowing increased replication of interferon-sensitive viruses. Despite having evolved numerous mechanisms to restrict the vertebrate host's IFN-I response, West Nile virus (WNV) replication is sensitive to pretreatment with IFN-I. However, the regulators and products of the IFN-I pathway that are important in regulating WNV replication are incompletely defined. Consistent with WNV's sensitivity to IFN-I, we found that in TRIM6 knockout (TRIM6-KO) A549 cells, WNV replication is significantly increased and IFN-I induction and signaling are impaired compared to wild-type (wt) cells. IFN-β pretreatment was more effective in protecting against subsequent WNV infection in wt cells than TRIM6-KO, indicating that TRIM6 contributes to the establishment of an IFN-induced antiviral response against WNV. Using next-generation sequencing, we identified VAMP8 as a potential factor involved in this TRIM6-mediated antiviral response. VAMP8 knockdown resulted in reduced JAK1 and STAT1 phosphorylation and impaired induction of several interferon-stimulated genes (ISGs) following WNV infection or IFN-β treatment. Furthermore, VAMP8-mediated STAT1 phosphorylation required the presence of TRIM6. Therefore, the VAMP8 protein is a novel regulator of IFN-I signaling, and its expression and function are dependent on TRIM6 activity. Overall, these results provide evidence that TRIM6 contributes to the antiviral response against WNV and identify VAMP8 as a novel regulator of the IFN-I system.IMPORTANCE WNV is a mosquito-borne flavivirus that poses a threat to human health across large discontinuous areas throughout the world. Infection with WNV results in febrile illness, which can progress to severe neurological disease. Currently, there are no approved treatment options to control WNV infection. Understanding the cellular immune responses that regulate viral replication is important in diversifying the resources available to control WNV. Here, we show that the elimination of TRIM6 in human cells results in an increase in WNV replication and alters the expression and function of other components of the IFN-I pathway through VAMP8. Dissecting the interactions between WNV and host defenses both informs basic molecular virology and promotes the development of host- and virus-targeted antiviral strategies.

Keywords: TRIM6; VAMP8; West Nile virus; flavivirus; immunology; type I interferon pathway; ubiquitin.

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Figures

FIG 1
FIG 1
Increased WNV replication in TRIM6-KO cells is associated with impaired IFN-I induction and signaling. TRIM6-KO or wt A549 cells were infected with WNV 385-99 at an MOI of 0.1 (A, C to I, and K) or 5.0 (B, J, and L). (A and B) The viral load in supernatants of infected cells was measured by a plaque assay on Vero ATCC CCL-81 cells. (C) Whole-cell lysates from WNV (MOI of 0.1)-infected cells were run on Western blots for analysis of protein expression and phosphorylation. (D to I) The densitometry area under the curve (AUC) for each band was calculated using Fiji (59). For the phosphorylated proteins, the AUC for the respective total protein was normalized to the actin AUC (AUC total/AUC actin), and the AUC for the phosphorylated protein was then normalized to the total/actin ratio. The AUC reported is the average from three separate measurements. (J) RNA isolated from mock- and WNV-infected cells was isolated to assess gene expression of Ifnb; the ISGs Isg54, Oas1, Irf7, and Stat1; and a non-IFN-I-regulated gene, Il6. Change in expression is represented as fold induction. (K and L) IFN-β was measured via an ELISA of irradiated supernatants infected with WNV at MOIs of 0.1 (K) and 5.0 (L). Error bars represent standard deviations (n = 3). For statistical analysis, two-way ANOVA with Tukey’s posttest for multiple comparisons was used (****, P < 0.0001; ***, P < 0.001; **, P < 0.01; *, P < 0.05). All experiments were performed in triplicate, and immunoblots are for representative samples. All experiments were repeated at least 2 times. LOD, limit of detection.
FIG 2
FIG 2
IFN-I pretreatment is less efficient in antagonizing WNV replication in TRIM6-KO cells. TRIM6-KO or wt A549 cells were treated with recombinant human IFN-β-1a (100 U) for 4 h prior to infection with WNV 385-99 (MOI of 5.0) for 24 h. Supernatants from infected cells were titrated, and the viral load was calculated via a plaque assay. Error bars represent standard deviations (n = 3). One-way ANOVA with Tukey’s posttest was performed to assess statistical significance (****, P < 0.0001; *, P < 0.05). Fold changes are reported in parentheses. All experiments were performed in triplicate.
FIG 3
FIG 3
Transcription of canonical interferon-stimulated genes (ISGs) and VAMP8 is downregulated in TRIM6 knockout cells. (A and B) Transcriptional profiling of cellular mRNA by next-generation sequencing of mock-infected (A) or WNV 385-99-infected (MOI of 5.0) (B) wt or TRIM6-KO A549 cells at 24 h postinfection. The log2-fold change was calculated as TRIM6-KO/wt, with genes downregulated in TRIM6-KO cells on the left (negative values) and those upregulated in TRIM6-KO cells on the right (positive values). The −log10 P value represents the significance. VAMP8 data points are represented as light gray squares, and ISGs are represented as dark gray triangles. (C and D) Validation of VAMP8 expression at the protein (immunoblotting) (C) or RNA (RT-qPCR) (D) levels in wt or TRIM6-KO cells. Error bars represent standard deviations (n = 3), and VAMP8 expression validation experiments were performed in triplicate and repeated three times.
FIG 4
FIG 4
Depletion of VAMP8 impairs STAT1 phosphorylation downstream of IFN-I signaling but does not alter WNV replication. (A and B) wt A549 cells were treated with nontargeting control (control) or VAMP8-targeting (VAMP8) siRNAs for 24 h, followed by infection with WNV 385-99 (MOI of 0.1) for 72 h. Supernatants and lysates of infected cells were collected at 1, 6, 24, 48, and 72 h p.i. to assess viral loads by plaque assays (A) and protein expression and phosphorylation by Western blotting (B). (C and D) The bands in the Western blot were quantified by densitometry as described in the legend of Fig. 1. (E to J) wt A549 (E to I) or ATCC HTB-15 (J) cells were treated with nontargeting control (siControl) or VAMP8-targeting (siVAMP8) siRNAs for 24 h, followed by treatment with recombinant human IFN-β-1a (500 U/ml) (E, F, I, and J) or human IFN-γ (500 U/ml) (G and H). IFN treatments shown in panels E, F, I, and J were done for 16 h. Cells were lysed, and either protein (E to H) or RNA (I and J) was isolated for analysis by Western blotting or qRT-PCR, respectively. Error bars represent standard deviations. Gene expression data were analyzed using one-way ANOVA with Tukey’s posttest to assess statistical significance (I and J) (****, P < 0.0001; **, P < 0.01. No statistical significance was found in panel A. The experiment was performed in triplicate.
FIG 5
FIG 5
VAMP8 regulates JAK1 phosphorylation downstream of IFN-I signaling. (A and B) wt A549 cells were treated with nontargeting control (control) or VAMP8-targeting (VAMP8) siRNAs for 24 h, followed by treatment with recombinant human IFN-β-1a (500 U/ml) for 0, 15, and 30 min. (C and D) FLAG-tagged VAMP8 or an empty vector was transfected into HEK293T cells for 24 h prior to treatment with human IFNβ-1a (1,000 U/ml) for 1 h, and protein lysates were collected to assess JAK1 activation. The bands in the Western blot were quantified by densitometry (B and D) as described in the legend of Fig. 1.
FIG 6
FIG 6
VAMP8 is important for efficient establishment of an anti-WNV response mediated by IFN-β. Wild-type A549 (A) or ATCC HTB-15 (B) cells were treated with nontargeting control (siControl) or VAMP8-targeting (siVAMP8) siRNAs for 24 h and then treated with recombinant human IFN-β-1a (500 U/ml) for 16 h prior to infection with WNV 385-99 (MOI of 5.0) for 24 h. Supernatants from infected cells were titrated, and the viral load was calculated via a plaque assay. Error bars represent standard deviations. One-way ANOVA with Tukey’s posttest was performed to assess statistical significance (****, P < 0.0001; *, P < 0.05). Fold changes are reported in parentheses. The experiment was completed in triplicate.
FIG 7
FIG 7
VAMP8 overexpression attenuates WNV replication. (A to E) Wild-type or TRIM6-KO A549 cells were transfected with 250 ng of an empty vector or FLAG-VAMP8 for 30 h and then infected with WNV 385-99 at an MOI of 5.0 for 24 h (A to C) or treated with IFN-β (500 U/ml) for 16 h (D and E). (A) Supernatants from infected cells were titrated, and the viral load was calculated via a plaque assay. (B and D) Protein lysates were collected to measure STAT1 activation [pSTAT1 (S708) (B) or pSTAT1 (Y701) (D)] and to confirm equal levels of VAMP8 overexpression in wt and TRIM6-KO cells. (C and E) The bands in the Western blots were quantified by densitometry as described in the legend of Fig. 1. (F and G) FLAG-tagged VAMP8 was cotransfected with an empty vector or HA-tagged TRIM6 into HEK293T cells for 24 h. Protein lysates from the cotransfected cells were immunoprecipitated (IP) with either anti-FLAG (F) or anti-HA (G) beads overnight. Whole-cell lysates (WCE) and IP samples were immunoblotted to assess expression and the VAMP8-TRIM6 interaction. Error bars represent standard deviations. Student’s t test was used to assess statistical significance (***, P < 0.001; **, P < 0.01; ns, no significance). The experiment was performed in triplicate.
FIG 8
FIG 8
VAMP8 expression is associated with the presence of TRIM6 but is IKKε independent. (A) Protein lysates from wt (+/+) or IKBKE knockout (−/−) murine embryonic fibroblasts were collected under basal conditions to evaluate the expression of IKKε, VAMP8, and actin via immunoblotting. (B and C) Next-generation sequencing of mRNA collected from mock-infected wt or TRIM6 KO A549 cells. Analysis of the experiment is shown in Fig. 3. The gene expression levels of two transcription factors, HNF4α (B) and HNF4γ (C), with binding sites within the VAMP8 gene region (GenBank accession number NG_022887.1) are presented. FPKM, fragments per kilobase per million. (D) Schematic showing the HNF4α and -γ binding sites within the VAMP8 gene (GenBank accession number NG_022887.1) related to the transcript (GenBank accession number NM_003761.5). The binding sites identified in the VAMP8 genetic region were compared to the consensus sequences of HNF4α (MA0114.1; http://jaspar.genereg.net/matrix/MA0114.1/) and HNF4γ (MA0484.1; http://jaspar.genereg.net/matrix/MA0484.1/) available through JASPAR (2018). nt, nucleotides. (E) The overexpression of wt TRIM6, but not the catalytic mutant (C15A), in TRIM6-KO A549 cells partially rescues the protein expression of VAMP8 to the levels observed in wt A549 cells.
FIG 9
FIG 9
Graphical summary. Following virus infection, viral RNA is recognized by pathogen recognition receptors (PRRs). PRRs then signal through their adaptors, triggering the activation of the kinases TBK1 and IKKε, which phosphorylate and activate the transcription factor IRF3. Once activated, IRF3 translocates to the nucleus and, in concert with other factors not indicated, promotes the transcription of IFN-β. IFN-β is then secreted and signals in an autocrine or paracrine manner through the type I IFN receptor (IFNAR). The kinases (JAK1 and Tyk2) associated with the IFNAR then facilitate the phosphorylation of STAT1 at tyrosine 701 (Y701) and STAT2 in an IKKε-independent manner. Phosphorylated STAT1 and STAT2 interact with IRF9 to form the ISGF3 complex, which translocates to the nucleus to promote the transcription of genes with interferon-stimulated response elements (ISREs), including Stat1, Oas1, and Isg54. In addition to IKKε-independent IFN-I signaling, the E3 ubiquitin ligase TRIM6 facilitates IKKε-dependent IFN-I signaling. TRIM6, in coordination with the ubiquitin-activating (UbE1) and -ligating (UbE2K) enzymes, facilitates the formation of K48-linked unanchored polyubiquitin chains, which act as a scaffold for the oligomerization and cross-phosphorylation of IKKε at threonine 501 (T501) (30). TRIM6 also facilitates the activation of IKKε during IFN-I induction. During IFN-I signaling, activated IKKε phosphorylates STAT1 at serine 708 (S708). STAT1 phosphorylation at S708, an IKKε-dependent modification, facilitates the formation of an ISGF3 complex with different biophysiological properties, which allows the ISGF3 complex to have enhanced binding to certain ISRE-containing promoters, ultimately inducing the complete ISG profile. When STAT1 is phosphorylated only at Y701 (in the absence of IKKε and/or TRIM6), IFN-I signaling results in the induction of a different and incomplete ISG profile (30, 31, 42). Although the mechanism is currently unknown (question mark), TRIM6 induces VAMP8 expression and VAMP8 activity. VAMP8, in turn, is important for the optimal activation of JAK1 and, subsequently, STAT1 (Y701) required for an efficient antiviral response.
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