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. 2022 Oct 26;10(5):e0232222.
doi: 10.1128/spectrum.02322-22. Epub 2022 Sep 29.

Interaction of HDAC2 with SARS-CoV-2 NSP5 and IRF3 Is Not Required for NSP5-Mediated Inhibition of Type I Interferon Signaling Pathway

Nenavath Gopal Naik #  1 , See-Chi Lee #  1 , Beatriz H S Veronese  2 , Zhe Ma  3   2 , Zsolt Toth  1   4   3
Affiliations

Interaction of HDAC2 with SARS-CoV-2 NSP5 and IRF3 Is Not Required for NSP5-Mediated Inhibition of Type I Interferon Signaling Pathway

Nenavath Gopal Naik et al. Microbiol Spectr. .

Abstract

Over the last 2 years, several global virus-host interactome studies have been published with SARS-CoV-2 proteins with the purpose of better understanding how specific viral proteins can subvert or utilize different cellular processes to promote viral infection and pathogenesis. However, most of the virus-host protein interactions have not yet been confirmed experimentally, and their biological significance is largely unknown. The goal of this study was to verify the interaction of NSP5, the main protease of SARS-CoV-2, with the host epigenetic factor histone deacetylase 2 (HDAC2) and test if HDAC2 is required for NSP5-mediated inhibition of the type I interferon signaling pathway. Our results show that NSP5 can significantly reduce the expression of a subset of immune response genes such as IL-6, IL-1β, and IFNβ, which requires NSP5's protease activity. We also found that NSP5 can inhibit Sendai virus-, RNA sensor-, and DNA sensor-mediated induction of IFNβ promoter, block the IFN response pathway, and reduce the expression of IFN-stimulated genes. We also provide evidence for HDAC2 interacting with IRF3, and NSP5 can abrogate their interaction by binding to both IRF3 and HDAC2. In addition, we found that HDAC2 plays an inhibitory role in the regulation of IFNβ and IFN-induced promoters, but our results indicate that HDAC2 is not involved in NSP5-mediated inhibition of IFNβ gene expression. Taken together, our data show that NSP5 interacts with HDAC2 but NSP5 inhibits the IFNβ gene expression and interferon-signaling pathway in an HDAC2-independent manner. IMPORTANCE SARS-CoV-2 has developed multiple strategies to antagonize the host antiviral response, such as blocking the IFN signaling pathway, which favors the replication and spreading of the virus. A recent SARS-CoV-2 protein interaction mapping revealed that the main viral protease NSP5 interacts with the host epigenetic factor HDAC2, but the interaction was not confirmed experimentally and its biological importance remains unclear. Here, we not only verified the interaction of HDAC2 with NSP5, but we also found that HDAC2 also binds to IRF3, and NSP5 can disrupt the IRF3-HDAC2 complex. Furthermore, our results show that NSP5 can efficiently repress the IFN signaling pathway regardless of whether viral infections, RNA, or DNA sensors activated it. However, our data indicate that HDAC2 is not involved in NSP5-mediated inhibition of IFNβ promoter induction and IFNβ gene expression.

Keywords: HDAC2; NSP5; SARS-CoV-2; interferons; protein-protein interactions; type I IFN signaling.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

FIG 1
FIG 1
Expression, subcellular localization, and the effect of SARS-CoV-2 nuclear proteins on SeV-induced cytokines in epithelial cells. (A) Immunoblot analysis of the expression of 2×-Strep tagged NSP5, NSP9, NSP10, and EGFP in transfected HEK293T cells. (B) IFA was performed on transfected HeLa cells using an anti-Strep antibody. Nucleus is indicated by DAPI staining. (C) Expression of the 2×-Strep tagged viral proteins and EGFP in A549 cells using lentiviral transduction. (D) Experimental flowchart. After 48 h postransduction, A549 cells were treated with SeV (2 HA units/mL) for 24 h. (E) Cytokine gene expressions are measured by RT-qPCR. t tests were performed compared to EGFP in +SeV samples, and P of <0.05 (*) was considered statistically significant.
FIG 2
FIG 2
Protease activity of NSP5 is required for repressing the induction of cytokine genes. A549 cells were transduced with lentiviruses expressing the indicated SARS-CoV-2 proteins or EGFP as a negative control. At 48 h postransduction, the cells were treated with SeV (2 HA units/mL) for 24 h. Total RNA was extracted, and the expression of cytokine genes was analyzed by RT-qPCR. t tests were performed compared to EGFP in +SeV samples, and P of <0.05 (*) was considered statistically significant.
FIG 3
FIG 3
Testing the effect of NSP5 on IFNβ promoter induced by viral infection or different RNA or DNA sensors. (A) Schematic of the different modes of IFNβ promoter induction that were tested. Each tested pathway transmits the activation through the transcription factor IRF3, which is required for the induction of IFNβ promoter. (B to F) Luciferase assays using HEK293T cells that were cotransfected with an IFNβ promoter luciferase reporter plasmid (IFNβ-Luc) and plasmids expressing NSP5, NSP5 mutant C145A, or EGFP (negative control). The fold change was calculated by comparing luciferase activities to the basal activity of the IFNβ-Luc plasmid cotransfected with EGFP or IFNβ-Luc alone (mock). t tests were performed between NSP5 and EGFP samples, and P of <0.05 (*) was considered statistically significant. (B) IFNβ promoter was induced with SeV infection (2 HA units/mL) for 24 h. (C) IFNβ promoter was induced with the expression of the constitutively active RIG-I 2CARD. (D) IFNβ promoter was induced with the expression of 3×FLAG-MDA5. HA-MeV-V was used as a positive control for the inhibition. (E) IFNβ promoter was induced with cotransfection of cGAS and STING. KSHV vIRF1 was used as a positive control for the inhibition. (F) A constitutively active mutant of IRF3 (IRF3sa) was used for IFNβ promoter induction.
FIG 4
FIG 4
NSP5 inhibits the expression of interferon-stimulated genes. (A) Luciferase assay to test if NSP5 inhibits IFNβ-mediated activation of an interferon-stimulated response element (ISRE)-driven promoter (ISRE-Luc). HEK293T cells were cotransfected with the ISRE promoter luciferase reporter vector and the indicated expression plasmids. At 48 h posttransfection, the cells were treated with IFNβ (1000 IU/mL) for 24 h before harvesting them for luciferase assay. (B) Experimental flowchart. (C) RT-qPCR analysis of the expression of two ISGs in IFNβ-treated A549 cells expressing EGFP or NSP5. (D) The expression of type I IFN pathway-related genes was analyzed by RT-qPCR array. Error bars represent standard deviation (n = 2). t tests in panels A and C were performed between NSP5 and EGFP samples, and P of <0.05 (*) was considered statistically significant.
FIG 5
FIG 5
NSP5 interacts with HDAC2 and IRF3. (A) HEK293T cells were transfected with NSP5-2×Strep, and the immunoprecipitation (IP) was performed with HDAC2 or IgG (rabbit) antibodies. (B) FLAG IP was performed using HEK293T cells transfected with 3×FLAG-NSP5 or 3×FLAG-NSP5 mutant C145A. (C) HEK293T cells were transfected with FLAG-IRF3, and the IPs were carried out with anti-FLAG antibody. (D) HEK293T cells were transfected with V5-IRF3 and 3×FLAG-NSP5 for 48 h and then treated with SeV for 24 h. The IPs were performed with anti-V5 antibody. (E) Schematic of interactions between NSP5, IRF3, and HDAC2. We note that these interactions are not necessarily direct–direct protein interactions, which have yet to be shown.
FIG 6
FIG 6
Testing the effect of HDAC2 on the activation of type I IFN signaling pathway. Luciferase assay with siControl- and siHDAC2-treated HEK293T cells that were transfected with IFNβ-Luc reporter plasmid, and the IFNβ promoter was induced with (A) cotransfection of RIG-I-2CARD or with (B) SeV infection (2 HA units/mL). (C and D) Luciferase assay with HEK293T cells that were transfected with ISRE-Luc reporter plasmid and also treated with HDAC2 siRNA (C) or HDAC2 inhibitor (D). The cells were induced with IFNβ (500 IU/mL) for 24 h before being harvested for the luciferase assay. (E and F) A549 cells treated with 10 μM HDAC2 inhibitor were induced with 500 IU/mL of IFNβ for 24 h. Subsequently, immunoblot analysis was performed (E), and RT-qPCR was used for analyzing the expression of ISGs (F). t tests were performed, and P of <0.05 (*) was considered statistically significant; ns, not significant.
FIG 7
FIG 7
HDAC2 is dispensable for NSP5-mediated inhibition of the type I interferon signaling pathway. siControl- and siHDAC2-treated HEK293T cells were cotransfected with NSP5 and IFNβ-Luc followed by SeV infection, and then (A) immunoblot analysis and (B) luciferase assay were performed. (C) Luciferase assay with ISRE-Luc in siHDAC2-treated 293T cells. We used 500 IU/mL of IFNβ for induction. (D) siControl- and siHDAC2-treated HEK293T cells were transfected with V5-IRF3, and the IPs were performed with an anti-V5 antibody. (E) HEK293T cells were cotransfected with V5-IRF3 and 3×FLAG-NSP5. The IPs were performed with an anti-V5 antibody. (F) siControl- and siHDAC2-treated A549 cells were transduced with lentivirus-expressing EGFP (negative control) or NSP5 followed by SeV infection (2 HA units/mL) for 24 h. IFNβ gene expression was determined by RT-qPCR.
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