Depletion of TAX1BP1 Amplifies Innate Immune Responses during Respiratory Syncytial Virus Infection

Abstract

Respiratory syncytial virus (RSV) is the main cause of acute respiratory infections in young children and also has a major impact on the elderly and immunocompromised people. In the absence of a vaccine or efficient treatment, a better understanding of RSV interactions with the host antiviral response during infection is needed. Previous studies revealed that cytoplasmic inclusion bodies (IBs), where viral replication and transcription occur, could play a major role in the control of innate immunity during infection by recruiting cellular proteins involved in the host antiviral response. We recently showed that the morphogenesis of IBs relies on a liquid-liquid-phase separation mechanism depending on the interaction between viral nucleoprotein (N) and phosphoprotein (P). These scaffold proteins are expected to play a central role in the recruitment of cellular proteins to IBs. Here, we performed a yeast two-hybrid screen using RSV N protein as bait and identified the cellular protein TAX1BP1 as a potential partner of this viral protein. This interaction was validated by pulldown and immunoprecipitation assays. We showed that TAX1BP1 suppression has only a limited impact on RSV infection in cell cultures. However, RSV replication is decreased in TAX1BP1-deficient (TAX1BP1 knockout [TAX1BP1^KO]) mice, whereas the production of inflammatory and antiviral cytokines is enhanced. In vitro infection of wild-type or TAX1BP1^KO alveolar macrophages confirmed that the innate immune response to RSV infection is enhanced in the absence of TAX1BP1. Altogether, our results suggest that RSV could hijack TAX1BP1 to restrain the host immune response during infection. IMPORTANCE Respiratory syncytial virus (RSV), which is the leading cause of lower respiratory tract illness in infants, remains a medical problem in the absence of a vaccine or efficient treatment. This virus is also recognized as a main pathogen in the elderly and immunocompromised people, and the occurrence of coinfections (with other respiratory viruses and bacteria) amplifies the risks of developing respiratory distress. In this context, a better understanding of the pathogenesis associated with viral respiratory infections, which depends on both viral replication and the host immune response, is needed. The present study reveals that the cellular protein TAX1BP1, which interacts with the RSV nucleoprotein N, participates in the control of the innate immune response during RSV infection, suggesting that the N-TAX1BP1 interaction represents a new target for the development of antivirals.

Keywords: RSV; TAX1BP1; innate immunity; interferons; lung; nucleoprotein; yeast two-hybrid screening.

FIG 1

Identification and validation of the TAX1BP1-N interaction. (A) Multiple-sequence alignment of sequencing reads obtained from the 40 yeast colonies matching TAXBP1. As the cDNA library used in the screen was built by oligo(dT) priming, TAX1BP1 fragments captured in the screen extend from the beginning of the sequencing reads (thick green line) to the end of the TAX1BP1 sequence. The shortest TAX1BP1 fragment captured with N^mono is depicted in blue. Below the alignment, a scheme of the TAX1BP1 structural organization is presented, with numbers indicating residues of TAX1B1P1: the SKIP carboxyl homology domain (SKICH), the LC3-interacting region (LIR), central coiled coils constituting the oligomerization domain, and the two C-terminal zinc fingers (ZF). (B) Validation of the N-TAX1BP1 interaction by GST pulldown with recombinant proteins. GST and GST-TAX1BP1 proteins were purified on glutathione-Sepharose beads and incubated in the presence of recombinant N protein, and interactions were analyzed by SDS-PAGE and Coomassie blue staining. The asterisks indicate the product of degradation of GST-TAX1BP1 corresponding to the deletion of the C-terminal domain. Molecular masses (MW) corresponding to the ladder’s bands are indicated. (C) Western blot analysis of the TAX1BP1-N interaction after an immunoprecipitation assay. Cells were transiently transfected with constructs allowing the expression of the Flag tag alone or the Flag-TAX1BP1 fusion protein with N protein. Immunoprecipitations (IP) were performed with an anti-Flag antibody.

FIG 2

Impact of TAX1BP1 depletion on RSV replication in cells. A549 cells were transfected with control siRNA (siCT) or siRNA targeting TAX1BP1 (siTAX1BP1) and then infected 24 h later with either rHRSV-mCherry or rHRSV-Luc at an MOI of 0.5. (A) RSV replication was quantified at 48 h postinfection by measurement of fluorescence (left) and luminescence (right), expressed in arbitrary units (A.U.), in cell lysates. Data are representative of results from three experiments made in quadruplicates. Data are means ± standard errors of the means (SEM). *, P < 0.05. (B) Western blot analysis of TAX1BP1 silencing and RSV-N expression in cells infected with either rHRSV-mCherry or rHRSV-Luc, at 48 h postinfection. (C) Titration of virions released into the culture media of cells treated with siCT (left) and siTAX1BP1 (right) and infected with rHRSV-mCherry (top) or rHRSV-Luc (bottom). Calculated viral titers in PFU per milliliter are indicated.

FIG 3

TAX1BP1-deficient mice infected with RSV present reduced virus replication in the lungs. (A) Kinetics of RSV infection in 129 mice. Wild-type (WT) strain 129 mice were infected with the HEp-2 supernatant (mock) (n = 1) or rHRSV-Luc (n = 4). (Left) Luciferase activity associated with viral replication was measured at different days postinfection (dpi) in lung lysates by quantification of photon emission (radiance in photons per second per square centimeter per steradian) and normalized to the amount of lysed tissue. (Right) In parallel, N-RSV gene expression was measured in the lung lysates by RT-qPCR and calculated by the formula 2^−ΔCT, where ΔC_T = C_TN-RSV − C_THPRT. Data are means ± SEM. *, P < 0.05; ns, not significant. (B) WT or TAX1BP1^KO 129 mice were infected with the HEp-2 supernatant (mock) or rHRSV-Luc. Luciferase activity associated with viral replication was measured at 2 or 4 dpi (left and right, respectively) in lung lysates by quantification of photon emission (radiance in photons per second per square centimeter per steradian) and normalized to the amount of lysed tissue. Data are means ± SEM from two independent experiments with 7 RSV-infected WT mice and 11 RSV-infected TAX1BP1^KO mice. (C) Quantification of N-RSV gene expression at 4 dpi in RSV-infected WT or TAX1BP1^KO mice (n = 4). N-RSV gene expression was measured in the lung lysates by RT-qPCR and calculated by the formula 2^−ΔCT, where ΔC_T = C_TN-RSV − C_THPRT (right). Data are means ± SEM. *, P < 0.05.

FIG 4

Study of antiviral/inflammatory immune responses in the lungs of infected TAX1BP1^KO mice. WT or TAX1BP1^KO mice were infected with the HEp-2 supernatant (mock) or rHRSV-Luc. (A and B) The levels of production of IFN-α and IFN-β were measured at 24 h postinfection in lung lysates using a ProcartaPlex immunoassay. (C and D) The levels of production of IL-6 and TNF-α were measured at 24 h postinfection in lung lysates using a MilliPlex Map immunoassay. The concentrations were normalized to lung weights. Data are means ± SEM (*, P < 0.05; **, P < 0.01) and are representative of results from two independent experiments with 5 to 6 mice per group.

FIG 5

Deletion of TAX1BP1 enhances the production of type I IFN and inflammatory cytokines in AMs following RSV infection. AMs from WT or TAX1BP1^KO mice were either not infected (mock) (black triangles) or exposed to rHRSV-mCherry (RSV) (inverted black triangles) or UV-inactivated rHRSV-mCherry (UV-RSV) (white circles) at an MOI of 5 for 2 h. (A and B) The levels of production of IFN-α and IFN-β were measured at 24 h postinfection in the supernatants using a ProcartaPlex immunoassay. (C and D) The levels of production of IL-6 and TNF-α were measured at 24 h postinfection in supernatants using a MilliPlex Map immunoassay. Data are means ± SEM from two independent experiments. ***, P < 0.001.