Abstract
Immune evasion and inhibition of apoptosis are required for successful virus infection. However, inhibition of apoptosis can increase antiviral immune responses, which can then clear viral infections. Here we show that human cytomegalovirus (HCMV)-encoded UL37 exon-1 protein (UL37x1) not only inhibits apoptosis but also suppresses the cGAS-STING immune pathway. Using co-immunoprecipitation assays, we found that UL37x1 binds to TBK1 to abrogate the TBK1-STING-IRF3 interaction. Although the anti-apoptosis function of UL37x1 increases immune signalling, the immunosuppressive role of UL37x1 counteracts this undesirable side-effect. Furthermore, we used mutational analyses to show that the loss of either immunosuppressive or anti-apoptotic function of UL37x1 significantly reduced HCMV replication in human primary foreskin fibroblasts and humanized mice by over twofold. Finally, loss of both functions resulted in over fourfold reduction of HCMV replication in the same cell type and mouse model, showing that both UL37x1 functions are crucial for HCMV infection. We conclude that this sophisticated mechanism enables HCMV to control innate immunity and apoptosis to ensure efficient infection.
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Data availability
All data supporting the findings of this study are available within the Article and its Supplementary Information. The nucleotide sequences of HCMV strains mentioned in this study are publicly available on NCBI GenBank AD169 (FJ527563.1) and Towne (GQ121041.1). Source data are provided with this paper.
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Acknowledgements
We thank Y. Fu, Y. Wang and B. Zhong (Wuhan, China) for reagents.
This work was supported by the National Natural Science Foundation of China (NSFC) (31970169 to X.Z. and 32100106 to Y.R.), Strategic Priority Research Program of CAS (XDB29010300 to X.Z.), National Key R&D Program of China (2021YFC2300700 to X.Z.) and NSFC (U21A20423 to X.Z. and 81873964 to Y.Q.); a grant from the CAS Youth Innovation Promotion Association (2020332 to Y.Q.), the Hubei Province Natural Science Funds for Distinguished Young Scholars (2021CFA047 to Y.Q.) and the Young Top-notch Talent Cultivation Program of Hubei Province (Y.Q.).
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Contributions
Y.R. performed most of the experiments. A.W., D.W., C.W., M.H., X.X. and L.J. performed specific experiments. Y.R. and X.Z. conceived the hypothesis. Y.R., W.Z., Y.Q. and X.Z. designed the experiments and analysed the data. Y.R., Y.Q. and X.Z. wrote the manuscript with inputs from all authors. X.Z. supervised the study.
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Extended data
Extended Data Fig. 1 Sequence comparison of UL37x1 proteins.
The amino acid sequence alignment of UL37x1 proteins of different HCMV strains.
Extended Data Fig. 2 UL37x1 inhibits the DNA-triggered innate immune signaling in 293 T cells.
(a) Luciferase reporter assay analyzing IFNβ, ISRE, or NF-κB promoter activity was conducted in 293 T cells transfected with the plasmid of FLAG-cGAS plus FLAG-STING or an empty vector together with the indicated amounts of FLAG-UL37x1 expression plasmid for 24 h. The lower blots showed the expression levels of these transfected proteins. (b) 293 T cells were transfected with the plasmid of FLAG-cGAS plus FLAG-STING or an empty vector together with the indicated amounts of FLAG-UL37x1 expression plasmid for 24 h, followed by qPCR of the indicated antiviral genes. The lower blots showed the expression levels of these transfected proteins. (c) 293 T cells stably expressing STING were transfected with the plasmid of FLAG-UL37x1 or an empty vector. After 24 h, cells were untreated (mock) or transfected with indicated DNA ligands (HSV60, DNA90, or HSV120). At 3 and 6 h post transfection (h.p.t.), total RNA was extracted and subject to qPCR of indicated antiviral genes. Graphs show mean ± SD (n = 3, biologically independent experiments). Statistical significance was determined by two-way ANOVA. Immunoblots are representative of three independent experiments.
Extended Data Fig. 3 UL37x1 inhibits TBK1-mediated immune signaling.
(a) 293 T cells were transfected with the indicated plasmid in the presence or absence of the plasmid of FLAG-UL37x1. At 24 h.p.t., qRT-PCR was performed to measure the transcription of indicated antiviral genes. (b) HFFs were infected with HCMV for indicated time, followed by qPCR analysis of UL37x1. (c-d) HFFs were transfected with siRNA specifically for UL37x1 (UL37x1-siRNA#1, UL37x1-siRNA#2, or UL37x1-siRNA#3) or a control siRNA (Control). After 24 h, cells were infected with HCMV. At 6 h.p.i., qPCR was performed to measure the transcription of UL37x1 (c). At 12 h.p.i., cell lysates were subject to immunoblots with indicated antibodies (d). Immunoblots are representative of three independent experiments. Graphs show mean ± SD (n = 3, biologically independent experiments). Statistical significance was determined by two-way ANOVA in a, or one-way ANOVA in c.
Extended Data Fig. 4 Identifying the key residues for UL37x1 inhibiting cGAS-STING axis.
(a-b) Upper, the schematic illustration of the UL37x1 truncations. Middle, 293 T cells were transfected with the plasmid of FLAG-cGAS plus FLAG-STING together with the plasmid of FLAG-UL37x1 or indicated truncation for 24 h, followed by qPCR analysis of IFNB1. The lower blots showed the expression levels of the indicated proteins. (c) 293 T cells were transfected with the plasmid of FLAG-UL37x1, FLAG-UL37x1△40-44, or FLAG-UL37x1K40R together with the plasmid of FLAG-TBK1, FLAG-IRF3-5D, or an empty vector. At 24 h.p.t., qPCR was performed to measure the transcription of indicated antiviral genes. (d) 293 T cells were transfected with the plasmid of HA-TBK1 together with the plasmid of FLAG-UL37x1 or FLAG-UL37x1△40-44. At 24 h.p.t., cell lysates were subject to immunoprecipitation with anti-FLAG antibody or IgG, followed by immunoblots with indicated antibodies. (e) HFFs stably expressing UL37x1, UL37x1K40R, or empty vector were transfected with HA-MAVS, Myc-TBK1. At 24 h.p.i., cell lysates were subject to immunoprecipitation with anti-HA antibody or IgG, followed by immunoblots with indicated antibodies. Immunoblots are representative of three independent experiments. (f) Luciferase reporter assay analyzing IFNβ, ISRE, or NF-κB promoter activity was conducted in 293 T cells transfected with or without the plasmid of FLAG-RIG-I CARD together with the plasmid of FLAG-UL37x1 or FLAG-UL37x1K40R for 24 h. (g) 293 T cells were transfected with or without the plasmid of FLAG-RIG-I CARD together with the plasmids of FLAG-UL37x1 or FLAG-UL37x1K40R for 24 h. Total RNAs were extracted and subject to qPCR of the indicated antiviral genes. Graphs show mean ± SD (n = 3, biologically independent experiments). Statistical significance was determined by two-way ANOVA.
Extended Data Fig. 5 The effects of UL37x1 on immune signaling and apoptosis.
(a) HFFs stably expressing UL37x1 or empty vector were infected with HCMV (MOI = 1). At 0, 6, and 12 h.p.i., qPCR was performed to measure the transcription of indicated antiviral genes. (b) HFFs stably expressing UL37x1 or empty vector were uninfected (mock) or infected with HCMV in the absence or presence of z-VAD-FMK. At 12 h.p.i., cells were collected and stained with Annexin V-FITC/PI for flow cytometry analysis (left, early apoptosis: Annexin V-FITC single positive, Q3; late apoptosis: Annexin V-FITC and PI double positive, Q2), and the percentage of apoptotic cells (Q2 + Q3) was measured (right). This gating strategy applies to all flow cytometry analyses in this study. (c) 293 T cells were transfected with the plasmid of FLAG-cGAS plus FLAG-STING or an empty vector together with or without the plasmid of FLAG-UL37x1. After 24 h, cells were collected and stained with Annexin V-FITC/PI for flow cytometry analysis, and the percentage of apoptotic cells was measured. (d) HFFs were transfected with HSV60, DNA90, or HSV120. After 12 h, cells were collected and stained with Annexin V-FITC/ PI for flow cytometry analysis, and the percentage of apoptotic cells was measured. (e-f) HFFs were transfected with UL37x1-siRNA#1, UL37x1-siRNA#2, UL37x1-siRNA#3 or control siRNA for 24 h, followed by infection with HCMV (MOI = 1). At 12 h.p.i., the mRNA levels of the indicated HCMV genes were measured by qPCR (e) and the protein level of HCMV-expressed reporter GFP was measured by immunoblot (f). (g) HFFs were transfected as in (b) for 24 h, followed by infection with HCMVTowne (MOI = 1). At 12 h.p.i., cell lysate was subject to immunoblot with anti-UL37x1 and anti-β-Actin. (h) HFFs were transfected with UL37x1-siRNA#1, UL37x1-siRNA#3 or control siRNA and infected with HCMVTowne (MOI = 1) in the absence or presence of z-VAD-FMK. At 12 h.p.i., qPCR was performed to measure the transcription of indicated antiviral genes. Immunoblots are representative of three independent experiments. Graphs show mean ± SD (n = 3 for a, e, and h, or n = 2, for b, c, and d biologically independent experiments). Statistical significance was determined by two-way ANOVA in a, b, c, d, and h, or one-way ANOVA in e.
Extended Data Fig. 6 The immunosuppressive and anti-apoptotic activities of UL37x1 are functionally separated.
(a) 293 T cells were transfected with the plasmid of FLAG-UL37x1 or FLAG-UL37x1△2-23. At 24 h.p.t., cells were stained with Mito Tracker Red (mitochondria staining reagent) at 37 °C. After 15 min, cells were fixed and stained with anti-FLAG and Alexa-488 conjugated anti-mouse IgG antibodies, followed by immunofluorescence microscopy. Scale bar, 10 μM. (b) 293 T cells were transfected with the plasmid of FLAG-UL37x1 or FLAG-UL37x1△2-23. At 24 h.p.t., cells were collected and the mitochondria was separated via gradient centrifugation. The indicated cell lysate fractions were subject to immunoblots with anti-FLAG, anti-β-Actin, and anti-GDH (glutamate dehydrogenase, used to mark mitochondria) antibodies. Immunoblots are representative of three independent experiments. (c) HFFs stably expressing UL37x1, UL37x1△2-23, or empty vector were uninfected or infected with HCMV (MOI = 1). At 12 h.p.i., cells were collected and stained with Annexin V-FITC/ PI for flow cytometry analysis, and the percentage of apoptotic cells was measured. (d) HFFs stably expressing FLAG-UL37x1, FLAG-UL37x1△40-44, FLAG-UL37x1K40R, or empty vector were uninfected or infected with HCMV (MOI = 1). At 12 h.p.i., cells were collected and stained with Annexin V-FITC/ PI for flow cytometry analysis, and the percentage of apoptotic cells was measured. Graphs show mean ± SD (n = 2, biologically independent experiments). Statistical significance was determined by two-way ANOVA.
Extended Data Fig. 7 A scheme for homologous recombination mediated genome editing of the UL37x1 gene locus.
(a, c and e) A scheme for homologous recombination mediated genome editing of the UL37x1 gene locus. (b, d and f) DNA sequence and reading frame of wild-type and edited UL37x1 alleles. (g) HFFs were infected with HCMVWT, HCMV△UL37x1, HCMV△2-23, or HCMVK40R. At 12 h.p.i., cell lysates were subject to immunoblots with anti-UL37x1 and anti-UL36 antibodies. Immunoblots are representative of two independent experiments.
Extended Data Fig. 8 Endogenous UL37x1 inhibits innate immunity and apoptosis.
(a) HFFs were infected with HCMVWT, HCMV△UL37x1, HCMV△2-23, or HCMVK40R (MOI = 1) in the presence of z-VAD-FMK. At 12 h.p.i., cells were collected and stained with Annexin V-FITC/PI for flow cytometry analysis, and the percentage of apoptotic cells was measured. (b-c) HFFs were infected with HCMVWT, HCMV△UL37x1, HCMV△2-23, or HCMVK40R (MOI = 1) in the presence of z-VAD-FMK. At 0, 6, and 12 h.p.i., total RNA were extracted and cell lysates were prepared, followed by qPCR to measure the transcription of indicated antiviral genes (b), and immunoprecipitation with anti-STING antibody followed by immunoblots with indicated antibodies (c), respectively. Immunoblots are representative of three independent experiments. (d-e) HFFs stably expressing UL37x1, UL37x1△2-23, UL37x1K40R, or empty vector were infected with HCMV△UL37x1 (MOI = 1). At 96 h.p.i., GFP signals were analyzed by fluorescence microscopy (d, left). The percentage of the infected cells (GFP positive) were measured and shown as the graph (d, right). The mRNA levels of the indicated HCMV genes in HFFs were measured by qPCR at 96 h.p.i (e). (f-g) STING-/- or cGAS-/- HFFs were infected with HCMVWT, HCMV△UL37x1, HCMV△2-23, or HCMVK40R. At 96 h.p.i., the mRNA levels of the indicated HCMV genes in HFFs were measured by qPCR. (h) HFFs were infected with HCMVWT, HCMV△UL37x1, HCMV△2-23, or HCMVK40R in the presence of z-VAD-FMK. At 96 h.p.i., the mRNA levels of the indicated HCMV genes in HFFs were measured by qPCR. Graphs show mean ± SD (n = 2 for a, or n = 3 for b and e-h, biologically independent experiments). Statistical significance was determined by one-way ANOVA in a and e-h, or two-way ANOVA in b.
Extended Data Fig. 9 Humanized mouse model for HCMV infection.
(a) The experimental workflow of in vivo HCMV infection experiment in humanized mice: NOD-scid IL2RGγcnull mice were engrafted with human CD34+ HPCs and then transfused with NHDFs infected with HCMVWT, HCMV△UL37x1, HCMV△2-23 or HCMVK40R (n = 6/group). Uninfected mice served as the negative control group (n = 6). At 28 days post infection, in each group, three mice were euthanized for tissue harvesting and three mice were treated with G-CSF and AMD-3100 for another 10 days and then euthanized for tissue harvesting and analysis. (b-c) NHDF cells were infected with wild-type or mutant HCMV. At 12 h.p.i., the mRNA levels of the indicated HCMV genes in NHDFs were measured by qPCR (b) and the protein level of HCMV-expressed reporter GFP was measured by immunoblot (c). Immunoblots are representative of three independent experiments. Graphs in b show mean ± SD (n = 3, biologically independent experiments). Statistical significance was determined by one-way ANOVA.
Extended Data Fig. 10 Model for the dual inhibition of innate immunity and apoptosis by HCMV UL37x1.
Left: Apoptotic caspases cleave key components of the cGAS-STING axis to inhibit innate immune signaling. Right: During HCMV infection, UL37x1 inhibits apoptosis, which enhances cGAS-STING immune signaling. On the other hand, UL37x1 can directly target to TBK1 to disrupt TBK1 interaction with STING as well as IRF3 recruitment to STING, thereby suppressing the cGAS-STING pathway. This immunosuppressing activity of UL37x1 counterbalances the immune enhancing “side-effect” of UL37x1’s anti-apoptotic activity.
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Ren, Y., Wang, A., Wu, D. et al. Dual inhibition of innate immunity and apoptosis by human cytomegalovirus protein UL37x1 enables efficient virus replication. Nat Microbiol 7, 1041–1053 (2022). https://doi.org/10.1038/s41564-022-01136-6
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DOI: https://doi.org/10.1038/s41564-022-01136-6
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