Fig 2: HCMV US7 and US8 target TLR3-mediated and TLR4-mediated antiviral responses. a Schematic representation of the HCMV genome and the US2-US11 region capable of targeting various cellular immune molecules. b Heat map showing expression of cellular targets of US7 and US8 in HFF cells expressing US7 or US8 after stimulation by dsDNA (Supplementary Data 1). c Change in cellular mRNA expression caused by US7 or US8 versus mRNA expression in empty vector-expressing HFF cells stimulated by dsDNA. Scatter plots of US7- or US8-upregulated ( > 1.5-fold change, red dots) or -downregulated genes ( < 1.5-fold change, blue dots) in dsDNA-stimulated HFF cells. d US7 and US8 inhibit DNA-induced innate antiviral response. HFF cells expressing empty vector, HA-US7, or HA-US8 were transfected with 500 ng ml-1 dsDNA for 12 h. The mRNA expression of the indicated genes was analyzed by qPCR or RT-PCR. *P < 0.001, **P < 0.05 (Student’s t-test). e US7 and US8 inhibit antiviral gene expression mediated by TLR3 and TLR4. TLR3-Myc- or TLR4-Myc-expressing HFF cells were transduced with empty vector, HA-US7, or HA-US8 and were then stimulated by 10 µg ml-1 poly(I:C) or 5 µg ml-1 LPS for 12 h. The indicated gene expression was measured by qPCR. *P < 0.001, **P < 0.05 (Student’s t-test) f US7 and US8 suppress ifnb and nfkb promoter activity. Luciferase assays of ifnb and nfkb promoter activity in TLR3- or TLR4/MD2-expressing HEK293T cells transfected with empty vector, HA-US7, or HA-US8 and incubated with 5 µg ml-1 LPS or 10 µg ml-1 poly(I:C) for 12 h. The protein over-expression of HA-US7 or HA-US8 was analyzed by immunoblot analysis with anti-HA antibody. *P < 0.001, **P < 0.05 (Student’s t-test). g US7 and US8 block IFN-ß production mediated by TLR3 and TLR4. THP-1 cells expressing empty vector, US7, or US8 were stimulated by 100 µg ml-1 poly(I:C) or 1 µg ml-1 LPS for 24 h. IFN-ß secretion levels or HA-US7/US8 overexpression levels were analyzed by ELISA or immunoblot analysis, respectively. *P < 0.001, **P < 0.05 (Student’s t-test). Data are representative of three independent experiments and are presented as the mean ± s.d. in d–g. Source data are provided as a Source Data file

Fig 3: US7 and US8 suppress innate antiviral response in vivo. a Schematic representation of the HCMV strains used. HCMV WT, wild-type HCMV AD169; HCMV?US7-16, HCMV deletion mutant lacking the US7-US16 region; HCMV?US7-16-Rev.US7 or -Rev.US8 HCMV mutant lacking all genes in the US7-US16 region restored with US7 or US8 expression. b, c HCMV, but not HCMV?US7-16, blocks antiviral response through destabilizing TLR3 and TLR4 proteins. HFF cells were infected with HCMV WT or HCMV?US7-16 at an MOI of 2 for the indicated times. The presence of HCMV-derived US7 or US8 and ifnb mRNA expressions were analyzed by RT-PCR. UL44 and GAPDH were used as HCMV infection and loading controls, respectively. d HCMV, but not HCMV?US7-16, degrades TLR3 and TLR4 proteins at late times. TLR3-Myc- or TLR4-Myc-expressing HFF cells were infected with HCMV WT or HCMV?US7-16 and the lysates were immunoblotted with indicated antibodies. RT-PCR analysis of UL44 and GAPDH was used as HCMV infection and loading controls, respectively. e US7 and US8 decreases TLR3 or TLR4 expression levels in vivo. HFF cells were infected with HCMV WT, HCMV?US7-16, HCMV?US7-16-Rev.US7, or HCMV?US7-16-Rev.US8. Lysates were immunoblotted with anti-TLR3, anti-TLR4, anti-GFP, anti-Calnexin, or anti-Tubulin antibody. RT-PCR analysis of UL44 and GAPDH was used as HCMV infection and loading controls, respectively. The intensity of TLRs bands was quantified as comparing the relative abundance of TLR3 or TLR4 to Tubulin (right graphs). f Both US7 and US8 block innate antiviral response in vivo. HFF cells were infected with HCMV WT, HCMV?US7-16, HCMV?US7-16-Rev.US7, or HCMV?US7-16-Rev.US8. The indicated gene expression was measured by qPCR. *P < 0.001, **P < 0.05 (Student’s t-test). Data are representative of three independent experiments and are presented as means ± s.d. in f. Source data are provided as a Source Data file

Fig 4: US7-mediated TLR3 or TLR4 degradation requires Derlin-1/Sec61 and ubiquitination. a US7 physically interacts with TLR3 and TLR4. HeLa cells were transfected with empty vector or HA-US7 and treated with 20 µM MG132 for 4 h. Lysates were immunoprecipitated with anti-TLR3 or anti-TLR4 antibody and then immunoblotted with anti-HA, anti-TLR3, anti-TLR4, or anti-Tubulin antibody. Asterisk denotes immunoglobulin (Ig) heavy chains. b US7 co-localizes with TLR3 or TLR4. HeLa cells expressing TLR3-Myc or Flag-TLR4/MD2-Myc were transfected with empty vector, US7-GFP or HA-US7 and then stained with anti-Myc, anti-Flag or anti-HA antibody. DAPI was used as a nuclear counterstain. Data are given as the average fluorescence intensity (FI) per cell in the selected fields. Scale bars, 10 µm. c US7 binds to both Sec61ß and Derlin-1. Lysates from HA-US7-expressing HEK 293 T cells were immunoprecipitated with anti-HA and anti-Derlin-1 and immunoblotted with anti-Sec61ß, anti-Derlin-1, or anti-HA antibody. HA-US2 or HA-US11 served as a control. *, Ig heavy chains; **, Ig light chains. d US7-mediated TLR3 and TLR4 degradation depends on Sec61ß and Derlin-1 expression. HeLa cells stably expressing control shRNA, Sec61ß, or Derlin-1 were transfected with HA-US7. Lysates were immunoblotted with the indicated antibodies. e US7 promotes TLR3 and TLR4 ubiquitination. Cells expressing TLR3-Myc or TLR4-Myc with or without HA-US7 were treated with 20 µM MG132 for 4 h and lysed. Cell extracts were immunoprecipitated with anti-Myc antibodies and then analyzed by immunoblotting with anti-Ubiquitin, anti-HA, anti-Tubulin and anti-Myc antibodies. Data are representative of at least three independent experiments. Source data are provided as a Source Data file

Fig 5: US8 promotes TLR3 and TLR4 destabilization by disrupting TLR3-UNC93B1 interaction and targeting TLR4 to lysosomes. a US8 interacts with TLR3 and TLR4. HeLa cells were transfected with empty vector or HA-US8 and then treated with 100 µM chloroquine for 4 h. Lysates were immunoprecipitated with anti-TLR3 or anti-TLR4 antibody and immunoblotted with anti-TLR3, anti-TLR4, anti-HA, or anti-Tubulin antibody. *, Ig heavy chains; **, Ig light chains. b US8 co-localizes with TLR3 or TLR4. HeLa cells expressing TLR3-Myc or Flag-TLR4/MD-2-Myc were transfected with empty vector, HA-US8 or US8-GFP treated with or without chloroquine, and then stained with anti-Myc, anti-Flag or anti-HA antibody. DAPI was used as a nuclear counterstain. Scale bars, 10 µm. c US8 promotes TLR4 ubiquitination. Cells expressing TLR4-Myc with or without HA-US8 were treated with 20 µM MG132 for 4 h and lysed. Cell extracts were immunoprecipitated with anti-Myc antibodies and then analyzed by immunoblotting with indicated antibodies. d, e US8 associates with UNC93B1. HeLa cells expressing UNC93B1-GFP were transfected with empty vector or HA-US8 and immunoblotted d or stained e with indicated antibodies. Scale bars, 10 µm. *, non-specific bands. f US8 reduces UNC93B1 stability in a lysosome-dependent manner. HeLa cells expressing UNC93B1-GFP were transfected with empty vector or HA-US8 and treated with 100 µM chloroquine for 4 h. Lysates were immunoblotted with the indicated antibodies. g US8 disrupts the TLR3-UNC93B1 interaction. TLR3-Myc-expressing HeLa cells were transfected with UNC93B1-GFP and empty vector or HA-US8 and then treated with 100 µM chloroquine. Lysates were immunoprecipitated with anti-Myc and immunoblotted with anti-GFP and anti-Myc or anti-HA antibody. *, Ig heavy chains. h US8 promotes TLR3 ubiquitination. Cells expressing TLR3-Myc with or without HA-US8 were treated with 20 µM MG132 for 4 h and lysed. Cell extracts were immunoprecipitated with anti-Myc antibodies and then analyzed by immunoblotting with indicated antibodies. Data are representative of at least three independent experiments. Source data are provided as a Source Data file