Fig 2: HCMV US9 inhibits MAVS- and STING-mediated IFN-β signaling. a US9 expression in HCMV WT-infected HFF cells. HFF cells were infected with HCMV WT at a multiplicity of infection (MOI) of 2. After incubating the infected cells for the indicated number of days post-infection (dpi), total RNA was isolated. The IE1 and GAPDH mRNA expressions were measured as virus infectivity and loading controls, respectively. b Schematic representation of the HCMV strains used. HCMV WT, wild-type HCMV AD169; HCMVΔUS7-16, HCMV deletion mutant lacking the US7–US16 region; HCMVΔUS7-16 + US9, HCMV mutant lacking all genes in the US7–US16 region restored with US9 expression. c HCMV US9 attenuates IFN-β expression. HFF cells were infected with HCMV WT, HCMVΔUS7-16, or HCMVΔUS7-16 + US9 at an MOI of 2 for 2 days (c–h). IFN-β mRNA levels were analyzed by RT-PCR. HFF cells transfected with poly(I:C) (20 μg ml−1) for 12 h were used as a positive control. d ER- and mitochondria-targeted US9 correlates with altered ER/mitochondrial staining patterns. HCMVΔUS7-16+US9-infected HFF cells were then immunostained with an ER marker (anti-PDI) or mitochondrial marker (anti-Tom20). The nuclei were stained with DAPI. Scale bars, 10 μm. e, f HCMV US9 is colocalized with MAVS, STING, and TBK1. HCMVΔUS7-16+US9-infected HFF cells were immunostained with an anti-MAVS (e), anti-STING, or anti-TBK1 (f). The nuclei were stained with DAPI. Scale bars, 10 μm. g HCMV US9 downregulates mitochondrial MAVS expression levels in vivo. Mitochondrial fraction from HCMV-infected HFF cells was isolated and immunoblotted with the indicated antibodies. The presence of HCMV-derived US9 and IE1 expressions were analyzed by RT-PCR. h STING–TBK1 association is disrupted by HCMV US9. HFF cells (−) and HEK293T cells with salmon sperm DNA (2 μg ml−1) for 12 h were used as negative and positive controls, respectively. US9 and IE1 expressions were analyzed by RT-PCR. i HCMV US9 blocks pIRF3 nuclear translocation in vivo. HFF cells infected with HCMV viruses at an MOI of 6 for 1.5, 2.5, or 3.5 days were subjected to nuclear fractionation, and then immunoblotted with anti-p-IRF3. Tubulin and Lamin A/C were analyzed as fraction loading controls. US9 and IE1 expressions were analyzed by RT-PCR. Data are representative of three independent experiments

Fig 3: ISGylation modifies ER and Golgi proteins and increases canonical secretion.(A) Gene ontology (GO) analysis of ISGylated proteins enriched following ISG15 overexpression (blue) relative to all proteins identified in this analysis (light gray) and all human proteins as annotated by the Uniprot/Swiss-prot database (dark gray). (B) Nickel affinity purification of ISG15 in empty-vector control or ISG15 overexpression cells followed by α-ISG15, α-MAGT1, and α-RTN4 immunoblot. Input is 0.1% of the initial affinity purification volume. Higher molecular weight species corresponding to ISGylated RTN4 or MAGT1 are indicated. (C) Cells were fixed and immunostained with α-PDI antibody (green), with α-TGN46 (red) and Hoechst (blue). Cells with four or more large puncta were enumerated as cells with PDI clusters, a minimum of 100 cells were counted per experiment, and data are shown as percentage of cells with PDI clusters; AVG ± SEM. Statistical significance determined using two-tailed t-test. (D) The indicated cells were treated with 20 ng/ml TNF-α for 24 hr in a 96-well format. 100 μl of supernatant was collected for each ELISA and assayed for the presence of IL-8 and IL-6. Statistical significance determined using two-tailed t-test. (E) The indicated cells were infected for 3 or 7 hr with Listeria (MOI 25) in a 96-well format, supernatants were collected and 100 μl of supernatant was used for an IL-8 ELISA. Statistical significance determined using two-tailed t-test. (F) The indicated cells were either infected with Listeria (MOI 25) for 24 hr in a 96-well format. 100 μl of supernatant was collected for each ELISA and assayed for the presence of IL-6 or IL-8. Statistical significance determined using two-tailed t-test. Statistical significance is indicated as follows: NS, nonsignificant; *p < 0.05; **p < 0.01; ***p < 0.001.DOI: http://dx.doi.org/10.7554/eLife.06848.013

Fig 4: Transient Expression and Intracellular Localization of SEC61A1_FLAG Variants in Human Embryonic Kidney 293 Cells(A) Western blot detection of transiently expressed wild-type and mutated SEC61A1_FLAG proteins and endogenously expressed SEC61A, SEC61B, and tubulin at 36 hr post-transfection.(B) Quantitative image analysis of wild-type and mutated SEC61A1_FLAG proteins demonstrating decreased amounts of mutated proteins compared to the wild-type. Results represent means of fold change ± SD of the relative signal intensities of mutated proteins to the wild-type protein from three biological replicates. Signal intensities of SEC61A1_FLAG proteins were normalized to that of α-tubulin. Statistical significance was assessed using Student’s t test; ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001.(C) Quantitative image analysis of the endogenously expressed SEC61A and SEC61B demonstrating that overexpression of neither protein significantly affected relative amounts of the endogeneously expressed SEC61A1 and SEC61B transient expression of SEC61A1. Results represent means of fold change ± SD from three biological replicates of the relative signal intensities of SE61A over SEC61B in cells expressing individual mutated SEC61A1_FLAG protein and empty vector compare to that of cells expressing the wild-type protein. Signal intensities of SEC61A and SEC61B were normalized to that of α-tubulin. Statistical significance was assessed using Student’s t test.(D) Immunofluorescence analysis showing that the wild-type SEC61A1_FLAG is present in a finely granular (subpanel A) or coarsely granular (subpanel D) structures. Co-staining of wt-SEC61A1_FLAG with Golgi matrix protein GM130 (subpanel B) and with Protein disulphide isomerase (PDI) (subpanel E), a marker of endoplasmic reticulum (ER), demonstrating absence of the wt-SEC61A1_FLAG in the Golgi (subpanel C) but presence in the ER (subpanel F). p.Val67Gly (subpanels G and J) and p.Thr185Ala (subpanels M and P) variants of SEC61A1_FLAG are present in a form of intracellular clumps that are more pronounced in the latter. Co-staining with GM130 (subpanels H and N) and PDI (subpanels K and Q) demonstrating localization of both mutant proteins in the Golgi (subpanels I and O) as well as in ER (subpanels L and R). The degree of SEC6A1A_FLAG colocalization with selected markers is demonstrated by the fluorescent signal overlap coefficient values that range from 0 to 1. The resulting overlap coefficient values are presented as the pseudo color which scale is shown in corresponding lookup table.

Fig 5: ER- and mitochondria-targeted US9 inhibits the MAVS- and STING-mediated IFN-β response. a Schematic representation of the HCMV genome and US9. MLS, mitochondrial localization signal. b US9 is localized to the ER and the mitochondria. Cytosolic/ER/mitochondrial fractions isolated from HA-US9-GFP-expressing HEK293T cells were immunoblotted with anti-GFP antibody. C/ER, cytosolic/ER fraction; Mito, mitochondrial fraction; Total, total cell lysate. Anti-Tom20, anti-Tubulin, and anti-PDI were used as the mitochondrial, cytosolic, and ER markers, respectively. c IFA of US9 subcellular localization. HEK293T cells expressing HA-US9-GFP were stained with an ER (anti-PDI) or mitochondrial marker (anti-Tom20). The nuclei were stained with DAPI. Scale bars, 10 μm. d US9 undergoes dimerization. Cell lysates from US9-GFP- and HA-US9-expressing HEK293T cells were immunoprecipitated with anti-GFP, and then immunoblotted with the indicated antibodies. e US9 forms high-molecular-weight oligomers. The cell extracts from HEK293T cells with or without HA-US9-GFP were resolved by SDD-AGE (top) and SDS-PAGE (bottom). f US9 undergoes ubiquitination. HEK293T cells expressing HA-US9-GFP were immunoprecipitated with anti-HA, and immunoblotted with anti-Ubiquitin. g US9 inhibits RNA- and DNA-induced IFN-β induction. HeLa or HEK293T cells were transfected with poly(I:C) (20 μg ml−1) or salmon sperm DNA (dsDNA) (2 μg ml−1) for 12 h. IFN-β mRNA levels were analyzed by RT-PCR. Right graph, quantification of IFN-β mRNA expression levels was shown in the graph. *P < 0.005 (Student’s t test). h US9 impedes MAVS-mediated IFN-β expression. HEK293T cells were transfected with the indicated plasmids and incubated for 21 h. Right graph, quantification of IFN-β mRNA levels was shown in the graph. *P < 0.005 (Student’s t test). i US9 suppresses STING–TBK1-induced IFN-β mRNA expression. HEK293T cells expressing STING-Myc and Flag-TBK1 were transfected mock or HA-US9-GFP. Right graph, quantification of IFN-β mRNA levels was shown in the graph. *P < 0.005 (Student’s t test). j US9 blocks MAVS- and STING-induced IFN-β production. HEK293T or HFF cells were transfected with MAVS or STING-Myc in the absence or presence of HA-US9-GFP, followed by transfection with salmon sperm DNA (1 μg ml−1). After 36 h incubation, IFN-β levels were analyzed by ELISA assay. *P < 0.005 (Student’s t test). Data are representative of three independent experiments and are presented as mean ± standard deviation (mean ± s.d.) in g, h, i, and j