Fig 1.
MCMV m139 is expressed with early kinetics and localizes to the cytoplasm and viral replication compartments in the nucleus.
(A) 10.1 fibroblasts were infected with MCMV m139-HA (MOI = 3) in the presence of CHX (5 μg/ml) or left untreated. Four hpi, the CHX-containing medium was replaced by normal medium or medium containing ActD (50 μg/ml). After 4 hours, viral protein expression was analyzed by immunoblotting. (B) 10.1 fibroblasts were infected with MCMV m139-HA (MOI = 5). Cell lysates were harvested at 5 and 24 hpi, separated into nuclear and cytoplasmic fractions, and analyzed by immunoblotting. (C) 10.1 fibroblast were infected with MCMV m139-HA (MOI = 1), fixed at 5 and 24 hpi, and subjected to immunofluorescence. Mock-infected cells were used as a control. m139 was detected with an anti-HA antibody and the viral E1 (encoded by M112-113) proteins with an E1-specific antibody. Nuclei were counterstained with Hoechst 33342. Representative images taken by confocal microscopy are shown. Scale bar, 10 μm. The results shown in this figure are representative of two (A) or three (B, C) independent experiments.
Fig 2.
MCMV m139 is important for viral replication in macrophages and endothelial cells.
Multistep replication kinetics of WT MCMV, MCMV m139-HA and MCMV m139stop in murine 10.1 fibroblasts (A), TCMK-1 epithelial cells (B), SVEC4-10 endothelial cells (C), and immortalized bone marrow-derived macrophages (iBMDM) (D). iBMDM were infected at an MOI of 0.025, all others at an MOI of 0.01. Virus released by infected cells into the supernatant was quantified by titration. Viral titers are shown as means ±SD of three biological replicates.
Fig 3.
m139 interacts with DDX3 and UBR5.
(A) SVEC4-10 cells were infected with MCMV m139-HA or M45-HA (MOI = 5). Cell lysates were collected 24 hpi and subjected to immunoprecipitation (IP) using an anti-HA affinity matrix. Co-precipitating proteins were detected by immunoblotting with specific antibodies. (B, C) HEK-293A cells were transfected with expression plasmids encoding HA-tagged MCMV proteins m139, m140, or m141. Cell lysates were subjected to immunoprecipitation (IP) using anti-DDX3 (B) or anti-UBR5 (C) antibodies. Co-precipitating proteins were detected by immunoblotting as in A. The results shown in this figure are representative of two (B, C) or three (A) independent experiments.
Table 1.
Proteins interacting with m139 identified by AP-MS.
Fig 4.
DDX3 and UBR5 are recruited to viral replication compartments.
SVEC4-10 endothelial cells were infected with MCMV m139-HA or MCMV m139stop (MOI = 1) and fixed at 8 hpi. The subcellular localization of DDX3 (A), UBR5 (B), and the viral m139 and E1 (M112-113) proteins was analyzed by immunofluorescence using protein-specific and anti-HA antibodies. Nuclei were counterstained with Hoechst 33342. Representative images acquired by confocal microscopy are shown. Scale bar, 10 μm. The results shown in this figure are representative of three independent experiments.
Fig 5.
Rescue of the MCMV m139stop replication defect in DDX3- and in UBR5-deficient SVEC4-10 cells.
(A, B) Ddx3x and Ubr5 ko SVEC4-10 cells were generated by CRISPR/Cas9 gene editing. DDX3 (A) and UBR5 (B) expression was verified by immunoblot analysis. Note that the Ddx3x ko was incomplete. (C, D, E) For multistep replication kinetics, WT (C), Ddx3x (D), and Ubr5 (E) ko SVEC4-10 cells were infected with MCMV m139-HA and MCMV m139stop (MOI = 0.01). At different days post infection, supernatants were collected for titration. Viral titers are shown as means ±SD of three biological replicates. (F, G). WT and Ddx3x ko (F) or Ubr5 ko (G) SVEC4-10 cells were infected with MCMV m139-HA or MCMV M45-HA (control) at an MOI of 5 and harvested 24 hpi. The m139 protein was immunoprecipitated using an anti-HA affinity matrix. Co-precipitating proteins were detected by immunoblotting. The immunoblots shown in this figure are representative of two (F) or three (A, B, G) independent experiments.
Fig 6.
MCMV m139 curtails type I IFN production.
(A) iBMDM macrophages expressing luciferase under the control of the IFN-β promoter were infected with MCMV m139-HA or m139stop. Luciferase activity was measured 8 hpi. Means ±SD of three biological replicates are shown. (B, C) iBMDM (B) or SVEC4-10 (C) cells were infected with MCMV m139-HA, MCMV m139stop or a recombinant vesicular stomatitis virus (VSV-GFP), a potent inducer of IFN-β transcription. RNA was harvested 6 hpi, and Ifnb transcripts were quantified by qRT-PCR. Means ±SD of three biological replicates are shown. (D, E, F) iBMDM were infected with MCMV m139-HA or MCMV m139stop. Cells treated with lipopolysaccharide (LPS, 100 ng/ml) were used as a positive control. RNA harvested 6 hpi was used to quantify Isg20 (D), Cxcl10 (E), and IL6 (F) transcripts by qRT-PCR. Means ±SD of three biological replicates are shown. (G) iBMDM were treated as above. Supernatants were collected 16 hpi to determine TNF-α levels by ELISA. Mean ±SD of three biological replicates are shown. (H, I) HEK-293A cells were co-transfected with DDX3 and IKKε (H) or DDX3 and IRF7 (I) expression plasmids, an IFNβ-luc (H) or an IFNα4-luc (I) reporter plasmid, and a renilla luciferase plasmid for normalization. Plasmids expressing MCMV m139, m140, VACV K7, or empty vector (EV) were co-transfected. Firefly and renilla luciferase activities were determined in the same samples. Values were normalized to those of cells co-transfected with EV. Means ±SD of three biological replicates are shown. (J, K) BALB/c mice were infected i.p. with 106 PFU MCMV m139-HA or MCMV m139stop. IFN-β (J) and IFN-α (K) levels in serum and spleen were measured 8 hpi by ELISA. Means ±SEM are shown. Significance was determined by ANOVA. ns, not significant; *, p<0.05; **, p<0.01; ***, p<0.001.
Fig 7.
Modulation DDX3-mediated IFN signaling by m139 is crucial for efficient replication in macrophages.
(A, B) Ddx3x and Ubr5 ko iBMDM (two independent clones each) were generated by CRISPR/Cas9 gene editing. DDX3 (A) and UBR5 (B) expression was verified by immunoblot analysis. (C, D, E) For multistep replication kinetics, WT (C) Ddx3x (D) and Ubr5 (E) ko iBMDM were infected with MCMV m139-HA or MCMV m139stop (MOI = 0.025). At different days post infection, supernatants were collected for titration. Viral titers are shown as means ±SD of three biological replicates. MCMV replication kinetics on the other Ddx3x and Ubr5 ko cell clones are shown in S5 Fig. (F) Schematic representation of WT MCMV and MCMV K7[Δm139], a substitution mutant expressing HA-tagged VACV K7 instead of m139. (G) 10.1 fibroblasts were infected with WT MCMV or K7[Δm139] (MOI = 5). Cell lysates were collected 24 hpi, and HA-K7 and E1 were detected by immunoblot analysis. (H, I) iBMDM were infected with MCMV m139-HA, m139stop, or K7[Δm139] (MOI = 0.1). At 16 hpi, IFN-β and IFN-α levels in the supernatant were measured by ELISA. Means ±SD of three biological replicates are shown. (J) For multistep replication kinetics, iBMDM were infected with MCMV m139-HA, m139stop, or K7[Δm139] (MOI = 0.025). Viral titers in the supernatant are shown as means ±SD of three biological replicates. (K, L) Multistep replication kinetics in (K) SVEC4-10 endothelial (MOI = 0.01) and (L) Ifnar-/- iBMDM (MOI = 0.05) with the same viruses as in J.
Fig 8.
m139 is crucial for MCMV dissemination.
BALB/c mice were infected by injection of 105 PFU MCMV m139-HA, m139stop, or K7[Δm139] into the footpad. Mice (5 per group) were sacrificed on days 3, 7, and 14 post infection, and MCMV titers in different organs were determined by plaque assay. Titers in (A) popliteal lymph on day 3, (B) lungs on day 7, and salivary glands on day7 (C) and 14 (D) post infection are shown as mean titers ±SEM. DL, detection limit; ns, not significant; *, p<0.05; **, p<0.01.
Fig 9.
Known functions of DDX3 and UBR5 and their presumed targeting by m139.
(A) Functions of DDX3 in IFN activating pathways. MCMV m139 interacts with DDX3 and inhibits IFN-β transcription either by targeting the interaction of DDX3 with IKKε (similar to VACV K7) or by blocking DDX3 binding to the IFN promoter; m139 also inhibits IFN-α induction in a similar way as K7. (B) Functions of DDX3 and UBR5 in RNA transcription, processing, and translation. MCMV m139 co-localizes with DDX3 and UBR5 in the nucleus, suggesting that m139 antagonizes DDX3 and UBR5-dependent functions in the nucleus.