Fig 1.
The MCMV M35 protein inhibits signaling of multiple pattern recognition receptors.
(A) NIH3T3 fibroblasts were co-transfected with a reporter plasmid containing firefly luciferase under the control of the murine IFNβ promoter (IFNβ-Luc) together with a Renilla luciferase normalization control (pRL-TK) and influenza NS1 or MCMV ORFs known or predicted to code for tegument or immediate-early proteins or their corresponding empty vector (pcDNA and pDEST40, respectively). At 24 hours post transfection, cells were treated with medium or stimulated by infection with Newcastle disease virus (NDV). 21 hours p.i. cells were lysed for analysis of luciferase activity. Luciferase fold induction was calculated based on firefly luciferase values normalized to Renilla luciferase from stimulated samples divided by corresponding values from unstimulated samples. Data set is combined from one to four independent experiments and represented as mean ± SD. (B) NIH3T3 fibroblasts were co-transfected with the IFNβ-Luc and pRL-TK luciferase plasmids described in (A) as well as V5-tagged M35 or pcDNA and luciferase assay was performed following stimulation as for (A). Data is combined from three independent experiments and shown as mean ± SD. (C) NIH3T3 fibroblasts were co-transfected as described in (B) and cells stimulated with 10 μg/ml of poly(I:C) in the presence of Lipofectamine 2000 or Lipofectamine 2000 alone for 6 hours before lysis for analysis by luciferase assay. Data is combined from three independent experiments and shown as mean ± SD. (D) 293T cells were co-transfected with expression plasmids for either cGAS (stimulated) or GFP (unstimulated) together with mCherry-STING, the IFNβ-Luc and pRL-TK luciferase plasmids, and either pcDNA, myc-tagged KSHV ORF36 or V5-tagged M35. At 20 hours post transfection, cells were lysed and luciferase production was analyzed. Data is combined from four independent experiments and shown as mean ± SD. (E) Immortalized BMDM stably expressing myc-tagged LacZ or M35 were stimulated by addition of 3 μg/ml cGAMP or left unstimulated. RNA was extracted at indicated time points and IFNβ induction was measured by quantitative RT-PCR and expressed as IFNβ induction normalized to the housekeeping gene Rpl8. Data is shown as mean ± SD and combined from two independent experiments. (F) Immortalized BMDM stably expressing myc-tagged LacZ or M35 were stimulated by addition of indicated amounts of cGAMP and supernatant was collected 16 hours later and analyzed by IFNβ ELISA. Data is shown as mean ± SD and representative of two independent experiments. (G) Immortalized BMDM stably expressing myc-tagged M35 or the corresponding empty vector (pMSCV) were stimulated by addition of 10 ng/ml LPS or 1 μM CpG-B 1826 and supernatant collected at 16 hours post stimulation for analysis by TNFα ELISA. Data is combined from three independent experiments and shown as mean ± SD.***p<0.001, ****p<0.0001.
Fig 2.
M35 does not target signaling downstream of the type I IFN receptor.
(A) 293T cells were co-transfected with a reporter plasmid containing the endogenous ISG56 promoter cloned upstream of the firefly luciferase gene, pRL-TK and V5-tagged versions of LacZ, M27 or M35. 24 hours post transfection, cells were stimulated by addition of 0.1 ng/ml recombinant human IFNβ or left unstimulated. At 16 hours post stimulation, cells were lysed for analysis of luciferase production (left panel). Simultaneously, 293T cells were co-transfected with expression constructs for cGAS (stimulated) or GFP (unstimulated) together with mCherry-STING, the IFNβ-Luc and pRL-TK luciferase plasmids, and V5-tagged versions of LacZ, M27 or M35. Cells were lysed at 20 hours post transfection for analysis of luciferase production (right panel). Luciferase fold induction was calculated based on luciferase values normalized to Renilla from stimulated samples divided by corresponding values from unstimulated samples. For both panels, data is combined from four independent experiments and shown as mean ± SD. (B) Immortalized BMDM stably expressing myc-tagged LacZ or M35 were stimulated by addition of 100 U/ml of recombinant murine IFNβ or left unstimulated. Cells were lysed for RNA extraction at indicated timepoints for analysis of ISG transcription by quantitative RT-PCR (left panel: CXCL10, right panel: IFIT3) and normalized to the housekeeping gene Rpl8. Data is shown as mean ± SD and combined from three independent experiments.
Fig 3.
M35 is localized to the nucleus, but excluded from nucleoli.
NIH3T3 fibroblasts stably expressing LacZ-myc or M35-myc or empty vector (pQCXIH) were fixed for immunolabeling with a mouse anti-myc antibody and either a rabbit anti-calnexin (A) or rabbit anti-fibrillarin (B) antibody and imaged by confocal microscopy. Nuclei were stained with Hoechst. Scale bars represent 10 μm.
Fig 4.
M35 does not target the phosphorylation or nuclear translocation of key transcription factors.
(A) Immortalized BMDM stably expressing LacZ-myc or M35-myc were stimulated by addition of 3 μg/ml cGAMP and cells lysed at indicated time points. Lysates were separated by SDS-PAGE and endogenous IRF3 and phospho-IRF3 were detected with specific antibodies. Expression of myc-tagged LacZ and M35 was verified with a myc-specific antibody. Immunoblot shown is representative of three independent experiments. (B) NIH3T3 fibroblasts stably expressing myc-tagged LacZ or M35 and eGFP-IRF3 were stimulated by transfection of 10 μg/ml poly(I:C) with Lipofectamine 2000. At indicated times post stimulation, cells were fixed for immunolabeling with a mouse anti-myc antibody and imaged by confocal microscopy (upper panel). Nuclei were stained with Hoechst. Scale bars represent 10 μm. Corresponding counts for cells showing nuclear IRF3 (lower panel) are represented as the percentage of total cells (with a minimum of 100 cells counted per timepoint). Data is shown as mean ± SD and combined from two independent experiments. (C) NIH3T3 fibroblasts stably expressing myc-tagged M35 or corresponding empty vector pQCXIH were stimulated by transfection of 10 μg/ml poly(I:C) in the presence of Lipofectamine 2000 and cells were lysed at indicated time points. Lysates were separated by SDS-PAGE and endogenous p65, phospho-p65 and tubulin were detected with specific antibodies. Expression of myc-tagged M35 was verified with a myc-specific antibody. Immunoblot shown is representative of three independent experiments. (D) NIH3T3 fibroblasts stably expressing myc-tagged M35 or corresponding empty vector pQCXIH were stimulated as for (B). At indicated times post stimulation, cells were fixed for immunolabeling with a mouse anti-myc antibody and a rabbit anti-p65 antibody and imaged by confocal microscopy (upper panel). Nuclei were stained with Hoechst. Scale bars represent 10 μm. Corresponding counts for cells showing nuclear p65 (lower panel) are represented as percentage of total cells (with a minimum of 300 cells counted per timepoint). Data is shown as mean ± SD and combined from two independent experiments.
Fig 5.
M35 targets NF-κB- but not IRF-mediated transcription.
(A) 293T cells were co-transfected with expression plasmids for either cGAS (stimulated) or GFP (unstimulated) together with mCherry-STING, the p125 and pRL-TK luciferase plasmids, and V5-tagged M35 or empty vector control (pcDNA). At 20 hours post transfection, cells were lysed for analysis of luciferase production. Luciferase fold induction was calculated based on firefly luciferase values normalized to Renilla luciferase from stimulated samples divided by corresponding values from unstimulated samples. The p125 reporter contains the IFNβ enhancer consisting of PRD-IV, -III, -I and -II (sequence of the IFNβ enhancer is shown below). (B) As for (A) except with the luciferase reporter pNF-κB instead of p125. (C) As for (A) except with the p125-AA luciferase plasmid instead of p125. p125-AA contains two nucleotide exchanges within the NF-κB binding site in the PRD-II region (highlighted in red). (D) As for (A) but with the p55-CIB luciferase plasmid instead of p125. p55-CIB contains 8 tandem repeat motifs (AAGTGA, highlighted in bold in the PRD-I region), corresponding to 7 repeats of an IRF binding element. Data is combined from three (A-C) or four (D) independent experiments and shown as mean ± SD. For all, n.s. indicates not significant, **p<0.01, ****p<0.0001.
Fig 6.
Upon MCMV infection, delivery of tegument M35 to the nucleus precedes translocation of p65.
(A) Schematic representation of recombinant MCMV constructed for this study. Numbers correspond to nucleotide locations in the genome of MCMV strain Smith (accession #GU305914). Left panel: MCMV-M35stop. ATG represents the start codon of M35 and STOP denotes the introduced stop cassette. Right panel: MCMV-M35-myc. Linker-myc-His represents an 18 amino acid linker fused to a 10 amino acid myc-tag and a 6x Histidine tag. (B) Nycodenz-purified virus preparations of MCMV-WT (WT), MCMV-M35stop-REV (REV), MCMV-M35stop (M35stop) and MCMV-M35-myc (M35-myc) adjusted to 5 x 104 infectious viral particles were lysed in SDS loading buffer and separated by SDS-PAGE. Mock denotes a Nycodenz-purified preparation of uninfected cells. Immunoblotting was performed with antibodies specific for M35, MCMV glycoprotein B (gB) or myc. (C) NIH3T3 fibroblasts were left untreated (left panel) or were treated with 5 μg/ml actinomycin D (right panel) 15 minutes prior to infection. MCMV-M35-myc or MCMV-WT was added at an MOI of 0.5 to the cells and infection was enhanced by centrifugation. The time point after centrifugation was defined as 0. After a 30 min incubation at 37°C to allow virus entry, unbound virus was removed with a citric acid buffer wash. Cells were then further incubated and then lysed at the indicated time points. For treatment with actinomycin D, cells were cultured in the presence of actinomycin D for the entire duration of the time course. Cell lysates were subjected to immunoblotting with antibodies specific for myc (to detect M35) as well as the MCMV proteins immediate-early protein 1 (IE1), early protein M45 or late protein gB. Tubulin levels were determined with a tubulin antibody. (D) NIH3T3 fibroblasts were either treated with media alone or infected with MCMV-M35-myc as described in (C). Whole cell lysates (WCL) were harvested at indicated time points and separated into cytoplasmic (C) and nuclear (N) fractions. Expression of myc-tagged M35 was detected by immunoblotting with an anti-myc antibody, the MCMV protein IE1 with an IE1-specific antibody, and p65 detected by an anti-p65 antibody. Tubulin and fibrillarin were used as controls for the cytoplasmic and nuclear fraction, respectively.
Fig 7.
Tegument M35 modulates type I IFN responses upon MCMV infection.
(A) Immortalized BMDM were infected with MCMV-WT (WT), MCMV-M35stop (M35stop) or UV-inactivated MCMV-WT (UV WT) at an MOI of 0.1 and infection was enhanced by centrifugation. Control cells were mock infected. After a 30 min incubation at 37°C followed by a citric acid buffer wash, cells were harvested for RNA extraction at 4 or 6 hours p.i. for quantification of IFNβ (left panel) and CXCL10 (right panel) transcription by quantitative RT-PCR, respectively. Values were normalized to the housekeeping gene Rpl8. Data is shown as mean ± SD and combined from three independent experiments. (B) Primary BMDM were infected with MCMV-M35stop-REV (REV) or MCMV-M35stop (M35stop) at an MOI of 0.1 or were mock infected. Supernatants were harvested 16 hours p.i. for quantification of IFNα (left panel) and IFNβ (right panel) levels by ELISA. Data is shown as mean ± SD and representative of three independent experiments. (C) Nycodenz-purified virus preparations of MCMV-WT (WT), MCMV-M35stop (M35stop) and M35-complemented MCMV-M35stop (M35stop-comp) adjusted to 5 x 104 infectious viral particles were lysed in SDS-loading buffer and separated by SDS-PAGE. Mock denotes a Nycodenz-purified preparation of uninfected cells. Immunoblotting was carried out with antibodies specific for M35 and MCMV glycoprotein B (gB). (D) Primary BMDM were infected at an MOI of 0.5 by addition of MCMV-M35stop-REV (REV), MCMV-M35stop (M35stop) or M35-complemented MCMV-M35stop (M35stop-comp) to the culture medium. Supernatants were harvested at indicated timepoints p.i. for quantification of IFNβ (top panel) and IFNα (bottom panel) levels by ELISA. Data is combined from three independent experiments performed in duplicates and shown as mean ± SD. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.
Fig 8.
M35 is a determinant for MCMV replication in macrophages.
(A) Multistep growth analysis of MCMV-M35stop-REV (REV) or MCMV-M35stop (M35stop) infection (MOI 0.05) in M2-10B4 stromal cells (left panel), SVEC-10 endothelial cells (central panel) or TCMK-1 epithelial cells (right panel). Supernatant was harvested at indicated days post infection (dpi) and viral titers were determined by standard plaque assay. The limit of detection is 10 PFU/ml. One representative of two independent experiments performed in triplicates is shown. Data is shown as mean ± SD. (B) Multistep growth analysis of MCMV-M35stop-REV (REV) or MCMV-M35stop (M35stop) infection (MOI 0.05) in immortalized WT BMDM as in (A). The limit of detection is 100 PFU/ml. One representative of two independent experiments performed in triplicates is shown. Data is shown as mean ± SD. (C) Same as for (B) but with immortalized BMDM derived from IFNAR1-/- mice.
Fig 9.
M35 inhibits the induction of type I IFN in vivo.
(A) In vivo whole-body imaging of luciferase activity in IFN-β+/Δβ-luc BALB/c reporter mice upon i.v. infection with 2 x 105 PFU MCMV-M35stop-REV (REV) or MCMV-M35stop (M35stop) (left panel). Imaging was performed at 0, 4 and 8 hours p.i. after i.v. injection of the luciferase substrate luciferin. Right panel shows corresponding quantification of luciferase activity by region of interest analysis of the liver. Data is shown as mean ± SD and representative of two independent experiments. Type I IFN levels in serum (B) and spleen (C) following i.v. infection with 4 x 105 PFU MCMV-M35stop-REV (REV) or MCMV-M35stop (M35stop) of BALB/c mice at 6 hours p.i. The levels of IFNα (respective left panel) and IFNβ (respective right panel) in the serum and spleen organ homogenates were quantified by ELISA. Mock denotes uninfected mice. Data shown is combined from two independent experiments. ***p<0.001 and ****p<0.0001.
Fig 10.
Infection with MCMV lacking M35 leads to lower levels of viral transcripts in vivo.
BALB/c mice were infected i.v. with 2 x 105 PFU MCMV-M35stop-REV (REV, black circle) or MCMV-M35-stop (M35stop, open square). Expression of viral transcripts in liver and spleen was quantitated 24 hours p.i. by quantitative RT-PCR specific for m123/IE1, m112/E1 and M86/MCP. Data were normalized to 107 cellular β-actin transcripts. *p<0.05.
Fig 11.
MCMV lacking M35 recruits antiviral CD3+ cells more efficiently to infected IE1+ tissue cells for the formation of protective nodular inflammatory foci (NIF).
(A) For the quantification of focal infiltrates in the liver, tissue sections were collected randomly from four BALB/c mice per group on day 3 after i.v. infection with 2 x 105 PFU of either MCMV-M35stop (M35stop) or MCMV-M35stop-REV (REV). Sections were stained by 2-color IHC (2C-IHC) for the expression of intranuclear viral IE1 protein (red staining) in infected liver tissue cells, as well as for the CD3ε molecule (black staining) expressed by T cells and NKT cells. Sections were counterstained with hematoxylin. Representative low-magnification overview images documenting a marked difference in the numbers of NIF (upper panels). Higher resolution images of representative foci that are marked by arrows in the overview images (lower panels). Scale bars represent 100 μm. (B) Data quantification and statistical evaluation of differences for representative tissue section areas of 40 mm2. Each dot symbol (n = 29 for M35stop and n = 149 for REV) represents a focus of infection or a NIF in case of CD3+ cell recruitment. P values were calculated by using the unpaired two-tailed Student’s t-test with Welch’s correction to account for unequal variances. Differences between data sets are considered statistically significant for *p< 0.05 and***p<0.001.
Fig 12.
MCMV lacking M35 has a growth defect in the spleen and cannot establish chronic infection in the salivary glands.
MCMV load in the spleen (left panel) and salivary glands (right panel) following i.v. infection of BALB/c mice with 2 x 105 PFU MCMV-M35stop-REV (REV) or MCMV-M35stop (M35stop). Spleens were analyzed 3 days post infection (dpi) and salivary glands analyzed at indicated time points. Viral titers were determined by standard plaque assay. Data shown is combined from two independent experiments. Each symbol corresponds to an individual mouse; horizontal bars indicate mean values. *p<0.05, **p<0.01.