Figure 1.
ΔM36 MCMV applied locally is avirulent in RAG1−/− mice.
RAG1−/− mice were infected by (A) intravenous, (B) intraperitoneal, (C) subcutaneous, or (D) intranasal administration with 105 PFU of ΔM36 (○) or M36rev (•) MCMV (n = 6–15/group) and monitored for weight loss and survival. Mortality also includes mice that were sacrificed because they had lost more than 20% of body weight.
Figure 2.
Apoptosis inhibition is required for viral dissemination to distant organs.
RAG1−/− mice were (A) i.p. or (B) s.c. infected with 105 PFU of indicated virus and monitored for survival (n = 4–6/group). Mortality also includes mice that were sacrificed because they had lost more than 20% of body weight. (C) Infectious virus was determined by plaque assay on MEF cells in spleen (top panel), lungs (middle panel), and salivary glands (SG, bottom panel) of i.p. infected mice on day 13 after infection with 105 PFU of indicated virus. Each symbol represents an individual mouse. Differences in median values are highlighted by grey shading. The dashed line shows the limit of detection.
Figure 3.
Macrophage, but not NK cell, depletion rescues ΔM36 MCMV in vivo.
In a combined experiment to elucidate the role of (A) NK cells and (B) macrophages in the control of ΔM36 MCMV growth, RAG1−/− and RAGγC−/− mice received injections of 200 µl liposome encapsulated (A) PBS or (B) clodronate 48 hours (i.v.) and 24 hours (i.p.) prior to viral infection. Following liposome injection mice were i.p. injected with 105 PFU ΔM36 (○) or M36rev (•) MCMV (n = 4–5/group). At day 3 post infection infectious virus was determined by plaque assay on MEF cells in spleen (top panels), lungs (middle panels) and liver (bottom panels). Each symbol represents an individual mouse. Differences in median values are highlighted by grey shading. The dashed line shows the limit of detection. *p<0.05; **p<0.01.
Figure 4.
Apoptosis of primary MEF cells inhibits ΔM36 growth.
(A) MEF cells and MEF preparations depleted of CD11b positive cells (MEF ΔCD11b) were infected with indicated virus at a MOI of 1, and 24 hours later analyzed for the induction of the active isoform of caspase-3 by flow cytometry. The percentage of caspase-3 positive cells in a representative experiment is indicated. (B) MEF or NIH-3T3 cells were infected at a MOI of 0.03 with ΔM36 (white bars) or M36rev (grey bars) and supernatants were titrated for infectious MCMV titer at day 4 post infection. Where indicated, Zymosan (30 µg/ml) and/or IFNγ (100 ng/ml) were added to the supernatant immediately following infection. Histograms indicate means from three experiments, error bars are standard deviations. (C) MEF cells were infected at a MOI of 5 with ΔM36, M36rev or mock-infected. Where indicated, IFNγ (100 ng/ml) and Zymosan (30 µg/ml) were added to the supernatant immediately following infection. Expression of the active isoform of caspase-3 was measured 24 hours post infection by flow cytometry and the percentage of caspase-3 positive cells in the total cell pool is indicated. (D) MEF cells were infected at a MOI of 0.03 with ΔM36 (white bars) or M36rev (grey bars) and supernatants were titrated for infectious MCMV at day 4 post infection. Where indicated, z-VAD-fmk (33 µM), Zymosan (30 µg/ml) and IFNγ (100 ng/ml) were added to the supernatant immediately following infection. Histograms indicate means from three experiments, error bars are standard deviations, * p<0.05.
Figure 5.
ΔM36 grows poorly in the presence of macrophages.
(A) CD11b positive cells were removed from MEF cell preparations by monoclonal antibodies coupled to magnetic beads, upon which the cells were infected with ΔM36 (white bars) or M36rev (grey bars), alone or in the presence of Zymosan (30 µg/ml) or IFNγ (100 ng/ml). Virus titer in the supernatant of cells depleted of macrophages was compared to macrophage-undepleted MEF preparations at day 4 post infection. (B) Upon macrophage depletion, primary fibroblasts were cultured with indicated amounts of ANA-I macrophages (MΦ), in the presence or absence of Zymosan (30 µg/ml) and IFNγ (100 ng/ml). Infectious virus titer in supernatants was established at day 4 post infection. Histograms indicate mean values from three separate experiments, error bars show standard deviation, * p<0.05.
Figure 6.
IFNγ controls ΔM36 growth by acting on the IFNγ receptor on fibroblasts, not on macrophages.
(A) Experimental setup: MEF cells depleted for CD11b positive cells (MEF ΔCD11b) and BM-derived macrophages (BMM) were obtained from IFNγRec−/− (IFNγR−/−) or wild-type (WT) mice and cocultured (10% of macrophages, 90% of fibroblasts in cell culture) in all possible combinations. Cells were infected with ΔM36 or M36rev in the presence of Zymosan (30 µg/ml) and IFNγ (100 ng/ml) and virus titer in the supernatants was established at day 4 post infection. (B) Infectious titer of ΔM36 (white bars) or M36rev MCMV (grey bars) is shown as mean+standard deviation from three independent experiments. The combination of cells used in the infectious experiment is indicated below the x-axis, * p<0.05.
Figure 7.
TNFα secreted from macrophages synergizes with IFNγ to impair ΔM36 growth by a caspase-dependent mechanism.
(A) Experimental setup: ANA-I cells were treated for 5 days with Zymosan and IFNγ in the presence of ΔM36 MCMV, M36rev MCMV, or no virus, upon which the supernatants were filtered to prevent virus carryover and transferred to CD11b-depleted MEF cells infected with ΔM36 or M36rev. (B) Infectious titer of ΔM36 (white bars) or M36rev-MCMV (grey bars) at 5 days post infection. Legends below the x-axis indicate the medium used during infection – control medium (DMEM), supernatant from ANA-I cells infected with ΔM36, M36rev or no virus (MOCK). Where indicated, the ANA-I supernatant was supplemented with neutralizing anti-TNFα (1 µg/ml) antibodies or z-VAD-fmk (33 µM). Histograms indicate mean values from three separate experiments, error bars show SD, * p<0.05.
Figure 8.
Diagram of the proposed mechanism of action.
Activated macrophages secrete TNFα (and possibly additional cytokines) which synergize with IFNγ in fibroblasts to block virus growth by a mechanism that is dependent on caspase signaling. M36 blocks the caspase-dependent signaling pathway and thus prevents apoptosis and rescues the virus growth.