Figure 1.
Figure 1. PTX3 binds to CMV, inhibits viral replication, and activates IRF3. Binding of biotin-labeled PTX3 (PTX3bio+) to MCMV (A) or HCMV (B) virus. Different concentrations of unbiotinylated PTX3 (PTX3bio–) were added for 2 hours at 37°C to MCMV Ag-coated or HCMV-coated plates followed by the addition of different concentrations of PTX3bio+ for an additional 2 hours at 37°C. The optical density at 450 nm was read using the Horseradish Peroxidase Substrate Kit. *P < .05, 1 or 0.5 μg/mL vs 5 μg/mL PTX3bio+;**P < .05, PTX3bio+ with and without PTX3bio–. Error bars indicate SE. For inhibition of viral replication, MEF or HEL cells were (0) infected with MCMV or HCMV, respectively; (V) untreated and infected with PTX3-treated CMV; or (C) pretreated with PTX3 and added to untreated CMV. MCMV gB transcript expression or amounts of genomic equivalents (GE)/mL of HCMV DNA were assessed by RT-PCR 72 hours after the infection. The results shown are from 1 representative experiment out of 3. (C) Electron microscopy of MEF cells after 30 or 120 minutes' exposure to untreated or PTX3-treated MCMVs (2 hours at 37°C; PTX3 + MCMVb). Arrows indicate virions. Magnification (right to left), × 36 000; × 23 000; × 29 000; and × 19 000. (D) CD11b+ DCs or pDCs, generated from bone marrow progenitors of BALB/c mice, were mock infected (–), infected with untreated (MCMV) or PTX3-treated virions (PTX3 + MCMVb), or pretreated with PTX3 (PTX3 + MCMVa) and then infected before being assessed, 48 hours later, for morphology by light microscopy and viral replication by RT-PCR. Images were visualized with a 100×/1.25 NA oil-immersion objective lens, using cedar oil. (E) IRF3 phosphorylation in CD11b+ DCs or pDCs mock-infected (lane 1) or after 3 hours' exposure to MCMV (lane 2), PTX3 (lane 3), or both (lane 4). The 2 nonactivated IRF3 forms (I and II) and the activated, C-terminally phosphorylated IRF3 (P) are indicated. (F) Induction of Ifnγ and Il12p35 mRNA in CD11b+ DCs after 3 hours' exposure to MCMV (lane 2), PTX3 (lane 3), or both (lane 4). Data are expressed as relative cytokine mRNA (ααCt) in treated cells compared with that of mock-infected cells.

PTX3 binds to CMV, inhibits viral replication, and activates IRF3. Binding of biotin-labeled PTX3 (PTX3bio+) to MCMV (A) or HCMV (B) virus. Different concentrations of unbiotinylated PTX3 (PTX3bio–) were added for 2 hours at 37°C to MCMV Ag-coated or HCMV-coated plates followed by the addition of different concentrations of PTX3bio+ for an additional 2 hours at 37°C. The optical density at 450 nm was read using the Horseradish Peroxidase Substrate Kit. *P < .05, 1 or 0.5 μg/mL vs 5 μg/mL PTX3bio+;**P < .05, PTX3bio+ with and without PTX3bio–. Error bars indicate SE. For inhibition of viral replication, MEF or HEL cells were (0) infected with MCMV or HCMV, respectively; (V) untreated and infected with PTX3-treated CMV; or (C) pretreated with PTX3 and added to untreated CMV. MCMV gB transcript expression or amounts of genomic equivalents (GE)/mL of HCMV DNA were assessed by RT-PCR 72 hours after the infection. The results shown are from 1 representative experiment out of 3. (C) Electron microscopy of MEF cells after 30 or 120 minutes' exposure to untreated or PTX3-treated MCMVs (2 hours at 37°C; PTX3 + MCMVb). Arrows indicate virions. Magnification (right to left), × 36 000; × 23 000; × 29 000; and × 19 000. (D) CD11b+ DCs or pDCs, generated from bone marrow progenitors of BALB/c mice, were mock infected (–), infected with untreated (MCMV) or PTX3-treated virions (PTX3 + MCMVb), or pretreated with PTX3 (PTX3 + MCMVa) and then infected before being assessed, 48 hours later, for morphology by light microscopy and viral replication by RT-PCR. Images were visualized with a 100×/1.25 NA oil-immersion objective lens, using cedar oil. (E) IRF3 phosphorylation in CD11b+ DCs or pDCs mock-infected (lane 1) or after 3 hours' exposure to MCMV (lane 2), PTX3 (lane 3), or both (lane 4). The 2 nonactivated IRF3 forms (I and II) and the activated, C-terminally phosphorylated IRF3 (P) are indicated. (F) Induction of Ifnγ and Il12p35 mRNA in CD11b+ DCs after 3 hours' exposure to MCMV (lane 2), PTX3 (lane 3), or both (lane 4). Data are expressed as relative cytokine mRNA (ααCt) in treated cells compared with that of mock-infected cells.

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