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Virus-Cell Interactions

Human Cytomegalovirus Inhibits Transcription of the CC Chemokine MCP-1 Gene

Alec J. Hirsch, Thomas Shenk
Alec J. Hirsch
Howard Hughes Medical Institute, Department of Molecular Biology, Princeton University, Princeton, New Jersey 08544-1014
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Thomas Shenk
Howard Hughes Medical Institute, Department of Molecular Biology, Princeton University, Princeton, New Jersey 08544-1014
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DOI: 10.1128/JVI.73.1.404-410.1999
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    Fig. 1.

    MCP-1 concentration in the culture medium of infected fibroblasts. (A) MCP-1 secreted into the culture medium during 4-h intervals. HFFs were infected with active (CMV) or UV-inactivated (UV-CMV) HCMV at an MOI of 3 PFU/cell. At the indicated times, fresh, serum-free DMEM was added, and supernatants were collected 4 h later. MCP-1 concentration in the medium was measured by ELISA. (B) Total MCP-1 secreted into the culture medium during infection. Supernatant was collected at the indicated times p.i., and the MCP-1 concentration was measured by ELISA. All values shown are the averages of data from three independent experiments. mock, supernatant from mock-infected cells.

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    Fig. 2.

    MCP-1 within infected cells. HFFs were infected with HCMV or UV-inactivated HCMV (UV HCMV). 35S-labeled proteins were immunoprecipitated by an antibody (Ab) specific to MCP-1, and the immunoprecipitate was analyzed by electrophoresis and autoradiography. The positions to which marker proteins migrated are indicated on the left (in kilodaltons), and bands corresponding to the chemokine are labeled MCP-1. M, mock-infected cells.

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    Fig. 3.

    MCP-1 mRNA accumulation during infection. HFFs were infected by HCMV (A) or UV-inactivated HCMV (B). RNA was collected at the indicated times and analyzed by Northern blotting with an MCP-1-specific cDNA probe. A cDNA probe for one of the cellular cPLA2 mRNAs was used as a loading control. Mock, mock-infected cells.

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    Fig. 4.

    MCP-1 is induced by a factor present in the medium of infected cells. (A) HFFs were infected and/or treated as described in the text, and RNA was collected 8 h later and analyzed by Northern blotting. Lanes: 1, mock-infected cells; 2, crude HCMV stock, MOI = 3; 3, purified HCMV stock, MOI = 3; 4, flowthrough fraction of crude virus stock filtered through a 100-kDa-cutoff filter; 5, crude HCMV stock plus heparin (10 μg/ml); 6, mock-infected cells treated with heparin (10 μg/ml). (B) Purified HCMV stock represses MCP-1 mRNA levels. Lanes: 1, mock-infected cells; 2, HCMV, 6 h p.i.; 3, HCMV, 24 h p.i.; 4, HCMV, 48 h p.i. The autoradiograph was exposed for a longer time than in other panels so that the MCP-1 mRNA in the mock lane would be clearly visible.

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    Fig. 5.

    HCMV infection prevents the induction of MCP-1 mRNA accumulation by TNF-α and IL-1β. RNA was collected at the indicated times p.i. and analyzed by Northern blotting with an MCP-1-specific cDNA probe. A cDNA probe for one of the cellular cPLA2 mRNAs was used as a control. (A) TNF-α treatment. Where indicated, HFFs were treated with TNF-α (10 ng/ml) and infected with HCMV. RNA was collected 2 h after addition of TNF-α. Lanes: 1, mock-infected cells; 2, TNF-α only; lanes 3 to 9, TNF-α added at the indicated times p.i. (B) IL-1β treatment. IL-1β was added (1 ng/ml) at the indicated times p.i. (lanes 3 to 9) or alone (lane 2). (C) PAA treatment. HFFs were infected with HCMV, treated (+) or not treated (−) with IL-1β or PAA (100 μg/ml, 1 h p.i.) as indicated. RNA was collected at 24 h p.i., 2 h after IL-1β addition.

  • Fig. 6.
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    Fig. 6.

    HCMV blocks the transcriptional induction of MCP-1 by IL-1β. (A) Nuclear run-on assay of mock-infected HFFs, mock-infected HFFs treated with IL-1β (1 ng/ml, 1.5 h), and HFFs infected with HCMV (24 h) and treated with IL-1β (1 ng/ml, 1.5 h). Newly transcribed, labeled RNAs for MCP-1, β-actin, and the viral gene encoding US28 were hybridized to specific cDNAs immobilized on a nitrocellulose filter. pSP72 is a plasmid lacking a virus-specific insert that was included to monitor nonspecific binding. (B) Relative rate of MCP-1 transcription after various HFF treatments. The bands in panel A were quantitated with a phosphorimager, and values are normalized to β-actin. Mock, mock-infected cells; IL-1β, treated with IL-1β only; HCMV + IL-1β, HCMV infected and treated with IL-1β.

  • Fig. 7.
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    Fig. 7.

    The US28 gene product is not required for the inhibition of MCP-1 mRNA induction. (A) Schematic representation of the US27-US29 region of the HCMV genome, the plasmid construct used to delete the US28 ORF, and probes used to characterize the mutant virus. (B) Southern blot of AD169 and ADsubUS28 viral DNA. Viral DNA was digested with BamHI, resulting in a 6.0-kb fragment containing the US28 ORF for the wild-type virus and in 3.9- and 2.8-kb fragments for the mutant, due to a BamHI site within the GFP marker. (C) Northern blot of RNA isolated from cells infected with AD169 or the AdsubUS28 mutant and treated with IL-1β (1 ng/ml). RNA was collected at various times p.i. and 2 h after IL-1β addition. An MCP-1-specific cDNA probe and a cDNA probe for a cellular cPLA2 RNA (control) were used. Lanes: 1, mock infection; 2, IL-1β only; 3, IL-1β and HCMV (AD169), 4 h p.i.; 4, IL-1β and HCMV (AD169), 24 h p.i.; 5, IL-1β and HCMV (AD169), 48 h p.i.; 6, IL-1β and HCMV (ADsubUS28), 4 h p.i.; 7, IL-1β and HCMV (ADsubUS28), 24 h p.i.; 8, IL-1β and HCMV (ADsubUS28), 48 h p.i.

  • Fig. 8.
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    Fig. 8.

    HCMV and TNF-α synergize to induce RANTES mRNA accumulation. HFFs were infected with HCMV (MOI = 3) and/or treated with TNF-α (10 ng/ml). RNA was collected at the indicated times p.i. Northern blots were probed with cDNA specific for RANTES, MCP-1, or cPLA2. M, mock-infected cells.

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Human Cytomegalovirus Inhibits Transcription of the CC Chemokine MCP-1 Gene
Alec J. Hirsch, Thomas Shenk
Journal of Virology Jan 1999, 73 (1) 404-410; DOI: 10.1128/JVI.73.1.404-410.1999

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Human Cytomegalovirus Inhibits Transcription of the CC Chemokine MCP-1 Gene
Alec J. Hirsch, Thomas Shenk
Journal of Virology Jan 1999, 73 (1) 404-410; DOI: 10.1128/JVI.73.1.404-410.1999
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KEYWORDS

Chemokine CCL2
cytomegalovirus
Transcription, Genetic

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