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. 2008 May;82(9):4250-6.
doi: 10.1128/JVI.02156-07. Epub 2008 Feb 20.

NF-kappaB-mediated activation of the chemokine CCL22 by the product of the human cytomegalovirus gene UL144 escapes regulation by viral IE86

Emma Poole  1 , Elizabeth Atkins, Takashi Nakayama, Osamu Yoshie, Ian Groves, Antonio Alcami, John Sinclair
Affiliations

NF-kappaB-mediated activation of the chemokine CCL22 by the product of the human cytomegalovirus gene UL144 escapes regulation by viral IE86

Emma Poole et al. J Virol. 2008 May.

Abstract

The product of the human cytomegalovirus (HCMV) gene UL144, expressed at early times postinfection, is located in the UL/b' region of the viral genome and is related to members of the tumor necrosis factor receptor superfamily, but it does not bind tumor necrosis factor superfamily ligands. However, UL144 does activate NF-kappaB, resulting in NF-kappaB-mediated activation of the cellular chemokine CCL22. Consistent with this finding, isolates of HCMV lacking the UL/b' region show no such activation of CCL22. Recently, it has been suggested that activation of NF-kappaB is repressed by the product of the viral gene IE86: IE86 appears to block NF-kappaB binding to DNA but not nuclear translocation of NF-kappaB. Intriguingly, IE86 is detectable throughout an infection with the virus, so how UL144 is able to activate NF-kappaB in the presence of continued IE86 expression is unclear. Here we show that although IE86 does repress the UL144-mediated activation of a synthetic NF-kappaB promoter, it is unable to block UL144-mediated activation of the CCL22 promoter, and this lack of responsiveness to IE86 appears to be regulated by binding of the CREB transcription factor.

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Figures

FIG. 1.
FIG. 1.
UL144-containing viruses induce NF-κB nuclear relocalization. HFF cells were infected with HCMV AD169 (a UL144-negative strain) or Toledo (a UL144-positive clinical isolate) for 48 h. Infection was detected with FITC-conjugated anti-IE72/86 (green), and a p65 subunit of NF-κB was detected with rabbit polyclonal antibody followed by anti-rabbit tetramethyl rhodamine isocyanate (red). Cells were visualized by confocal microscopy, and merges are shown (right panel).
FIG. 2.
FIG. 2.
IE86 blocks UL144-induced activation of NF-κB in a dose-dependent manner. (A and B) HFF cells were transfected with p(PRDII)5tk-lucter and pJATlac and either a mammalian expression plasmid driving the overexpression of the HCMV protein IE86 or UL144 or the control empty vector, as indicated by plus or minus signs. (B) The results of titration of IE86 are indicated by a triangular bar, with increasing amounts of IE86 shown from left to right. Cells were transfected for 48 h, and cell extracts were prepared. Luciferase and β-galactosidase activities were determined from cellular extracts, and relative expression values were calculated accordingly (expressed relative to the induced level of the vector-only sample [1.0]). The histogram represents the results of three independent experiments, with bars denoting standard deviations. (C) Alternatively, cells were harvested in Laemmli buffer, and the expression levels of IE86, UL144, and actin were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting.
FIG. 3.
FIG. 3.
IE86 does not prevent the induction of CCL22 by UL144. (A and B) HFF (A) or macrophage (B) cells were transfected with pGL3-CCL22 and pJATlac (A), expression plasmids for IE86 or UL144, or the control empty vector, as indicated by plus and minus signs. Cells were transfected with Lipofectamine (A) or by electroporation (B), and after 48 h, cell extracts were prepared for panel A and supernatants were collected for panel B. For panel A, luciferase and β-galactosidase activities were determined from cellular extracts, and relative expression values were calculated accordingly (expressed relative to the induced level of the vector-only sample [1.0]). The histogram represents the results of three independent experiments with standard deviations. For panel B, the supernatants were cleared by centrifugation and analyzed for CCL22 content by ELISA. The results from one experiment with quadruplet samples are shown, with error bars.
FIG. 4.
FIG. 4.
Inhibition of UL144-mediated activation of the CCL22 promoter by IE86 is dependent upon the absence of a CREB binding site. HFF cells were transfected with pGL3-MDC (A and B) or deletion mutant −553 (lanes 5 to 8), −479 (lanes 9 to 12), −289 (lanes 13 to 16), −114 (lanes 17 to 20), or −80 (lanes 21 to 24) and pJATlac (A) with expression vectors for IE86, UL144, or the control empty vector, as indicated. Cells were transfected for 48 h, and cell extracts were prepared for luciferase assays (A) or DNA immunoprecipitation (IP) (B to E). For panel A, luciferase and β-galactosidase activities were determined from cellular extracts, and relative expression values were calculated accordingly (expressed relative to the induced level of the vector-only sample [1.0]). The histogram represents the results of two independent experiments with triplicate samples, and standard deviations are shown. For panels B, C (endogenous CCL22), and E, CCL22 promoter DNA/protein complexes were immunoprecipitated with mouse monoclonal antibodies to NF-κB p65, CREB, or an IgG control. For panel D, the PACT promoter was immunoprecipitated with the same antibodies. The immunoprecipitates were amplified by PCR for the MDC promoter and analyzed by gel electrophoresis and densitometry. The histogram represents the results of two independent experiments with triplicate samples. Standard errors are shown.
FIG. 5.
FIG. 5.
Inhibition of UL144-mediated activation of the CCL22 promoter by IE86 is dependent upon the absence of a CREB binding site. HFF cells were transfected with pGL3-CCL22 with deletion mutations in the NF-κB (A and B) or CREB (A and C) binding sites and pJATlac (A) with expression vectors for IE86, UL144, or the control empty vector, as indicated. Cells were transfected for 48 h, and cell extracts were prepared for luciferase assays (A) or DNA immunoprecipitation (IP) (B and C). For panel A, luciferase and β-galactosidase activities were determined from cellular extracts, and relative expression values were calculated accordingly (expressed relative to the induced level of the vector-only sample [1.0]). The histogram represents the results of two independent experiments with triplicate samples, and standard deviations are shown. For panels B and C, CCL22 promoter DNA/protein complexes were immunoprecipitated with mouse monoclonal antibodies to NF-κB p65, CREB, or an IgG control. Immunoprecipitates were amplified by PCR for the CCL22 promoter and analyzed by gel electrophoresis and densitometry. The histogram represents the results of two independent experiments with triplicate samples, and standard errors are shown.
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