Neutrality of the canonical NF-kappaB-dependent pathway for human and murine cytomegalovirus transcription and replication in vitro

Abstract

Cytomegalovirus (CMV) is known to rapidly induce activation of nuclear factor kappaB (NF-kappaB) after infection of fibroblast and macrophage cells. NF-kappaB response elements are present in the enhancer region of the CMV major immediate-early promoter (MIEP), and activity of the MIEP is strongly upregulated by NF-kappaB in transient-transfection assays. Here we investigate whether the NF-kappaB-dependent pathway is required for initiating or potentiating human and murine CMV replication in vitro. We show that expression of a dominant negative mutant of the inhibitor of NF-kappaB-alpha (IkappaBalphaM) does not alter the replication kinetics of human or mouse CMV in cultured cells. In addition, mouse embryo fibroblasts genetically deficient for p65/RelA actually showed elevated levels of MCMV replication. Mutation of all NF-kappaB response elements within the enhancer of the MIEP in a recombinant mouse CMV containing the human MIEP (hMCMV-ES), which we have previously shown to replicate in murine fibroblasts with kinetics equivalent to that of wild-type mouse CMV, did not negatively affect replication in fibroblasts. Taken together, these data show that, for CMV replication in cultured fibroblasts activation of the canonical NF-kappaB pathway and binding of NF-kappaB to the MIEP are dispensable, and in the case of p65 may even interfere, thus uncovering a previously unrecognized level of complexity in the host regulatory network governing MIE gene expression in the context of a viral infection.

FIG. 1.

Mutagenesis of the HCMV-MIEP NF-κB binding sites in hMCMV-ES. (A) Organization of the MIE regions of the constructed MCMV BAC genomes. The HindIII map of the MCMV genome is given at the top. The expanded map below represents the MIE region of MCMV with the enhancer (gray box) and the exon-intron structure of the ie1, ie2, and ie3 genes indicated. Coding and noncoding exons are depicted as black and white boxes, respectively. The BAC plasmids hMCMV-ES and hMCMV-ES.NF-κB carry a 616-bp fragment (white rectangle) from the HCMV MIE promoter in replacement of the MCMV enhancer. In hMCMV-ES.NF-κB, the NF-κB binding sites were mutated, introducing unique restriction sites (−98 to −103 StuI, −161 to −166 KpnI, and −265 to −270 and −416 to −421 BglII), which are indicated by asterisks or black bars. The sizes of the expected HindIII L fragments of the MCMV BAC genomes and the sizes of the expected BglII and KpnI fragments of the PCR-amplified HCMV enhancer (with primers shown with short arrows flanking the enhancer) are indicated by the arrows. Coordinates are given relative to the MCMV ie1/ie2 transcription start site. The diagrams are not drawn to scale. (B) Ethidium bromide-stained agarose gel of HindIII-digested MCMV BAC plasmid (1), hMCMV-ES (2), and hMCMV-ES.NF-κB (3). The names and sizes of the HindIII fragments of the MCMV genome are indicated in the margins. (C) To verify successful mutagenesis of NF-κB sites, enhancer sequences were amplified from MCMV-ES (lanes 1 and 3), and hMCMV-ES.NF-κB (lanes 2 and 4)BACs by PCR. The amplified products were digested with restriction enzyme BglII (lanes 1 and 2) or KpnI (lanes 3 and 4), and the resulting DNA fragments were separated by gel electrophoresis.

FIG. 2.

The MIEP NF-κB binding sites are not required for hMCMV-ES replication in NIH 3T3 cells. NIH 3T3 cells were infected at an MOI of 0.1 with either hMCMV-ES or hMCMV-ES.NF-κB recombinant virus. Cell supernatants were collected at various days postinfection, and titers (PFU/ml) were analyzed in triplicate by plaque assays on NIH 3T3 cells. Error bars represent standard errors of the means.

FIG. 3.

IκBαM overexpression inhibits p50/p65 NF-κB activation in NIH 3T3 and J774 cells. NIH 3T3 or J774 cells were transduced with either control (LXSN) or IκBαM retroviral vector. (A) To verify that the IκBαM-expressing cells were inhibited for p50/p65 NF-κB-mediated transcription, J774 and 3T3 cells were incubated with ±1 nM TNF or LTα for 24 h, respectively, and cell surface levels of ICAM-1 (a NF-κB responsive gene) were analyzed by flow cytometry. Dotted histograms represent binding of isotype control antibody, gray histograms are cells untreated with cytokine, and black histograms are cytokine-treated cells. (B) Translocation of p50/p65 NF-κB to the nucleus is inhibited in IκBαM-expressing cells. NIH 3T3 LXSN or IκBαM (+) cells were treated with LTα (1 nM) or MCMV (MOI = 2) for 2 h; nuclear extracts were prepared and subjected to EMSA. The arrow identifies the p50/p65 heterodimer of NF-κB. M, mock treated; cp, addition of unlabeled competitor, NF-κB-binding oligonucleotide probe.

FIG. 4.

Replication of MCMV is normal in cells expressing a dominant negative inhibitor of IκBα. NIH 3T3 (upper two panels) and J774 (lower two panels) control (LXSN) and IκBαM cells were infected at either high or low MOI with MCMV, and supernatant was collected at different times (days or hours) postinfection (dpi and hpi, respectively). MCMV titers (PFU/ml) were determined in triplicate by limit dilution in NIH 3T3 cells. Error bars represent standard errors of the means.

FIG.5.

Mouse CMV replication is increased in p65-deficient fibroblasts. (A) p65-deficient (p65^−/−) and control (p65^+/+) immortalized MEFs were infected with MCMV at several different MOIs, and supernatant was collected at various days postinfection for analysis of viral PFU produced. Titers were determined in triplicate in NIH 3T3 cells, and error bars represent standard errors of the means. (B) The relative level of MCMV ie1 and ie3 expression in p65^−/− MEFs was compared to that of p65^+/+ MEFs at various times postinfection at several different MOI by real-time PCR. Values are representative of three independent experiments, and error bars represent the standard errors of the means.

FIG. 6.

The canonical NF-κB pathway is not required for replication of human CMV in fibroblasts. (A) NHDF were transduced with control (LXSN) or IκBαM retroviral vector. Cells were examined for upregulation of ICAM-1 by flow cytometry after incubation with ±1 nM TNF. Dotted histogram represents binding of isotype control antibody. (B) Nuclear extracts from NHDF-LXSN or -IκBαM (+) cells treated with TNF (1 nM) or LTα (1 nM) or infected with HCMV or UV light-inactivated HCMV (uvHCMV; MOI, 5 or 2) for 2 h were subjected to EMSA to analyze the level of p50/p65 NF-κB (indicated by the arrow) translocation to the nucleus. (C) NHDF-LXSN or -IκBαM cells were infected at an MOI of 0.01 or 2 with HCMV strain AD169 or Toledo. Cell supernatant was collected at various days postinfection, and viral titers (PFU/ml) were determined in triplicate by plaque assays in NHDF. Error bars represent standard errors of the means.

FIG. 7.

Inhibition of the canonical NF-κB pathway does not inhibit expression of HCMV IE1. (A) NHDF-LXSN or -IκBαM cells were infected at an MOI of 0.005 (dark bars) or 0.1 (light bars) and were harvested at various hours postinfection (hpi) for analysis of ie1 expression by real-time PCR. Results represent the relative amount of ie1 expression in IκBαM cells compared to that of control cells (LXSN) at the various times. Values are averaged data from three independent experiments, and error bars represent the standard errors of the means. (B) NHDF-LXSN or -IκBαM cells were infected with HCMV at an MOI of 0.5 or 0.02. Cells were harvested at various hpi for analysis of IE1 protein levels by Western blotting. For samples treated at an MOI of 0.02, LXSN-infected cells are shown in the left lane, and IκBαM cells are shown in the right. (C) NHDF-LXSN (first two lanes of each time point) or -IκBαM cells (second two lanes of each time point) were infected with HCMV at an MOI of 2, and cells were harvested at various hpi for analysis of IE1 protein levels by Western blot. For each time point, cells were either treated with (+) 1 nM TNF or were not (−).