The Activator Protein 1 Binding Motifs within the Human Cytomegalovirus Major Immediate-Early Enhancer Are Functionally Redundant and Act in a Cooperative Manner with the NF- (cid:1) B Sites during Acute Infection (cid:1) §

Human cytomegalovirus (HCMV) infection causes a rapid induction of c-Fos and c-Jun, the major subunits of activator protein 1 (AP-1), which in turn have been postulated to activate the viral immediate-early (IE) genes. Accordingly, the major IE promoter (MIEP) enhancer, a critical control region for initiating lytic HCMV infection and reactivation from the latent state, contains one well-characterized AP-1 site and a second candidate interaction site. In this study we explored the role of these AP-1 elements in the context of the infection. We ﬁrst show that the distal candidate AP-1 motif binds c-Fos/c-Jun heterodimers (AP-1 complex) and confers c-Fos/c-Jun-mediated activity to a core promoter. Site-directed mutagenesis studies indicate that both AP-1 response elements are critical for 12- O -tetradecanoylphorbol-13-acetate (TPA)-enhanced MIEP activity in transient-transfection assays. In marked contrast to the results obtained with the isolated promoter, disruption of the AP-1 recognition sites of the MIEP in the context of the infectious HCMV genome has no signiﬁcant inﬂuence on the expression of the MIE protein IE1 or viral replication in different cell types. Moreover, a chimeric murine CMV driven by the HCMV MIEP (hMCMV-ES) with the two AP-1 binding sites mutated is not compromised in virulence, is able to grow and disseminate to different organs of the newborn mice as efﬁciently as the parental virus, and is competent in reactivation. We show, however, that combined inactivation of the enhancer AP-1 and NF- (cid:1) B recognition sites leads to an attenuation of the hMCMV-ES in the neonatal murine infection model, not observed when each single element is abolished. Altogether, these results underline the functional redundancy of the MIEP elements, highlighting the plasticity of this region, which probably evolved to ensure maximal transcriptional performance across many diverse environments. were analyzed Filters were incubated with anti-HCMV IE1 or anti- (cid:7) -actin speciﬁc monoclonal antibodies, MAB810 (Chemicon) and A2066 (Sigma), respectively. As secondary antibodies, horseradish peroxidase-conjugated goat anti-mouse immunoglobulin G (W402B; Promega) or peroxidase-conjugated goat anti-rabbit immunoglobulin G (111-035-003; Jackson) was used. Blots were developed by using the Immuno-Star horseradish peroxidase (HRP) substrate (Bio-Rad) according to the manufacturer’s protocol. Mouse infections. Three-day-old BALB/cOlaHsd mice were obtained from Harlan (Netherlands) and housed in the vivarium (University of Barcelona) under speciﬁc-pathogen-free conditions. To assess levels of virulence, 3-day-old BALB/c mice were inoculated intraperitoneally (i.p.) with increasing doses of tissue culture-derived hMCMV-ES recombinants (ranging from 1 (cid:8) 10 3 to 2 (cid:8) 10 6 PFU per mouse in a ﬁnal volume of 40 (cid:5) l) and their survival was monitored daily for at least 40 days after infection. Data points were ﬁtted to a sigmoidal function available at Origin software, and the 50% lethal dose (LD 50 ) values were estimated from these curves. To analyze the replication of the hMCMV-ES recombinants in vivo , mice were i.p. inoculated with 5 (cid:8) 10 4 PFU of tissue culture-propagated virus. At designated times after infection, mice were sacriﬁced, and speciﬁc organs were removed and harvested as a 10% (wt/vol) tissue homogenate. Tissue homogenates were sonicated and centrifuged, and viral titers from the supernatants were determined by standard plaque assays, includ- ing centrifugal enhancement of infectivity (29) on MEFs. An ex vivo assay was utilized to detect reactivated virus from spleen