Antagonism between Members of the CNC-bZIP Family and the Immediate-Early Protein IE2 of Human Cytomegalovirus

Abstract

The HCMV IE2 protein negatively autoregulates its own expression as well as represses the transactivation activity of p53. Using the repression domain of IE2 as bait in the yeast two-hybrid system, Nrf1 and Nrf2, members of the CNC-bZIP family, were found to be IE2-interacting proteins. Residues 331-448 encompassing the DNA-binding and the dimerization domains of Nrf1 are sufficient for the interaction. The interaction was further confirmed in vitro by a glutathione S-transferase pull-down assay and in vivo by co-immunoprecipitation. In transient transfection studies, transcription driven by six copies of an NF-E2 site or by chimeric proteins between the DNA-binding domain of LexA and members of the CNC-bZIP family is repressed by IE2. Importantly, the DNA binding activity of the Nrf1/MafK heterodimer is not impeded by IE2. In a parallel study, CNC-bZIP factors attenuate the negative autoregulation of IE2. The attenuation could be explained by the finding that Nrf1 functions alone and synergistically with its heterodimerization partner, MafK, in inhibiting the DNA binding activity of IE2. Taken together, these results demonstrate the existence of antagonism between members of the CNC-bZIP family and IE2.Human cytomegalovirus (HCMV), 1 a member of the ␤ subgroup of herpesviruses, has a double-stranded DNA genome of 229,354 base pairs with a potential to encode more than 200 proteins (1). A number of immediate-early (IE) proteins of HCMV are made following entry of the virus into cells (2). Among them the IE2 86-kDa protein (referred hereafter as IE2) is most studied. IE2 appears to be a promiscuous transactivator of viral and cellular gene expression (3-5). IE2 stimulates transcription by interacting with general transcription factors and/or gene-specific factors (6 -9). In addition, IE2 negatively autoregulates its own expression by binding to a short nucleotide sequence, termed the cis repression signal (CRS), located immediately downstream of the TATA box of the HCMV major immediate early promoter (MIEP) (10, 11). Binding of IE2 to CRS prevents the recruitment of RNA polymerase II to the promoter via steric hindrance (12). Thus, IE2-mediated, CRSdependent transcriptional repression is both passive and position-dependent. The efficiency of IE2-mediated repression of MIEP seems to be cell-dependent (4, 10, 13), strongly suggesting that the IE2 activity may be modulated by post-translational modification and/or by interaction with cellular proteins. Indeed, phosphorylation has been shown to be one of these factors (14).Furthermore, recent studies demonstrate that IE2 can be converted into a trans-repressor following binding to p53 (15,16). A transcriptional repression domain has been mapped to the C terminus of IE2 (16), which is also required for its transcriptional activation, DNA binding, and interaction with CREB, c-Jun, RB, TBP, and TFIIB (6 -9). The DNA-binding and the repression domains of IE2 seem to be overlapping, because an IE2 mutant devoid of DNA binding activity also loses its repression activity (1...

Host-viral effects of chromatin assembly factor 1 interaction with HCMV IE2

“…Yeast two-hybrid assays were performed as described [45]. GST pull-down, IP and western analyses were all carried out as indicated [54,73].…”

“…Thus, recruitment of p48 to the viral DNA replication center cannot ensure the involvement of chromatin assembly activity. In our previous yeast two-hybrid assay to screen for IE2-interacting proteins [45], one of positive clones identified using the C-terminal half (aa 291-579) of IE2 as bait is the p150 fragment containing amino acids 87 to 938 (full-length p150: 938 residues). Here, we show that CAF1 interacts with HCMV IE2 through p150 in HCMV-infected cells and by this interaction CAF1 is mislocated to viral DNA replication foci to facilitate HCMV genome replication.…”

YY1AP, A Novel Co-activator of YY1

“…The templates were pCITE-YY1AP and its YY1AP deletion mutants. The protocol of GST pull-down has been described previously (28,29). After being washed with Buffer D (28), the bound proteins were eluted and analyzed by electrophoresis on a 10% SDS-PAGE.…”

Gene-specific Transcriptional Activation Mediated by the p150 Subunit of the Chromatin Assembly Factor 1

“…We identified that the p150 fragment aa 87-938 interacted with the C-terminal half (aa 291-579) of IE2. Because proteins obtained using this strategy, such as Nrf1 and Nrf2 (24), antagonized the IE2-mediated transcriptional repression of its own promoter, the major IE promoter (MIEP), we examined if p150 has a similar effect. Unexpectedly, in the absence of IE2, p150, but not p60, increased the MIEP activity in a dose-dependent manner.…”

“…Previously, a yeast two-hybrid assay was used to screen for proteins associating with the immediate early protein 2 (IE2) of the human cytomegalovirus (HCMV) (24). We identified that the p150 fragment aa 87-938 interacted with the C-terminal half (aa 291-579) of IE2.…”