[PMC free article] [PubMed] [CrossRef] [Google Scholar] 12. replication, while overexpression enhances viral genome numbers in infected cells. Additionally, Nek9 was found to colocalize with the viral E4 orf3 protein, a repressor of cellular stress response. Significantly, Nek9 was also shown to associate with viral and cellular promoters and appears to function as a transcriptional repressor, representing the first instance of Nek9 playing a role in gene regulation. Overall, these results highlight the complexity of virus-host interactions and identify a new role for the cellular protein Nek9 during infection, suggesting a role for Nek9 in regulating p53 target gene expression. IMPORTANCE In the arms race that exists between a pathogen and its host, Sapacitabine (CYC682) each has continually evolved mechanisms to either promote or prevent infection. In order to successfully replicate and spread, a virus must overcome every mechanism that a cell can assemble to block infection. On the other hand, to counter viral spread, cells must have multiple mechanisms to stifle viral replication. In the present study, we add to our understanding of how the human adenovirus is able to circumvent cellular roadblocks to replication. We show Rabbit Polyclonal to HP1gamma (phospho-Ser93) that the virus uses a cellular protein, Nek9, in order to block activation of p53-regulated gene as a protein responsible for regulation of mitotic progression (1). There is only one NimA kinase in (promoter together with E1A. This represents, as far as we are aware, the first report of Nek9 playing a role in transcriptional regulation. Together, these results highlight a novel function for Nek9 in the innate antiviral response via its role in the downregulation of expression and identify a new pathway on which E1A impinges in order to enable a productive viral infection. Importantly, our study highlights the complexity and importance of silencing p53 target genes by HAdV and Sapacitabine (CYC682) identifies a cellular factor, Nek9, coopted by the virus for this purpose. MATERIALS AND METHODS Antibodies. Mouse monoclonal anti-E1A M73 and M58 antibodies were previously described (18) and were grown in-house and used as the hybridoma supernatant. For immunoprecipitations (IPs), 25 l was used, and for Western blot assays a dilution of 1 1:400 was used. 12CA5 antihemagglutinin (anti-HA) mouse monoclonal antibody was previously described (19); 25 l of hybridoma supernatant was used in chromatin immunoprecipitation (ChIP) experiments. Mouse monoclonal anti-myc 9E10 antibody was previously described (20) and was grown in-house. For IPs, 50 l of 9E10 hybridoma supernatant was used, while for Western blots the supernatant was used at a 1:100 dilution. Mouse monoclonal anti-72k DNA-binding protein (DBP) antibody was previously described (21) and was used at a dilution of 1 1:400 for Western blotting. Anti-adenovirus type 5 (ab6982) and anti-Nek9 (ab138488) antibodies were purchased from Abcam and were used at recommended dilutions. Rabbit polyclonal anti-Nek9 antibody was previously described (6) and was a generous gift from Peter Whyte. Rat monoclonal anti-E4 orf3 antibody was previously described (22) and was a generous gift from Thomas Dobner. Cell and virus culture. IMR-90 (ATCC CCL-186), HT1080 (ATCC CCL-121), and MEF/3T3-Nek9V5 cells were grown in Dulbecco’s modified Eagle’s medium (HyClone) supplemented with 10% fetal bovine serum (Invitrogen) and streptomycin-penicillin (HyClone). All virus infections were carried out in serum-free medium for 1 h, after Sapacitabine (CYC682) which saved complete medium was added without removal of the Sapacitabine (CYC682) infection medium. MEF/3T3-Nek9V5 cells were a generous gift from Peter Whyte; these cells express a tetracycline (Tet)-regulated murine Nek9 with a C-terminal V5 tag. To induce the expression of Nek9, doxycycline medium was removed from the cells, cells were washed with phosphate-buffered saline (PBS) three times, and Tet-free medium was applied to the cells for 2 h, washed off again, and replaced with Tet-free medium. Cells were then incubated for at least 24 h prior to viral infection in order to overexpress Nek9. Cells were maintained in Tet-free medium for the duration of the experiment in order to maintain Nek9 overexpression. Chromatin immunoprecipitation. ChIP was carried out essentially as previously described (23). IMR-90 cells were infected with the indicated adenoviruses at a multiplicity of infection (MOI) of 5 and harvested 24 h after infection for ChIP analysis. For immunoprecipitation of E1A, monoclonal M73 and M58 antibodies were used. For immunoprecipitation of Nek9, the polyclonal anti-Nek9 antibody was used (6). Mouse monoclonal 12CA5 anti-HA antibody was used as a negative IgG control. PCRs were carried out for HAdV5 early and major late promoters using SYBR Select master mix for CFX (Applied Biosystems) according to the manufacturer’s directions,.