DNA-PK phosphorylation at Ser2056 during adenovirus E4 mutant infection is promoted by viral DNA replication and independent of the MRN complex.

Adenovirus (Ad) early region 4 (E4) mutants activate cellular DNA damage responses (DDRs) that include non-homologous end joining (NHEJ) pathways mediated by the DNA repair kinase DNA-PK and its associated factors Ku70/Ku86. NHEJ results in concatenation of the viral linear double-stranded DNA genome and inhibits a productive infection. E4 proteins normally prevent activation of cellular DDRs in wild-type Ad type 5 (Ad5) infections, thereby promoting efficient viral growth. The purpose of this study was to evaluate the factors that govern DNA-PK activation during adenovirus infection. Our data indicate that viral DNA replication promotes DNA-PK activation, which is required for genome concatenation by NHEJ. Although the Mre11/Rad50/Nbs1 (MRN) DDR sensor complex is not required for DNA-PK activation, Mre11 is important for recruitment of the NHEJ factor Ku86 to viral replication centers. Our study addresses the interplay between the DNA-PK and MRN complexes during viral genome concatenation by NHEJ.

An Adenovirus early region 4 deletion mutant induces G2/M arrest via ATM activation and reduces expression of the mitotic marker phosphorylated (ser10) histone 3.

Adenovirus (Ad) type 5 (Ad5) early region 4 (E4) proteins inhibit the DNA damage response (DDR) including activation of the DDR kinase ATM and its substrates, which can induce G2/M cell cycle arrest. Infection with Ad5 or the E4 deletion mutant H5dl1007 (1007) resulted in the accumulation of post G1 cells with > 2 N cellular DNA content. A greater fraction of cells with 4 N DNA content was observed in 1007 infections compared to Ad5; this population was dependent on activation of ATM. G2/M checkpoint kinases, phosphorylated Chk2 (pChk2), and phosphorylated Cdk1 (pCdk1) were upregulated in 1007 infections, and 1007 showed reduced levels of the mitosis marker phosphorylated (Ser10) histone 3 compared to Ad5. Our results show that E4 mutant activation of ATM induces G2/M arrest via activation of checkpoint kinases, thereby contributing to viral-mediated regulation of the cell cycle.

Localization of the kinase Ataxia Telangiectasia Mutated to Adenovirus E4 mutant DNA replication centers is important for its inhibitory effect on viral DNA accumulation.

Adenovirus (Ad) type 5 (Ad5) E4 deletion mutants including H5dl1007 (E4-) induce a DNA damage response (DDR) that activates the kinase ataxia-telangiectasia mutated (ATM), which can interfere with efficient viral DNA replication. We find that localization of active phosphorylated ATM (pATM) to E4- viral replication centers (VRCs) is important for its inhibitory effect. ATM is necessary for localization of RNF8 and 53BP1 to E4 mutant VRCs, while recruitment of DDR factors Mre11, Mdc1 and γH2AX is ATM-independent, raising the possibility that ATM may affect viral chromatin at VRCs. We assessed E4- and Ad5 chromatin organization by micrococcal nuclease (MN) digestion. A significant fraction of Ad5 DNA is somewhat resistant to MN digestion, whereas E4- DNA is more susceptible. ATM inhibition increases the fraction of E4- DNA that is resistant to MN digestion. Our results address possible mechanisms through which ATM inhibits E4- DNA replication.

The kinase activity of ataxia-telangiectasia mutated interferes with adenovirus E4 mutant DNA replication.

Adenovirus (Ad) mutants that lack early region 4 (E4) are unable to produce the early regulatory proteins that normally inactivate the Mre11/Rad50/Nbs1 (MRN) sensor complex, which is a critical component for the ability of cells to respond to DNA damage. E4 mutant infection therefore activates a DNA damage response, which in turn interferes with a productive viral infection. MRN complex proteins localize to viral DNA replication centers in E4 mutant-infected cells, and this complex is critical for activating the kinases ataxia-telangiectasia mutated (ATM) and ATM and Rad3-related (ATR), which phosphorylate numerous substrates important for DNA repair, cell cycle checkpoint activation, and apoptosis. E4 mutant growth defects are substantially rescued in cells lacking an intact MRN complex. We have assessed the role of the downstream ATM and ATR kinases in several MRN-dependent E4 mutant phenotypes. We did not identify a role for either ATM or ATR in "repair" of E4 mutant genomes to form concatemers. ATR was also not observed to contribute to E4 mutant defects in late protein production. In contrast, the kinase activity of ATM was important for preventing efficient E4 mutant DNA replication and late gene expression. Our results suggest that the MRN complex interferes with E4 mutant DNA replication at least in part through its ability to activate ATM.

Differential activation of cellular DNA damage responses by replication-defective and replication-competent adenovirus mutants.

Adenovirus (Ad) mutants that lack early region 4 (E4) activate the phosphorylation of cellular DNA damage response proteins. In wild-type Ad type 5 (Ad5) infections, E1b and E4 proteins target the cellular DNA repair protein Mre11 for redistribution and degradation, thereby interfering with its ability to activate phosphorylation cascades important during DNA repair. The characteristics of Ad infection that activate cellular DNA repair processes are not yet well understood. We investigated the activation of DNA damage responses by a replication-defective Ad vector (AdRSVßgal) that lacks E1 and fails to produce the immediate-early E1a protein. E1a is important for activating early gene expression from the other viral early transcription units, including E4. AdRSVßgal can deliver its genome to the cell, but it is subsequently deficient for viral early gene expression and DNA replication. We studied the ability of AdRSVßgal-infected cells to induce cellular DNA damage responses. AdRSVßgal infection does activate formation of foci containing the Mdc1 protein. However, AdRSVßgal fails to activate phosphorylation of the damage response proteins Nbs1 and Chk1. We found that viral DNA replication is important for Nbs1 phosphorylation, suggesting that this step in the viral life cycle may provide an important trigger for activating at least some DNA repair proteins.

Nbs1-dependent binding of Mre11 to adenovirus E4 mutant viral DNA is important for inhibiting DNA replication.

Adenovirus (Ad) infections stimulate the activation of cellular DNA damage response and repair pathways. Ad early regulatory proteins prevent activation of DNA damage responses by targeting the MRN complex, composed of the Mre11, Rad50 and Nbs1 proteins, for relocalization and degradation. In the absence of these viral proteins, Mre11 colocalizes with viral DNA replication foci. Mre11 foci formation at DNA damage induced by ionizing radiation depends on the Nbs1 component of the MRN complex and is stabilized by the mediator of DNA damage checkpoint protein 1 (Mdc1). We find that Nbs1 is required for Mre11 localization at DNA replication foci in Ad E4 mutant infections. Mre11 is important for Mdc1 foci formation in infected cells, consistent with its role as a sensor of DNA damage. Chromatin immunoprecipitation assays indicate that both Mre11 and Mdc1 are physically bound to viral DNA, which could account for their localization in viral DNA containing foci. Efficient binding of Mre11 to E4 mutant DNA depends on the presence of Nbs1, and is correlated with a significant E4 mutant DNA replication defect. Our results are consistent with a model in which physical interaction of Mre11 with viral DNA is mediated by Nbs1, and interferes with viral DNA replication.

Simultaneous detection of adenovirus RNA and cellular proteins by fluorescent labeling in situ.

Investigating the cell biology of gene expression requires methodologies for localizing RNA relative to proteins involved in RNA transcription, processing, and export. Adenovirus is an important model system for the analysis of eukaryotic gene expression and is also being used to investigate the organization of gene expression within the nucleus. Here are described the combined in situ hybridization and immunofluorescence staining techniques that have been used to study the localization of viral RNA relative to nuclear structures that contain splicing factors.

The cellular Mre11 protein interferes with adenovirus E4 mutant DNA replication.

Adenovirus type 5 (Ad5) relocalizes and degrades the host DNA repair protein Mre11, and efficiently initiates viral DNA replication. Mre11 associates with Ad E4 mutant DNA replication centers and is important for concatenating viral genomes. We have investigated the role of Mre11 in the E4 mutant DNA replication defect. RNAi-mediated knockdown of Mre11 dramatically rescues E4 mutant DNA replication in cells that do or do not concatenate viral genomes, suggesting that Mre11 inhibits DNA replication independent of genome concatenation. The mediator of DNA damage checkpoint 1 (Mdc1) protein is involved in recruiting and sustaining Mre11 at sites of DNA damage following ionizing radiation. We observe foci formation by Mdc1 in response to viral infection, indicating that this damage response protein is activated. However, knockdown of Mdc1 does not prevent Mre11 from localizing at viral DNA replication foci or rescue E4 mutant DNA replication. Our results are consistent with a model in which Mre11 interferes with DNA replication when it is localized at viral DNA replication foci.

Genome concatenation contributes to the late gene expression defect of an adenovirus E4 mutant.

Adenovirus mutants that lack the entire E4 region are severely defective for late gene expression. E4 mutant genomes are also concatenated by host double strand break repair (DSBR) proteins. We find that E4 mutant late gene expression improves in MO59J cells that fail to form genome concatemers. DSBR kinase inhibitors interfere with genome concatenation and also stimulate late gene expression. Concatenation of E4 mutant genomes interferes with cytoplasmic accumulation of viral late messages and leads to reduced late protein levels and poor viral yields following high multiplicity infection. However, failure to concatenate viral genomes did not rescue either the DNA replication defect or virus yield following low multiplicity E4 mutant infection. Our results indicate that if the E4 mutant DNA replication defect is overcome by high multiplicity infection, concatenation of the replicated genomes by host DSBR interferes with viral late gene expression.

Evaluating the role of CRM1-mediated export for adenovirus gene expression.

A complex of the Adenovirus (Ad) early region 1b 55-kDa (E1b-55kDa) and early region 4 ORF6 34-kDa (E4-34kDa) proteins promotes viral late gene expression. E1b-55kDa and E4-34kDa have leucine-rich nuclear export signals (NESs) similar to that of HIV Rev. It was proposed that E1b-55kDa and/or E4-34kDa might promote the export of Ad late mRNA via their Rev-like NESs, and the transport receptor CRM1. We treated infected cells with the cytotoxin leptomycin B to inhibit CRM1-mediated export; treatment initially delays the onset of late gene expression, but this activity completely recovers as the late phase progresses. We find that the E1b-55kDa NES is not required to promote late gene expression. Previous results showed that E4-34kDa-mediated late gene expression does not require an intact NES (J. Virol. 74 (2000), 6684-6688). Our results indicate that these Ad regulatory proteins promote late gene expression without intact NESs or active CRM1.

Adenovirus E4-34kDa requires active proteasomes to promote late gene expression.

A complex of the Adenovirus (Ad) early region 1b 55-kDa protein (E1b-55kDa) and the early region 4 ORF6 34-kDa protein (E4-34kDa) promotes viral late RNA accumulation in the cytoplasm while inhibiting the transport of most newly synthesized cellular mRNA. The E4 ORF3 11-kDa protein (E4-11kDa) functionally compensates for at least some of the activities of this complex. We find that the same large central region of E4-34kDa that is required for proteasome-mediated degradation of p53 (J. Virol. 75, (2001) 699-709) is also required to promote viral late gene expression in a complementation assay. E4-34kDa does not promote late gene expression in complementation assays performed in the presence of proteasome inhibitors. A proteasome inhibitor also dramatically reduced late gene expression by a virus that lacks the E4-11kDa gene and therefore relies on E4-34kDa for late gene expression. Our results suggest that E4-34kDa activity in promoting late gene expression depends on the proteasome.

Adenovirus early region 4 promotes the localization of splicing factors and viral RNA in late-phase interchromatin granule clusters.

Adenovirus early region 4 (E4) mutants are defective for late gene expression and show reduced levels of late RNA in both the cytoplasm and the nucleus. These reductions reflect a posttranscriptional defect in the production of viral late RNA. We find that E4 mutants form replication centers during the initial stages of infection and are able to redistribute splicing factors to transcription sites that surround viral replication centers. However, E4 mutant infected cultures have reduced numbers of cells with splicing factors localized in enlarged interchromatin granule clusters during the late phase. Although the late-phase interchromatin granule clusters that formed in wild-type and E4 mutant infected cells had similar levels of poly(A) RNA, they contained reduced levels of viral RNA. These results suggest that E4 mutants do not efficiently accumulate viral late RNA in late-phase interchromatin granule clusters following the onset of late RNA transcription.

Release of snRNP and RNA from transcription sites in adenovirus-infected cells.

Small nuclear ribonucleoprotein (snRNP) splicing factors colocalize with nascent RNA in the nucleus of adenovirus-infected cells in a pattern that appears as a series of rings surrounding viral replication centers. We have studied the release of snRNP and RNA from transcription sites following transcription inhibition by actinomycin D. SnRNP, poly(A) RNA, and viral RNA were no longer detected in the ring pattern following transcription inhibition and were instead detected in nuclear clusters. Release of snRNP from transcription sites was blocked when transcription was inhibited at 4 degrees C, suggesting that release requires temperature-dependent processes. Release of snRNP was also inhibited when transcription was blocked in the presence of 9-beta-D-arabinofuranosyladenine, to inhibit 3'-end cleavage and polyadenylation, or staurosporine, to inhibit kinases. By contrast, release of snRNP was not inhibited when transcription was blocked in the presence of cordycepin, to inhibit RNA polyadenylation without affecting 3'-end cleavage, or okadaic acid, to inhibit phosphatase activity. Our results suggest that temperature-dependent processes involved in the release of splicing factors from transcription sites could include 3'-end cleavage of pre-mRNA and phosphorylation events inhibited by stauropsorine.