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.     

E1B 55k-independent dissociation of the DNA ligase IV/XRCC4 complex by E4 34k during adenovirus infection

The ligase IV/XRCC4 complex plays a central role in DNA double-strand break repair by non-homologous end joining (NHEJ). During adenovirus infection, NHEJ is inhibited by viral proteins E4 34k and E1B 55k, which redirect the Cul5/Rbx1/Elongin BC ubiquitin E3 ligase to polyubiquitinate and promote degradation of ligase IV. In cells infected with E1B 55k-deficient adenovirus, ligase IV could not be found in XRCC4-containing complexes and was observed in a novel ligase IV/E4 34k/Cul5/Elongin BC complex. These observations suggest that dissociation of the ligase IV/XRCC4 complex occurs at an early stage in E4 34k-mediated degradation of ligase IV and indicate a role for E4 34k in dissociation of the ligase IV/XRCCC4 complex. Expression of E4 34k alone was not sufficient to dissociate the ligase IV/XRCC4 complex, which indicates a requirement for an additional, as yet unidentified, factor in E1B 55k-independent dissociation of the ligase IV/XRCC4 complex.     

Human papillomavirus E1 and E2 mediated DNA replication is not arrested by DNA damage signalling

Integration of human papillomaviruses into that of the host promotes genomic instability and progression to cancer; factors that promote integration remain to be fully identified. DNA damage agents can promote double strand breaks during DNA replication providing substrates for integration and we investigated the ability of DNA damage to regulate HPV E1 and E2 mediated DNA replication. Results demonstrate that HPV E1 and E2 replication is not arrested following DNA damage, both in vivo and in vitro, while replication by SV40 Large T antigen is arrested and ATR is the candidate kinase for mediating the arrest. LTAg is a target for PIKK DNA damage signalling kinases, while E1 is not. We propose that the failure of E1 to be targeted by PIKKs allows HPV replication in the presence of DNA damaging agents. Such replication will result in double strand breaks in the viral genome ultimately promoting viral integration and cervical cancer.     

The human papillomavirus type 11 E1/\E4 protein is not essential for viral genome amplification

An abundant human papillomavirus (HPV) protein E1/\E4 is expressed late in the virus life cycle in the terminally differentiated layers of epithelia. The expression of E1/\E4 usually coincides with the onset of viral DNA amplification. However, the function of E1/\E4 in viral life cycle is not completely understood. To examine the role of E1/\E4 in the virus life cycle, we introduced a single nucleotide change in the HPV-11 genome to result in a truncation of E1/\E4 protein without affecting the E2 amino acid sequence. This mutated HPV-11 genome was introduced into a human foreskin keratinocyte cell line immortalized by the catalytic subunit of human telomerase, deficient in p16(INK4a) expression, and previously shown to support the HPV-11 life cycle when grown in organotypic raft culture. We have demonstrated that E1/\E4 is dispensable for HPV-11 viral DNA amplification in the late stages of the viral life cycle.     

Matching complementing functions of transformed cells with stable expression of selected viral genes for production of E1-deleted adenovirus vectors

Production of E1-deleted adenovirus (rAd) vectors requires complementation by E1A and E1B functions provided by the production cell line. The two cell lines most commonly used for production of rAd vectors, 293 and Per.C6, were derived from human primary cells and contain contiguous E1A and E1B sequences from the Ad genome. As an alternative system, we tested complementation of rAd vectors using sequential transfection of individual E1A and E1B expression cassettes into A549 human lung tumor cells, which support highly efficient replication of wild type adenovirus. We found that E1A function could be complemented in A549 cells by the mutant E1Adl01/07, and that E1B function could be provided in such cells using only the 55K E1B gene. Production yields in the resulting producer cell line, designated SL0003, were similar to those obtained from 293 cells without generation of detectable recombinant replication competent adenovirus.     

ATR phosphorylates SMARCAL1 to prevent replication fork collapse

The DNA damage response kinase ataxia telangiectasia and Rad3-related (ATR) coordinates much of the cellular response to replication stress. The exact mechanisms by which ATR regulates DNA synthesis in conditions of replication stress are largely unknown, but this activity is critical for the viability and proliferation of cancer cells, making ATR a potential therapeutic target. Here we use selective ATR inhibitors to demonstrate that acute inhibition of ATR kinase activity yields rapid cell lethality, disrupts the timing of replication initiation, slows replication elongation, and induces fork collapse. We define the mechanism of this fork collapse, which includes SLX4-dependent cleavage yielding double-strand breaks and CtIP-dependent resection generating excess single-stranded template and nascent DNA strands. Our data suggest that the DNA substrates of these nucleases are generated at least in part by the SMARCAL1 DNA translocase. Properly regulated SMARCAL1 promotes stalled fork repair and restart; however, unregulated SMARCAL1 contributes to fork collapse when ATR is inactivated in both mammalian and Xenopus systems. ATR phosphorylates SMARCAL1 on S652, thereby limiting its fork regression activities and preventing aberrant fork processing. Thus, phosphorylation of SMARCAL1 is one mechanism by which ATR prevents fork collapse, promotes the completion of DNA replication, and maintains genome integrity.     

The full-length E1E4 protein of human papillomavirus type 18 modulates differentiation-dependent viral DNA amplification and late gene expression

Activation of the productive phase of the human papillomavirus (HPV) life cycle in differentiated keratinocytes is coincident with high-level expression of E1E4 protein. To determine the role of E1E4 in the HPV replication cycle, we constructed HPV18 mutant genomes in which expression of the full-length E1E4 protein was abrogated. Undifferentiated keratinocytes containing mutant genomes showed enhanced proliferation when compared to cells containing wildtype genomes, but there were no differences in maintenance of viral episomes. Following differentiation, cells with mutant genomes exhibited reduced levels of viral DNA amplification and late gene expression, compared to wildtype genome-containing cells. This indicates that HPV18 E1E4 plays an important role in regulating HPV late functions, and it may also function in the early phase of the replication cycle. Our finding that full-length HPV18 E1E4 protein plays a significant role in promoting viral genome amplification concurs with a similar report with HPV31, but is in contrast to an HPV11 study where viral DNA amplification was not dependent on full-length E1E4 expression, and to HPV16 where only C-terminal truncations in E1E4 abrogated vegetative genome replication. This suggests that type-specific differences exist between various E1E4 proteins.     

The adenovirus E4 11 k protein binds and relocalizes the cytoplasmic P-body component Ddx6 to aggresomes

The adenovirus E4 11 k protein, product of E4 ORF3, is required in infection for processes including normal accumulation of viral late mRNAs. 11 k restructures both the nucleus and cytoplasm of infected cells by relocalizing specific host cell target proteins, most strikingly components of nuclear PML oncogenic domains. It is likely that in many cases relocalization inactivates target proteins to produce 11 k's effects, although the mechanism and targets for stimulation of late mRNA accumulation is unknown. We have identified a new set of proteins relocalized by 11 k: at least five protein components of cytoplasmic mRNA processing bodies (p-bodies) are found in 11 k-induced cytoplasmic aggresomes, sites where proteins are inactivated or destroyed. One of these p-body proteins, RNA helicase Ddx6, binds 11 k, suggesting a mechanism for relocalization. Because p-bodies are sites for mRNA degradation, their modification by 11 k may provide an explanation for the role of 11 k in viral late mRNA accumulation.     

JC virus agnoprotein enhances large T antigen binding to the origin of viral DNA replication: Evidence for its involvement in viral DNA replication

Agnoprotein is required for the successful completion of the JC virus (JCV) life cycle and was previously shown to interact with JCV large T-antigen (LT-Ag). Here, we further characterized agnoprotein's involvement in viral DNA replication. Agnoprotein enhances the DNA binding activity of LT-Ag to the viral origin (Ori) without directly interacting with DNA. The predicted amphipathic α-helix of agnoprotein plays a major role in this enhancement. All three phenylalanine (Phe) residues of agnoprotein localize to this α-helix and Phe residues in general are known to play critical roles in protein-protein interaction, protein folding and stability. The functional relevance of all Phe residues was investigated by mutagenesis. When all were mutated to alanine (Ala), the mutant virus (F31AF35AF39A) replicated significantly less efficiently than each individual Phe mutant virus alone, indicating the importance of Phe residues for agnoprotein function. Collectively, these studies indicate a close involvement of agnoprotein in viral DNA replication.     

Construction of human cell lines which contain and express the adenovirus DNA binding protein gene by cotransformation with the HSV-1 tk gene

We have introduced the DNA binding protein (DBP) gene of human adenovirus type 5 (Ad5) into high molecular weight DNA of permissive human cells by cotransformation of tk- cells with the cloned DBP and HSV-1 thymidine kinase genes. 110 tk+ cell lines were isolated after selection in HAT medium. The amount and arrangement of adenovirus sequences in the tk+ cell lines were analyzed by restriction endonuclease digestion and filter hybridization. Twelve of the 110 lines carry at least a segment of the DBP gene while only three of these contain the entire DBP gene at approximately one copy per cell. Cytoplasmic, polyadenylated DBP mRNA is made in all three cell lines though the amount is very low compared to that present in infected HeLa cells. The cell line U13-2 which contains approximately 1/30 the steady-state level of DBP mRNA found in infected HeLa cells produces a few percent of the amount of DBP made during the peak period of DBP synthesis in infected cells. The other two lines contain lower levels of DBP mRNA and do not synthesize detectable levels of the protein. When these DBP-tk+ cell lines are infected with adenovirus mutants containing temperature-sensitive (ts) mutations in the DBP gene, only U13-2 permits some viral DNA replication (and hence late gene expression) at the nonpermissive temperature, indicating that sufficient quantities of DBP from the integrated gene are produced to allow complementation of the ts mutation in this cell line. However, growth of these ts mutants (as measured by virus production) is only partially complemented in U13-2 at the nonpermissive temperature.     

SUMO-targeted ubiquitin E3 ligase RNF4 is required for the response of human cells to DNA damage

Here we demonstrate that RNF4, a highly conserved small ubiquitin-like modifier (SUMO)-targeted ubiquitin E3 ligase, plays a critical role in the response of mammalian cells to DNA damage. Human cells in which RNF4 expression was ablated by siRNA or chicken DT40 cells with a homozygous deletion of the RNF4 gene displayed increased sensitivity to DNA-damaging agents. Recruitment of RNF4 to double-strand breaks required its RING and SUMO interaction motif (SIM) domains and DNA damage factors such as NBS1, mediator of DNA damage checkpoint 1 (MDC1), RNF8, 53BP1, and BRCA1. In the absence of RNF4, these factors were still recruited to sites of DNA damage, but 53BP1, RNF8, and RNF168 displayed delayed clearance from such foci. SILAC-based proteomics of SUMO substrates revealed that MDC1 was SUMO-modified in response to ionizing radiation. As a consequence of SUMO modification, MDC1 recruited RNF4, which mediated ubiquitylation at the DNA damage site. Failure to recruit RNF4 resulted in defective loading of replication protein A (RPA) and Rad51 onto ssDNA. This appeared to be a consequence of reduced recruitment of the CtIP nuclease, resulting in inefficient end resection. Thus, RNF4 is a novel DNA damage-responsive protein that plays a role in homologous recombination and integrates SUMO modification and ubiquitin signaling in the cellular response to genotoxic stress.     

The human autoantigen MCP1 is required during early stages of DNA replication

Metaphase chromosome protein 1 (MCP1) is a nuclear autoantigen that is associated with condensed chromosomes throughout mitosis. During interphase, this antigen shows a speckle distribution in the nucleus, excluding the nucleolus. Additionally, MCP1 binds tightly to the scaffold/matrix component of nuclei and isolated chromosomes. In order to determine the in-vivo localization of the antigen, we have expressed MCP1 fused to EGFP in tissue culture cells. The results demonstrate that MCP1 is located in the nucleus during interphase and during mitosis associates tightly to condensed chromosomes. Furthermore, microinjection of specific antibody confirms these results. We have used a specific monoclonal antibody (mAb 402) against MCP1 to assess the function of this antigen during cell cycle progression. HeLa and Ptk-2 cells that were microinjected into the nucleus and/or cytoplasm at G1/S and very early S phase were not able to progress and complete DNA replication. However, injection of mAb 402 at mid or late S phase does not prevent completion of DNA replication and subsequent progression into mitosis. Microinjection of mAb 402 in Ptk-2 cells synchronized in mitosis did not interfere with progression of mitosis and cells divided. Our results suggest that MCP1 is required at the G1/S transition and during early S phase. This revised version was published online in July 2006 with corrections to the Cover Date.     

The ribonuclease domain function is dispensable for SLFN11 to mediate cell fate decision during replication stress response

The SLFN11 gene participates in cell fate decision following cancer chemotherapy and encodes the N-terminal ribonuclease (RNase) domain and the C-terminal helicase/ATPase domain. How these domains contribute to the chemotherapeutic response remains controversial. Here, we expressed SLFN11 containing mutations in two critical residues required for RNase activity in SLFN11^−/− cells. We found that this mutant was still able to suppress DNA damage tolerance, destabilized the stalled replication forks, and perturbed recruitment of the fork protector RAD51. In contrast, we confirmed that the helicase domain was essential to accelerate fork degradation. The fork degradation by the RNase mutant was dependent on both DNA2 and MRE11 nuclease, but not on MRE11's novel interactor FXR1. Collectively, these results supported the view that the RNase domain function is dispensable for SLFN11 to mediate cell fate decision during replication stress response.     

Expression of eukaryotic initiation factor 4E and 4E binding protein 1 in colorectal carcinogenesis

Cap dependent translation is mainly regulated at the level of the eukaryotic initiation factor 4E (eIF4E), the activity of which is controlled by phosphorylation and sequestration by its well established regulator, 4E binding protein 1 (4E-BP1). Both eIF4E and 4E-BP1 have been shown to be involved in the malignant progression of multiple human cancers, including colorectal cancer. However, the data on determining the expression of eIF4E, 4E-BP1 and their phosphorylated forms simultaneously in a single patient with colorectal cancer is lacking. Therefore the aim of our study was to explore the roles of these factors in colorectal carcinogenesis by immunohistostaining colorectal tissues (normal, low grade adenoma, high grade adenoma, and adenocarcinoma). Our results showed that the expression levels of eIF4E increased steadily as the cancer progressed from the case of benign dysplasia to an adenocarcinoma; all the while maintaining an unphosphorylated form. On the other hand, total expression levels of 4E-BP1 increased only in the premalignant state of the disease and decreased (but highly phosphorylated or inactivated) or abolished upon malignancy. Taken together, our findings suggest that strong correlations exist between the expression of eIF4E (not p-eIF4E) and tumor grade providing evidence that eIF4E expression plays a pivotal role in the malignant progression of colorectal cancer. Moreover, 4E-BP1 showed a bi-phasic level of expression during carcinogenesis, which is expressed only in hyperplasic or dysplastic tissues as an endogenous tumor suppressor molecule.     

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.     

High-affinity Rep-binding is not required for the replication of a geminivirus DNA and its satellite

The 682-nt satellite DNA (sat-DNA) of Tomato leaf curl virus (TLCV) depends on the helper virus for its replication. In contrast to the strict specificity that exists in each geminivirus for its cognate replication associated protein (Rep), TLCV sat-DNA can utilize Rep encoded by distinct geminiviruses. We have used a combination of protein-binding assays and mutagenesis to show that repeat motifs in TLCV and sat-DNA are essential for Rep-binding in vitro. Surprisingly, mutants of TLCV and sat-DNA impaired in their ability to bind TLCV Rep in vitro were infectious in tomato. Thus, in contrast to other geminiviruses reported, TLCV and sat-DNA replication is independent of the high-affinity in vitro Rep binding. These results prompt a reassessment of the current model of geminivirus replication where Rep/DNA interaction is a highly specific step in the initiation of rolling circle replication.