Andrew S. Turnell

Adenovirus E1A: remodelling the host cell, a life or death experience

It came as a considerable surprise to virologists in 1962 when John Trentin and his colleagues (Trentin et al., 1962) reported that a human adenovirus was oncogenic. At Baylor University (Houston, Texas) they had been inoculating newborn hamsters with human adenoviruses and found that adenovirus serotype 12 (Ad12) induced tumours in a high proportion of the animals injected. Other Ad serotypes (Ads) were found to be non-tumorigenic (this included Ad2, which with its closely related non-oncogenic serotype Ad5, have been the most extensively studied Ads; referred to in this review as Ad2/5). This important observation was initially greeted with some scepticism (see Gross, 1966). However, Ad12 oncogenesis was rapidly con®rmed by others (Huebner et al., 1962) and lead to more extensive in vivo and in vitro studies on the capacity of human Ads to disrupt normal cell growth control. It is not possible to review all of the literature on this subject here but there are a number of reviews that cover this subject (Gross, 1966; Gallimore et al., 1984; Williams et al., 1995). From the in vivo studies in the 1960s it soon became clear that there were indeed tumorigenic (Huebner et al., 1962; Trentin et al., 1962, Girardi et al., 1964; Pereira et al., 1965) and nontumorigenic adenoviruses (Trentin et al., 1962, 1968). As well as Ad serotype, adenovirus induced tumorigenicity was found to be dependent on virus dose (Yabe et al., 1962), host genetic constitution (Yabe et al., 1964, Yohn et al., 1965; Allison et al., 1967), age at inoculation (Yabe et al., 1962) and the hosts immune status (Yohn et al., 1965; Allison et al., 1967). Around this time tissue culture studies revealed that rodent cells were susceptible to adenovirus transformation and that both tumorigenic (McBride and Wiener, 1964) and non-tumorigenic Ads (Freeman et al., 1967) induced morphological transformation from which immortal cell lines could be easily derived. Studies on immortal rodent cell lines showed that the cells transformed by non-oncogenic viruses could be tumorigenic, but only in immunocompromised hosts (Gallimore, 1972, Gallimore et al., 1977). The next logical step in adenovirus research was to identify the virus genes responsible for transformation and/or tumorigenicity. Unlike today, no restriction enzymes were readily available and there was no Southern blotting technique (Southern, 1975). Fujinaga and Green used membrane hybridization to show that adenovirus sequences were indeed retained and expressed in Ad transformed cells (Fujinaga and Green, 1970). This was followed by the ®rst attempt to utilize restriction endonucleases and Cot analysis (Pettersson and Sambrook, 1973) to map the viral sequences in an Ad2 transformed line, Ad2/8617. In the following year, a more extensive study found that a minimal region of the virus genome representing the left hand 14% was common to nine independently isolated Ad2 transformed rat embryo cell lines (Gallimore et al., 1974). This strongly suggested, but did not prove, that the Ad transforming gene resided in this region of the Ad genome. With the development of the calcium phosphate DNA transfection technique (Graham and van der Eb, 1973) and the use of de®ned Ad DNA fragments, it was convincingly shown that this region was responsible for the induction of transformation (Graham et al., 1974a,b). RNA mapping was carried out (Sharp et al., 1974; Flint et al., 1975) which de®ned the early region transcripts (reviewed by Shenk and Flint, 1991; Shenk, 1996) and later designated E1 (E1A+E1B) E2, E3 and E4 (Kitchingman et al., 1977). Viral RNA selected on speci®c Ad DNA fragments was then used in transcription/translation assays to identify the proteins expressed from these regions and their molecular weights (Lewis et al., 1976; Harter and Lewis, 1978; Halbert et al., 1979). The E1A proteins were found to have a molecular weight range from approximately 28 to 58 kDa. The next major step in our understanding of E1A was facilitated by the isolation of the now famous 293 cells, human embryo kidney cells transformed by Ad5 DNA (Graham et al., 1977). These cells expressed the Ad5 E1 transforming proteins that provided a permissive environment for replicationand transformation-defective E1 region mutants (Frost and Williams, 1978; Graham et al., 1978). Similar cell lines were developed for the isolation of Ad12 mutants (Byrd et al., 1982). The E1 regions of Ad2/5 and Ad12 Oncogene (2001) 20, 7824 ± 7835 ã 2001 Nature Publishing Group All rights reserved 0950 ± 9232/01

DNA viruses and the cellular DNA-damage response.

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The APC/C and CBP/p300 cooperate to regulate transcription and cell-cycle progression

It is clear that a number of host-cell factors facilitate virus replication and, conversely, a number of other factors possess inherent antiviral activity. Research, particularly over the last decade or so, has revealed that there is a complex inter-relationship between viral infection and the host-cell DNA-damage response and repair pathways. There is now a realization that viruses can selectively activate and/or repress specific components of these host-cell pathways in a temporally coordinated manner, in order to promote virus replication. Thus, some viruses, such as simian virus 40, require active DNA-repair pathways for optimal virus replication, whereas others, such as adenovirus, go to considerable lengths to inactivate some pathways. Although there is ever-increasing molecular insight into how viruses interact with host-cell damage pathways, the precise molecular roles of these pathways in virus life cycles is not well understood. The object of this review is to consider how DNA viruses have evolved to manage the function of three principal DNA damage-response pathways controlled by the three phosphoinositide 3-kinase (PI3K)-related protein kinases ATM, ATR and DNA-PK and to explore further how virus interactions with these pathways promote virus replication.

Regulation of DNA-end resection by hnRNPU-like proteins promotes DNA double-strand break signaling and repair

The anaphase-promoting complex/cyclosome (APC/C) is a multicomponent E3 ubiquitin ligase that, by targeting protein substrates for 26S proteasome-mediated degradation through ubiquitination, coordinates the temporal progression of eukaryotic cells through mitosis and the subsequent G1 phase of the cell cycle. Other functions of the APC/C are, however, less well defined. Here we show that two APC/C components, APC5 and APC7, interact directly with the coactivators CBP and p300 through protein–protein interaction domains that are evolutionarily conserved in adenovirus E1A. This interaction stimulates intrinsic CBP/p300 acetyltransferase activity and potentiates CBP/p300-dependent transcription. We also show that APC5 and APC7 suppress E1A-mediated transformation in a CBP/p300-dependent manner, indicating that these components of the APC/C may be targeted during cellular transformation. Furthermore, we establish that CBP is required in APC/C function; specifically, gene ablation of CBP by RNA-mediated interference markedly reduces the E3 ubiquitin ligase activity of the APC/C and the progression of cells through mitosis. Taken together, our results define discrete roles for the APC/C–CBP/p300 complexes in growth regulation.

Regulation of the 26S proteasome by adenovirus E1A

DNA double-strand break (DSB) signaling and repair are critical for cell viability, and rely on highly coordinated pathways whose molecular organization is still incompletely understood. Here, we show that heterogeneous nuclear ribonucleoprotein U-like (hnRNPUL) proteins 1 and 2 play key roles in cellular responses to DSBs. We identify human hnRNPUL1 and -2 as binding partners for the DSB sensor complex MRE11-RAD50-NBS1 (MRN) and demonstrate that hnRNPUL1 and -2 are recruited to DNA damage in an interdependent manner that requires MRN. Moreover, we show that hnRNPUL1 and -2 stimulate DNA-end resection and promote ATR-dependent signaling and DSB repair by homologous recombination, thereby contributing to cell survival upon exposure to DSB-inducing agents. Finally, we establish that hnRNPUL1 and -2 function downstream of MRN and CtBP-interacting protein (CtIP) to promote recruitment of the BLM helicase to DNA breaks. Collectively, these results provide insights into how mammalian cells respond to DSBs.

Adenovirus 12 E4orf6 inhibits ATR activation by promoting TOPBP1 degradation

We have identified the N‐terminus of adenovirus early region 1A (AdE1A) as a region that can regulate the 26S proteasome. Specifically, in vitro and in vivo co‐precipitation studies have revealed that the 19S regulatory components of the proteasome, Sug1 (S8) and S4, bind through amino acids (aa) 4–25 of Ad5 E1A. In vivo expression of wild‐type (wt) AdE1A, in contrast to the N‐terminal AdE1A mutant that does not bind the proteasome, reduces ATPase activity associated with anti‐S4 immunoprecipitates relative to mock‐infected cells. This reduction in ATPase activity correlates positively with the ability of wt AdE1A, but not the N‐terminal deletion mutant, to significantly reduce the ability of HPV16 E6 to target p53 for ubiquitin‐mediated proteasomal degradation. AdE1A/proteasomal complexes are present in both the cytoplasm and the nucleus, suggesting that AdE1A interferes with both nuclear and cytoplasmic proteasomal degradation. We have also demonstrated that wt AdE1A and the N‐terminal AdE1A deletion mutant are substrates for proteasomal‐mediated degradation. AdE1A degradation is not, however, mediated through ubiquitylation, but is regulated through phosphorylation of residues within a C‐terminal PEST region (aa 224–238).

A novel mechanism of sodium iodide symporter repression in differentiated thyroid cancer.

Activation of the cellular DNA damage response is detrimental to adenovirus (Ad) infection. Ad has therefore evolved a number of strategies to inhibit ATM- and ATR-dependent signaling pathways during infection. Recent work suggests that the Ad5 E4orf3 protein prevents ATR activation through its ability to mislocalize the MRN complex. Here we provide evidence to indicate that Ad12 has evolved a different strategy from Ad5 to inhibit ATR. We show that Ad12 utilizes a CUL2/RBX1/elongin C-containing ubiquitin ligase to promote the proteasomal degradation of the ATR activator protein topoisomerase-IIβ–binding protein 1 (TOPBP1). Ad12 also uses this complex to degrade p53 during infection, in contrast to Ad5, which requires a CUL5-based ubiquitin ligase. Although Ad12-mediated degradation of p53 is dependent upon both E1B-55K and E4orf6, Ad12-mediated degradation of TOPBP1 is solely dependent on E4orf6. We propose that Ad12 E4orf6 has two principal activities: to recruit the CUL2-based ubiquitin ligase and to act as substrate receptor for TOPBP1. In support of the idea that Ad12 E4orf6 specifically prevents ATR activation during infection by targeting TOPBP1 for degradation, we demonstrate that Ad12 E4orf6 can inhibit the ATR-dependent phosphorylation of CHK1 in response to replication stress. Taken together, these data provide insights into how Ad modulates ATR signaling pathways during infection.

Roles for APIS and the 20S proteasome in adenovirus E1A-dependent transcription

Differentiated thyroid cancers and their metastases frequently exhibit reduced iodide uptake, impacting on the efficacy of radioiodine ablation therapy. PTTG binding factor (PBF) is a proto-oncogene implicated in the pathogenesis of thyroid cancer. We recently reported that PBF inhibits iodide uptake, and have now elucidated a mechanism by which PBF directly modulates sodium iodide symporter (NIS) activity in vitro. In subcellular localisation studies, PBF overexpression resulted in the redistribution of NIS from the plasma membrane into intracellular vesicles, where it colocalised with the tetraspanin CD63. Cell-surface biotinylation assays confirmed a reduction in plasma membrane NIS expression following PBF transfection compared with vector-only treatment. Coimmunoprecipitation and GST-pull-down experiments demonstrated a direct interaction between NIS and PBF, the functional consequence of which was assessed using iodide-uptake studies in rat thyroid FRTL-5 cells. PBF repressed iodide uptake, whereas three deletion mutants, which did not localise within intracellular vesicles, lost the ability to inhibit NIS activity. In summary, we present an entirely novel mechanism by which the proto-oncogene PBF binds NIS and alters its subcellular localisation, thereby regulating its ability to uptake iodide. Given that PBF is overexpressed in thyroid cancer, these findings have profound implications for thyroid cancer ablation using radioiodine.

Roles for the coactivators CBP and p300 and the APC/C E3 ubiquitin ligase in E1A-dependent cell transformation

We have determined distinct roles for different proteasome complexes in adenovirus (Ad) E1A‐dependent transcription. We show that the 19S ATPase, S8, as a component of 19S ATPase proteins independent of 20S (APIS), binds specifically to the E1A transactivation domain, conserved region 3 (CR3). Recruitment of APIS to CR3 enhances the ability of E1A to stimulate transcription from viral early gene promoters during Ad infection of human cells. The ability of CR3 to stimulate transcription in yeast is similarly dependent on the functional integrity of yeast APIS components, Sug1 and Sug2. The 20S proteasome is also recruited to CR3 independently of APIS and the 26S proteasome. Chromatin immunoprecipitation reveals that E1A, S8 and the 20S proteasome are recruited to both Ad early region gene promoters and early region gene sequences during Ad infection, suggesting their requirement in both transcriptional initiation and elongation. We also demonstrate that E1A CR3 transactivation and degradation sequences functionally overlap and that proteasome inhibitors repress E1A transcription. Taken together, these data demonstrate distinct roles for APIS and the 20S proteasome in E1A‐dependent transactivation.

Serotype-specific inactivation of the cellular DNA damage response during adenovirus infection.

Adenovirus early region 1A (E1A) possesses potent transforming activity when expressed in concert with activated ras or E1B genes in in vitro tissue culture systems such as embryonic human retinal neuroepithelial cells or embryonic rodent epithelial and fibroblast cells. Early region 1A has thus been used extensively and very effectively as a tool to determine the molecular mechanisms that underlie the basis of cellular transformation. In this regard, roles for the E1A-binding proteins pRb, p107, p130, cyclic AMP response element-binding protein (CBP)/p300, p400, TRRAP and CtBP in cellular transformation have been established. However, the mechanisms by which E1A promotes transformation through interaction with these partner proteins are not fully delineated. In this review, we focus on recent advances in our understanding of CBP/p300 function, particularly with regard to its relationship to the anaphase-promoting complex/cyclosome E3 ubiquitin ligase, which has recently been shown to interact and affect the activity of CBP/p300 through interaction domains that are evolutionarily conserved in E1A.