Transforming Potential of the Adenovirus Type 5 E4orf3 Protein

Previous observations that the adenovirus type 5 (Ad5) E4orf6 and E4orf3 gene products have redundant effects in viral lytic infection together with the recent findings that E4orf6 possesses transforming potential prompted us to investigate the effect of E4orf3 expression on the transformation of primary rat cells in combination with adenovirus E1 oncogene products. Our results demonstrate for the first time that E4orf3 can cooperate with adenovirus E1A and E1A plus E1B proteins to transform primary baby rat kidney cells, acting synergistically with E4orf6 in the presence of E1B gene products. Transformed rat cells expressing E4orf3 exhibit morphological alterations, higher growth rates and saturation densities, and increased tumorigenicity compared with transformants expressing E1 proteins only. Consistent with previous results for adenovirus-infected cells, the E4orf3 protein is immunologically restricted to discrete nuclear structures known as PML oncogenic domains (PODs) in transformed rat cells. As opposed to E4orf6, the ability of E4orf3 to promote oncogenic cell growth is probably not linked to a modulation of p53 functions and stability. Instead, our results indicate that the transforming activities of E4orf3 are due to combinatorial effects that involve the binding to the adenovirus 55-kDa E1B protein and the colocalization with PODs independent from interactions with the PML gene product. These data fit well with a model in which the reorganization of PODs may trigger a cascade of processes that cause uncontrolled cell proliferation and neoplastic growth. In sum, our results provide strong evidence for the idea that interactions with PODs by viral proteins are linked to oncogenic transformation.     

Adenovirus early region 4 encodes two gene products with redundant effects in lytic infection.

In order to assign specific functions to individual gene products encoded by adenovirus type 5 early region 4 (E4), we have constructed and analyzed a set of mutant viruses that express individual E4 open reading frames or combinations of open reading frames. The results of these analyses demonstrate that the gene products of E4 open reading frames 3 and 6 have redundant effects in viral lytic infection. These E4 products independently augment viral DNA replication, viral late protein synthesis, the shutoff of host cell protein synthesis, and the production of infectious virus. The product of open reading frame 6 is more efficient in the regulation of these processes than is the product of open reading frame 3. The regulation of viral DNA replication and the control of viral and cellular protein synthesis appear to be separable functions associated with both E4 gene products. The role of early region 4 in adeno-associated virus helper function, however, is mediated only by the product of open reading frame 6. Finally, we demonstrate that E4 mutant viruses display a multiplicity-leakiness phenotype which is consistent with the regulatory role that this region plays in viral infection.     

Interactions of minute virus of mice and adenovirus with host nucleoli.

Biochemical evidence is presented that both minute virus of mice (MVM) and adenovirus interact with the nucleolus during lytic growth and that MVM can also target specific changes involving nucleolar components in adenovirus-infected cells. These virus-nucleolus interactions were studied by analysis of intranuclear compartmentalization of both viral DNAs and host nucleolar proteins: (i) MVM in mouse cells (its normal host) replicates its DNA in the host nucleoli; (ii) specific nucleolar proteins as well as small nuclear ribonucleoprotein antigens are recompartmentalized to multiple intranuclear foci in adenovirus-infected HeLa cells; and (iii) when adenovirus helps MVM DNA replication in a nonpermissive human cell (HeLa), the MVM DNA is also recompartmentalized for synthesis. The data suggest mechanisms for disruption of nucleolar function common to oncogenic or oncolytic virus lytic growth and cell transformation.     

The E3 ubiquitin ligase activity associated with the adenoviral E1B-55K-E4orf6 complex does not require CRM1-dependent export

The adenovirus type 5 (Ad5) E1B-55K and E4orf6 (E1B-55K/E4orf6) proteins are multifunctional regulators of Ad5 replication, participating in many processes required for virus growth. A complex containing the two proteins mediates the degradation of cellular proteins through assembly of an E3 ubiquitin ligase and induces shutoff of host cell protein synthesis through selective nucleocytoplasmic viral late mRNA export. Both proteins shuttle between the nuclear and cytoplasmic compartments via leucine-rich nuclear export signals (NES). However, the role of their NES-dependent export in viral replication has not been established. It was initially shown that mutations in the E4orf6 NES negatively affect viral late gene expression in transfection/infection complementation assays, suggesting that E1B-55K/E4orf6-dependent viral late mRNA export involves a CRM1 export pathway. However, a different conclusion was drawn from similar studies showing that E1B-55K/E4orf6 promote late gene expression without active CRM1 or functional NES. To evaluate the role of the E1B-55K/E4orf6 NES in viral replication in the context of Ad-infected cells and in the presence of functional CRM1, we generated virus mutants carrying amino acid exchanges in the NES of either or both proteins. Phenotypic analyses revealed that mutations in the NES of E1B-55K and/or E4orf6 had no or only moderate effects on viral DNA replication, viral late protein synthesis, or viral late mRNA export. Significantly, such mutations also did not interfere with the degradation of cellular substrates, indicating that the NES of E1B-55K or E4orf6 is dispensable both for late gene expression and for the activity associated with the E3 ubiquitin ligase.     

Hybrids of human and monkey adenoviruses (adeno-adeno hybrids) that can reproduce in monkey cells: biological and molecular genetic peculiarities

A highly oncogenic monkey adenovirus SA7(C8) facilitates the reproduction of human adenovirus type 2 (Ad2) in monkey cells. Upon mixed infection of monkey cells with both viruses, these viruses recombine producing defective adeno-adeno hybrids Ad2C8 serologically identical to Ad2 and capable of assisting Ad2 to reproduce in monkey cells. Ad2C8 and Ad2 form an intercomplementary pair inseparable in monkey cells. Unlike oncogenic SA7(C8), Ad2C8 is a nononcogenic virus for hamsters but is able to induce tumor antigens of this virus (T and TSTA). Molecular genetic analysis of 68 clones of adeno-adeno hybrids revealed that the left part of their genome consists of Ad2 DNA, and the right part contains no less than 40% of the viral SA7(C8) genome where E2A, E3, and E4 genes are located. Apparently, the products of these genes contribute to the composition of adenoviral tumor antigens, while the E4 gene is involved in complementation of monkey and human adenoviruses and makes a contribution to host range determination of these viruses.     

Oncogenicity by adenovirus is not determined by the transforming region only.

We have constructed a nondefective recombinant virus between the nononcogenic adenovirus 5 (Ad5) and the highly oncogenic Ad12. The recombinant genome consists essentially of Ad5 sequences, with the exception of the transforming early region 1 (E1) which is derived from Ad12. HeLa cells infected with the recombinant virus were shown to contain the Ad12-specific E1 proteins of 41 kilodaltons (E1a) and 19 and 54 kilodaltons (both encoded by E1b). The recombinant virus replicated efficiently in human embryonic kidney cells and HeLa cells, showing that the transforming regions of Ad5 and Ad12 had similar functions in productive infection. After the recombinant virus was injected into newborn hamsters, no tumors were produced during an observation period of 200 days. Thus, despite the fact that all products required for oncogenic transformation in vitro were derived from the highly oncogenic Ad12, the recombinant virus did not produce tumors in vivo. These data show that tumor induction by adenovirus virions is not determined only by the gene products of the transforming region.     

Chromatin structure of adenovirus DNA throughout infection

For more than half a century, researchers have studied the basic biology of Adenovirus (Ad), unraveling the subtle, yet profound, interactions between the virus and the host. These studies have uncovered previously unknown proteins and pathways crucial for normal cell function that the virus manipulates to achieve optimal virus replication and gene expression. In the infecting virion, the viral DNA is tightly condensed in a virally encoded protamine-like protein which must be remodeled within the first few hours of infection to allow for efficient expression of virus-encoded genes and subsequent viral DNA replication. This review discusses our current knowledge of Ad DNA-protein complex within the infected cell nucleus, the cellular proteins the virus utilizes to achieve chromatinization, and how this event contributes to efficient gene expression and progression of the virus life cycle.     

A carboxy-terminal region required by the adenovirus type 9 E4 ORF1 oncoprotein for transformation mediates direct binding to cellular polypeptides.

Human adenovirus type 9 (Ad9) is unique among oncogenic adenoviruses in that it elicits exclusively mammary tumors in rats and requires the viral E4 region open reading frame 1 (9ORF1) gene for tumorigenicity. The 9ORF1 oncogenic determinant codes for a 14-kDa transforming protein, and three separate regions of this polypeptide, including one at the extreme C terminus, are necessary for transforming activity. In this study, we investigated whether the 9ORF1 transforming protein interacts with cellular factors. Following incubation with cell extracts, a glutathione S-transferase (GST)-9ORF1 fusion protein associated with several cellular phosphoproteins (p220, p180, p160, p155), whereas GST fusion proteins of transformation-defective 9ORF1 C-terminal mutants did not. Similar interactions requiring the 9ORF1 C terminus were revealed with protein-blotting assays, in which a GST-9ORF1 protein probe reacted specifically with cellular polypeptides having gel mobilities resembling those of the 9ORF1-associated cellular phosphoproteins, as well as with additional cellular polypeptides designated p140/p130. In addition, GST fusion proteins containing 9ORF1 C-terminal fragments associated with some of the 9ORF1-associated cellular polypeptides, as did GST fusion proteins of full-length wild-type Ad5 and Ad12 E4 ORF1 transforming proteins. Significantly, the results of coimmunoprecipitation analyses suggested that the same cellular polypeptides also associate with wild-type but not C-terminal-mutant 9ORF1 proteins in vivo. Together, these findings suggest that the 9ORF1 C terminus, which is essential for transformation, participates in specific and direct binding of the 9ORF1 oncoprotein to multiple cellular polypeptides. We propose that interactions with these cellular factors may be responsible, at least in part, for the transforming activity of the 9ORF1 viral oncoprotein.     

DNA Damage Signaling Is Induced in the Absence of Epstein—Barr Virus (EBV) Lytic DNA Replication and in Response to Expression of ZEBRA

Epstein Barr virus (EBV), like other oncogenic viruses, modulates the activity of cellular DNA damage responses (DDR) during its life cycle. Our aim was to characterize the role of early lytic proteins and viral lytic DNA replication in activation of DNA damage signaling during the EBV lytic cycle. Our data challenge the prevalent hypothesis that activation of DDR pathways during the EBV lytic cycle occurs solely in response to large amounts of exogenous double stranded DNA products generated during lytic viral DNA replication. In immunofluorescence or immunoblot assays, DDR activation markers, specifically phosphorylated ATM (pATM), H2AX (γH2AX), or 53BP1 (p53BP1), were induced in the presence or absence of viral DNA amplification or replication compartments during the EBV lytic cycle. In assays with an ATM inhibitor and DNA damaging reagents in Burkitt lymphoma cell lines, γH2AX induction was necessary for optimal expression of early EBV genes, but not sufficient for lytic reactivation. Studies in lytically reactivated EBV-positive cells in which early EBV proteins, BGLF4, BGLF5, or BALF2, were not expressed showed that these proteins were not necessary for DDR activation during the EBV lytic cycle. Expression of ZEBRA, a viral protein that is necessary for EBV entry into the lytic phase, induced pATM foci and γH2AX independent of other EBV gene products. ZEBRA mutants deficient in DNA binding, Z(R183E) and Z(S186E), did not induce foci of pATM. ZEBRA co-localized with HP1β, a heterochromatin associated protein involved in DNA damage signaling. We propose a model of DDR activation during the EBV lytic cycle in which ZEBRA induces ATM kinase phosphorylation, in a DNA binding dependent manner, to modulate gene expression. ATM and H2AX phosphorylation induced prior to EBV replication may be critical for creating a microenvironment of viral and cellular gene expression that enables lytic cycle progression.     

Differential requirements of the C terminus of Nbs1 in suppressing adenovirus DNA replication and promoting concatemer formation

Adenoviruses (Ad) with the early region E4 deleted (E4-deleted virus) are defective for DNA replication and late protein synthesis. Infection with E4-deleted viruses results in activation of a DNA damage response, accumulation of cellular repair factors in foci at viral replication centers, and joining together of viral genomes into concatemers. The cellular DNA repair complex composed of Mre11, Rad50, and Nbs1 (MRN) is required for concatemer formation and full activation of damage signaling through the protein kinases Ataxia-telangiectasia mutated (ATM) and ATM-Rad3-related (ATR). The E4orf3 and E4orf6 proteins expressed from the E4 region of Ad type 5 (Ad5) inactivate the MRN complex by degradation and mislocalization, and prevent the DNA damage response. Here we investigated individual contributions of the MRN complex, concatemer formation, and damage signaling to viral DNA replication during infection with E4-deleted virus. Using virus mutants, short hairpin RNA knockdown and hypomorphic cell lines, we show that inactivation of MRN results in increased viral replication. We demonstrate that defective replication in the absence of E4 is not due to concatemer formation or DNA damage signaling. The C terminus of Nbs1 is required for the inhibition of Ad DNA replication and recruitment of MRN to viral replication centers. We identified regions of Nbs1 that are differentially required for concatemer formation and inhibition of Ad DNA replication. These results demonstrate that targeting of the MRN complex explains the redundant functions of E4orf3 and E4orf6 in promoting Ad DNA replication. Understanding how MRN impacts the adenoviral life cycle will provide insights into the functions of this DNA damage sensor.