Expression of the adenovirus E4 34k oncoprotein inhibits repair of double strand breaks in the cellular genome of a 293-based inducible cell line

The dependence of adenovirus on the host pre-RNA splicing machinery for expression of its complete genome potentially makes it vulnerable to modulators of RNA splicing, such as digoxin and digitoxin. Both drugs reduced the yields of four human adenoviruses (HAdV-A31, -B35, and -C5 and a species D conjunctivitis isolate) by at least 2 to 3 logs by affecting one or more steps needed for genome replication. Immediate early E1A protein levels are unaffected by the drugs, but synthesis of the delayed protein E4orf6 and the major late capsid protein hexon is compromised. Quantitative reverse transcription-PCR (qRT-PCR) analyses revealed that both drugs altered E1A RNA splicing (favoring the production of 13S over 12S RNA) early in infection and partially blocked the transition from 12S and 13S to 9S RNA at late stages of virus replication. Expression of multiple late viral protein mRNAs was lost in the presence of either drug, consistent with the observed block in viral DNA replication. The antiviral effect was dependent on the continued presence of the drug and was rapidly reversible. RIDK34, a derivative of convallotoxin, although having more potent antiviral activity, did not show an improved selectivity index. All three drugs reduced metabolic activity to some degree without evidence of cell death. By blocking adenovirus replication at one or more steps beyond the onset of E1A expression and prior to genome replication, digoxin and digitoxin show potential as antiviral agents for treatment of serious adenovirus infections. Furthermore, understanding the mechanism(s) by which digoxin and digitoxin inhibit adenovirus replication will guide the development of novel antiviral therapies.IMPORTANCE Despite human adenoviruses being a common and, in some instances, life-threating pathogen in humans, there are few well-tolerated therapies. In this report, we demonstrate that two cardiotonic steroids already in use in humans, digoxin and digitoxin, are potent inhibitors of multiple adenovirus species. A synthetic derivative of the cardiotonic steroid convallotoxin was even more potent than digoxin and digitoxin when tested with HAdV-C5. These drugs alter the cascade of adenovirus gene expression, acting after initiation of early gene expression to block viral DNA replication and synthesis of viral structural proteins. These findings validate a novel approach to treating adenovirus infections through the modulation of host cell processes.

Section: Discussionmentioning

confidence: 99%

Suppression of Adenovirus Replication by Cardiotonic Steroids

Grosso

Stoilov

Lingwood

et al. 2017

“…These include double-strand breaks introduced into the cellular genome by ionizing radiation (27), internal breaks in the viral genome (31), and Rag1-Rag2-induced double-strand breaks in V(D)J recombination substrate plasmids (6). In contrast, E4 11k, while effectively preventing genome concatenation, fails to inhibit repair of internal breaks in the viral genome (31) and does not inhibit V(D)J recombination (6).…”

mentioning

confidence: 99%

Adenovirus E4 34k and E1b 55k Oncoproteins Target Host DNA Ligase IV for Proteasomal Degradation

Baker

Rohleder²,

Hanakahi

et al. 2007

149

164

Cells infected by adenovirus E4 mutants accumulate end-to-end concatemers of the viral genome that are assembled from unit-length viral DNAs by nonhomologous end joining (NHEJ). Genome concatenation can be prevented by expression either of E4 11k (product of E4orf3) or of the complex of E4 34k (product of E4orf6) and E1b 55k. Both E4 11k and the E4 34k/E1b 55k complex prevent concatenation at least in part by inactivation of the host protein Mre11: E4 11k sequesters Mre11 in aggresomes, while the E4 34k/E1b 55k complex participates in a virus-specific E3 ubiquitin ligase that mediates ubiquitination and proteasomal degradation. The E4 34k/E1b 55k complex, but not E4 11k, also inhibits NHEJ activity on internal breaks in the viral genome and on V(D)J recombination substrate plasmids, suggesting that it may interfere with NHEJ independently of its effect on Mre11. We show here that DNA ligase IV, which performs the joining step of NHEJ, is degraded as a consequence of adenovirus infection. Degradation is dependent upon E4 34k and E1b 55k, functional proteasomes, and the activity of cellular cullin 5, a component of the adenoviral ubiquitin ligase. DNA ligase IV also interacts physically with E1b 55k. The data demonstrate that DNA ligase IV, like Mre11, is a substrate for the adenovirus-specific E3 ubiquitin ligase; identify an additional viral approach to prevention of genome concatenation; and provide a mechanism for the general inhibition of NHEJ by adenoviruses.The linear, double-stranded adenovirus genome remains predominantly monomeric throughout a wild-type adenovirus infection. In contrast, most of the viral DNA present in cells infected by mutants that lack early region 4 (E4) is in the form of end-to-end concatemers containing two to five or more unit-length genomes (6,36,37). The viral genomes present in concatemers are joined in all orientations (head to tail, head to head, and tail to tail), and the joints between genomes typically lack a few to hundreds of nucleotides from each of the component molecules (37). Concatemer formation does not occur in mutant human cells that lack either DNA-dependent protein kinase (DNA-PK) (6) or DNA ligase IV (36), each of which is a central component of the nonhomologous endjoining (NHEJ) DNA repair system, and concatemers presumably are assembled from monomeric genomes by NHEJ. Concatemer formation is also blocked in cells that lack the host Mre11 or NBS1 protein, each of which is a member with the Rad50 protein of the MRN complex, which is central in DNA damage signal transduction in eukaryotes.Genome concatenation is redundantly antagonized by two virus-encoded systems in wild-type adenovirus infections: the presence of either the E4 11k (E4orf3) protein or the complex formed by the E4 34k (E4orf6) and E1b 55k proteins prevents concatemer formation (36, 37). These systems act by different mechanisms, but inhibition of concatenation by both is at least partly explained by inactivation of the Mre11 protein. E4 11k induces sequestration of Mre11 in aggresomes, inhibit...

“…The linear double-stranded adenoviral genomes act as targets for the MRN complex (10,24,53,75,78,81). This repair complex, when uninhibited, functions with additional nonhomologous end-joining DNA-double-strand-break repair proteins to concatemerize adenoviral genomes, making them too long to package into adenovirus capsids (24,53,78,81). The MRN complex also interferes with viral DNA replication even when concatemerization of the viral genomes is blocked in mutant cells with defects in other cellular proteins required for double-strand break repair (24,75).…”

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confidence: 99%

Adenovirus Ubiquitin-Protein Ligase Stimulates Viral Late mRNA NuclearExport

Woo

Berk

2007

102

The adenovirus type 5 (Ad5) E1B-55K and E4orf6 proteins are required together to stimulate viral late nuclear mRNA export to the cytoplasm and to restrict host cell nuclear mRNA export during the late phase of infection. Previous studies have shown that these two viral proteins interact with the cellular proteins elongins B and C, cullin 5, RBX1, and additional cellular proteins to form an E3 ubiquitin-protein ligase that polyubiquitinates p53 and probably one or more subunits of the MRE11-RAD50-NBS1 (MRN) complex, directing their proteasomal degradation. The MRN complex is required for cellular DNA double-strand break repair and induction of the DNA damage response by adenovirus infection. To determine if the ability of E1B-55K and E4orf6 to stimulate viral late mRNA nuclear export requires the ubiquitin-protein ligase activity of this viral ubiquitin-protein ligase complex, we designed and expressed a dominant-negative mutant form of cullin 5 in HeLa cells before infection with wild-type Ad5 or the E1B-55K null mutant dl1520. The dominantnegative cullin 5 protein stabilized p53 and the MRN complex, indicating that it inhibited the viral ubiquitinprotein ligase but had no effect on viral early mRNA synthesis, early protein synthesis, or viral DNA replication. However, expression of the dominant-negative cullin 5 protein caused a decrease in viral late protein synthesis and viral nuclear mRNA export similar to the phenotype produced by mutations in E1B-55K. We conclude that the stimulation of adenovirus late mRNA nuclear export by E1B-55K and E4orf6 results from the ubiquitin-protein ligase activity of the adenovirus ubiquitin-protein ligase complex.Two adenovirus type 5 (Ad5) early proteins, E1B-55K and E4orf6, function to stimulate nuclear export and translation of viral late mRNAs, inhibit host cell mRNA nuclear export, and inhibit the functions of p53 and the MRE11-RAD50-NBS1 (MRN) DNA double-strand break repair complex during wildtype (WT) Ad5 infection (3,4,6,11,18,30,35,47,50,64,83). Initial insight into how these two proteins are involved in these diverse processes came from characterizing adenoviral mutant forms of E1B-55K and/or E4orf6. Mutants defective in expressing either E1B-55K or E4orf6 or with a mutation in genes for both show similar phenotypes: They all have a defect in viral late nuclear message export to the cytoplasm and cannot inhibit host cell mRNA nuclear export to the cytoplasm, and synthesis of viral late proteins is reduced compared to that of WT Ad5 (3,4,18,29,30,47,64). These mutants are also unable to induce the degradation of p53 or subunits of the MRN complex (56,65,73,78).Much information has accumulated that is relevant to the functions of E1B-55K and E4orf6. The two viral proteins associate in vivo (68) and colocalize in Ad5-infected cell nuclei at viral DNA replication-transcription centers, as well as other regions of the nucleoplasm and cytoplasm during the late phase of infection (59). Both E1B-55K and E4orf6 have nuclear export signals and nuclear localization signals req...