Transcriptional Activation of the Adenoviral Genome Is Mediated by Capsid Protein VI

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

“…Recently, our group showed that the constitutive PML NB protein Daxx represses Ad5 gene expression and productive infection (80). This is counteracted by capsid protein pVI-dependent Daxx relocalization from the nuclear bodies at immediate-early times of infection, followed by E1B-55K-dependent proteasomal degradation (67,80).…”

Section: Discussionmentioning

confidence: 99%

“…For dual-luciferase assays, subconfluent H1299 cells were transfected as described above, using 1 g of reporter (pGL-C3G5-luc or pGL-E2-early promoter), 1 g of pRL-TK (Promega), which expresses Renilla luciferase under the control of the herpes simplex virus (HSV) thymidine kinase promoter, and 1 g of effector plasmids (E1A-12S, E1A-13S, PML-II, and pG4-p300). Cell extracts were prepared, measured, and normalized as described recently (67).…”

Section: Methodsmentioning

confidence: 99%

The Adenoviral Oncogene E1A-13S Interacts with a Specific Isoform of the Tumor Suppressor PML To Enhance Viral Transcription

Berscheminski

Groitl

Dobner

et al. 2013

Self Cite

PML nuclear bodies (PML NBs), also called ND10, are matrix-bound nuclear structures that have been implicated in a variety of functions, including DNA repair, transcriptional regulation, protein degradation, and tumor suppression. These domains are also known for their potential to mediate an intracellular defense mechanism against many virus types. This is likely why they are targeted and subsequently manipulated by numerous viral proteins. Paradoxically, the genomes of various DNA viruses become associated with PML NBs, and initial sites of viral transcription/replication centers are often juxtaposed to these domains. The question is why viruses start their transcription and replication next to their supposed antagonists. Here, we report that PML NBs are targeted by the adenoviral (Ad) transactivator protein E1A-13S. Alternatively spliced E1A isoforms (E1A-12S and E1A-13S) are the first proteins expressed upon Ad infection. E1A-13S is essential for activating viral transcription in the early phase of infection. Coimmunoprecipitation assays showed that E1A-13S preferentially interacts with only one (PML-II) of at least six nuclear human PML isoforms. Deletion mapping located the interaction site within E1A conserved region 3 (CR3), which was previously described as the transcription factor binding region of E1A-13S. Indeed, cooperation with PML-II enhanced E1A-mediated transcriptional activation, while deleting the SUMO-interacting motif (SIM) of PML proved even more effective. Our results suggest that in contrast to PML NB-associated antiviral defense, PML-II may help transactivate viral gene expression and therefore play a novel role in activating Ad transcription during the early viral life cycle. Human adenovirus type 5 (Ad5) early region 1A (E1A) is the first protein expressed upon infection and plays an essential role in transcriptional activation and induction of cell cycle progression (1). At early times of infection, two major E1A proteins, E1A-12S (243R) and E1A-13S (289R), are synthesized from alternatively spliced mRNA transcripts of the E1A gene (2, 3). The proteins are identical except for a 46-amino-acid (aa) conserved region (CR3) that is unique to the larger E1A protein (3). Despite being very similar, these proteins show significant differences in their biological activities. The larger E1A isoform (E1A-13S) is considered to be primarily responsible for transactivating viral gene expression by interacting with a wide range of transcription factors via its CR3 (4-8).The PML (promyelocytic leukemia) protein was first described in acute promyelocytic leukemia (APL), showing that PML was fused to retinoic acid receptor alpha (RAR␣) due to a chromosomal translocation (t15;17) (9-20). The PML gene encodes multiple isoforms, derived from alternative mRNA splicing, differing only in the C-terminal parts of the proteins (21). PML seems to be responsible for the assembly of nuclear bodies by recruiting other constitutive components such as Daxx (death domain-associated protein), Sp100 (speckled protein ...

“…Human foreskin fibroblasts (HFFs) were obtained from J. Dechanet (CIRID University of Bordeaux) and maintained as described above. Recombinant replication-competent human adenovirus type 5 (Ad5), replication-deficient E1-deleted green fluorescent protein (GFP)-expressing Ad5 vector (Ad5-GFP), and Ad5-GFP-M1, in which the PPxY motif of protein VI is mutated (33), were amplified and purified as described previously (33,34). Ad infection was carried out at a multiplicity of infection (MOI) of 100 PFU/cell.…”

Section: Methodsmentioning

confidence: 99%

An Adenovirus DNA Replication Factor, but Not Incoming Genome Complexes, Targets PML Nuclear Bodies

Komatsu

Nagata

Wodrich

2016

Self Cite

Promyelocytic leukemia protein nuclear bodies (PML-NBs) are subnuclear domains implicated in cellular antiviral responses. Despite the antiviral activity, several nuclear replicating DNA viruses use the domains as deposition sites for the incoming viral genomes and/or as sites for viral DNA replication, suggesting that PML-NBs are functionally relevant during early viral infection to establish productive replication. Although PML-NBs and their components have also been implicated in the adenoviral life cycle, it remains unclear whether incoming adenoviral genome complexes target PML-NBs. Here we show using immunofluorescence and live-cell imaging analyses that incoming adenovirus genome complexes neither localize at nor recruit components of PML-NBs during early phases of infection. We further show that the viral DNA binding protein (DBP), an early expressed viral gene and essential DNA replication factor, independently targets PML-NBs. We show that DBP oligomerization is required to selectively recruit the PML-NB components Sp100 and USP7. Depletion experiments suggest that the absence of one PML-NB component might not affect the recruitment of other components toward DBP oligomers. Thus, our findings suggest a model in which an adenoviral DNA replication factor, but not incoming viral genome complexes, targets and modulates PML-NBs to support a conducive state for viral DNA replication and argue against a generalized concept that PML-NBs target incoming viral genomes. IMPORTANCEThe immediate fate upon nuclear delivery of genomes of incoming DNA viruses is largely unclear. Early reports suggested that incoming genomes of herpesviruses are targeted and repressed by PML-NBs immediately upon nuclear import. Genome localization and/or viral DNA replication has also been observed at PML-NBs for other DNA viruses. Thus, it was suggested that PML-NBs may immediately sense and target nuclear viral genomes and hence serve as sites for deposition of incoming viral genomes and/or subsequent viral DNA replication. Here we performed a detailed analyses of the spatiotemporal distribution of incoming adenoviral genome complexes and found, in contrast to the expectation, that an adenoviral DNA replication factor, but not incoming genomes, targets PML-NBs. Thus, our findings may explain why adenoviral genomes could be observed at PML-NBs in earlier reports but argue against a generalized role for PML-NBs in targeting invading viral genomes.