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Adenovirus 12 E4orf6 inhibits ATR activation by promoting TOPBP1 degradation.

ABSTRACT: 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-IIbeta-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.

SUBMITTER: Blackford AN

PROVIDER: S-EPMC2901489 | biostudies-literature | 2010 Jul

REPOSITORIES: biostudies-literature

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Adenovirus 12 E4orf6 inhibits ATR activation by promoting TOPBP1 degradation.

Blackford Andrew N AN Patel Rakesh N RN Forrester Natalie A NA Theil Kathrin K Groitl Peter P Stewart Grant S GS Taylor A Malcolm R AM Morgan Iain M IM Dobner Thomas T Grand Roger J A RJ Turnell Andrew S AS

Proceedings of the National Academy of Sciences of the United States of America 20100621 27

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 ...[more]

PMID: 20566845

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Project description:Protein kinase R (PKR) is an interferon-induced antiviral protein activated by autophosphorylation in response to double strand DNA (dsRNA) and other stimuli. Activated PKR causes translation inhibition and apoptosis, and it contributes to proinflammatory responses, cell growth, and differentiation. Mouse adenovirus type 1 (MAV-1) counteracts PKR by causing its degradation via a viral protein, early region 4 open reading frame 6 (E4orf6). Degradation is dependent on E4orf6 binding to Cullin 2, a component of the MAV-1 E4orf6 ubiquitin ligase. We investigated the importance of E4orf6 for induction of PKR degradation by exploiting the ability to infect the natural host with the adenovirus MAV-1. First, we used a new PKR-deficient mouse strain, PKR-TKO. PKR-TKO mouse embryo fibroblasts (MEFs) produced higher levels of MAV-1 upon infection than did wild-type (WT) MEFs. PKR-TKO mice had significantly reduced survival, and MAV-1 had a lower LD50 than in WT control mice. However, virus loads in brains and spleens, key organs infected by MAV-1, were similar between PKR-TKO and WT mice. Second, we constructed a virus, E4orf6TMC2, that has three amino acid changes in the E4orf6 domain involved in Cullin 2 binding. In cell culture infection, compared to WT virus, E4orf6TMC2 resulted in reduced PKR degradation, but its growth was equivalent to WT virus. However, E4orf6TMC2 was avirulent in three mouse strains, including the PKR-TKO mice. The results indicate that PKR is an essential antiviral protein that protects against MAV-1 infection. We confirmed that the viral E4orf6 protein is a virulence protein important for PKR degradation during virus infection, and our results suggest its function is not limited to PKR degradation.IMPORTANCEProtein kinase R (PKR) is a host protein that is central to many aspects of the cellular stress response. PKR protects against viral infection by inhibiting viral and host protein synthesis. Most animal viruses have developed ways to circumvent PKR effects by at least one of a variety of means, including inducing its degradation. A new mouse strain knocked out for PKR expression has enabled us to show the importance of PKR for protection from mouse adenovirus type 1 infection in the natural host, which is not possible for human adenoviruses. Mouse adenovirus type 1 induces degradation of PKR through an interaction with host protein Cullin 2. We generated a mutant virus that is defective in its ability to interact with Cullin 2 and showed that the virus does not cause pathogenesis in mice. This work provides critical evidence from mouse studies supporting the importance of PKR for adenovirus pathogenesis.

Dataset Information

Adenovirus 12 E4orf6 inhibits ATR activation by promoting TOPBP1 degradation.

Publications

Adenovirus 12 E4orf6 inhibits ATR activation by promoting TOPBP1 degradation.

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