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Viral interactions with the cellular DNA repair machinery

Mira Chaurushiya

Appointment Period: 2007-2010, Grant Years: [23,24,25]

Mira ChaurushiyaThe cellular DNA repair pathway is integral to maintaining genomic integrity and preventing the spread of deleterious mutations that can lead to cancer. The rise of cancer-predisposing disorders such as Ataxia-telangeitasia (A-T), Ataxia-telangeitasia-like disorder (ATLD), and Nijmeigen Breakage Syndrome (NBS) have been traced to mutations in the DNA repair proteins ATM, Mre11 and Nbs1, respectively. Herpes simplex virus (HSV-1) infection activates an ATM-mediated DNA damage response and activated forms repair factors (ATM, Mre11, and Nbs1) are recruited to viral replication centers where they enhance viral replication. However, other parts of the DNA damage response are inactivated, demonstrating the finesse with which HSV-1 manipulates the DNA repair.

Our studies revealed that the HSV-1 protein ICP0, which is required for efficient viral replication and reactivation from latency, prevents recruitment of DNA repair proteins to sites of damage (irradiation induced foci, IRIF). This may occur in order to redirect DNA repair proteins from IRIF to viral replication centers. ICP0 is an E3 ubiquitin ligase that mediates the ubiquitination and degradation of several cellular proteins. I hypothesized that ICP0 may be interacting with DNA repair proteins to mediate its effects on the DNA repair pathway. To identify cellular proteins that interact with ICP0, I tandem-affinity purified ICP0 from cell lysates and identified co-purifying cellular proteins via mass spectrometry analysis. I found that ICP0 interacts with cellular pathways involved in RNA processing, histone /chromatin modification, DNA repair, and ubiquitination. Using these results, I identified two cellular proteins, RNF8 and RNF168, which are targeted for degradation by ICP0 Overexpression of RNF8 and RNF168 in the presence of ICP0 is able to rescue IRIF formation, indicating that degradation of these to proteins is the likely mechanism by which ICP0 disrupts IRIF. I have gone on to identify the mechanism by which ICP0 targets RNF8 for degradation. Similarly, ICP0 mutants were analyzed for their association with RNF8. Interestingly, I found that ICP0 mimics a cellular phosphorylation mark that would normally bind RNF8 during a DNA damage response, thus redirecting RNF8 from its cellular binding partners to bind ICP0. Analysis of other proteins from the TAP-MS experiments have identified and confirmed the interaction of two cancer-associated proteins with ICP0. We seek to determine whether these proteins may represent new degradation targets of ICP0, and if so, ICP0 may be useful in understanding the underlying functions of these proteins, both at the cellular level and in the context of HSV-1 infection.

Chaurushiya MS, and Weitzman MD. Viral manipulation of DNA repair and cell cycle checkpoints. DNA Repair (Amst). (2009) 8:1166-76. PMID: 19473887; PMC2725192

Lilley CE, Chaurushiya MS, and Weitzman MD. Chromatin at the intersection of viral infection and DNA damage. Biochim Biophys Acta. (2010) 1799:319-27. PMID: 19616655; PMC2838936.

Lilley CE, Chaurushiya MS, Boutell C, Landry S, Suh J, Panier S, Everett RD, Stewart GS, Durocher D, and Weitzman MD. A viral E3 ligase targets RNF8 and RNF168 to control histone ubiquitination and the DNA damage responses. EMBO J. (2010) 29:943-55. PMID: 20075863; PMC2837166.

Weitzman MD, Lilley CE, Chaurushiya MS. Genomes in conflict: maintaining genome integrity during virus infection. Annu Rev Microbiol. (2010) 64:61-81. PMID: 20690823.

Weitzman MD, Lilley CE, Chaurushiya MS. Changing the ubiquitin landscape during viral manipulation of the DNA damage response. FEBS Lett. (2011) 585:2897-906. PMID: 21549706.

Lilley CE, Chaurushiya MS, Boutell C, Everett RD, Weitzman MD. The intrinsic antiviral defense to incoming HSV-1 genomes includes specific DNA repair proteins and is counteracted by the viral protein ICP0. PLoS Pathog. (2011) 7:e1002084. PMID: 21698222; PMCID: PMC3116817.

Chaurushiya MS, Lilley CE, Aslanian A, Meisenhelder J, Scott DC, Landry S,Ticau S, Boutell C, Yates JR 3rd, Schulman BA, Hunter T, Weitzman MD. ViralE3Ubiquitin Ligase-Mediated Degradation of a Cellular E3: Viral Mimicry of aCellular Phosphorylation Mark Targets the RNF8 FHA Domain. Mol Cell. 2012 Apr13;46(1):79-90. Epub 2012 Mar 7. PubMed PMID: 22405594.