Mechanism and Therapeutic Implication of Host Cell Telomerase Modulation by Human Cytomegalovirus

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Project Summary Human Cytomegalovirus (HCMV) is a herpesvirus whose seroprevalence and disease burden remain high worldwide. While disease is often mild in immunocompetent individuals, it is often severe for immunocompromised individuals, and congenital infection remains a leading cause of birth defects. There is currently no vaccine or permanent effective anti-viral therapy against HCMV. The host enzyme telomerase, which maintains telomeres to protect the integrity of DNA, is key for cell survival and has been implicated in several diseases including cancer. Telomerase has been implicated as an important agent in the life cycles and potential oncogenic activity of several herpesviruses, and recently has been shown to be hyperactive in response to active HCMV infection. Furthermore, telomerase inhibition has been associated with a significant reduction of viral infectivity in laboratory and clinical strains of HCMV. However, the significance of telomerase in the HCMV viral life cycle, and the potential therapeutic implications of this relationship, remain unexplored. We hypothesize host telomerase is important for the HCMV viral life cycle. We will first establish telomerase activity upregulation upon HCMV infection, and reduced HCMV replication following telomerase inhibition, in multiple strains and cell lines using several approaches to telomerase repression. Assays of telomerase activity in the presence of pharmaceutical telomerase inhibitors BIBR1532 and MST-312 will establish a reduction of viral infectivity following telomerase inhibition in both laboratory and clinical strains. Subsequent assays using inducible genetic inhibitors, specifically siRNA and inducible shRNA constructs against hTERT, will test whether reduction of viral replication is a result of telomerase inhibition and not off-target effects. To define the mechanism of action by which HCMV upregulates telomerase activity, we will assess telomerase activity following ectopic expression and shRNA-mediated knockdown of key viral genes, and implicated gene products will be subjected to binding assays in infected and telomerase-inhibited infected conditions to assess association with DNA via ChiP and QTIP assays, and with other proteins via IP and mass spectrometry. ChiP assays in infected and telomerase-inhibited infected conditions will also be performed on host proteins previously shown to influence telomerase activity. To define the function of telomerase in the viral life cycle, we will conduct differentially-timed inhibition of telomerase and assess the viral gene class(es) the activity of which is constrained by the absence of telomerase. Overall, these experiments will establish the potential clinical relevance of telomerase in HCMV infection and define both the mechanism of action by which HCMV upregulates telomerase activity and the function of telomerase in the viral life cycle. Increased understanding of these relationships may provide insight into other herpesviruses, inform clinical HCMV management, and lead to new anti-viral therapies.