Treatment options remain limited for Human Cytomegalovirus (HCMV). Host telomerase has been implicated in the pathogenesis and oncogenesis of multiple herpesviruses, most recently including HCMV. In this study, we investigated the effect of telomerase inhibition on HCMV replication, as well as the mechanism of the interaction between HCMV and host telomerase in vitro. We found that lytic HCMV infection increases host telomerase activity, at least in part through modulation of hTERT expression during earlier phases of the HCMV replication cycle. We found telomerase inhibition strongly reduced viral titer for two HCMV strains in a dose-specific manner. Both post-translational pharmaceutical telomerase inhibition and siRNA-mediated knockdown of hTERT reduce HCMV yield. Telomerase inhibition results in both reduction of viral gene and protein expression across the HCMV replication cycle, and suppressed viral genome replication and viral infectivity, suggesting interference with at least early steps of the HCMV viral life cycle. Altogether, our findings indicate telomerase plays an important, perhaps non-canonical role in lytic HCMV infection which includes the support of viral replication and infectivity.

Targeted MS analysis was performed via LC-MS/MS using a Dionex Ultimate 3000 nanoRSLC coupled to a Q Exactive HF mass spectrometer (Thermo Fisher). Peptides were separated by reverse-phase chromatography on an EASY-Spray HPLC Column (Thermo Fisher, 500 mm length, 2 µm particle size, 75 µm diameter) using a 60-minute gradient (3% to 35% B) with 250 nL/min flow rate. Mobile phase A consisted of 0.1% formic acid in water and mobile phase B consisted of 0.1% formic acid in 97% ACN. The PRM method consisted of targeted MS2 scans recorded in profile mode performed at a resolution of 30,000, with an AGC target of 1e5, maximum inject time of 200 ms, isolation window of 1.2, and normalized collision energy of 27% controlled by a peptide inclusion list derived from the TRUSTED targeted MS assay. An MS1 scan was performed at a resolution of 15,000 across a mass range of 400-2,000 with an automatic gain control (AGC) of 3e6, and max injection time of 15 ms. Label-free quantitation was performed using previously described methods, and Skyline Daily software for targeted proteomics. A summed area under the curve of 3 transitions per peptide was used for quantitation. MS1 intensity-based normalization, with peak intensity determined using RawMeat (Vast Scientific), was performed for normalization across samples and replicates. Individual peptides were normalized to their average abundance across all time points/sample conditions. Following normalization, if there were multiple peptides per protein, they were averaged. Average protein abundance was then calculated across replicates and proteins were organized by temporality.

Samples contained MRC5 cells infected with MOI 3 TB40E HCMV, and MRC5 cells infected with MOI 3 TB40E HCMV and treated with 0.5µM MST-312. Sample lysates were harvested at timepoints 12, 24, 72, and 96 hpi.