Investigating a Potential Role of Telomerase in Resolving Non-Canonical Structures During Human Cytomegalovirus Infection

Abstract

Human cytomegalovirus (HCMV) is a prevalent herpesvirus, with a significant global health burden. Recent work done in our lab has shown that HCMV infection upregulates host cell telomerase, and that inhibiting telomerase stunts HCMV replication. As current treatment options to combat HCMV have their limitations, the potential of telomerase inhibition as an anti-viral therapy remains an important field to be studied. Mechanistically, we hypothesized that host telomerase may be resolving G-quadruplexes (G4s) that have been shown to exist in HCMV DNA, allowing for replication and transcription of crucial viral genes. To test this, we first formed G4s from HCMV and human genomic oligonucleotides, using circular dichroism (CD) spectroscopy to confirm the folded structure and topology of the G4s. We grew cells with different levels of telomerase activity and hTERT expression, which was verified using a TRAP assay for telomerase catalytic activity and qPCR for hTERT, and took their lysates to mix with the folded G4s. We found that this assay could not replicate results in the literature where telomerase resolves telomeric G4s, so we tested, in the presence and absence of telomerase inhibition, the prevalence of transcripts predicted to form G4s. We found, from RNA-sequencing, that inhibition of telomerase leads to an overall drop in viral gene expression 48 hours post infection, but that this drop is not unique to G4-associated genes. We also identified two genes for future study whose transcription levels were most affected by telomerase inhibition. Altogether, this study offers initial insight into the relationship between telomerase and viral gene expression and gives direction for future clarification of the mechanisms involved.