Abstract
G-quadruplex (G4), formed by repetitive guanosine-rich sequences, is known to play various key regulatory roles in cells. Herpesviruses containing a large double-stranded DNA genome show relatively higher density of G4-forming sequences in their genomes compared to human and mouse. However, it remains poorly understood whether all of these sequences form G4 and how they play a role in the virus life cycle. In this study, we performed genome-wide analyses of G4s present in the putative promoter or gene regulatory regions of a 235-kb human cytomegalovirus (HCMV) genome and investigated their roles in viral gene expression. We evaluated 36 putative G4-forming sequences associated with 20 genes for their ability to form G4 and for the stability of G4s in the presence or absence of G4-stabilizing ligands, by circular dichroism and melting temperature analyses. Most identified sequences formed a stable G4; 28 sequences formed parallel G4s, one formed an antiparallel G4, and four showed mixed conformations. However, when we assessed the effect of G4 on viral promoters by cloning the 20 putative viral promoter regions containing 36 G4-forming sequences into the luciferase reporter and monitoring the expression of luciferase reporter gene in the presence of G4-stabilizing chemicals, we found that only 9 genes were affected by G4 formation. These results revealed promoter context-dependent gene suppression by G4 formation. Mutational analysis of two potential regulatory G4s also demonstrated gene suppression by the sequence-specific G4 formation. Furthermore, the analysis of a mutant virus incapable of G4 formation in the UL35 promoter confirmed promoter regulation by G4 in the context of virus infection. Our analyses provide a platform for assessing G4 functions at the genomic level and demonstrate the properties of the HCMV G4s and their regulatory roles in viral gene expression.
Highlights
Repetitive guanosine-rich (G-rich) sequences connected by short stretches of nucleotides in the genome of an organism can fold into a distinct type of tertiary structure known as a Gquadruplex (G4)
It was found that only a few human cytomegalovirus (HCMV) G4s suppress viral gene expression when the effect of G4 on gene expression was examined by in vivo reporter assay
This study provides evidence that G4 plays a regulatory role in gene expression during HCMV infection
Summary
Repetitive guanosine-rich (G-rich) sequences connected by short stretches of nucleotides in the genome of an organism can fold into a distinct type of tertiary structure known as a Gquadruplex (G4). G4 formation and functions in cells can be greatly influenced by proteins that can stabilize or resolve G4 structures [14, 15]. G4 stability can be enhanced by several ligands that recognize and bind G4 structures [2, 16]. In this regard, G4-stabilizing ligands have been extensively studied for therapeutic purposes [17, 18], mostly targeting G4s present in the promoters of oncogenes such as C-MYC, K-RAS, and BCL2 [7, 19,20,21,22]. G4-binding ligands have been studied for the treatment of neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS), motor neuron disease (MND), and frontotemporal dementia (FTD) [23]
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