Genome-wide analysis of regulatory G-quadruplexes affecting gene expression in human cytomegalovirus

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.

LANA and hnRNP A1 Regulate the Translation of LANA mRNA through G-Quadruplexes

“…G-quadruplexes have gained recognition as regulatory nucleic acid structures that influence a number of biological processes. Many members of the Herpesviridae family have been shown to form G-quadruplexes that regulate viral gene expression, viral DNA replication, and virion production (52,53). Moreover, G-quadruplexes have gained recognition as immunomodulatory structures in a few gammaherpesviruses, such as EBV and alcelaphine herpesvirus 1 (AIHV), in which these structures abrogate protein synthesis and restrict antigen presentation, thereby helping in immune evasion (30,54).…”

Viral G-quadruplexes: New frontiers in virus pathogenesis and antiviral therapy

“…Interestingly, in a luciferase reporter system that included different PQSs in viral promoters, NMM was shown to suppress the expression of several mRNAs and to reduce both intracellular and extracellular viral DNA levels. 20 Recently, the anti-CMV activity of a polymerase inhibitor CX-5461, a known G4 ligand currently in phase I clinical trial, was also demonstrated. Interestingly, the compound induced a 2.0 log reduction in viral titer, together with a significant decrease in the amount of viral DNA and pUL44, the viral processivity factor: these data indicate that the effect of CX-5461 is exerted at the viral DNA replication stage of the viral life cycle.…”

Enriched G-quadruplexes on the Drosophila Male X Chromosome Function as Insulators of Dosage Compensation Complex

“…We also classified pG4s into genic and intergenic PG4s based on General Transfer Format (GTF) format gene annotation of D. melanogaster (Flybase r6. 19) downloaded from Flybase (http://flybase.org/; last access Jan 27, 2018). We used the software bedtools command intersect (27) (v2.25.0) to search pG4s which had at least 1 nt overlap with any protein-coding genes, non-coding genes or psuedogenes and classified them into genic pG4s.…”

“…G4s were reported to suppress gene expression (18,19). To test whether the enrichment of intergenic pG4s was responsible for the male-specific fine-tuning of X chromosome in dosage compensation, we then took advantage of RNA-seq data from fly tissues to measure the expression of pG4s.…”