Abstract
Human Alphaherpesviruses comprise three members, herpes simplex virus (HSV) 1 and 2 and varicella zoster virus (VZV). These viruses are characterized by a lytic cycle in epithelial cells and latency in the nervous system, with lifelong infections that may periodically reactivate and lead to serious complications, especially in immunocompromised patients. The mechanisms that regulate viral transcription have not been fully elucidated, but the master role of the immediate early (IE) genes has been established. G-quadruplexes are non-canonical nucleic-acid structures that control transcription, replication, and recombination in many organisms including viruses and that represent attractive antiviral targets. In this work, we investigate the presence, conservation, folding and activity of G-quadruplexes in the IE promoters of the Alphaherpesviruses. Our analysis shows that all IE promoters in the genome of HSV-1, HSV-2 and VZV contain fully conserved G-quadruplex forming sequences. These comprise sequences with long loops and bulges, and thus deviating from the classic G-quadruplex motifs. Moreover, their location is both on the leading and lagging strand and in some instances they contain exuberant G-tracts. Biophysical and biological analysis proved that all sequences actually fold into G-quadruplex under physiological conditions and can be further stabilized by the G-quadruplex ligand BRACO-19, with subsequent impairment of viral IE gene transcription in cells. These results help shed light on the control of viral transcription and indicate new viral targets to design drugs that impair the early steps of Alphaherpesviruses. In addition, they validate the significance of G-quadruplexes in the general regulation of viral cycles.
Highlights
In the last years the fact that the DNA can adopt complex secondary structures other than the double-stranded form wrapped around histones and packaged as chromatin [1] has become evident [2,3,4]
The herpes simplex virus (HSV)-1 infective cycle is predominately driven by the five immediate early (IE) genes, namely ICP0, ICP4, ICP27 (UL54), ICP22 and ICP47 (US12) [34]
Our previous investigation showed that the GGG-island type putative G4 sequences (PQS) in HSV-1 were distributed along four defined genomic features, i.e., coding sequences (CDS), repeat regions (RR), 50 - and 30 -untranslated (UTR), and promoter regions, with a high concentration in the RR and 50 –regulatory region [33]
Summary
In the last years the fact that the DNA can adopt complex secondary structures other than the double-stranded (ds) form wrapped around histones and packaged as chromatin [1] has become evident [2,3,4]. Structures, comprising G-tetrads linked by loop nucleotides. Each G-tetrad is composed of four G residues connected through Hoogsteen-type hydrogen bonds. G4s are highly polymorphic structures, the topology of which depends on the strand stoichiometry and polarity, the nature and length of the loops and their location in the sequence [5]. The G4 parallel, antiparallel or mixed topology is directly correlated to the syn and anti conformational state of the glycosidic bond between the G base and the sugar [2]. The anti conformation characterizes a parallel folding, while antiparallel
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