Stabilization of Telomere G-Quadruplexes Interferes with Human Herpesvirus 6A Chromosomal Integration

Abstract

Human herpesviruses 6A and 6B (HHV-6A/B) can integrate their genomes into the telomeres of human chromosomes using a mechanism that remains poorly understood. To achieve a better understanding of the HHV-6A/B integration mechanism, we made use of BRACO-19, a compound that stabilizes G-quadruplex secondary structures and prevents telomere elongation by the telomerase complex. First, we analyzed the folding of telomeric sequences into G-quadruplex structures and their binding to BRACO-19 using G-quadruplex-specific antibodies and surface plasmon resonance. Circular dichroism studies indicate that BRACO-19 modifies the conformation and greatly stabilizes the G-quadruplexes formed in G-rich telomeric DNA. Subsequently we assessed the effects of BRACO-19 on the HHV-6A initial phase of infection. Our results indicate that BRACO-19 does not affect entry of HHV-6A DNA into cells. We next investigated if stabilization of G-quadruplexes by BRACO-19 affected HHV-6A's ability to integrate its genome into host chromosomes. Incubation of telomerase-expressing cells with BRACO-19, such as HeLa and MCF-7, caused a significant reduction in the HHV-6A integration frequency (P Ͻ 0.002); in contrast, BRACO-19 had no effect on HHV-6 integration frequency in U2OS cells that lack telomerase activity and elongate their telomeres through alternative lengthening mechanisms. Our data suggest that the fluidity of telomeres is important for efficient chromosomal integration of HHV-6A and that interference with telomerase activity negatively affects the generation of cellular clones containing integrated HHV-6A.IMPORTANCE HHV-6A/B can integrate their genomes into the telomeres of infected cells. Telomeres consist of repeated hexanucleotides (TTAGGG) of various lengths (up to several kilobases) and end with a single-stranded 3= extension. To avoid recognition and induce a DNA damage response, the single-stranded overhang folds back on itself and forms a telomeric loop (T-loop) or adopts a tertiary structure, referred to as a G-quadruplex. In the current study, we have examined the effects of a G-quadruplex binding and stabilizing agent, BRACO-19, on HHV-6A chromosomal integration. By stabilizing G-quadruplex structures, BRACO-19 affects the ability of the telomerase complex to elongate telomeres. Our results indicate that BRACO-19 reduces the number of clones harboring integrated HHV-6A. This study is the first of its kind and suggests that telomerase activity is essential to restore a functional telomere of adequate length following HHV-6A integration.

G-Quadruplex DNA and RNA

“…Particularly, G-quadruplexes are found in genomes of human pathogens [144]. Many examples of G-quadruplexes were identified in viruses [145,146], including human immunodeficiency virus (HIV) [147][148][149][150], multiple species of herpes virus [151][152][153][154][155], human papillomavirus (HPV) [156], hepatitis C [157], Zika [158], Ebola [159], and a G-quadruplexbinding protein was found in severe acute respiratory syndrome (SARS) coronavirus [160]. In bacteria, G-quadruplexes are found in Escherichia coli [161], Neisseria gonorrhoeae [162], Neisseria meningitidis [163], Mycobacterium tuberculosis [164], and Deinococcus radiodurans [165].…”

G-quadruplex forming sequences in the genome of all known human viruses: A comprehensive guide

“…The presence of G4s in viruses and their involvement in virus key steps is increasingly evident in most of the Baltimore groups [10,11]. In the dsDNA group, G4s were described in both Herpesviridae and Papillomaviridae families [12][13][14][15][16][17][18][19][20]. In ssDNA viruses, the presence of G4s was reported in the adeno-associated virus genome [21].…”

Role of PML-Nuclear Bodies in Human Herpesvirus 6A and 6B Genome Integration

“…We have previously shown that U2OS and Hela cells are semi-permissive to infection, as the HHV-6A/B initiates replication in only a minority of cells despite considerable expression of IE and E proteins (45), (47). Both cell lines have been extensively used to assess HHV-6A/B integration (48), (49), (50). To determine if IE proteins might contribute to HHV-6A/B integration, we first determined whether they localize to sites of integration, the telomeres.…”