Classification of HHV-6A and HHV-6B as distinct viruses

Self Cite

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.

mentioning

confidence: 99%

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

Gilbert-Girard

Gravel

Artusi

et al. 2017

Self Cite

“…They share more than 90% nucleotide sequence identity but differ in several genetic features and clinical manifestations (1,2). HHV-6B has long been identified as the etiological agent for exanthem subitum, a common infant febrile illness (3), while primary infection with HHV- 6A has not yet been conclusively associated with any specific disease.…”

mentioning

confidence: 99%

Human Herpesvirus 6A Infection in CD46 Transgenic Mice: Viral Persistence in the Brain and Increased Production of Proinflammatory Chemokines via Toll-Like Receptor 9

Reynaud

Jégou

Welsch

et al. 2014

Human herpesvirus 6 (HHV-6) is widely spread in the human population and has been associated with several neuroinflammatory diseases, including multiple sclerosis. To develop a small-animal model of HHV-6 infection, we analyzed the susceptibility of several lines of transgenic mice expressing human CD46, identified as a receptor for HHV-6. We showed that HHV-6A (GS) infection results in the expression of viral transcripts in primary brain glial cultures from CD46-expressing mice, while HHV-6B (Z29) infection was inefficient. HHV-6A DNA persisted for up to 9 months in the brain of CD46-expressing mice but not in the nontransgenic littermates, whereas HHV-6B DNA levels decreased rapidly after infection in all mice. Persistence in the brain was observed with infectious but not heat-inactivated HHV-6A. Immunohistological studies revealed the presence of infiltrating lymphocytes in periventricular areas of the brain of HHV-6A-infected mice. Furthermore, HHV-6A stimulated the production of a panel of proinflammatory chemokines in primary brain glial cultures, including CCL2, CCL5, and CXCL10, and induced the expression of CCL5 in the brains of HHV-6A-infected mice. HHV-6A-induced production of chemokines in the primary glial cultures was dependent on the stimulation of toll-like receptor 9 (TLR9). Finally, HHV-6A induced signaling through human TLR9 as well, extending observations from the murine model to human infection. Altogether, this study presents a first murine model for HHV-6A-induced brain infection and suggests a role for TLR9 in the HHV-6A-initiated production of proinflammatory chemokines in the brain, opening novel perspectives for the study of virus-associated neuropathology. IMPORTANCEHHV-6 infection has been related to neuroinflammatory diseases; however, the lack of a suitable small-animal infection model has considerably hampered further studies of HHV-6-induced neuropathogenesis. In this study, we have characterized a new model for HHV-6 infection in mice expressing the human CD46 protein. Infection of CD46 transgenic mice with HHV-6A resulted in long-term persistence of viral DNA in the brains of infected animals and was followed by lymphocyte infiltration and upregulation of the CCL5 chemokine in the absence of clinical signs of disease. The secretion of a panel of chemokines was increased after infection in primary murine brain glial cultures, and the HHV-6-induced chemokine expression was inhibited when TLR9 signaling was blocked. These results describe the first murine model for HHV-6A-induced brain infection and suggest the importance of the TLR9 pathway in HHV-6A-initiated neuroinflammation.

“…Initially, HHV-6B and HHV-6A were classified as two variants of HHV-6; however, they are now classified as two virus species due to their different bio-characteristics (1)(2)(3)(4)(5). One of the striking differences between these two viruses is receptor ligand usage.…”

mentioning

confidence: 99%

Determinants of Human CD134 Essential for Entry of Human Herpesvirus 6B

Tang

Mori

2015

Self Cite

bWe identified two key amino acid residues within human CD134 (hCD134) that are required for its interaction with human herpesvirus 6B (HHV-6B) and for HHV-6B entry into cells. One of the residues (K79) allows access of the HHV-6B ligand to hCD134. Murine CD134 (mCD134) functioned as an HHV-6B receptor when these two amino acid residues were replaced with homologous human residues. This study identifies both the HHV-6B receptor-ligand interaction and the species-specific determinants of hCD134 essential for HHV-6B entry. Human herpesvirus 6B (HHV-6B) is a betaherpesvirus. Initially, HHV-6B and HHV-6A were classified as two variants of HHV-6; however, they are now classified as two virus species due to their different bio-characteristics (1-5). One of the striking differences between these two viruses is receptor ligand usage. The HHV-6A ligand, glycoprotein H (gH)/gL/gQ1/gQ2 complex, binds to human CD46, a ubiquitously expressed molecule that may contribute to the relatively broad cell tropism of HHV-6A (6-8). In contrast, human CD134 (hCD134) is the specific receptor for HHV-6B (9, 10).In a previous study, we performed a detailed analysis of the interaction between the HHV-6B ligand and its cellular receptor, hCD134. We found that cysteine-rich domain 2 (CRD 2) of hCD134 is critical for HHV-6B ligand binding and that a key amino acid residue (E127) within HHV-6B gQ1 is required for its function. The E127Q mutation within HHV-6B gQ1 completely abolishes binding to hCD134; however, the homologous mutation (Q135E) in HHV-6A gQ1 does not enable it to bind to CD134, even though these two gQ1s share high amino acid sequence similarity (10). Interestingly, the E (glutamic acid) and Q (glutamine) residues have similar structures in terms of their side chains, although only glutamic acid has a negative electric charge under physiological conditions. These results led us to speculate that the negative electric charge of E might be important for HHV-6B gQ1 function and that there should be a corresponding residue(s) with a positive electric charge in hCD134 (most likely in the CRD 2 domain) that facilitates the interaction between hCD134 and the HHV-6B ligand. To test this, we focused on two amino acid residues in the CRD 2 domain of hCD134 (K79 and R95), both of which have a positive electric charge; neither of these residues is conserved in murine CD134 (mCD134) (Fig. 1A). Also, mCD134 did not interact with the HHV-6B gH/gL/gQ1/gQ2 complex (Fig. 1E).We replaced these two residues with the homologous residues in mCD134 (K79D and R95L) and then examined the interaction between the CD134 mutants and the HHV-6B gH/gL/gQ1/gQ2 complex by immunoprecipitation with an anti-gH antibody followed by Western blotting (in our previous study, we confirmed that both immunoprecipitation and pulldown assays could be used for the analysis of the HHV-6 receptor-ligand interaction [10,11]). The interaction between the HHV-6B complex and hCD134 was detected when R95, but not K79, was mutated. Since K79 is located within the loop structure of...