Inherited chromosomally integrated human herpesvirus 6 as a predisposing risk factor for the development of angina pectoris.

Inherited chromosomally integrated human herpesvirus-6 (iciHHV-6) results in the germ-line transmission of the HHV-6 genome. Every somatic cell of iciHHV-6+ individuals contains the HHV-6 genome integrated in the telomere of chromosomes. Whether having iciHHV-6 predisposes humans to diseases remains undefined. DNA from 19,597 participants between 40 and 69 years of age were analyzed by quantitative PCR (qPCR) for the presence of iciHHV-6. Telomere lengths were determined by qPCR. Medical records, hematological, biochemical, and anthropometric measurements and telomere lengths were compared between iciHHV-6+ and iciHHV-6- subjects. The prevalence of iciHHV-6 was 0.58%. Two-way ANOVA with a Holm-Bonferroni correction was used to determine the effects of iciHHV6, sex, and their interaction on continuous outcomes. Two-way logistic regression with a Holm-Bonferroni correction was used to determine the effects of iciHHV6, sex, and their interaction on disease prevalence. Of 50 diseases monitored, a single one, angina pectoris, is significantly elevated (3.3×) in iciHHV-6+ individuals relative to iciHHV-6- subjects (P = 0.017; 95% CI, 1.73-6.35). When adjusted for potential confounding factors (age, body mass index, percent body fat, and systolic blood pressure), the prevalence of angina remained three times greater in iciHHV-6+ subjects (P = 0.015; 95%CI, 1.23-7.15). Analyses of telomere lengths between iciHHV-6- without angina, iciHHV-6- with angina, and iciHHV-6+ with angina indicate that iciHHV-6+ with angina have shorter telomeres than age-matched iciHHV-6- subjects (P = 0.006). Our study represents, to our knowledge, the first large-scale analysis of disease association with iciHHV-6. Our results are consistent with iciHHV-6 representing a risk factor for the development of angina.

Characterization of human herpesvirus 6A/B U94 as ATPase, helicase, exonuclease and DNA-binding proteins.

Human herpesvirus-6A (HHV-6A) and HHV-6B integrate their genomes into the telomeres of human chromosomes, however, the mechanisms leading to integration remain unknown. HHV-6A/B encode a protein that has been proposed to be involved in integration termed U94, an ortholog of adeno-associated virus type 2 (AAV-2) Rep68 integrase. In this report, we addressed whether purified recombinant maltose-binding protein (MBP)-U94 fusion proteins of HHV-6A/B possess biological functions compatible with viral integration. We could demonstrate that MBP-U94 efficiently binds both dsDNA and ssDNA containing telomeric repeats using gel shift assay and surface plasmon resonance. MBP-U94 is also able to hydrolyze adenosine triphosphate (ATP) to ADP, providing the energy for further catalytic activities. In addition, U94 displays a 3' to 5' exonuclease activity on dsDNA with a preference for 3'-recessed ends. Once the DNA strand reaches 8-10 nt in length, the enzyme dissociates it from the complementary strand. Lastly, MBP-U94 compromises the integrity of a synthetic telomeric D-loop through exonuclease attack at the 3' end of the invading strand. The preferential DNA binding of MBP-U94 to telomeric sequences, its ability to hydrolyze ATP and its exonuclease/helicase activities suggest that U94 possesses all functions required for HHV-6A/B chromosomal integration.

Human herpesvirus 6B immediate-early I protein contains functional HLA-A*02, HLA-A*03, and HLA-B*07 class I restricted CD8(+) T-cell epitopes.

Human herpesvirus 6B (HHV-6B) is a ubiquitous pathogen with frequent reactivation observed in immunocompromised patients such as BM transplant (BMT) recipients. Adoptive immunotherapy is a promising therapeutic avenue for the treatment of opportunistic infections, including herpesviruses. While T-cell immunotherapy can successfully control CMV and EBV reactivations in BMT recipients, such therapy is not available for HHV-6 infections, in part due to a lack of identified protective CD8(+) T-cell epitopes. Our goal was to identify CD8(+) T-cell viral epitopes derived from the HHV-6B immediate-early protein I and presented by common human leukocyte Ag (HLA) class I alleles including HLA-A*02, HLA-A*03, and HLA-B*07. These epitopes were functionally tested for their ability to induce CD8(+) T-cell expansion and kill HHV-6-infected autologous cells. Cross-reactivity of specific HHV-6B-expanded T cells against HHV-6A-infected cells was also confirmed for a conserved epitope presented by HLA-A*02 molecule. Our findings will help push forward the field of adoptive immunotherapy for the treatment and/or the prevention of HHV-6 reactivation in BMT recipients.

Inhibition of interleukin-2 gene expression by human herpesvirus 6B U54 tegument protein.

Human herpesvirus 6B (HHV-6B) is a ubiquitous pathogen causing lifelong infections in approximately 95% of humans worldwide. To persist within its host, HHV-6B has developed several immune evasion mechanisms, such as latency, during which minimal proteins are expressed, and the ability to disturb innate and adaptive immune responses. The primary cellular targets of HHV-6B are CD4(+) T cells. Previous studies by Flamand et al. (L. Flamand, J. Gosselin, I. Stefanescu, D. Ablashi, and J. Menezes, Blood 85:1263-1271, 1995) reported on the capacity of HHV-6A as well as UV-irradiated HHV-6A to inhibit interleukin-2 (IL-2) synthesis in CD4(+) lymphocytes, suggesting that viral structural components could be responsible for this effect. In the present study, we identified the HHV-6B U54 tegument protein (U54) as being capable of inhibiting IL-2 expression. U54 binds the calcineurin (CaN) phosphatase enzyme, causing improper dephosphorylation and nuclear translocation of NFAT (nuclear factor of activated T cells) proteins, resulting in suboptimal IL-2 gene transcription. The U54 GISIT motif (amino acids 293 to 297), analogous to the NFAT PXIXIT motif, contributed to the inhibition of NFAT activation. IMPORTANCE Human herpesvirus 6A (HHV-6A) and HHV-6B are associated with an increasing number of pathologies. These viruses have developed strategies to avoid the immune response allowing them to persist in the host. Several studies have illustrated mechanisms by which HHV-6A and HHV-6B are able to disrupt host defenses (reviewed in L. Dagna, J. C. Pritchett, and P. Lusso, Future Virol. 8:273-287, 2013, doi:10.2217/fvl.13.7). Previous work informed us that HHV-6A is able to suppress synthesis of interleukin-2 (IL-2), a key immune growth factor essential for adequate T lymphocyte proliferation and expansion. We obtained evidence that HHV-6B also inhibits IL-2 gene expression and identified the mechanisms by which it does so. Our work led us to the identification of U54, a virion-associated tegument protein, as being responsible for suppression of IL-2. Consequently, we have identified HHV-6B U54 protein as playing a role in immune evasion. These results further contribute to our understanding of HHV-6 interactions with its human host and the efforts deployed to ensure its long-term persistence.

Cation trapping by cellular acidic compartments: beyond the concept of lysosomotropic drugs.

"Lysosomotropic" cationic drugs are known to concentrate in acidic cell compartments due to low retro-diffusion of the protonated molecule (ion trapping); they draw water by an osmotic mechanism, leading to a vacuolar response. Several aspects of this phenomenon were recently reexamined. (1) The proton pump vacuolar (V)-ATPase is the driving force of cationic drug uptake and ensuing vacuolization. In quantitative transport experiments, V-ATPase inhibitors, such as bafilomycin A1, greatly reduced the uptake of cationic drugs and released them in preloaded cells. (2) Pigmented or fluorescent amines are effectively present in a concentrated form in the large vacuoles. (3) Consistent with V-ATPase expression in trans-Golgi, lysosomes and endosomes, a fraction of the vacuoles is consistently labeled with trans-Golgi markers and protein secretion and endocytosis are often inhibited in vacuolar cells. (4) Macroautophagic signaling (accumulation of lipidated and membrane-bound LC3 II) and labeling of the large vacuoles by the autophagy effector LC3 were consistently observed in cells, precisely at incubation periods and amine concentrations that cause vacuolization. Vacuoles also exhibit late endosome/lysosome markers, because they may originate from such organelles or because macroautophagosomes fuse with lysosomes. Autophagosome persistence is likely due to the lack of resolution of autophagy, rather than to nutritional deprivation. (5) Increased lipophilicity decreases the threshold concentration for the vacuolar and autophagic cytopathology, because simple diffusion into cells is limiting. (6) A still unexplained mitotic arrest is consistently observed in cells loaded with amines. An extended recognition of relevant clinical situations is proposed for local or systemic drug administration.

Herpesviruses and chromosomal integration.

Herpesviruses are members of a diverse family of viruses that colonize all vertebrates from fish to mammals. Although more than one hundred herpesviruses exist, all are nearly identical architecturally, with a genome consisting of a linear double-stranded DNA molecule (100 to 225 kbp) protected by an icosahedral capsid made up of 162 hollow-centered capsomeres, a tegument surrounding the nucleocapsid, and a viral envelope derived from host membranes. Upon infection, the linear viral DNA is delivered to the nucleus, where it circularizes to form the viral episome. Depending on several factors, the viral cycle can proceed either to a productive infection or to a state of latency. In either case, the viral genetic information is maintained as extrachromosomal circular DNA. Interestingly, however, certain oncogenic herpesviruses such as Marek's disease virus and Epstein-Barr virus can be found integrated at low frequencies in the host's chromosomes. These findings have mostly been viewed as anecdotal and considered exceptions rather than properties of herpesviruses. In recent years, the consistent and rather frequent detection (in approximately 1% of the human population) of human herpesvirus 6 (HHV-6) viral DNA integrated into human chromosomes has spurred renewed interest in our understanding of how these viruses infect, replicate, and propagate themselves. In this review, we provide a historical perspective on chromosomal integration by herpesviruses and present the current state of knowledge on integration by HHV-6 with the possible clinical implications associated with viral integration.

Dissociation of the vacuolar and macroautophagic cytopathology from the cytotoxicity induced by the lipophilic local anesthetic bupivacaine.

Local anesthetics, like many other cationic drugs, induce a vacuolar and macroautophagic cytopathology that has been observed in vivo and in various cell types; some also induce cytotoxicity of mitochondrial origin (apoptosis and necrosis) and it is not known whether the 2 types of toxicity overlap or interact. We compared bupivacaine with a more hydrophilic agent, lidocaine, for morphological, functional, and toxicological responses in a previously exploited nonneuronal system, primary smooth muscle cells. Bupivacaine induced little vacuolization (≥2.5 mmol/L, 4 h), but elicited autophagic accumulation (≥0.5 mmol/L, 4 h) and was massively cytotoxic at 2.5-5 mmol/L (4-24 h), the latter effect being unabated by the V-ATPase inhibitor bafilomycin A1. Lidocaine exerted little cytotoxicity at and below 5 mmol/L for 24 h, but intensely induced the V-ATPase-dependent vacuolar and autophagic cytopathology. Bupivacaine was more potent than lidocaine in disrupting mitochondrial potential, as judged by Mitotracker staining (significant proportions of cells affected in the 1-5 and 5-10 mmol/L concentration ranges, respectively). The addition of mitochondrial-inactivating toxins antimycin A and oligomycin to lidocaine (2.5 mmol/L) reproduced the profile of bupivacaine action (low intensity of vacuolization and retained autophagic accumulation). The high potency of bupivacaine as a mitochondrial toxicant eclipses the benign vacuolar and autophagic response seen with more hydrophilic local anesthetics.

[Human herpesviruses HHV-6 and chromosomal integration: pathological and medical associated consequences]. / L'herpèsvirus humain de type 6 et l'intégration chromosomique : conséquences biologiques et médicales associées.

Herpesviruses are extremely well adapted to their hosts and cause persistent infections that span over decades. Typically, these viruses establish latency by maintaining their viral genomes as extrachromosomal episomes and restricting their gene expression to a minimum. Human herpesvirus 6 (HHV-6) is perhaps one of the most well-adapted human herpesvirus with a prevalence approaching 100%. Unlike any other human herpesviruses, HHV-6 has developed the ability to integrate its genome into the host chromosomes by targeting the telomeric region. Whether integration represents a true mode of latency for this virus or whether is constitutes a viral dead-end remains uncertain. The present review aims to familiarize the readers with the concept of viral integration and delineates the various biological, pathological and medical consequences associated with HHV-6 chromosomal integration.

Vacuolar ATPase-mediated sequestration of local anesthetics in swollen macroautophagosomes.

PURPOSE: Local anesthetics in their therapeutic concentration range cause a vacuolar cytopathology that has been observed in vivo and in various types of mammalian cells. We examined whether active concentration ranges of drugs and the kinetics of the vacuolar response are clinically relevant and whether this phenomenon is associated with cytotoxicity, autophagy, and cell stress signalling. METHODS: We compared procaine and lidocaine for morphological, functional, and signalling responses in a previously exploited non-neuronal system, primary smooth muscle cells. Several markers conjugated to fluorescent proteins allowed morphological and functional analysis of vacuolar cells. Signalling related to autophagy and cell stress was addressed (immunoblotting of cell lysates). RESULTS: Within 2-4 hr, lidocaine and procaine (> or = 1 mM) induced massive cell vacuolization, a response abated by the V-ATPase inhibitor, bafilomycin A1, and activated macroautophagic signalling (LC3 II formation) but not other stress signalling (p38, ERK1/2, p53, no influence on serum-controlled Akt phosphorylation). Novel aspects of the morphological analysis include reduced LC3 labelling of the large vacuoles in cells treated with 3-methyl-adenine, inhibition of CD63 labelling of these vacuoles by co-expression of dominant negative Rab7, retention of secretory green fluorescent protein (GFP) possessing a signal sequence in vacuolar cells, and partial vacuole labelling with lysosomal-associated membrane protein 1 (LAMP1). Lidocaine (2.5-5 mM) was not overtly cytotoxic but arrested cell division over 48 hr. CONCLUSIONS: V-ATPase-mediated sequestration of clinically relevant concentrations of local anesthetics sequentially involves vacuolization, macroautophagic signalling, and lysosome fusion to large vacuoles. Disruption of the secretory pathway and mitotic arrest were also observed over several hours without major cytotoxicity.

Bradykinin receptors: Agonists, antagonists, expression, signaling, and adaptation to sustained stimulation.

Bradykinin-related peptides, the kinins, are blood-derived peptides that stimulate 2 G protein-coupled receptors, the B1 and B2 receptors (B1R, B2R). The pharmacologic and molecular identities of these 2 receptor subtypes will be succinctly reviewed herein, with emphasis on drug development, receptor expression, signaling, and adaptation to persistent stimulation. Peptide and non-peptide antagonists and fluorescent ligands have been produced for each receptor. The B2R is widely and constitutively expressed in mammalian tissues, whereas the B1R is mostly inducible under the effect of cytokines during infection and immunopathology. The B2R is temporarily desensitized by a cycle of phosphorylation/endocytosis followed by recycling, whereas the nonphosphorylable B1R is relatively resistant to desensitization and translocated to caveolae on activation. Both receptor subtypes, mainly coupled to protein G Gq, phospholipase C and calcium signaling, mediate the vascular aspects of inflammation (vasodilation, edema formation). On this basis, icatibant, a peptide antagonist of the B2R, is approved in the management of hereditary angioedema attacks. This disease is the therapeutic showcase of the kallikrein-kinin system, with an orally bioavailable B2R antagonist under development, as well as other agents that inhibit the kinin forming protease, plasma kallikrein. Other clinical applications are still elusive despite the maturity of the medicinal chemistry efforts applied to kinin receptors.