The Telomeric Repeats of Human Herpesvirus 6A (HHV-6A) Are Required for Efficient Virus Integration

Human herpesvirus 6A (HHV-6A) and 6B (HHV-6B) are ubiquitous betaherpesviruses that infects humans within the first years of life and establishes latency in various cell types. Both viruses can integrate their genomes into telomeres of host chromosomes in latently infected cells. The molecular mechanism of viral integration remains elusive. Intriguingly, HHV-6A, HHV-6B and several other herpesviruses harbor arrays of telomeric repeats (TMR) identical to human telomere sequences at the ends of their genomes. The HHV-6A and HHV-6B genomes harbor two TMR arrays, the perfect TMR (pTMR) and the imperfect TMR (impTMR). To determine if the TMR are involved in virus integration, we deleted both pTMR and impTMR in the HHV-6A genome. Upon reconstitution, the TMR mutant virus replicated comparable to wild type (wt) virus, indicating that the TMR are not essential for HHV- 6A replication. To assess the integration properties of the recombinant viruses, we established an in vitro integration system that allows assessment of integration efficiency and genome maintenance in latently infected cells. Integration of HHV-6A was severely impaired in the absence of the TMR and the virus genome was lost rapidly, suggesting that integration is crucial for the maintenance of the virus genome. Individual deletion of the pTMR and impTMR revealed that the pTMR play the major role in HHV-6A integration, whereas the impTMR only make a minor contribution, allowing us to establish a model for HHV-6A integration. Taken together, our data shows that the HHV-6A TMR are dispensable for virus replication, but are crucial for integration and maintenance of the virus genome in latently infected cells

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 integratio

ライフサイクルとヒューマンケア: 高齢者への健康支援(平成23年度教養コア科目) 授業資料ナビゲータ(PathFinder)

担当教員:黒田久美子,野地有子,今村恵美子,永野みどり平成23年度(2011)教養コア科目授業B(こころと発達),授業コード:G14B1410

Die Rolle der Telomersequenzen und des Proteins U94 in der Integration des humanen Herpesvirus 6

Human herpesvirus 6 (HHV-6) is a betaherpesvirus related to the human cytomegalovirus. It is the causative agent of roseola infantum, a febrile illness in infants, and has a seroprevalence of over 90 % worldwide. Upon primary infection, HHV-6 establishes a persistent infection in the host for life termed latency, mostly in bone marrow progenitor cells, monocytes and macrophages. Reactivation from latency preferentially occurs in immunocompromised individuals and is associated with several diseases including encephalitis, multiple sclerosis, graft rejection as well as a more rapid AIDS progression. HHV-6 has previously been shown to integrate its genetic material into telomeres of human chromosomes, a mechanism that allows vertical transmission of the virus via the germline, resulting in individuals that harbor the integrated virus in every single cell of their body. This condition is termed ciHHV-6 (chromosomally integrated HHV-6) and is present in roughly 1 % of the human population. The molecular mechanism and the factors involved in HHV-6 integration remain completely unknown. Intriguingly, HHV-6 and several other herpesviruses harbor arrays of telomeric repeats (TMRs) at their genome termini that are identical to human telomere sequences. The TMRs in HHV-6 have been termed perfect TMRs (pTMRs) and imperfect TMRs (impTMRs), and have been proposed to facilitate homologous recombination (HR). Furthermore, HHV-6 encodes the U94 gene that contains all conserved domains of the Rep recombinase of Adeno-associated virus 2 (AAV-2). Expression of U94 restores replication of a Rep-deficient AAV-2, suggesting that both proteins have similar functions. Indeed, recently it was confirmed that a purified MBP-U94 fusion protein has DNA-binding, ATPase, helicase and exonuclease activities as described for Rep. However, the actual role of U94 and the TMRs in HHV-6 replication and integration remains elusive. To determine whether the TMRs are involved in HHV-6 integration, I deleted the two distinct sets of TMRs, individually or simultaneously, in a bacterial artificial chromosome (BAC) of HHV-6A by en passant mutagenesis. Upon reconstitution, the TMR mutant viruses replicated comparable to wild type (wt) and revertant viruses, indicating that the TMRs are not essential for HHV-6A replication. To assess the integration properties of the recombinant viruses, I established an in vitro latency system that allows assessment of integration efficiency and genome maintenance in latently infected U2OS cells. Fluorescence in situ hybridization (FISH) analyses revealed that integration is severely impaired in the absence of the TMRs. The genome of the TMR mutants was poorly maintained in latently infected cells, suggesting that integration is crucial for the maintenance of the virus genome. To investigate the role of the putative HHV-6 recombinase, the ORF U94 was either deleted entirely from the HHV-6A BAC or its expression was abrogated by introducing a premature stop codon. Integration efficiencies of the U94 mutants were not altered compared to wt and revertant viruses in the U2OS integration assay, suggesting that U94 is not essential for HHV-6A integration. In addition, inhibiting the cellular recombinase Rad51, using a specific inhibitor, also did not significantly change the integration frequencies of the viruses, indicating that other viral or cellular recombinases can complement the function of U94 and Rad51 in HHV- 6A integration.Das Humane Herpesvirus 6 (HHV-6) gehört zu den Betaherpesviren und ist eng verwandt mit dem humanen Cytomegalievirus. HHV-6 verursacht die Kinderkrankheit Roseola Infantum und hat eine weltweite Seroprävalenz von über 90 %. Nach überstandener Primärinfektion kann HHV-6 im Wirt eine persistierende Infektion ausbilden, die sich durch lebenslange Latenz des Virus in Knochenmark-Vorläuferzellen, Monozyten und Makrophagen auszeichnet. Eine Reaktivierung des Virus tritt häufig bei immunsupprimierten Patienten auf und wird in diesen Fällen mit Krankheitsbildern wie Enzephalitis, Multipler Sklerose, Transplantatabstoßung und einem schnelleren Vorschreiten von HIV zu AIDS in Verbindung gebracht. Es wurde gezeigt, dass HHV-6 sein genetisches Material in die Telomere von humanen Chromosomen einbauen kann. Dieser Mechanismus erlaubt die vertikale Übertragung des Virus durch die Keimbahn, was dazu führt, dass in betroffenen Individuen das integrierte Virus in jeder Körperzelle vorzufinden ist. Dieser Zustand wird als chromosomal integriertes HHV-6 (ciHHV-6) bezeichnet und kann in circa 1 % der Weltbevölkerung nachgewiesen werden. Bis heute sind der Integrationsmechanismus, sowie die benötigten Faktoren gänzlich unbekannt. Interessanterweise besitzen HHV-6 und einige andere Herpesviren Telomersequenzen (TMRs) an ihren Genomenden, die identisch mit den Sequenzen humaner Telomere sind. In HHV-6 werden diese als perfekte TMRs (pTMRs) und nicht-perfekte TMRs (impTMRs) bezeichnet und es wird angenommen, dass sie homologe Rekombination zwischen Virus und Wirt ermöglichen. Des Weiteren kodiert HHV-6 für das Gen U94, das alle konservierten Domänen der Rekombinase Rep des Adeno-assoziierten Virus 2 (AAV-2) aufweist. Die Expression von U94 konnte nachweislich die Replikation eines Rep-defizienten AAV-2 wiederherstellen, was darauf hindeutet, dass beide Proteine vergleichbare Funktionen ausüben. In der Tat wurde vor kurzem gezeigt, dass U94 in vitro DNA-bindungs-, ATPase-, Helikase- und Exonuklease- Aktivitäten besitzt, wie sie auch für Rep beschrieben ist. Die tatsächliche Rolle dieser beiden viralen Faktoren im Verlauf der Integration von HHV-6 ist aber immer noch ungeklärt. Um die Funktion der TMRs während der HHV-6 Integration aufzuschlüsseln, wurden beide TMR Sequenzen, entweder einzeln oder gleichzeitig, in einem bakteriellen artifiziellen Chromosom (BAC) des HHV-6A mit Hilfe der en passant Mutagenese Methode beseitigt. Nach erfolgreicher Rekonstitution, replizierten die TMR Mutanten vergleichbar mit dem Ursprungsvirus (wt), was dafür spricht, dass die TMR Sequenzen nicht zwingend notwendig für die Replikation von HHV-6A sind. Um die Integrationseigenschaften der rekombinanten Viren zu untersuchen, habe ich ein in vitro Latenzsystem entwickelt, dass es mir ermöglicht die Integrationseffizienz, sowie den Erhalt des viralen Genoms in latent infizierten U2OS Zellen zu analysieren. Fluoreszenz in situ Hybridisierungs- Analysen (FISH) zeigten, dass die Integration der TMR Mutanten stark beeinträchtigt war im Vergleich zum Wildtyp Virus. Das Genom der TMR Mutanten wurde in den latent infizierten Zellen nur unzureichend erhalten, was dafür spricht, dass Integration essenziell für den Erhalt des Virusgenoms ist. Um Aufschluss über die Rolle der vermeintlichen Rekombinase zu erhalten, wurde zudem der offene Leserahmen von U94 gänzlich aus dem HHV-6A BAC beseitigt oder die Proteinexpression durch das Einfügen eines verfrühten Stopkodons außer Kraft gesetzt. Die resultierenden U94 Mutanten zeigten in meinen Integrationsanalysen einen vergleichbaren Phänotyp wie das Wildtyp Virus. Dies deutet darauf hin, dass U94 nicht essentiell für die Integration von HHV-6A ist. Zusätzlich zeigte auch eine Inhibierung der zellulären Rekombinase Rad51, mit Hilfe eines spezifischen Hemmstoffes, keine signifikanten Unterschiede im Bezug auf die Integrationsfrequenzen meiner Viren. Diese Ergebnisse legen nahe, dass es weitere zelluläre oder virale Rekombinasen gibt, die an der Integration von HHV-6A beteiligt sind

The ND10 complex represses lytic human herpesvirus 6A replication and promotes silencing of the viral genome

Human herpesvirus 6A (HHV-6A) replicates in peripheral blood mononuclear cells (PBMCs) and various T-cell lines in vitro. Intriguingly, the virus can also establish latency in these cells, but it remains unknown what influences the decision between lytic replication and the latency of the virus. Incoming virus genomes are confronted with the nuclear domain 10 (ND10) complex as part of an intrinsic antiviral response. Most herpesviruses can efficiently subvert ND10, but its role in HHV-6A infection remains poorly understood. In this study, we investigated if the ND10 complex affects HHV-6A replication and contributes to the silencing of the virus genome during latency. We could demonstrate that ND10 complex was not dissociated upon infection, while the number of ND10 bodies was reduced in lytically infected cells. Virus replication was significantly enhanced upon knock down of the ND10 complex using shRNAs against its major constituents promyelocytic leukemia protein (PML), hDaxx, and Sp100. In addition, we could demonstrate that viral genes are more efficiently silenced in the presence of a functional ND10 complex. Our data thereby provides the first evidence that the cellular ND10 complex plays an important role in suppressing HHV-6A lytic replication and the silencing of the virus genome in latently infected cells

Viral Proteins U41 and U70 of Human Herpesvirus 6A Are Dispensable for Telomere Integration

Human herpesvirus-6A and -6B (HHV-6A and -6B) are two closely related betaherpesviruses that infect humans. Upon primary infection they establish a life-long infection termed latency, where the virus genome is integrated into the telomeres of latently infected cells. Intriguingly, HHV-6A/B can integrate into germ cells, leading to individuals with inherited chromosomally-integrated HHV-6 (iciHHV-6), who have the HHV-6 genome in every cell. It is known that telomeric repeats flanking the virus genome are essential for integration; however, the protein factors mediating integration remain enigmatic. We have previously shown that the putative viral integrase U94 is not essential for telomere integration; thus, we set out to assess the contribution of potential viral recombination proteins U41 and U70 towards integration. We could show that U70 enhances dsDNA break repair via a homology-directed mechanism using a reporter cell line. We then engineered cells to produce shRNAs targeting both U41 and U70 to inhibit their expression during infection. Using these cells in our HHV-6A in vitro integration assay, we could show that U41/U70 were dispensable for telomere integration. Furthermore, additional inhibition of the cellular recombinase Rad51 suggested that it was also not essential, indicating that other cellular and/or viral factors must mediate telomere integration

Poised RNA Polymerase II Changes over Developmental Time and Prepares Genes for Future Expression

Poised RNA polymerase II (Pol II) is predominantly found at developmental control genes and is thought to allow their rapid and synchronous induction in response to extracellular signals. How the recruitment of poised RNA Pol II is regulated during development is not known. By isolating muscle tissue from Drosophila embryos at five stages of differentiation, we show that the recruitment of poised Pol II occurs at many genes de novo and this makes them permissive for future gene expression. A comparison with other tissues shows that these changes are stage specific and not tissue specific. In contrast, Polycomb group repression is tissue specific, and in combination with Pol II (the balanced state) marks genes with highly dynamic expression. This suggests that poised Pol II is temporally regulated and is held in check in a tissue-specific fashion. We compare our data with findings in mammalian embryonic stem cells and discuss a framework for predicting developmental programs on the basis of the chromatin state