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

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

Mammalian deltavirus without hepadnavirus coinfection in the neotropical rodent Proechimys semispinosus.

Hepatitis delta virus (HDV) is a human hepatitis-causing RNA virus, unrelated to any other taxonomic group of RNA viruses. Its occurrence as a satellite virus of hepatitis B virus (HBV) is a singular case in animal virology for which no consensus evolutionary explanation exists. Here we present a mammalian deltavirus that does not occur in humans, identified in the neotropical rodent species Proechimys semispinosus The rodent deltavirus is highly distinct, showing a common ancestor with a recently described deltavirus in snakes. Reverse genetics based on a tandem minus-strand complementary DNA genome copy under the control of a cytomegalovirus (CMV) promoter confirms autonomous genome replication in transfected cells, with initiation of replication from the upstream genome copy. In contrast to HDV, a large delta antigen is not expressed and the farnesylation motif critical for HBV interaction is absent from a genome region that might correspond to a hypothetical rodent large delta antigen. Correspondingly, there is no evidence for coinfection with an HBV-related hepadnavirus based on virus detection and serology in any deltavirus-positive animal. No other coinfecting viruses were detected by RNA sequencing studies of 120 wild-caught animals that could serve as a potential helper virus. The presence of virus in blood and pronounced detection in reproductively active males suggest horizontal transmission linked to competitive behavior. Our study establishes a nonhuman, mammalian deltavirus that occurs as a horizontally transmitted infection, is potentially cleared by immune response, is not focused in the liver, and possibly does not require helper virus coinfection

Investigation of different virus species in neotropical rodents and the influence of ecological factors

Im Rahmen dieser Dissertation wurde ein breites Spektrum an Nage- und Beuteltierproben analysiert. Dies führte zu vielen Interessanten Entdeckungen, wie etwa die große Prävalenz an Picobirnaviren oder die Detektion von mindestens zwei, bis dato unbekannten, neuen Genera aus der Familie Picornaviridae. Zudem gelang erstmaligen Nachweis eines Deltavirus aus einem Säugetier abseits vom Menschen, sowie dessen molekularbiologische Charakterisierung und Unterschied im Replikationsverhalten gegenüber Hepatitis Deltaviren. Darüber hinaus brachte die Detektion und bioinformatische Analyse der Hepaciviren einen enorm großen Wissensgewinn. Darüber hinaus konnte die Theorie, die hinter dem ökologischen „Verdünnungseffekt“, nämlich die Abhängigkeit von Speziesdiversität zu Prävalenz, klar bestätigt werden. All diese Untersuchungen verschiedener Proben um den Gatún See in Panama zeigten viele, menschengemachte Einflüsse auf die ansässigen Viruspopulationen auf. Durch den Bau des Panama Kanals wurden weite Teile des Urwalds überflutet, woraufhin der Gatún See entstand. Zusätzlich zu diesem initialen Terraforming, kam es durch den wirtschaftlichen Aufschwung zu einer Ausbreitung urbaner Flächen. All dies führte dazu, dass der Mensch mehr Kontakt zu diversifizierenden, potenziell zoonotischen, sowie bislang noch unbekannten Viruspopulationen hat. Und wie uns die jüngste Zeit zeigt, ist die Gefahr einer Pandemie mit zoonotischen Ursprung allgegenwärtig und sollte nicht unterschätzt werden. Aus diesem Grund ist es wichtiger denn je die Zusammenhänge zwischen verschiedenen Einflussfaktoren zu verstehen und eine Ausbreitung beziehungsweise das Erscheinen neuer zoonotischer Erreger vorhersagen zu können. Die aktuelle Pandemie zeigt wie relevant das Wissen um die Auswirkungen dieser Faktoren auf Pathogenität und Infektiosität (bzw. Infektionsrisiko) ist. Die Ergebnisse dieser Arbeit sollen dazu dienen, wie im Fall der Deltaviren, den bislang unbekannten Ursprung dieser Familie Deltaviren zu ergründen. All diese Grundlagen können in weiterführenden Studien nicht nur helfen, Pathogen-Wirts Interaktionen besser zu verstehen, sondern auch mögliche medizinische und ökologische Relevanzen für den Menschen abzuschätzen.In this dissertation, a wide range of rodent and marsupial samples were analyzed. This led to many interesting discoveries, such as the high prevalence of picobirnaviruses or the detection of at least two, previously unknown, new genera from the family Picornaviridae. In addition, the first detection of deltavirus outside of humans was achieved, as well as its molecular characterization and difference in replication behavior compared to hepatitis deltaviruses. Furthermore, the detection and bioinformatic analysis of hepaciviruses provided a tremendous increase in knowledge. Moreover, the theory behind the ecological "dilution effect", namely the dependence of species diversity on prevalence, was clearly confirmed. All these studies of different samples around Lake Gatún in Panama revealed many, man-made influences on the resident virus populations. The construction of the Panama Canal flooded large areas of virgin forest, resulting in the creation of Lake Gatún. In addition to this initial terraforming, an expansion of urban areas occurred as a result of the economic boom. All of this led to humans having more contact with diversifying, potentially zoonotic, as well as yet unknown viral populations. And as recent times have shown us, the threat of a pandemic of zoonotic origin is ever-present and should not be underestimated. For this reason, it is more important than ever to understand the interrelationships between different influencing factors and to be able to predict the spread or emergence of new zoonotic pathogens. The current pandemic shows how relevant the knowledge of the impact of these factors on pathogenicity and infectivity (or risk of infection) is. The results of this work will be used, as in the case of deltaviruses, to elucidate the hitherto unknown origin of this family of deltaviruses. All these fundamentals can help in further studies not only to better understand pathogen-host interactions, but also to assess possible medical and ecological relevance for humans

Primers and probes for qPCR and generation of recombinant viruses.

<p>Primers and probes for qPCR and generation of recombinant viruses.</p

Integration efficiency and genome maintenance of the ΔTMR mutant in the U2OS integration system.

<p><b>(A)</b> Integration frequency was quantified by determining the integration status of at least 90 metaphases. Significant differences between wt and ΔTMR (Mann-Whitney U-test, p < 0.05) are indicated with an asterisk (*). Results are shown as means of three independent experiments with standard errors. Representative metaphase images are shown on the right. Scale bar corresponds to 10μm. <b>(B)</b> 300 interphase nuclei were examined for the presence of HHV-6A. Significant differences between wt and ΔTMR (Mann-Whitney U-test, p < 0.05) are indicated with an asterisk (*). Results are shown as means of three independent experiments with standard errors. Representative interphase images are shown on the right. Scale bar corresponds to 10μm. <b>(C)</b> Maintenance of the HHV-6A genome was determined by qPCR analysis at d0 and d14 post sorting. Copy numbers per 1x 10⁶ cells are shown as means of three independent experiments with corresponding standard errors. Significant differences between wt and ΔTMR (Mann-Whitney U-test, p < 0.01) are indicated with asterisks (**).</p

Generation and characterization of the ΔTMR mutant.

<p><b>(A)</b> Schematic representation of the HHV-6A genome with deletion of the TMR (ΔTMR). <b>(B)</b> RFLP pattern of the wt, the ΔpTMR intermediate and the double deletion mutant ΔTMR upon digestion with <i>Sac</i>I analyzed on a 0.8% agarose gel o/n at 65 V. M = marker. Sizes of the marker fragments are indicated on the left. Red boxes highlight the fragments containing the target regions, where the expected band shifts can be observed. <b>(C)</b> Corresponding southern blot analysis detecting TMR sequences of the impTMR (upper panel) and pTMR (lower panel) after <i>Sac</i>I digestion of the indicated BAC clones using a DIG-labeled TMR probe. <b>(D)</b> Growth kinetics comparing replication properties of wt and ΔTMR mutant virus in JJHan cells. HHV-6A genome copy numbers were detected by qPCR. Copy numbers per 1x 10⁶ cells are shown as means of three independent experiments with standard errors.</p