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

Viruses ◽  
2018 ◽  
Vol 10 (11) ◽  
pp. 656 ◽  
Author(s):  
Darren Wight ◽  
Nina Wallaschek ◽  
Anirban Sanyal ◽  
Sandra Weller ◽  
Louis Flamand ◽  
...  
Keyword(s):  
Primary Infection ◽  
Human Herpesvirus ◽  
Viral Proteins ◽  
Virus Genome ◽  
Reporter Cell ◽  
Viral Recombination ◽  
Recombination Proteins ◽  
Latently Infected ◽  
Infected Cells

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.

scholarly journals The ND10 Complex Represses Lytic Human Herpesvirus 6A Replication and Promotes Silencing of the Viral Genome

Viruses ◽  
2018 ◽  
Vol 10 (8) ◽  
pp. 401 ◽  
Author(s):  
Anirban Sanyal ◽  
Nina Wallaschek ◽  
Mandy Glass ◽  
Louis Flamand ◽  
Darren Wight ◽  
...  
Keyword(s):  
Mononuclear Cells ◽  
Human Herpesvirus ◽  
Virus Genome ◽  
Lytic Replication ◽  
Peripheral Blood Mononuclear ◽  
Latently Infected ◽  
Infected Cells ◽  
T Cell Lines ◽  
Virus Genomes

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.

scholarly journals Modulation of Nuclear Localization of the Influenza Virus Nucleoprotein through Interaction with Actin Filaments

1999 ◽  
Vol 73 (3) ◽  
pp. 2222-2231 ◽  
Author(s):  
Paul Digard ◽  
Debra Elton ◽  
Konrad Bishop ◽  
Elizabeth Medcalf ◽  
Alan Weeds ◽  
...  
Keyword(s):  
Influenza Virus ◽  
Nuclear Localization ◽  
Virus Genome ◽  
Actin Binding ◽  
Filamentous Actin ◽  
Nuclear Localization Signals ◽  
Infected Cells ◽  
Cytoplasmic Accumulation ◽  
High Level

ABSTRACT The influenza virus genome is transcribed in the nuclei of infected cells but assembled into progeny virions in the cytoplasm. This is reflected in the cellular distribution of the virus nucleoprotein (NP), a protein which encapsidates genomic RNA to form ribonucleoprotein structures. At early times postinfection NP is found in the nucleus, but at later times it is found predominantly in the cytoplasm. NP contains several sequences proposed to act as nuclear localization signals (NLSs), and it is not clear how these are overridden to allow cytoplasmic accumulation of the protein. We find that NP binds tightly to filamentous actin in vitro and have identified a cluster of residues in NP essential for the interaction. Complexes containing RNA, NP, and actin could be formed, suggesting that viral ribonucleoproteins also bind actin. In cells, exogenously expressed NP when expressed at a high level partitioned to the cytoplasm, where it associated with F-actin stress fibers. In contrast, mutants unable to bind F-actin efficiently were imported into the nucleus even under conditions of high-level expression. Similarly, nuclear import of NLS-deficient NP molecules was restored by concomitant disruption of F-actin binding. We propose that the interaction of NP with F-actin causes the cytoplasmic retention of influenza virus ribonucleoproteins.

scholarly journals In vitro cellular tropism of human B-lymphotropic virus (human herpesvirus-6).

1988 ◽  
Vol 167 (5) ◽  
pp. 1659-1670 ◽  
Author(s):  
P Lusso ◽  
P D Markham ◽  
E Tschachler ◽  
F di Marzo Veronese ◽  
S Z Salahuddin ◽  
...  
Keyword(s):  
T Cell ◽  
T Lymphocytes ◽  
Cell Population ◽  
Mononuclear Cells ◽  
Human Herpesvirus ◽  
Human Herpesvirus 6 ◽  
Infected Cells ◽  
Cellular Tropism ◽  
Herpesvirus 6

We investigated the cellular tropism of human B-lymphotropic virus (HBLV) (also designated Human Herpesvirus-6) in vitro by infecting fresh MN cells from normal human adult peripheral blood, umbilical cord blood, bone marrow, tonsil, and thymus. Cultures from all the sources examined contained infectable cells, as shown by the appearance of characteristic enlarged, round-shaped, short-lived cells expressing HBLV-specific markers. Detailed immunological analysis demonstrated that the vast majority of these cells expressed T cell-associated antigens (i.e., CD7, CD5, CD2, CD4, and to a lesser extent, CD8). The CD3 antigen and the TCR-alpha/beta heterodimer were not detectable on the surface membrane, but were identified within the cytoplasm of HBLV-infected cells, by both immunofluorescence and radioimmunoprecipitation assay. A proportion of the HBLV-infected cell population also expressed the CD15 and class II MHC DR antigens. By means of immunoselection procedures it was possible to show that a consistent proportion of HBLV-infectable cells were contained within the CD3-depleted immature T cell population, while the depletion of CD2+ cells completely abrogated the infectability of the cultures. Northern blot analysis confirmed the T cell origin of HBLV-infected cells, demonstrating the expression of full size TCR-alpha and -beta chain mRNA. In addition to fresh T cells, HBLV was able to infect normal T lymphocytes expanded in vitro with IL-2 for greater than 30 d. These results indicate that HBLV is selectively T cell tropic in the course of the in vitro infection of normal mononuclear cells and may therefore be directly involved in the pathogenesis of T cell related hematological disorders. In particular, in light of the cytopathic effect exerted in vitro on CD4+ T lymphocytes, a possible role of HBLV in immune deficiency conditions should be considered.

scholarly journals Nascent Transcriptomics Reveal Cellular Prolytic Factors Upregulated Upstream of the Latent-to-Lytic Switch Protein of Epstein-Barr Virus

2020 ◽  
Vol 94 (7) ◽  
Author(s):  
Tiffany R. Frey ◽  
Jozan Brathwaite ◽  
Xiaofan Li ◽  
Sandeepta Burgula ◽  
Ibukun A. Akinyemi ◽  
...  
Keyword(s):  
Epstein Barr Virus ◽  
Human Herpesvirus ◽  
Lytic Cycle ◽  
Barr Virus ◽  
Viral Spread ◽  
Cellular Genes ◽  
Latently Infected ◽  
Infected Cells ◽  
Host Shutoff ◽  
Epstein Barr

ABSTRACT Lytic activation from latency is a key transition point in the life cycle of herpesviruses. Epstein-Barr virus (EBV) is a human herpesvirus that can cause lymphomas, epithelial cancers, and other diseases, most of which require the lytic cycle. While the lytic cycle of EBV can be triggered by chemicals and immunologic ligands, the lytic cascade is activated only when expression of the EBV latent-to-lytic switch protein ZEBRA is turned on. ZEBRA then transcriptionally activates other EBV genes and, together with some of those gene products, ensures completion of the lytic cycle. However, not every latently infected cell exposed to a lytic trigger turns on the expression of ZEBRA, resulting in responsive and refractory subpopulations. What governs this dichotomy? By examining the nascent transcriptome following exposure to a lytic trigger, we find that several cellular genes are transcriptionally upregulated temporally upstream of ZEBRA. These genes regulate lytic susceptibility to various degrees in latently infected cells that respond to mechanistically distinct lytic triggers. While increased expression of these cellular genes defines a prolytic state, such upregulation also runs counter to the well-known mechanism of viral-nuclease-mediated host shutoff that is activated downstream of ZEBRA. Furthermore, a subset of upregulated cellular genes is transcriptionally repressed temporally downstream of ZEBRA, indicating an additional mode of virus-mediated host shutoff through transcriptional repression. Thus, increased transcription of a set of host genes contributes to a prolytic state that allows a subpopulation of cells to support the EBV lytic cycle. IMPORTANCE Transition from latency to the lytic phase is necessary for herpesvirus-mediated pathology as well as viral spread and persistence in the population at large. Yet, viral genomes in only some cells in a population of latently infected cells respond to lytic triggers, resulting in subpopulations of responsive/lytic and refractory cells. Our investigations into this partially permissive phenotype of the herpesvirus Epstein-Barr virus (EBV) indicate that upon exposure to lytic triggers, certain cellular genes are transcriptionally upregulated, while viral latency genes are downregulated ahead of expression of the viral latent-to-lytic switch protein. These cellular genes contribute to lytic susceptibility to various degrees. Apart from indicating that there may be a cellular “prolytic” state, our findings indicate that (i) early transcriptional upregulation of cellular genes counters the well-known viral-nuclease-mediated host shutoff and (ii) subsequent transcriptional downregulation of a subset of early upregulated cellular genes is a previously undescribed mode of host shutoff.

scholarly journals The Latency-Associated Nuclear Antigen of Rhesus Monkey Rhadinovirus Inhibits Viral Replication through Repression of Orf50/Rta Transcriptional Activation

2005 ◽  
Vol 79 (5) ◽  
pp. 3127-3138 ◽  
Author(s):  
Scott M. DeWire ◽  
Blossom Damania
Keyword(s):  
Rhesus Monkey ◽  
Transcriptional Activation ◽  
Trichostatin A ◽  
Human Herpesvirus ◽  
Lytic Replication ◽  
Lytic Cycle ◽  
Viral Reactivation ◽  
Nuclear Antigen ◽  
Latently Infected

ABSTRACT Rhesus monkey rhadinovirus (RRV) is a gamma-2-herpesvirus that is closely related to Kaposi's sarcoma-associated herpesvirus/human herpesvirus-8. We have previously reported that the transcript for RRV latency-associated nuclear antigen (R-LANA) is expressed during lytic replication in rhesus fibroblasts. In this article, we report the development of a latent culture system for RRV and show that mRNA specific for R-LANA is expressed during latency as well. We have characterized the R-LANA protein and demonstrate that it exhibits a nuclear speckled localization and possesses the ability to homodimerize. When expressed in rhesus fibroblasts, R-LANA can inhibit RRV lytic replication in vitro. We have investigated the mechanism behind this inhibition and find that, while R-LANA itself has very little effect on lytic promoters, it can dramatically decrease the transactivation function of RRV Orf50 (Rta), which is the major viral transcription factor. We further show that the mechanism for this repression involves the recruitment of histone deacetylase complexes (HDACs), because R-LANA's ability to repress Orf50 transactivation is completely reversed by the addition of the HDAC inhibitor trichostatin A (TSA). We also report that TSA alone can significantly reactivate RRV from latently infected cells. We propose that the repressive effects of R-LANA on RRV Orf50 transactivation serve to downregulate the transcription of early genes at late times during the lytic cycle and also help to maintain viral latency by preventing viral reactivation.

Calcium modulation activates Epstein-Barr virus genome in latently infected cells

Science ◽  
1986 ◽  
Vol 232 (4757) ◽  
pp. 1554-1556 ◽  
Author(s):  
A Faggioni ◽  
C Zompetta ◽  
S Grimaldi ◽  
G Barile ◽  
L Frati ◽  
...  
Keyword(s):  
Epstein Barr Virus ◽  
Virus Genome ◽  
Barr Virus ◽  
Calcium Modulation ◽  
Latently Infected ◽  
Infected Cells ◽  
Epstein Barr

scholarly journals Characterization of Entry Mechanisms of Human Herpesvirus 8 by Using an Rta-Dependent Reporter Cell Line

2003 ◽  
Vol 77 (14) ◽  
pp. 8147-8152 ◽  
Author(s):  
Naoki Inoue ◽  
Jörn Winter ◽  
Renu B. Lal ◽  
Margaret K. Offermann ◽  
Shin Koyano
Keyword(s):  
Cell Line ◽  
Primary Effusion Lymphoma ◽  
Human Herpesvirus ◽  
Human Herpesvirus 8 ◽  
Dependent Manner ◽  
Reporter Cell Line ◽  
Reporter Cell ◽  
Herpesvirus 8 ◽  
Intracellular Expression ◽  
Infected Cells

ABSTRACT To analyze the mechanisms of entry of human herpesvirus 8 (HHV-8), we established a reporter cell line T1H6 that contains the lacZ gene under the control of the polyadenylated nuclear RNA promoter, known to be strongly activated by a viral transactivator, Rta. We found that infection with cell-free virus, as well as cocultivation with HHV-8-positive primary effusion lymphoma cell lines, activated the lacZ gene of T1H6 in a sensitive and dose-dependent manner. Addition of Polybrene and centrifugation enhanced, but polysulfonate compounds inhibited, the HHV-8 infectivity. RGD-motif-containing polypeptides and integrins did not decrease the infectivity, suggesting the presence of an additional cellular receptor other than the reported one. The entry was dependent on pH acidification but not on the clathrin pathway. Although conditioned media obtained from human immunodeficiency virus (HIV)-infected cells did not have any effect on the early steps of HHV-8 infection, intracellular expression of a proviral HIV type 1, but not of Tat alone, increased the HHV-8-dependent reporter activation slightly, suggesting a potential of HIV-mediated enhancement of an early step of HHV-8 infection.

scholarly journals Human Herpesvirus 6B Induces Hypomethylation on Chromosome 17p13.3, Correlating with Increased Gene Expression and Virus Integration

2017 ◽  
Vol 91 (11) ◽  
Author(s):  
Elin Engdahl ◽  
Nicky Dunn ◽  
Pitt Niehusmann ◽  
Sarah Wideman ◽  
Peter Wipfler ◽  
...  
Keyword(s):  
Integration Site ◽  
Acute Infection ◽  
Human Herpesvirus ◽  
Epigenetic Modifications ◽  
Host Cells ◽  
Integration Process ◽  
450K Array ◽  
Infected Cells ◽  
Virus Integration

ABSTRACT Human herpesvirus 6B (HHV-6B) is a neurotropic betaherpesvirus that achieves latency by integrating its genome into host cell chromosomes. Several viruses can induce epigenetic modifications in their host cells, but no study has investigated the epigenetic modifications induced by HHV-6B. This study analyzed methylation with an Illumina 450K array, comparing HHV-6B-infected and uninfected Molt-3 T cells 3 days postinfection. Bisulfite pyrosequencing was used to validate the Illumina results and to investigate methylation over time in vitro. Expression of genes was investigated using quantitative PCR (qPCR), and virus integration was investigated with PCR. A total of 406 CpG sites showed a significant HHV-6B-induced change in methylation in vitro. Remarkably, 86% (351/406) of these CpGs were located <1 Mb from chromosomal ends and were all hypomethylated in virus-infected cells. This was most evident at chromosome 17p13.3, where HHV-6B had induced CpG hypomethylation after 2 days of infection, possibly through TET2, which was found to be upregulated by the virus. In addition, virus-induced cytosine hydroxymethylation was observed. Genes located in the hypomethylated region at 17p13.3 showed significantly upregulated expression in HHV-6B-infected cells. A temporal experiment revealed HHV-6B integration in Molt-3 cell DNA 3 days after infection. The telomere at 17p has repeatedly been described as an integration site for HHV-6B, and we show for the first time that HHV-6B induces hypomethylation in this region during acute infection, which may play a role in the integration process, possibly by making the DNA more accessible. IMPORTANCE The ability to establish latency in the host is a hallmark of herpesviruses, but the mechanisms differ. Human herpesvirus 6B (HHV-6B) is known to establish latency through integration of its genome into the telomeric regions of host cells, with the ability to reactivate. Our study is the first to show that HHV-6B specifically induces hypomethylated regions close to the telomeres and that integrating viruses may use the host methylation machinery to facilitate their integration process. The results from this study contribute to knowledge of HHV-6B biology and virus-host interaction. This in turn will lead to further progress in our understanding of the underlying mechanisms by which HHV-6B contributes to pathological processes and may have important implications in both disease prevention and treatment.

scholarly journals A Therapeutic Strategy to Combat HIV-1 Latently Infected Cells With a Combination of Latency-Reversing Agents Containing DAG-Lactone PKC Activators

2021 ◽  
Vol 12 ◽  
Author(s):  
Kouki Matsuda ◽  
Takuya Kobayakawa ◽  
Ryusho Kariya ◽  
Kiyoto Tsuchiya ◽  
Shoraku Ryu ◽  
...  
Keyword(s):  
Whole Body ◽  
Therapeutic Effects ◽  
Combined Antiretroviral Therapy ◽  
Latently Infected ◽  
Shock And Kill ◽  
Infected Cells ◽  
Hiv 1 ◽  
Reversing Agents

Advances in antiviral therapy have dramatically improved the therapeutic effects on HIV type 1 (HIV-1) infection. However, even with potent combined antiretroviral therapy, HIV-1 latently infected cells cannot be fully eradicated. Latency-reversing agents (LRAs) are considered a potential tool for eliminating such cells; however, recent in vitro and in vivo studies have raised serious concerns regarding the efficacy and safety of the “shock and kill” strategy using LRAs. In the present study, we examined the activity and safety of a panel of protein kinase C (PKC) activators with a diacylglycerol (DAG)-lactone structure that mimics DAG, an endogenous ligand for PKC isozymes. YSE028, a DAG-lactone derivative, reversed HIV-1 latency in vitro when tested using HIV-1 latently infected cells (e.g., ACH2 and J-Lat cells) and primary cells from HIV-1-infected individuals. The activity of YSE028 in reversing HIV-1 latency was synergistically enhanced when combined with JQ1, a bromodomain and extra-terminal inhibitor LRA. DAG-lactone PKC activators also induced caspase-mediated apoptosis, specifically in HIV-1 latently infected cells. In addition, these DAG-lactone PKC activators showed minimal toxicity in vitro and in vivo. These data suggest that DAG-lactone PKC activators may serve as potential candidates for combination therapy against HIV-1 latently infected cells, especially when combined with other LRAs with a different mechanism, to minimize side effects and achieve maximum efficacy in various reservoir cells of the whole body.

scholarly journals Protective Role of Combined Polyphenols and Micronutrients against Influenza A Virus and SARS-CoV-2 Infection In Vitro

Biomedicines ◽  
2021 ◽  
Vol 9 (11) ◽  
pp. 1721
Author(s):  
Marta De Angelis ◽  
David Della-Morte ◽  
Gabriele Buttinelli ◽  
Angela Di Martino ◽  
Francesca Pacifici ◽  
...  
Keyword(s):  
Influenza Virus ◽  
Influenza A ◽  
Viral Proteins ◽  
Antiviral Effect ◽  
Inhibitory Effect ◽  
Protective Role ◽  
Virus Infections ◽  
Infected Cells ◽  
Respiratory Virus Infections

Polyphenols have been widely studied for their antiviral effect against respiratory virus infections. Among these, resveratrol (RV) has been demonstrated to inhibit influenza virus replication and more recently, it has been tested together with pterostilbene against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. In the present work, we evaluated the antiviral activity of polydatin, an RV precursor, and a mixture of polyphenols and other micronutrients, named A5+, against influenza virus and SARS-CoV-2 infections. To this end, we infected Vero E6 cells and analyzed the replication of both respiratory viruses in terms of viral proteins synthesis and viral titration. We demonstrated that A5+ showed a higher efficacy in inhibiting both influenza virus and SARS-CoV-2 infections compared to polydatin treatment alone. Indeed, post infection treatment significantly decreased viral proteins expression and viral release, probably by interfering with any step of virus replicative cycle. Intriguingly, A5+ treatment strongly reduced IL-6 cytokine production in influenza virus-infected cells, suggesting its potential anti-inflammatory properties during the infection. Overall, these results demonstrate the synergic and innovative antiviral efficacy of A5+ mixture, although further studies are needed to clarify the mechanisms underlying its inhibitory effect.

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