scholarly journals Viral FGARAT Homolog ORF75 of Rhesus Monkey Rhadinovirus Effects Proteasomal Degradation of the ND10 Components SP100 and PML

2016 ◽  
Vol 90 (17) ◽  
pp. 8013-8028 ◽  
Author(s):  
Alexander S. Hahn ◽  
Anna K. Großkopf ◽  
Doris Jungnickl ◽  
Brigitte Scholz ◽  
Armin Ensser
Keyword(s):  
Rhesus Monkey ◽  
De Novo ◽  
Human Herpesvirus ◽  
Proteasomal Degradation ◽  
Lytic Replication ◽  
Intrinsic Immunity ◽  
Protein Levels ◽  
Short Palindromic Repeat ◽  
Infected Cells ◽  
The Individual

ABSTRACTNuclear domain 10 (ND10) components restrict herpesviral infection, and herpesviruses antagonize this restriction by a variety of strategies, including degradation or relocalization of ND10 proteins. The rhesus monkey rhadinovirus (RRV) shares many key biological features with the closely related Kaposi's sarcoma-associated herpesvirus (KSHV; human herpesvirus 8) and readily infects cells of both human and rhesus monkey origin. We used the clustered regularly interspaced short palindromic repeat-Cas9 (CRISPR-Cas9) technique to generate knockout (ko) cells for each of the four ND10 components, PML, SP100, DAXX, and ATRX. These ko cells were analyzed with regard to permissiveness for RRV infection. In addition, we analyzed the fate of the individual ND10 components in infected cells by immunofluorescence and Western blotting. Knockout of the ND10 component DAXX markedly increased RRV infection, while knockout of PML or SP100 had a less pronounced effect. In line with these observations, RRV infection resulted in rapid degradation of SP100, followed by degradation of PML and the loss of ND10 structures, whereas the protein levels of ATRX and DAXX remained constant. Notably, inhibition of the proteasome but not inhibition ofde novogene expression prevented the loss of SP100 and PML in cells that did not support lytic replication, compatible with proteasomal degradation of these ND10 components through the action of a viral tegument protein. Expression of the RRV FGARAT homolog ORF75 was sufficient to effect the loss of SP100 and PML in transfected or transduced cells, implicating ORF75 as the viral effector protein.IMPORTANCEOur findings highlight the antiviral role of ND10 and its individual components and further establish the viral FGARAT homologs of the gammaherpesviruses to be important viral effectors that counteract ND10-instituted intrinsic immunity. Surprisingly, even closely related viruses like KSHV and RRV evolved to use different strategies to evade ND10-mediated restriction. RRV first targets SP100 for degradation and then targets PML with a delayed kinetic, a strategy which clearly differs from that of other gammaherpesviruses. Despite efficient degradation of these two major ND10 components, RRV is still restricted by DAXX, another abundant ND10 component, as evidenced by a marked increase in RRV infection and replication upon knockout of DAXX. Taken together, our findings substantiate PML, SP100, and DAXX as key antiviral proteins, in that the first two are targeted for degradation by RRV and the last one still potently restricts replication of RRV.

scholarly journals Kinetics of Expression of Rhesus Monkey Rhadinovirus (RRV) and Identification and Characterization of a Polycistronic Transcript Encoding the RRV Orf50/Rta, RRV R8, and R8.1 Genes

2002 ◽  
Vol 76 (19) ◽  
pp. 9819-9831 ◽  
Author(s):  
Scott M. DeWire ◽  
Michael A. McVoy ◽  
Blossom Damania
Keyword(s):  
Rhesus Monkey ◽  
De Novo ◽  
Human Herpesvirus ◽  
Lytic Replication ◽  
Nuclear Antigen ◽  
Wild Type Virus ◽  
In Vivo Model ◽  
Close Relative ◽  
Kinetics Of

ABSTRACT Rhesus monkey rhadinovirus (RRV) is a close relative of Kaposi's sarcoma-associated herpesvirus (KSHV; human herpesvirus 8). RRV serves as an in vitro and an in vivo model for KSHV, and the mapping of its transcription program during lytic replication is significant since it represents de novo infection in the absence of stimulation with phorbol esters. Further, the RRV lytic system facilitates the making of recombinant viruses, and hence transcription profiling of the wild-type virus is important. Currently, the kinetics of lytic gene expression of RRV, the function of the RRV Orf50/Rta gene, and the presence of the RRV R8 and R8.1 genes are not known. This study details the transcription profile seen during RRV lytic replication and shows that RRV latency-associated nuclear antigen, viral FLIP (vFLIP), and vCyclin are transcribed during the RRV lytic phase. In addition, this study describes the identification of three new spliced products of the RRV Orf50, R8, and R8.1 genes, which are structural homologs of the KSHV Orf50, K8, and K8.1 genes, respectively. Characterization of the RRV Orf50 protein identifies it as a strong transcriptional transactivator capable of activating three early RRV promoters. Interestingly, the KSHV Orf50 transactivator can also activate these simian virus promoters, suggesting that there exists a conservation of gene function between the key transcription factors of KSHV and RRV.

scholarly journals Inhibition of Infection and Replication of Human Herpesvirus 8 in Microvascular Endothelial Cells by Alpha Interferon and Phosphonoformic Acid

2004 ◽  
Vol 78 (15) ◽  
pp. 8359-8371 ◽  
Author(s):  
Laurie T. Krug ◽  
Veronika P. Pozharskaya ◽  
Yimin Yu ◽  
Naoki Inoue ◽  
Margaret K. Offermann
Keyword(s):  
Endothelial Cells ◽  
Infectious Virus ◽  
Human Herpesvirus ◽  
Lytic Replication ◽  
Alpha Interferon ◽  
Human Herpesvirus 8 ◽  
Microvascular Endothelial Cells ◽  
Herpesvirus 8 ◽  
Phosphonoformic Acid ◽  
Infected Cells

ABSTRACT Infection of endothelial cells with human herpesvirus 8 (HHV-8) is an essential event in the development of Kaposi's sarcoma. When primary microvascular endothelial cells (MECs) were infected with HHV-8 at a low multiplicity of infection, considerable latent replication of HHV-8 occurred, leading to a time-dependent increase in the percentage of virus-infected cells that was accompanied by cellular spindling and growth to a high density with loss of contact inhibition. Only a low percentage of MECs supported lytic replication of HHV-8 and produced infectious virus. Phosphonoformic acid blocked production of infectious virus but did not inhibit the rapid expansion of latently infected MECs. Pretreatment of MECs with alpha interferon (IFN-α) prior to infection effectively reduced HHV-8 viral gene expression, latent replication, and production of infectious virus. High levels of the double-stranded RNA activated protein kinase (PKR) were expressed in HHV-8-infected cells, and incubation with IFN-α increased PKR expression more in virus-infected cells than in uninfected cells. MECs that were immortalized with simian virus 40 large-T antigen differed from nonimmortalized MECs in their response to infection with HHV-8 and demonstrated that cells with elevated levels of expression of antiviral transcripts expressed viral transcripts at reduced levels. These studies demonstrate that MECs respond to HHV-8 with enhanced expression of cellular antiviral genes and that augmentation of innate antiviral defenses with IFN-α is a more effective strategy than inhibition of viral lytic replication to protect MECs from infection with HHV-8 and to restrict proliferation of virus-infected MECs.

scholarly journals A Systems Biology Approach To Identify the Combination Effects of Human Herpesvirus 8 Genes on NF-κB Activation

2009 ◽  
Vol 83 (6) ◽  
pp. 2563-2574 ◽  
Author(s):  
Andreas Konrad ◽  
Effi Wies ◽  
Mathias Thurau ◽  
Gaby Marquardt ◽  
Elisabeth Naschberger ◽  
...  
Keyword(s):  
Systems Biology ◽  
Primary Effusion Lymphoma ◽  
Human Herpesvirus ◽  
Etiologic Agent ◽  
Lytic Replication ◽  
Fine Tuning ◽  
Human Herpesvirus 8 ◽  
Systematic Analysis ◽  
Herpesvirus 8 ◽  
Infected Cells

ABSTRACT Human herpesvirus 8 (HHV-8) is the etiologic agent of Kaposi's sarcoma and primary effusion lymphoma. Activation of the cellular transcription factor nuclear factor-kappa B (NF-κB) is essential for latent persistence of HHV-8, survival of HHV-8-infected cells, and disease progression. We used reverse-transfected cell microarrays (RTCM) as an unbiased systems biology approach to systematically analyze the effects of HHV-8 genes on the NF-κB signaling pathway. All HHV-8 genes individually (n = 86) and, additionally, all K and latent genes in pairwise combinations (n = 231) were investigated. Statistical analyses of more than 14,000 transfections identified ORF75 as a novel and confirmed K13 as a known HHV-8 activator of NF-κB. K13 and ORF75 showed cooperative NF-κB activation. Small interfering RNA-mediated knockdown of ORF75 expression demonstrated that this gene contributes significantly to NF-κB activation in HHV-8-infected cells. Furthermore, our approach confirmed K10.5 as an NF-κB inhibitor and newly identified K1 as an inhibitor of both K13- and ORF75-mediated NF-κB activation. All results obtained with RTCM were confirmed with classical transfection experiments. Our work describes the first successful application of RTCM for the systematic analysis of pathofunctions of genes of an infectious agent. With this approach, ORF75 and K1 were identified as novel HHV-8 regulatory molecules on the NF-κB signal transduction pathway. The genes identified may be involved in fine-tuning of the balance between latency and lytic replication, since this depends critically on the state of NF-κB activity.

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.

scholarly journals Open Reading Frame 33 of a Gammaherpesvirus Encodes a Tegument Protein Essential for Virion Morphogenesis and Egress

2009 ◽  
Vol 83 (20) ◽  
pp. 10582-10595 ◽  
Author(s):  
Haitao Guo ◽  
Lili Wang ◽  
Li Peng ◽  
Z. Hong Zhou ◽  
Hongyu Deng
Keyword(s):  
De Novo ◽  
Human Herpesvirus ◽  
Lytic Replication ◽  
Open Reading Frame ◽  
Tegument Protein ◽  
Late Gene ◽  
Viral Dna ◽  
Reading Frame ◽  
Murine Gammaherpesvirus ◽  
Virion Morphogenesis

ABSTRACT Tegument is a unique structure of herpesvirus, which surrounds the capsid and interacts with the envelope. Morphogenesis of gammaherpesvirus is poorly understood due to lack of efficient lytic replication for Epstein-Barr virus and Kaposi's sarcoma-associated herpesvirus/human herpesvirus 8, which are etiologically associated with several types of human malignancies. Murine gammaherpesvirus 68 (MHV-68) is genetically related to the human gammaherpesviruses and presents an excellent model for studying de novo lytic replication of gammaherpesviruses. MHV-68 open reading frame 33 (ORF33) is conserved among Alpha-, Beta-, and Gammaherpesvirinae subfamilies. However, the specific role of ORF33 in gammaherpesvirus replication has not yet been characterized. We describe here that ORF33 is a true late gene and encodes a tegument protein. By constructing an ORF33-null MHV-68 mutant, we demonstrated that ORF33 is not required for viral DNA replication, early and late gene expression, viral DNA packaging or capsid assembly but is required for virion morphogenesis and egress. Although the ORF33-null virus was deficient in release of infectious virions, partially tegumented capsids produced by the ORF33-null mutant accumulated in the cytoplasm, containing conserved capsid proteins, ORF52 tegument protein, but virtually no ORF45 tegument protein and the 65-kDa glycoprotein B. Finally, we found that the defect of ORF33-null MHV-68 could be rescued by providing ORF33 in trans or in an ORF33-null revertant virus. Taken together, our results indicate that ORF33 is a tegument protein required for viral lytic replication and functions in virion morphogenesis and egress.

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 Evidence for both Lytic Replication and Tightly Regulated Human Herpesvirus 8 Latency in Circulating Mononuclear Cells, with Virus Loads Frequently below Common Thresholds of Detection

2004 ◽  
Vol 78 (21) ◽  
pp. 11707-11714 ◽  
Author(s):  
Elisa Martró ◽  
Michael J. Cannon ◽  
Sheila C. Dollard ◽  
Thomas J. Spira ◽  
A. Scott Laney ◽  
...  
Keyword(s):  
Infected Cell ◽  
Mononuclear Cells ◽  
Human Herpesvirus ◽  
Lytic Replication ◽  
Human Herpesvirus 8 ◽  
Genome Copy ◽  
Herpesvirus 8 ◽  
Virus Load ◽  
Copy Numbers ◽  
Infected Cells

ABSTRACT To address whether human herpesvirus 8 (HHV-8) DNA in peripheral blood mononuclear cells (PBMCs) might be the product of latent or lytic infection and to shed light on sporadic detection of HHV-8 DNA in individuals seropositive for the virus, we studied the frequency of infected cells, total virus load, and virus load per infected cell in PBMCs from men coinfected with HHV-8 and human immunodeficiency virus (HIV), some of whom had Kaposi's sarcoma. The low frequencies of infected cells detected (fewer than one per million cells in some individuals) suggest that the prevalence of the virus in circulating leukocytes was underestimated in previous studies that employed more conventional sampling methods (single, small-volume specimens). Mean virus loads ranged from 3 to 330 copies per infected PBMC; these numbers can represent much higher loads in individual lytically infected cells (>103 genomes/cell) in mixtures that consist predominantly of latently (relatively few genomes) infected cells. The presence in some subjects of high HHV-8 mean genome copy numbers per infected cell, together with viral DNA being found in plasma only from subjects with positive PBMCs, supports earlier suggestions that the virus can actively replicate in PBMCs. In some individuals, mean virus loads were less than 10 genomes per infected cell, suggesting a tightly controlled purely latent state. HHV-8 genome copy numbers are substantially higher in latently infected cells derived from primary effusion lymphomas; thus, it appears that HHV-8 is able to adopt more than one latency program, perhaps analogous to the several types of Epstein-Barr virus latency.

scholarly journals Temporal Mapping of Transcripts in Herpesvirus 6 Variants

1998 ◽  
Vol 72 (5) ◽  
pp. 3837-3844 ◽  
Author(s):  
Prisco Mirandola ◽  
Paola Menegazzi ◽  
Stefania Merighi ◽  
Tullia Ravaioli ◽  
Enzo Cassai ◽  
...  
Keyword(s):  
Human Herpesvirus ◽  
Lytic Replication ◽  
Lymphoid Cells ◽  
Sequencing Data ◽  
Regulation Of Expression ◽  
Temporal Regulation ◽  
Reverse Transcription Pcr ◽  
Molecular Features ◽  
Infected Cells ◽  
Herpesvirus 6

ABSTRACT To define the molecular features characteristic of the early stages of infection of lymphocytes with human herpesvirus 6 (HHV-6) variant A or B, we studied the temporal regulation of expression of selected sets of viral genes. Thus, U42, U94, U89-U90, U73, and U39 are α genes since their transcripts (i) were made in the presence of inhibitors of protein synthesis and (ii) were detected 3 h after infection of untreated cells. U41, U53, U31, and U19 are β genes since their expression is inhibited by cycloheximide but not by phosphonoacetate, an inhibitor of DNA synthesis. U100 is a γ gene since its spliced transcript encoding the structural glycoprotein gp82/105 was first detected 16 h after infection of untreated cells but could not be detected in cells treated with phosphonoacetate. HHV-6 variants differ in the transcription patterns of their genes. U16-U17 originates a splice transcript and is regulated as α in HHV-6B and as β in HHV-6A. U91 generates two transcripts, amplified as 476- and 374-bp PCR fragments. The 476-bp fragment is α in HHV-6A-infected cells but β in HHV-6B-infected cells. Conversely, the 374-bp fragment is β in HHV-6A-infected cells and α in HHV-6B-infected cells. Furthermore, the spliced product of U18-U19-U20 (526 bp) is β in HHV-6A-infected cells, but only a partially spliced form (1.9 kb) was detected at late stages of infection in HHV-6B. HHV-6 transcription was also studied in nonproductive lymphoid cells, and the same transcription pattern detected during lytic infection was observed. Also, HHV-6 variants maintain the differences in U91, U16-17, and U18-U19-U20. We conclude that, as expected from the sequencing data, gene expression is generally similar in HHV-6 variants. However, transcription of selected genes in HHV-6A and HHV-6B differs with respect to temporal regulation and splicing pattern. Furthermore, the identification of viral functions expressed during the different stages of lytic replication suggests that reverse transcription-PCR for HHV-6 genes is a useful diagnostic approach to differentiate between latent and productive HHV-6 infection.

scholarly journals Whole-Genome Transcription Profiling of Rhesus Monkey Rhadinovirus

2005 ◽  
Vol 79 (13) ◽  
pp. 8637-8650 ◽  
Author(s):  
Dirk P. Dittmer ◽  
Carlos M. Gonzalez ◽  
Wolfgang Vahrson ◽  
Scott M. DeWire ◽  
Rebecca Hines-Boykin ◽  
...  
Keyword(s):  
Rhesus Monkey ◽  
De Novo ◽  
Sequence Similarity ◽  
Human Herpesvirus ◽  
High Specificity ◽  
Productive Infection ◽  
Transcription Profile ◽  
Genome Wide

ABSTRACT Rhesus monkey rhadinovirus (RRV) and Kaposi's sarcoma-associated herpesvirus (KSHV; also called human herpesvirus 8) belong to the gamma-2 grouping of herpesviruses. RRV and KSHV share a high degree of sequence similarity, and their genomes are organized in a similar fashion. RRV serves as an excellent animal model system to study the gamma herpesvirus life cycle both in vitro and in vivo. We have developed a high-sensitivity, high-throughput, high-specificity real-time quantitative reverse transcriptase-based PCR assay for RRV and have used this assay to profile transcription from the whole RRV genome during de novo productive infection of rhesus fibroblasts. Using this assay, we demonstrate that the genome-wide transcription profile for RRV closely parallels the genome-wide transcription profile for KSHV.

scholarly journals Surface Downregulation of Major Histocompatibility Complex Class I, PE-CAM, and ICAM-1 following De Novo Infection of Endothelial Cells with Kaposi's Sarcoma-Associated Herpesvirus

2003 ◽  
Vol 77 (17) ◽  
pp. 9669-9684 ◽  
Author(s):  
Costin Tomescu ◽  
Wai K. Law ◽  
Dean H. Kedes
Keyword(s):  
Endothelial Cells ◽  
Major Histocompatibility Complex ◽  
Cell Surface ◽  
Mhc Class I ◽  
Immune Evasion ◽  
De Novo ◽  
Lytic Replication ◽  
Class I ◽  
Histocompatibility Complex ◽  
Infected Cells

ABSTRACT Under selective pressure from host cytotoxic T lymphocytes, many viruses have evolved to downregulate major histocompatibility complex (MHC) class I and/or T-cell costimulatory molecules from the surface of infected cells. Kaposi's sarcoma-associated herpesvirus (KSHV) encodes two proteins, MIR-1 and MIR-2, that serve this function during lytic replication. In vivo, however, KSHV exists in a predominantly latent state, with less than 5% of infected cells expressing discernible lytic gene products. Thus, mechanisms of immune evasion that depend on genes expressed only during lytic replication are unlikely to be active in most KSHV-infected cells. As a result, we searched for evidence of similar defensive strategies extant during latency, employing culture systems that strongly favor latent KSHV infection. We measured cell surface levels of immunomodulatory proteins on both primary dermal microvascular endothelial cells (pDMVEC) infected through coculture with induced primary effusion lymphoma cells and telomerase-immortalized DMVEC infected directly with cell-free virus. Employing a panel of antibodies against several endothelial cell surface proteins, we show that de novo infection with KSHV leads to the downregulation of MHC class I, CD31 (PE-CAM), and CD54 (ICAM-I) but not CD58 (LFA-3) or CD95 (Fas). Furthermore, flow cytometry with a fluorescently labeled monoclonal antibody to the latency-associated nuclear antigen (LANA) revealed that downregulation occurred predominantly on KSHV-infected (LANA-positive) cells. Although the vast majority of infected cells displayed this downregulation, less than 1% expressed either immediate-early or late lytic proteins detectable by immunofluorescence. Together, these results suggest that downregulation of immunomodulatory proteins on the surface of target cells may represent a constitutive mode of immune evasion employed by KSHV following de novo infection.

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