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 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.

Human herpesvirus 8 enhances human immunodeficiency virus replication in acutely infected cells and induces reactivation in latently infected cells

Blood ◽  
2005 ◽  
Vol 106 (8) ◽  
pp. 2790-2797 ◽  
Author(s):  
Elisabetta Caselli ◽  
Monica Galvan ◽  
Enzo Cassai ◽  
Arnaldo Caruso ◽  
Laura Sighinolfi ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Endothelial Cells ◽  
Mononuclear Cells ◽  
Human Herpesvirus ◽  
Human Herpesvirus 8 ◽  
Human Immunodeficiency Virus Replication ◽  
Hiv Replication ◽  
Herpesvirus 8 ◽  
Immunodeficiency Virus ◽  
Infected Cells

AbstractHuman herpesvirus 8 (HHV-8) is etiologically associated with Kaposi sarcoma (KS), the most common AIDS-associated malignancy. Previous results indicate that the HHV-8 viral transactivator ORF50 interacts synergistically with Tat protein in the transactivation of human immunodeficiency virus (HIV) long terminal repeat (LTR), leading to increased cell susceptibility to HIV infection. Here, we analyze the effect of HHV-8 infection on HIV replication in monocyte-macrophage and endothelial cells, as potential targets of coinfection. Primary or transformed monocytic and endothelial cells were infected with a cell-free HHV-8 inoculum and subsequently infected with lymphotropic or monocytotropic strains of HIV. The results show that HHV-8 coinfection markedly increases HIV replication in both cell types. HHV-8 infection induces also HIV reactivation in chronically infected cell lines and in peripheral blood mononuclear cells (PBMCs) from patients with asymptomatic HIV, suggesting the possibility that similar interactions might take place also in vivo. Furthermore, coinfection is not an essential condition, since contiguity of differently infected cells is sufficient for HIV reactivation. The results suggest that HHV-8 might be a cofactor for HIV progression and that HHV-8-infected endothelial cells might play a relevant role in transendothelial HIV spread. (Blood. 2005;106:2790-2797)

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

2005 ◽  
Vol 79 (5) ◽  
pp. 3226-3226
Author(s):  
Elisa Martró ◽  
Michael J. Cannon ◽  
Sheila C. Dollard ◽  
Thomas J. Spira ◽  
A. Scott Laney ◽  
...  
Keyword(s):  
Mononuclear Cells ◽  
Human Herpesvirus ◽  
Lytic Replication ◽  
Human Herpesvirus 8 ◽  
Herpesvirus 8

scholarly journals Transcription Activation of Polyadenylated Nuclear RNA by Rta in Human Herpesvirus 8/Kaposi's Sarcoma-Associated Herpesvirus

2001 ◽  
Vol 75 (7) ◽  
pp. 3129-3140 ◽  
Author(s):  
Moon Jung Song ◽  
Helen J. Brown ◽  
Ting-Ting Wu ◽  
Ren Sun
Keyword(s):  
Gene Expression ◽  
Kaposi's Sarcoma ◽  
Human Herpesvirus ◽  
Kaposi’S Sarcoma ◽  
Lytic Replication ◽  
Human Herpesvirus 8 ◽  
Rna Expression ◽  
Herpesvirus 8 ◽  
Infected Cells ◽  
Kaposi’S Sarcoma Associated Herpesvirus

ABSTRACT Human herpesvirus 8 (HHV-8) (also known as Kaposi's sarcoma-associated herpesvirus) encodes a novel noncoding polyadenylated nuclear (PAN) RNA (also known as T1.1 or nut-1) during the early phase of lytic replication. PAN RNA is the most abundant transcript of HHV-8, comprising 80% of total poly(A)-selected transcripts in HHV-8-infected cells during lytic replication. We directly measured the abundance of PAN RNA by visualizing 1.1- to 1.2- kb PAN RNA in an ethidium bromide-stained gel from poly(A)-selected RNA. We further pursued the mechanisms by which PAN RNA expression is induced to such high levels.rta, an immediate-early gene of HHV-8, is a transactivator that is sufficient and necessary to activate lytic gene expression in latently infected cells. Ectopic expression of Rta was previously shown to induce PAN RNA expression from the endogenous viral genome and activate the PAN promoter in a reporter system. Here, we have identified the Rta-responsive element (RRE) in the PAN promoter. Deletion analysis revealed that the RRE is present in a region between nucleotides −69 and −38 of the PAN promoter. A promoter construct containing the 69 nucleotides upstream of the transcription start site of the PAN promoter was activated by Rta in the absence or presence of the HHV-8 genome. Rta activated the PAN promoter up to 7,000-fold in 293T cells and 2,000-fold in B cells. Electrophoretic mobility shift assays demonstrated that Rta formed a highly stable complex with the RRE of the PAN promoter. Our study suggests that Rta can induce PAN RNA expression by direct binding of Rta to the RRE of the PAN promoter. This study has highlighted an important mechanism controlling PAN RNA expression and also provides a model system for investigating how Rta transactivates gene expression during lytic replication.

scholarly journals Short Duration of Elevated vIRF-1 Expression during Lytic Replication of Human Herpesvirus 8 Limits Its Ability To Block Antiviral Responses Induced by Alpha Interferon in BCBL-1 Cells

2004 ◽  
Vol 78 (12) ◽  
pp. 6621-6635 ◽  
Author(s):  
Veronika P. Pozharskaya ◽  
Laura L. Weakland ◽  
James C. Zimring ◽  
Laurie T. Krug ◽  
Elizabeth R. Unger ◽  
...  
Keyword(s):  
Infectious Virus ◽  
Human Herpesvirus ◽  
Lytic Replication ◽  
Human Herpesvirus 8 ◽  
Herpesvirus 8 ◽  
Antiviral Responses ◽  
Short Period ◽  
Pml Bodies ◽  
Latently Infected ◽  
Low Levels

ABSTRACT Human herpesvirus 8 (HHV-8) encodes multiple proteins that disrupt the host antiviral response, including viral interferon (IFN) regulatory factor 1 (vIRF-1). The product of the vIRF-1 gene blocks responses to IFN when overexpressed by transfection, but the functional consequence of vIRF-1 that is expressed during infection with HHV-8 is not known. These studies demonstrate that BCBL-1 cells that were latently infected with HHV-8 expressed low levels of vIRF-1 that were associated with PML bodies, whereas much higher levels of vIRF-1 were transiently expressed during the lytic phase of HHV-8 replication. The low levels of vIRF-1 that were associated with PML bodies were insufficient to block alpha IFN (IFN-α)-induced alterations in gene expression, whereas cells that expressed high levels of vIRF-1 were resistant to some changes induced by IFN-α, including the expression of the double-stranded-RNA-activated protein kinase. High levels of vIRF-1 were expressed for only a short period during the lytic cascade, so many cells with HHV-8 in the lytic phase responded to IFN-α with increased expression of antiviral genes and enhanced apoptosis. Furthermore, the production of infectious virus was severely compromised when IFN-α was present early during the lytic cascade. These studies indicate that the transient expression of high levels of vIRF-1 is inadequate to subvert many of the antiviral effects of IFN-α so that IFN-α can effectively induce apoptosis and block production of infectious virus when present early in the lytic cascade of HHV-8.

scholarly journals Differential Expression of Viral Bcl-2 Encoded by Kaposi's Sarcoma-Associated Herpesvirus and Human Bcl-2 in Primary Effusion Lymphoma Cells and Kaposi's Sarcoma Lesions

2002 ◽  
Vol 76 (5) ◽  
pp. 2551-2556 ◽  
Author(s):  
Isabelle Widmer ◽  
Marion Wernli ◽  
Felix Bachmann ◽  
Fred Gudat ◽  
Gieri Cathomas ◽  
...  
Keyword(s):  
Kaposi's Sarcoma ◽  
Mononuclear Cells ◽  
Primary Effusion Lymphoma ◽  
Human Herpesvirus ◽  
Kaposi’S Sarcoma ◽  
Human Herpesvirus 8 ◽  
Lymphoma Cells ◽  
Herpesvirus 8 ◽  
Primary Effusion Lymphoma Cell ◽  
Spindle Cells

ABSTRACT Expression of human herpesvirus 8 viral Bcl-2 protein was demonstrated in spindle cells of late-stage Kaposi's sarcoma lesions but not in primary effusion lymphoma cell lines. In contrast, strong expression of human Bcl-2 was found in stimulated primary effusion lymphoma cells, whereas in Kaposi's sarcoma lesions preferential mononuclear cells, and to a lesser extent spindle cells, stained positive.

scholarly journals Kaposi's Sarcoma-Associated Herpesvirus (Human Herpesvirus 8) Contains Hypoxia Response Elements: Relevance to Lytic Induction by Hypoxia

2003 ◽  
Vol 77 (12) ◽  
pp. 6761-6768 ◽  
Author(s):  
Muzammel Haque ◽  
David A. Davis ◽  
Victoria Wang ◽  
Isabelle Widmer ◽  
Robert Yarchoan
Keyword(s):  
Promoter Region ◽  
Kaposi's Sarcoma ◽  
Human Herpesvirus ◽  
Kaposi’S Sarcoma ◽  
Lytic Replication ◽  
Human Herpesvirus 8 ◽  
Viral Gene ◽  
Hypoxia Response ◽  
Herpesvirus 8 ◽  
Response Elements

ABSTRACT Kaposi's sarcoma (KS)-associated herpesvirus (KSHV), also known as human herpesvirus 8, is an etiologic agent of KS, primary effusion lymphoma (PEL), and multicentric Castleman's disease. We recently demonstrated that hypoxia can induce lytic replication of KSHV in PEL cell lines. Hypoxia induces the accumulation of hypoxia-inducible factors (HIF), and we hypothesized that the KSHV genome may respond to hypoxia through functional hypoxia response elements (HREs). Here, we demonstrate the presence of at least two promoters within the KSHV genome that are activated by hypoxia or hypoxia mimics. One is in the promoter region of the gene for Rta, the main lytic switch gene, and the other is within the promoter region of ORF34, a lytic gene of unknown function. The ORF34 promoter contains three putative consensus HREs oriented in the direction of the gene. Dissection and site-directed mutagenesis studies confirmed that one of the HREs of the ORF34 promoter is functional. Under conditions of hypoxia, the ORF34 promoter was strongly upregulated by HIF-1α and HIF-2α. By contrast, the promoter of the gene for Rta appeared to be preferentially upregulated by HIF-2α. Reverse transcription-PCR analysis revealed that specific messages for ORF34 and ORF50 are upregulated in BCBL-1 cells exposed to hypoxia. An HIF-1 binding and competition assay demonstrated that the HRE sequence from the ORF34 promoter can compete for HIF-1α binding to an erythropoietin HRE oligonucleotide while a mutant sequence cannot. Thus, we demonstrated that a viral gene can be activated by hypoxia through activation of a functional viral HRE. To our knowledge, this is the first example of a functional HRE in a viral promoter.

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