Carboxy Terminus of Human Herpesvirus 8 Latency-Associated Nuclear Antigen Mediates Dimerization, Transcriptional Repression, and Targeting to Nuclear Bodies

ABSTRACT Human herpesvirus 8 (HHV-8; also known as Kaposi's sarcoma-associated herpesvirus) is the causative agent of Kaposi's sarcoma and certain B-cell lymphomas. In most infected cells, HHV-8 establishes a latent infection characterized by the expression of latency-associated nuclear antigen (LANA) encoded by open reading frame 73. Although unrelated by sequence, there are functional similarities between LANA and the EBNA-1 protein of Epstein-Barr virus. Both accumulate as subnuclear speckles and are required for maintenance of the viral episome. EBNA-1 also regulates viral gene expression and is required for cell immortalization, suggesting that LANA performs similar functions in the context of HHV-8 infection. Here we show that LANA forms stable dimers, or possibly higher-order multimers, and that this is mediated by a conserved region in the C terminus. By expressing a series of truncations, we show that both the N- and C-terminal regions localize to the nucleus, although only the C terminus accumulates as nuclear speckles characteristic of the intact protein. Lastly, we show that LANA can function as a potent transcriptional repressor when tethered to constitutively active promoters via a heterologous DNA-binding domain. Domains in both the N and C termini mediate repression. This suggests that one function of LANA is to suppress the expression of the viral lytic genes or cellular genes involved in the antiviral response.

Download Full-text

Latent nuclear antigen of Kaposi’s sarcoma herpesvirus/human herpesvirus-8 induces and relocates RING3 to nuclear heterochromatin regions

Journal of General Virology ◽

10.1099/0022-1317-83-1-179 ◽

2002 ◽

Vol 83 (1) ◽

pp. 179-188 ◽

Cited By ~ 46

Author(s):

Karin Mattsson ◽

Csaba Kiss ◽

Georgina M. Platt ◽

Guy R. Simpson ◽

Elena Kashuba ◽

...

Keyword(s):

Kaposi's Sarcoma ◽

Human Herpesvirus ◽

Kaposi’S Sarcoma ◽

Body Cavity ◽

Nuclear Bodies ◽

Human Herpesvirus 8 ◽

Nuclear Antigen ◽

Lymphoma Cells ◽

Herpesvirus 8 ◽

C Terminus

LANA, the major latency-associated nuclear antigen of Kaposi’s sarcoma herpesvirus/human herpesvirus-8 (KSHV/HHV-8), binds RING3 protein, one of five human homologues of the fsh (female sterile homeotic) gene product of Drosophila. In KSHV/HHV-8-infected cells LANA and the viral episomes accumulate in heterochromatin-associated nuclear bodies. Here we show that in several KSHV/HHV-8-negative cell lines derived from carcinomas, sarcomas and lymphomas, RING3 was expressed at low levels, primarily localized to the euchromatin, and dissociated from the chromosomes during mitosis. In contrast, in KSHV/HHV-8-infected body cavity lymphoma cells the bulk of RING3 localizes to the LANA nuclear bodies and remains associated with the chromosomes during cell division. KSHV/HHV-8-infected body cavity lymphoma cells expressed RING3 at much higher levels than cells without the virus. Transfection of full-length LANA, but not the C terminus alone, greatly induced RING3 gene expression, and LANA and RING3 co-localized even in the transfected cells, in the absence of KSHV/HHV-8 viral DNA. High levels of LANA expression led to the disappearance of heterochromatin in both human and mouse cells. We suggest that LANA and RING3 may create a local euchromatic microenvironment around the viral episomes that are anchored to the heterochromatin.

Download Full-text

Characterization of Monoclonal Antibodies Raised against the Latent Nuclear Antigen of Human Herpesvirus 8

Journal of Virology ◽

10.1128/jvi.73.6.5149-5155.1999 ◽

1999 ◽

Vol 73 (6) ◽

pp. 5149-5155 ◽

Cited By ~ 64

Author(s):

Paul Kellam ◽

Dimitra Bourboulia ◽

Nicolas Dupin ◽

Chris Shotton ◽

Cyril Fisher ◽

...

Keyword(s):

Monoclonal Antibodies ◽

Kaposi's Sarcoma ◽

Human Herpesvirus ◽

Kaposi’S Sarcoma ◽

Human Herpesvirus 8 ◽

Nuclear Antigen ◽

Viral Gene ◽

Herpesvirus 8 ◽

Latent Nuclear Antigen

ABSTRACT Human herpesvirus 8 (HHV-8; also designated Kaposi’s sarcoma-associated herpesvirus) is the likely etiological agent of Kaposi’s sarcoma (KS). HHV-8 encodes a latent nuclear antigen (LNA) which is the product of the viral gene orf 73. LNA is recognized by most infected patient sera and is the basis of current immunofluorescence assays used in epidemiological studies of HHV-8 infection. Here we describe the characterization of four monoclonal antibodies raised to the C-terminal third of LNA–glutathioneS-transferase fusion proteins. These monoclonal antibodies recognized discrete linear epitopes within the C terminus and repetitive region of LNA, detected antigen in primary effusion lymphoma (PEL) cells, and precipitated a 220- to 230-kDa protein doublet corresponding to LNA from HHV-8-infected PEL cell lines. In situ immunocytochemistry of KS lesions with these antibodies show that LNA is extensively expressed in KS spindle cells.

Download Full-text

Faculty Opinions recommendation of Seroreactivity to Kaposi's sarcoma-associated herpesvirus (human herpesvirus 8) latent nuclear antigen in AIDS-associated Kaposi's sarcoma patients depends on CD4+ T-cell count.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1091864.545185 ◽

2007 ◽

Author(s):

Patrick S Moore

Keyword(s):

T Cell ◽

Kaposi's Sarcoma ◽

Human Herpesvirus ◽

Kaposi’S Sarcoma ◽

Cd4 T Cell ◽

Human Herpesvirus 8 ◽

Nuclear Antigen ◽

Herpesvirus 8 ◽

Latent Nuclear Antigen ◽

Cd4 T Cell Count

Download Full-text

Fragment-Based Discovery of a Qualified Hit Targeting the Latency-Associated Nuclear Antigen of the Oncogenic Kaposi’s Sarcoma-Associated Herpesvirus/Human Herpesvirus 8

Journal of Medicinal Chemistry ◽

10.1021/acs.jmedchem.8b01827 ◽

2019 ◽

Vol 62 (8) ◽

pp. 3924-3939 ◽

Cited By ~ 9

Author(s):

Philine Kirsch ◽

Valentin Jakob ◽

Kevin Oberhausen ◽

Saskia C. Stein ◽

Ivano Cucarro ◽

...

Keyword(s):

Kaposi's Sarcoma ◽

Human Herpesvirus ◽

Kaposi’S Sarcoma ◽

Human Herpesvirus 8 ◽

Nuclear Antigen ◽

Herpesvirus 8 ◽

Kaposi's Sarcoma Associated Herpesvirus ◽

Kaposi’S Sarcoma Associated Herpesvirus

Download Full-text

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

Journal of Virology ◽

10.1128/jvi.77.12.6761-6768.2003 ◽

2003 ◽

Vol 77 (12) ◽

pp. 6761-6768 ◽

Cited By ~ 78

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.

Download Full-text

Transcription Program of Human Herpesvirus 8 (Kaposi's Sarcoma-Associated Herpesvirus)

Journal of Virology ◽

10.1128/jvi.75.10.4843-4853.2001 ◽

2001 ◽

Vol 75 (10) ◽

pp. 4843-4853 ◽

Cited By ~ 168

Author(s):

Mini Paulose-Murphy ◽

Nguyen-Khoi Ha ◽

Chunsheng Xiang ◽

Yidong Chen ◽

Laura Gillim ◽

...

Keyword(s):

Kaposi's Sarcoma ◽

Primary Effusion Lymphoma ◽

Human Herpesvirus ◽

Viral Gene Expression ◽

Kaposi’S Sarcoma ◽

Lytic Replication ◽

Human Herpesvirus 8 ◽

Viral Gene ◽

Herpesvirus 8 ◽

Transcription Program

ABSTRACT Human herpesvirus 8 (HHV-8), a gammaherpesvirus implicated in Kaposi's sarcoma, primary effusion lymphoma, and Castleman's disease, encodes several pathogenically important cellular homologs. To define the HHV-8 transcription program, RNA obtained from latently infected body cavity-based lymphoma 1 cells induced to undergo lytic replication was used to query a custom HHV-8 DNA microarray containing nearly every known viral open reading frame. The patterns of viral gene expression offer insights into the replication and pathogenic strategies of HHV-8.

Download Full-text