scholarly journals Acetylation of the Latency-Associated Nuclear Antigen Regulates Repression of Kaposi's Sarcoma-Associated Herpesvirus Lytic Transcription

2006 ◽  
Vol 80 (11) ◽  
pp. 5273-5282 ◽  
Author(s):  
Fang Lu ◽  
Latasha Day ◽  
S.-J. Gao ◽  
Paul M. Lieberman
Keyword(s):  
Kaposi's Sarcoma ◽  
Kaposi’S Sarcoma ◽  
Lytic Cycle ◽  
Early Gene ◽  
Nuclear Antigen ◽  
Lysine Acetylation ◽  
Mrna Levels ◽  
Latently Infected ◽  
Kaposi's Sarcoma Associated Herpesvirus ◽  
Kaposi’S Sarcoma Associated Herpesvirus

ABSTRACT Reactivation of the Kaposi's sarcoma-associated herpesvirus (KSHV) lytic cycle can be initiated by transcription activation of the ORF50 immediate early gene (Rta). We show that ORF50 transcription is actively repressed by the KSHV latency-associated nuclear antigen (LANA) during latency. Depletion of LANA by small interfering RNA derepressed ORF50 transcription in the latently infected BCBL1 pleural effusion lymphoma-derived cell line. In contrast, overexpression of LANA suppressed ORF50 mRNA levels in BCBL1 cells. ORF50 transcription was significantly elevated during primary infection with recombinant virus lacking LANA, further indicating that LANA plays a role in lytic gene silencing during the establishment of latency. Chromatin immunoprecipitation assays indicated that LANA interacts with the ORF50 promoter region in latently infected cells. Histone deacetylase inhibitors, including sodium butyrate (NaB) and trichostatin A, caused the rapid dissociation of LANA from the ORF50 promoter. NaB treatment of latently infected BCBL1 cells disrupted a stable interaction between LANA and the cellular proteins Sp1 and histone H2B. We also found immunological and radiochemical evidence that LANA is subject to lysine acetylation after NaB treatment. These findings support the role of LANA as a transcriptional repressor of lytic reactivation and provide evidence that lysine acetylation regulates LANA interactions with chromatin, Sp1, and ORF50 promoter DNA.

scholarly journals Activation of Kaposi's Sarcoma-Associated Herpesvirus (Human Herpesvirus 8) Lytic Replication by Human Cytomegalovirus

2001 ◽  
Vol 75 (3) ◽  
pp. 1378-1386 ◽  
Author(s):  
Jeffrey Vieira ◽  
Patricia O'Hearn ◽  
Louise Kimball ◽  
Bala Chandran ◽  
Lawrence Corey
Keyword(s):  
Human Cytomegalovirus ◽  
Kaposi's Sarcoma ◽  
Human Fibroblasts ◽  
Human Herpesvirus ◽  
Kaposi’S Sarcoma ◽  
Lytic Replication ◽  
Lytic Cycle ◽  
Nuclear Antigen ◽  
Kaposi's Sarcoma Associated Herpesvirus ◽  
Kaposi’S Sarcoma Associated Herpesvirus

ABSTRACT The majority of Kaposi's sarcoma-associated herpesvirus (KSHV)-infected cells identified in vivo contain latent KSHV, with lytic replication in only a few percent of cells, as is the case for the cells of Kaposi's sarcoma (KS) lesions. Factors that influence KSHV latent or lytic replication are not well defined. Because persons with KS are often immunosuppressed and susceptible to many infectious agents, including human cytomegalovirus (HCMV), we have investigated the potential for HCMV to influence the replication of KSHV. Important to this work was the construction of a recombinant KSHV, rKSHV.152, expressing the green fluorescent protein (GFP) andneo (conferring resistance to G418). The expression of GFP was a marker of KSHV infection in cells of both epithelial and endothelial origin. The rKSHV.152 virus was used to establish cells, including human fibroblasts (HF), containing only latent KSHV, as demonstrated by latency-associated nuclear antigen expression and Gardella gel analysis. HCMV infection of KSHV latently infected HF activated KSHV lytic replication with the production of infectious KSHV. Dual-color immunofluorescence detected both the KSHV lytic open reading frame 59 protein and the HCMV glycoprotein B in coinfected cells, and UV-inactivated HCMV did not activate the production of infectious KSHV-GFP. In addition, HCMV coinfection increased the production of KSHV from endothelial cells and activated lytic cycle gene expression in keratinocytes. These data demonstrate that HCMV can activate KSHV lytic replication and suggest that HCMV could influence KSHV pathogenesis.

scholarly journals Latency-Associated Nuclear Antigen of Kaposi's Sarcoma-Associated Herpesvirus (KSHV) Upregulates Survivin Expression in KSHV-Associated B-Lymphoma Cells and Contributes to Their Proliferation

2009 ◽  
Vol 83 (14) ◽  
pp. 7129-7141 ◽  
Author(s):  
Jie Lu ◽  
Subhash C. Verma ◽  
Masanao Murakami ◽  
Qiliang Cai ◽  
Pankaj Kumar ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Kaposi's Sarcoma ◽  
Survivin Expression ◽  
Kaposi’S Sarcoma ◽  
Nuclear Antigen ◽  
Mrna Levels ◽  
Survivin Promoter ◽  
Infected Cells ◽  
Kaposi's Sarcoma Associated Herpesvirus ◽  
Kaposi’S Sarcoma Associated Herpesvirus

ABSTRACT Survivin is a master regulator of cell proliferation and cell viability and is highly expressed in most human tumors. The molecular network linked to survivin expression in tumors has not been completely elucidated. In this study, we show that latency-associated nuclear antigen (LANA), a multifunctional protein of Kaposi's sarcoma-associated herpesvirus (KSHV) that is found in Kaposi's sarcoma tumors, upregulates survivin expression and increases the proliferation of KSHV-infected B cells. Analysis of pathway-specific gene arrays showed that survivin expression was highly upregulated in BJAB cells expressing LANA. The mRNA levels of survivin were also upregulated in HEK 293 and BJAB cells expressing LANA. Similarly, protein levels of survivin were significantly higher in LANA-expressing, as well as KSHV-infected, cells. Survivin promoter activity assays identified GC/Sp1 and p53 cis-acting elements within the core promoter region as being important for LANA activity. Gel mobility shift assays revealed that LANA forms a complex with Sp1 or Sp1-like proteins bound to the GC/Sp1 box of the survivin promoter. In addition, a LANA/p53 complex bound to the p53 cis-acting element within the survivin promoter, indicating that upregulation of survivin expression can also occur through suppression of p53 function. Furthermore, immunohistochemistry analyses revealed that survivin expression was upregulated in KSHV-associated Kaposi's sarcoma tissue, suggesting that LANA plays an important role in the upregulation of survivin expression in KSHV-infected endothelial cells. Knockdown of survivin expression by lentivirus-delivered small hairpin RNA resulted in loss of cell proliferation in KSHV-infected cells. Therefore, upregulation of survivin expression in KSHV-associated human cells contributes to their proliferation.

scholarly journals Latent Nuclear Antigen of Kaposi’s Sarcoma-Associated Herpesvirus Interacts with RING3, a Homolog of theDrosophila Female Sterile Homeotic (fsh) Gene

1999 ◽  
Vol 73 (12) ◽  
pp. 9789-9795 ◽  
Author(s):  
Georgina M. Platt ◽  
Guy R. Simpson ◽  
Sibylle Mittnacht ◽  
Thomas F. Schulz
Keyword(s):  
Protein Interactions ◽  
Kaposi's Sarcoma ◽  
Primary Effusion Lymphoma ◽  
Kaposi’S Sarcoma ◽  
Nuclear Antigen ◽  
Latently Infected ◽  
Kaposi's Sarcoma Associated Herpesvirus ◽  
Carboxy Terminal ◽  
Kaposi’S Sarcoma Associated Herpesvirus ◽  
Latent Nuclear Antigen

ABSTRACT Kaposi’s sarcoma-associated herpesvirus (KSHV/HHV-8) is the likely infectious cause of Kaposi’s sarcoma, primary effusion lymphoma, and some cases of multicentric Castleman’s disease. Its latent nuclear antigen (LANA) is expressed in the nuclei of latently infected cells and may play a role in the persistence of episomal viral DNA in dividing cells. Here we report that LANA interacts with RING3, a nuclear protein and member of the Drosophila fsh (female sterile homeotic) family of proteins, some of which have previously been implicated in controlling gene expression. Binding of RING3 to LANA involves the ET domain, characteristic of fsh-related proteins, suggesting that this highly conserved region is involved in protein-protein interactions. The interaction between RING3 and LANA results in phosphorylation of serine and threonine residues located between amino acids 951 and 1107 in the carboxy-terminal region of LANA. However, RING3 is not itself a kinase but appears to recruit an as yet unidentified serine/threonine protein kinase into the complex which it forms with LANA.

scholarly journals Reduction of Kaposi’s Sarcoma-Associated Herpesvirus Latency Using CRISPR-Cas9 To Edit the Latency-Associated Nuclear Antigen Gene

2019 ◽  
Vol 93 (7) ◽  
Author(s):  
For Yue Tso ◽  
John T. West ◽  
Charles Wood
Keyword(s):  
Kaposi's Sarcoma ◽  
Kaposi’S Sarcoma ◽  
Adenovirus Type ◽  
Nuclear Antigen ◽  
Latently Infected ◽  
Kaposi's Sarcoma Associated Herpesvirus ◽  
Kaposi’S Sarcoma Associated Herpesvirus ◽  
Adenovirus Type 5 ◽  
Hiv 1 ◽  
Over Time

ABSTRACTKaposi’s sarcoma-associated herpesvirus (KSHV) is the etiologic agent of Kaposi’s sarcoma (KS), an AIDS-defining cancer in HIV-1-infected individuals or immune-suppressed transplant patients. The prevalence for both KSHV and KS are highest in sub-Saharan Africa where HIV-1 infection is also epidemic. There is no effective treatment for advanced KS; therefore, the survival rate is low. Similar to other herpesviruses, KSHV’s ability to establish latent infection in the host presents a major challenge to KS treatment or prevention. Strategies to reduce KSHV episomal persistence in latently infected cells might lead to approaches to prevent KS development. The CRISPR-Cas9 system is a gene editing technique that has been used to specifically manipulate the HIV-1 genome but also Epstein-Barr virus (EBV) which, similar to KSHV, belongs to theGammaherpesvirusfamily. Among KSHV gene products, the latency-associated nuclear antigen (LANA) is absolutely required in the maintenance, replication, and segregation of KSHV episomes during mitosis, which makes LANA an ideal target for CRISPR-Cas9 editing. In this study, we designed a replication-incompetent adenovirus type 5 to deliver a LANA-specific Cas9 system (Ad-CC9-LANA) into various KSHV latent target cells. We showed that KSHV latently infected epithelial and endothelial cells transduced with Ad-CC9-LANA underwent significant reductions in the KSHV episome burden, LANA RNA and protein expression over time, but this effect is less profound in BC3 cells due to the low infection efficiency of adenovirus type 5 for B cells. The use of an adenovirus vector might confer potentialin vivoapplications of LANA-specific Cas9 against KSHV infection and KS.IMPORTANCEThe ability for Kaposi’s sarcoma-associated herpesvirus (KSHV), the causative agent of Kaposi’s sarcoma (KS), to establish and maintain latency has been a major challenge to clearing infection and preventing KS development. This is the first study to demonstrate the feasibility of using a KSHV LANA-targeted CRISPR-Cas9 and adenoviral delivery system to disrupt KSHV latency in infected epithelial and endothelial cell lines. Our system significantly reduced the KSHV episomal burden over time. Given the safety record of adenovirus as vaccine or delivery vectors, this approach to limit KSHV latency may also represent a viable strategy against other tumorigenic viruses and may have potential benefits in developing countries where the viral cancer burden is high.

scholarly journals Differential Regulation of the Overlapping Kaposi's Sarcoma-Associated Herpesvirus vGCR (orf74) and LANA (orf73) Promoters

2001 ◽  
Vol 75 (4) ◽  
pp. 1798-1807 ◽  
Author(s):  
Joseph Jeong ◽  
James Papin ◽  
Dirk Dittmer
Keyword(s):  
Kaposi's Sarcoma ◽  
Regulatory Region ◽  
Kaposi’S Sarcoma ◽  
Lytic Replication ◽  
Lytic Cycle ◽  
Nuclear Antigen ◽  
Barr Virus ◽  
Kaposi's Sarcoma Associated Herpesvirus ◽  
High Level ◽  
Kaposi’S Sarcoma Associated Herpesvirus

ABSTRACT Similar to that of other herpesviruses, Kaposi's sarcoma-associated herpesvirus (KSHV/HHV-8) lytic replication destroys the host cell, while the virus can persist in a latent state in synchrony with the host. During latency only a few genes are transcribed, and the question becomes one of what determines latent versus lytic gene expression. Here we undertake a detailed analysis of the latency-associated nuclear antigen (LANA [orf73]) promoter (LANAp). We characterized a minimal region that is necessary and sufficient to maintain high-level transcription in all tissues tested, including primary endothelial cells and B cells, which are the suspected natural host for KSHV. We show that in transient-transfection assays LANAp mimics the expression pattern observed for the authentic promoter in the context of the KSHV episome. Unlike other KSHV promoters tested thus far, LANAp is not affected by tetradecanoyl phorbol acetate or viral lytic cycle functions. It is, however, subject to control by LANA itself and cellular regulatory factors, such as p53. This is in contrast to the K14/vGCR (orf74) promoter, which overlaps LANAp and directs transcription on the opposite strand. We isolated a minimal cis-regulatory region sufficient for K14/vGCR promoter activity and show that it, too, mimics the regulation observed for the authentic viral promoter. In particular, we demonstrate that its activity is absolutely dependent on the immediate-early transactivator orf50, the KSHV homolog of the Epstein-Barr virus Rta transactivator.

scholarly journals Full-Length Isoforms of Kaposi's Sarcoma-Associated Herpesvirus Latency-Associated Nuclear Antigen Accumulate in the Cytoplasm of Cells Undergoing the Lytic Cycle of Replication

2017 ◽  
Vol 91 (24) ◽  
Author(s):  
H. Jacques Garrigues ◽  
Kellie Howard ◽  
Serge Barcy ◽  
Minako Ikoma ◽  
Ashlee V. Moses ◽  
...  
Keyword(s):  
Gene Expression ◽  
Nuclear Localization ◽  
Primary Infection ◽  
Kaposi's Sarcoma ◽  
Kaposi’S Sarcoma ◽  
Lytic Replication ◽  
Lytic Cycle ◽  
Nuclear Antigen ◽  
Kaposi's Sarcoma Associated Herpesvirus ◽  
Kaposi’S Sarcoma Associated Herpesvirus

ABSTRACT The latency-associated nuclear antigen (LANA) of the Kaposi's sarcoma-associated herpesvirus (KSHV) performs a variety of functions to establish and maintain KSHV latency. During latency, LANA localizes to discrete punctate spots in the nucleus, where it tethers viral episomes to cellular chromatin and interacts with nuclear components to regulate cellular and viral gene expression. Using highly sensitive tyramide signal amplification, we determined that LANA localizes to the cytoplasm in different cell types undergoing the lytic cycle of replication after de novo primary infection and after spontaneous, tetradecanoyl phorbol acetate-, or open reading frame 50 (ORF50)/replication transactivator (RTA)-induced activation. We confirmed the presence of cytoplasmic LANA in a subset of cells in lytically active multicentric Castleman disease lesions. The induction of cellular migration by scratch-wounding confluent cell cultures, culturing under subconfluent conditions, or induction of cell differentiation in primary cultures upregulated the number of cells permissive for primary lytic KSHV infection. The induction of lytic replication was characterized by high-level expression of cytoplasmic LANA and nuclear ORF59, a marker of lytic replication. Subcellular fractionation studies revealed the presence of multiple isoforms of LANA in the cytoplasm of ORF50/RTA-activated Vero cells undergoing primary infection. Mass spectrometry analysis demonstrated that cytoplasmic LANA isoforms were full length, containing the N-terminal nuclear localization signal. These results suggest that trafficking of LANA to different subcellular locations is a regulated phenomenon, which allows LANA to interact with cellular components in different compartments during both the latent and the replicative stages of the KSHV life cycle. IMPORTANCE Kaposi's sarcoma-associated herpesvirus (KSHV) causes AIDS-related malignancies, including lymphomas and Kaposi's sarcoma. KSHV establishes lifelong infections using its latency-associated nuclear antigen (LANA). During latency, LANA localizes to the nucleus, where it connects viral and cellular DNA complexes and regulates gene expression, allowing the virus to maintain long-term infections. Our research shows that intact LANA traffics to the cytoplasm of cells undergoing permissive lytic infections and latently infected cells in which the virus is induced to replicate. This suggests that LANA plays important roles in the cytoplasm and nuclear compartments of the cell during different stages of the KSHV life cycle. Determining cytoplasmic function and mechanism for regulation of the nuclear localization of LANA will enhance our understanding of the biology of this virus, leading to therapeutic approaches to eliminate infection and block its pathological effects.

scholarly journals Kaposi's Sarcoma-Associated Herpesvirus Latency-Associated Nuclear Antigen Induces a Strong Bend on Binding to Terminal Repeat DNA

2005 ◽  
Vol 79 (21) ◽  
pp. 13829-13836 ◽  
Author(s):  
Lai-Yee Wong ◽  
Angus C. Wilson
Keyword(s):  
Dna Replication ◽  
Kaposi's Sarcoma ◽  
Kaposi’S Sarcoma ◽  
Nuclear Antigen ◽  
Major Groove ◽  
Repeat Dna ◽  
Initiation Of Dna Replication ◽  
Kaposi's Sarcoma Associated Herpesvirus ◽  
Kaposi’S Sarcoma Associated Herpesvirus ◽  
Herpesvirus Genome

ABSTRACT During latency, the Kaposi's sarcoma-associated herpesvirus genome is maintained as a circular episome, replicating in synchrony with host chromosomes. Replication requires the latency-associated nuclear antigen (LANA) and an origin of latent DNA replication located in the viral terminal repeats, consisting of two LANA binding sites (LBSs) and a GC-rich sequence. Here, we show that the recruitment of a LANA dimer to high-affinity site LBS-1 bends DNA by 57° and towards the major groove. The cooccupancy of LBS-1 and lower-affinity LBS-2 induces a symmetrical bend of 110°. By changing the origin architecture, LANA may help to assemble a specific nucleoprotein structure important for the initiation of DNA replication.

scholarly journals Bub1 in Complex with LANA Recruits PCNA To Regulate Kaposi's Sarcoma-Associated Herpesvirus Latent Replication and DNA Translesion Synthesis

2015 ◽  
Vol 89 (20) ◽  
pp. 10206-10218 ◽  
Author(s):  
Zhiguo Sun ◽  
Hem Chandra Jha ◽  
Erle S. Robertson
Keyword(s):  
Dna Replication ◽  
Kaposi's Sarcoma ◽  
Kaposi’S Sarcoma ◽  
Kinase Domain ◽  
Cellular Protein ◽  
Nuclear Antigen ◽  
Infected Cells ◽  
Kaposi's Sarcoma Associated Herpesvirus ◽  
Kaposi’S Sarcoma Associated Herpesvirus ◽  
Molecular Bridge

ABSTRACTLatent DNA replication of Kaposi's sarcoma-associated herpesvirus (KSHV) initiates at the terminal repeat (TR) element and requirestrans-acting elements, both viral and cellular, such as ORCs, MCMs, and latency-associated nuclear antigen (LANA). However, how cellular proteins are recruited to the viral genome is not very clear. Here, we demonstrated that the host cellular protein, Bub1, is involved in KSHV latent DNA replication. We show that Bub1 constitutively interacts with proliferating cell nuclear antigen (PCNA) via a highly conserved PIP box motif within the kinase domain. Furthermore, we demonstrated that Bub1 can form a complex with LANA and PCNA in KSHV-positive cells. This strongly indicated that Bub1 serves as a scaffold or molecular bridge between LANA and PCNA. LANA recruited PCNA to the KSHV genome via Bub1 to initiate viral replication in S phase and interacted with PCNA to promote its monoubiquitination in response to UV-induced damage for translesion DNA synthesis. This resulted in increased survival of KSHV-infected cells.IMPORTANCEDuring latency in KSHV-infected cells, the viral episomal DNA replicates once each cell cycle. KSHV does not express DNA replication proteins during latency. Instead, KSHV LANA recruits the host cell DNA replication machinery to the replication origin. However, the mechanism by which LANA mediates replication is uncertain. Here, we show that LANA is able to form a complex with PCNA, a critical protein for viral DNA replication. Furthermore, our findings suggest that Bub1, a spindle checkpoint protein, serves as a scaffold or molecular bridge between LANA and PCNA. Our data further support a role for Bub1 and LANA in PCNA-mediated cellular DNA replication processes as well as monoubiquitination of PCNA in response to UV damage. These data reveal a therapeutic target for inhibition of KSHV persistence in malignant cells.

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