scholarly journals Identification of a Novel Cellular Transcriptional Repressor Interacting with the Latent Nuclear Antigen of Kaposi's Sarcoma-Associated Herpesvirus

2003 ◽  
Vol 77 (18) ◽  
pp. 9758-9768 ◽  
Author(s):  
Hong-Yi Pan ◽  
Yan-Jin Zhang ◽  
Xin-Ping Wang ◽  
Jian-Hong Deng ◽  
Fu-Chun Zhou ◽  
...  
Keyword(s):  
Nuclear Localization ◽  
Kaposi's Sarcoma ◽  
Promoter Activity ◽  
Kaposi’S Sarcoma ◽  
Nuclear Antigen ◽  
Northern Blot Hybridization ◽  
Nuclear Localization Signals ◽  
Kaposi's Sarcoma Associated Herpesvirus ◽  
Kaposi’S Sarcoma Associated Herpesvirus ◽  
Latent Nuclear Antigen

ABSTRACT The latent nuclear antigen (LNA) of Kaposi's sarcoma-associated herpesvirus (KSHV) has an essential role in viral latent infection. LNA maintains the stability of KSHV episomes and modulates the expression of cellular genes. A novel cellular protein KLIP1 was identified to interact with LNA through yeast two-hybrid screening, and confirmed by a glutathione S-transferase pull down assay. Domain mapping showed that KLIP1 interacted with the N-terminal domain of LNA. Northern blot hybridization with a KLIP1 probe identified a major transcript of 1.8 kb and a minor transcript of 2.8 kb. cDNA library screening and 5′-RACE revealed that the major transcript encoded an open-reading-frame of 1,257 bp and had a 5′-untranslated region of 73 nucleotides. The major KLIP1 transcript was ubiquitously present in different cell types examined. A KLIP1 synthetic peptide antibody detected a doublet of 58-kDa and 63-kDa proteins in a Western blot assay. KLIP1 had two putative nuclear localization signals and showed punctate nuclear localization when expressed as a GFP-fusion protein. KLIP1 interacted with LNA in vivo, as demonstrated by coimmunoprecipitation using KSHV-infected cells and colocalization when they were expressed as GFP- and DsRed-fusion proteins, respectively. Consistent with its interaction with LNA, nuclear localization, and possession of two leucine zipper motifs, KLIP1 behaved like a transcriptional factor and repressed herpes simplex virus thymidine kinase (TK) promoter activity in a mammalian one-hybrid assay. In addition, cotransfection with LNA alleviated the transcriptional repression effect of KLIP1 on TK promoter activity. These results suggest that KLIP1 is a new member of cellular transcriptional repressors, and that LNA is involved in deregulating cellular transcription process.

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 Disruption of Kaposi's Sarcoma-Associated Herpesvirus Latent Nuclear Antigen Leads to Abortive Episome Persistence

2004 ◽  
Vol 78 (20) ◽  
pp. 11121-11129 ◽  
Author(s):  
Feng-Chun Ye ◽  
Fu-Chun Zhou ◽  
Seung Min Yoo ◽  
Jian-Ping Xie ◽  
Philip J. Browning ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Kaposi's Sarcoma ◽  
Fluorescent Protein ◽  
Early Stage ◽  
Kaposi’S Sarcoma ◽  
Nuclear Antigen ◽  
Bac Clone ◽  
Kaposi's Sarcoma Associated Herpesvirus ◽  
Kaposi’S Sarcoma Associated Herpesvirus ◽  
Latent Nuclear Antigen

ABSTRACT Latent nuclear antigen (LNA) is implicated in Kaposi's sarcoma-associated herpesvirus (KSHV) episome persistence. LNA colocalizes with KSHV episomes on chromosomes in metaphase, and it maintains the stability and replication of KSHV terminal repeat-containing plasmids. In this study, we examined the function of LNA in episome persistence in the context of full-length KSHV genome by mutagenesis analysis. We generated a KSHV mutant, BAC36-ΔLNA, with LNA disrupted by transposon-based mutagenesis with a KSHV BAC clone, BAC36, as a template. Immunofluorescence antibody staining revealed that the insertion of a transposon cassette into LNA disrupted its expression but had no effect on the expression of two adjacent genes, the vCyclin and vFLIP genes. Using a green fluorescent protein (GFP) cassette as a tracking marker for the KSHV episome, we found 8.7-fold-fewer GFP-positive cells in BAC36-ΔLNA cultures than in wild-type BAC36 cultures at the early stage following episome delivery into 293 cells by transfection, which could be partially rescued by cotransfection with a LNA expression plasmid but not a control plasmid. Cells harboring BAC36-ΔLNA with or without transient complementation rapidly lost episomes and became virus-free after 2 weeks of culture based on GFP expression and Gardella gel analysis and quantitative PCR assays for detecting KSHV genomes. In contrast, BAC36 episomes were stably maintained during the same period. Stable cultures with close to 100% of cells harboring KSHV episomes were readily established by hygromycin selection for BAC36 but not for BAC36-ΔLNA. These results conclusively indicate that LNA is essential for the establishment and persistence of KSHV episomes in mammalian cells.

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.

scholarly journals Identification of the Nuclear Export and Adjacent Nuclear Localization Signals for ORF45 of Kaposi's Sarcoma-Associated Herpesvirus

2008 ◽  
Vol 83 (6) ◽  
pp. 2531-2539 ◽  
Author(s):  
Xiaojuan Li ◽  
Fanxiu Zhu
Keyword(s):  
Subcellular Localization ◽  
Nuclear Localization ◽  
Nuclear Export ◽  
Kaposi's Sarcoma ◽  
Nuclear Export Signal ◽  
Kaposi’S Sarcoma ◽  
Lytic Replication ◽  
Wild Type ◽  
Kaposi's Sarcoma Associated Herpesvirus ◽  
Kaposi’S Sarcoma Associated Herpesvirus

ABSTRACT Open reading frame 45 (ORF45) of Kaposi's sarcoma-associated herpesvirus 8 (KSHV) is an immediate-early phosphorylated tegument protein and has been shown to play important roles at both early and late stages of viral infection. Homologues of ORF45 exist only in gammaherpesviruses, and their homology is limited. These homologues differ in their protein lengths and subcellular localizations. We and others have reported that KSHV ORF45 is localized predominantly in the cytoplasm, whereas its homologue in murine herpesvirus 68 is localized exclusively in the nucleus. We observed that ORF45s of rhesus rhadinovirus and herpesvirus saimiri are found exclusively in the nucleus. As a first step toward understanding the mechanism underlying the distinct intracellular distribution of KSHV ORF45, we identified the signals that control its subcellular localization. We found that KSHV ORF45 accumulated rapidly in the nucleus in the presence of leptomycin B, an inhibitor of CRM1 (exportin 1)-dependent nuclear export, suggesting that it could shuttle between the nucleus and cytoplasm. Mutational analysis revealed that KSHV ORF45 contains a CRM1-dependent, leucine-rich-like nuclear export signal and an adjacent nuclear localization signal. Replacement of the key residues with alanines in these motifs of ORF45 disrupts its shuttling between the cytoplasm and nucleus. The resulting ORF45 mutants have restricted subcellular localizations, being found exclusively either in the cytoplasm or in the nucleus. Recombinant viruses were reconstituted by introduction of these mutations into KSHV bacterial artificial chromosome BAC36. The resultant viruses have distinct phenotypes. A mutant virus in which ORF45 is restricted to the cytoplasm behaves as an ORF45-null mutant and produces 5- to 10-fold fewer progeny viruses than the wild type. In contrast, mutants in which the ORF45 protein is mostly restricted to the nucleus produce numbers of progeny viruses similar to those produced by the wild type. These data suggest that the subcellular localization signals of ORF45 have important functional roles in KSHV lytic replication.

scholarly journals Kaposi's Sarcoma-Associated Herpesvirus Latency-Associated Nuclear Antigen 1 Mimics Epstein-Barr Virus EBNA1 Immune Evasion through Central Repeat Domain Effects on Protein Processing

2007 ◽  
Vol 81 (15) ◽  
pp. 8225-8235 ◽  
Author(s):  
Hyun Jin Kwun ◽  
Suzane Ramos da Silva ◽  
Ishita M. Shah ◽  
Neil Blake ◽  
Patrick S. Moore ◽  
...  
Keyword(s):  
Antigen Processing ◽  
Kaposi's Sarcoma ◽  
Epstein Barr Virus ◽  
Human Herpesvirus ◽  
Kaposi’S Sarcoma ◽  
Nuclear Antigen ◽  
Barr Virus ◽  
Kaposi's Sarcoma Associated Herpesvirus ◽  
Epstein Barr ◽  
Kaposi’S Sarcoma Associated Herpesvirus

ABSTRACT Kaposi's sarcoma-associated herpesvirus (KSHV/human herpesvirus 8 [HHV8]) and Epstein-Barr virus (EBV/HHV4) are distantly related gammaherpesviruses causing tumors in humans. KSHV latency-associated nuclear antigen 1 (LANA1) is functionally similar to the EBV nuclear antigen-1 (EBNA1) protein expressed during viral latency, although they have no amino acid similarities. EBNA1 escapes cytotoxic lymphocyte (CTL) antigen processing by inhibiting its own proteosomal degradation and retarding its own synthesis to reduce defective ribosomal product processing. We show here that the LANA1 QED-rich central repeat (CR) region, particularly the CR2CR3 subdomain, also retards LANA1 synthesis and markedly enhances LANA1 stability in vitro and in vivo. LANA1 isoforms have half-lives greater than 24 h, and fusion of the LANA1 CR2CR3 domain to a destabilized heterologous protein markedly decreases protein turnover. Unlike EBNA1, the LANA1 CR2CR3 subdomain retards translation regardless of whether it is fused to the 5′ or 3′ end of a heterologous gene construct. Manipulation of sequence order, orientation, and composition of the CR2 and CR3 subdomains suggests that specific peptide sequences rather than RNA structures are responsible for synthesis retardation. Although mechanistic differences exist between LANA1 and EBNA1, the primary structures of both proteins have evolved to minimize provoking CTL immune responses. Simple strategies to eliminate these viral inhibitory regions may markedly improve vaccine effectiveness by maximizing CTL responses.

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.

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