scholarly journals Kaposi's Sarcoma-Associated Herpesvirus Latency-Associated Nuclear Antigen Inhibits Major Histocompatibility Complex Class II Expression by Disrupting Enhanceosome Assembly through Binding with the Regulatory Factor X Complex

2015 ◽  
Vol 89 (10) ◽  
pp. 5536-5556 ◽  
Author(s):  
Suhani Thakker ◽  
Pravinkumar Purushothaman ◽  
Namrata Gupta ◽  
Shanthan Challa ◽  
Qiliang Cai ◽  
...  
Keyword(s):  
Gene Expression ◽  
Kaposi's Sarcoma ◽  
Kaposi’S Sarcoma ◽  
Class Ii ◽  
Nuclear Antigen ◽  
Factor X ◽  
Mhc Ii ◽  
Kaposi's Sarcoma Associated Herpesvirus ◽  
Rfx Complex ◽  
Kaposi’S Sarcoma Associated Herpesvirus

ABSTRACTMajor histocompatibility complex class II (MHC-II) molecules play a central role in adaptive antiviral immunity by presenting viral peptides to CD4+T cells. Due to their key role in adaptive immunity, many viruses, including Kaposi's sarcoma-associated herpesvirus (KSHV), have evolved multiple strategies to inhibit the MHC-II antigen presentation pathway. The expression of MHC-II, which is controlled mainly at the level of transcription, is strictly dependent upon the binding of the class II transactivator (CIITA) to the highly conserved promoters of all MHC-II genes. The recruitment of CIITA to MHC-II promoters requires its direct interactions with a preassembled MHC-II enhanceosome consisting of cyclic AMP response element-binding protein (CREB) and nuclear factor Y (NF-Y) complex and regulatory factor X (RFX) complex proteins. Here, we show that KSHV-encoded latency-associated nuclear antigen (LANA) disrupts the association of CIITA with the MHC-II enhanceosome by binding to the components of the RFX complex. Our data show that LANA is capable of binding to all three components of the RFX complex, RFX-associated protein (RFXAP), RFX5, and RFX-associated ankyrin-containing protein (RFXANK),in vivobut binds more strongly with the RFXAP component inin vitrobinding assays. Levels of MHC-II proteins were significantly reduced in KSHV-infected as well as LANA-expressing B cells. Additionally, the expression of LANA in a luciferase promoter reporter assay showed reduced HLA-DRA promoter activity in a dose-dependent manner. Chromatin immunoprecipitation assays showed that LANA binds to the MHC-II promoter along with RFX proteins and that the overexpression of LANA disrupts the association of CIITA with the MHC-II promoter. These assays led to the conclusion that the interaction of LANA with RFX proteins interferes with the recruitment of CIITA to MHC-II promoters, resulting in an inhibition of MHC-II gene expression. Thus, the data presented here identify a novel mechanism used by KSHV to downregulate the expressions of MHC-II genes.IMPORTANCEKaposi's sarcoma-associated herpesvirus is the causative agent of multiple human malignancies. It establishes a lifelong latent infection and persists in infected cells without being detected by the host's immune surveillance system. Only a limited number of viral proteins are expressed during latency, and these proteins play a significant role in suppressing both the innate and adaptive immunities of the host. Latency-associated nuclear antigen (LANA) is one of the major proteins expressed during latent infection. Here, we show that LANA blocks MHC-II gene expression to subvert the host immune system by disrupting the MHC-II enhanceosome through binding with RFX transcription factors. Therefore, this study identifies a novel mechanism utilized by KSHV LANA to deregulate MHC-II gene expression, which is critical for CD4+T cell responses in order to escape host immune surveillance.

scholarly journals DNA Binding and Modulation of Gene Expression by the Latency-Associated Nuclear Antigen of Kaposi's Sarcoma-Associated Herpesvirus

2001 ◽  
Vol 75 (17) ◽  
pp. 7882-7892 ◽  
Author(s):  
Alexander C. Garber ◽  
Marla A. Shu ◽  
Jianhong Hu ◽  
Rolf Renne
Keyword(s):  
Gene Expression ◽  
Dna Binding ◽  
Kaposi's Sarcoma ◽  
Primary Effusion Lymphoma ◽  
Kaposi’S Sarcoma ◽  
Nuclear Antigen ◽  
Viral Gene ◽  
Mobility Shift ◽  
Kaposi's Sarcoma Associated Herpesvirus ◽  
Kaposi’S Sarcoma Associated Herpesvirus

ABSTRACT Kaposi's sarcoma-associated herpesvirus (KSHV) is associated with Kaposi's sarcoma, primary effusion lymphoma, and multicentric Castleman's disease. The latency-associated nuclear antigen (LANA) is highly expressed in these malignancies and has been shown to play an important role in episomal maintenance, presumably by binding to a putative oriP. In addition, LANA modulates cellular and viral gene expression and interacts with the cellular tumor suppressors p53 and retinoblastoma suppressor protein. Many of these features are reminiscent of Epstein-Barr virus nuclear antigens (EBNAs), a family of six proteins expressed during latency. EBNA-1 is required for episome maintenance, binds to oriP, and strongly activates transcription from two promoters, including its own. We have previously shown that LANA can transactivate its own promoter and therefore asked whether LANA, like EBNA-1, activates transcription by direct binding to DNA. By using recombinant LANA expressed from vaccinia virus vectors for electrophoretic mobility shift assays, we found that LANA does not bind to its own promoter. In contrast, LANA binds specifically to sequences containing an imperfect 20-bp palindrome in the terminal repeat (TR) of KSHV. We further show that the C-terminal domain of LANA is sufficient for site-specific DNA binding. Unlike EBNA-1, which activates transcription through binding of oriP, we found that LANA inhibits transcription from a single TR binding site. A multimerized TR as found in the viral genome results in strong transcriptional suppression when linked to a heterologous promoter. These data suggest that LANA, although fulfilling functions similar to those of EBNA-1, does so by very different mechanisms.

scholarly journals Modulation of Cellular and Viral Gene Expression by the Latency-Associated Nuclear Antigen of Kaposi's Sarcoma-Associated Herpesvirus

2001 ◽  
Vol 75 (1) ◽  
pp. 458-468 ◽  
Author(s):  
Rolf Renne ◽  
Chris Barry ◽  
Dirk Dittmer ◽  
Nicole Compitello ◽  
Patrick O. Brown ◽  
...  
Keyword(s):  
Gene Expression ◽  
Reporter Gene ◽  
Kaposi's Sarcoma ◽  
Primary Effusion Lymphoma ◽  
Viral Gene Expression ◽  
Kaposi’S Sarcoma ◽  
Nuclear Antigen ◽  
Viral Gene ◽  
Kaposi's Sarcoma Associated Herpesvirus ◽  
Kaposi’S Sarcoma Associated Herpesvirus

ABSTRACT Kaposi's sarcoma-associated herpesvirus (KSHV), also called human herpesvirus 8 (HHV-8), is the likely etiological agent of Kaposi's sarcoma and primary effusion lymphoma. Common to these malignancies is that tumor cells are latently infected with KSHV. Viral gene expression is limited to a few genes, one of which is the latency-associated nuclear antigen (LANA), the product of ORF73. Examination of the primary sequence of LANA reveals some structural features reminiscent of transcription factors, leading us to hypothesize that LANA may regulate viral and cellular transcription during latency. In reporter gene-based transient transfection assays, we found that LANA can have either positive or negative effects on gene expression. While expression of a reporter gene from several synthetic promoters was increased in the presence of LANA, expression from the human immunodeficiency virus (HIV) long terminal repeat (LTR)—and from NF-κB-dependent reporter genes—was reduced by LANA expression. In addition, the promoter of KSHV ORF73 itself is activated up to 5.5-fold by LANA. This autoregulation may be important in tumorigenesis, because two other genes (v-cyclin and v-FLIP) with likely roles in cell growth and survival are also controlled by this element. To identify cellular genes influenced by LANA, we employed cDNA array-based expression profiling. Six known genes (and nine expressed sequence tags) were found to be upregulated in LANA-expressing cell lines. One of these, Staf-50, is known to inhibit expression from the HIV LTR; most of the other known genes are interferon inducible, although the interferon genes themselves were not induced by LANA. These data demonstrate that LANA expression has effects on cellular and viral gene expression. We suggest that, whether direct or indirect in origin, these effects may play important roles in the pathobiology of KSHV infection.

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 Major Histocompatibility Complex Class II HLA-DRα Is Downregulated by Kaposi's Sarcoma-Associated Herpesvirus-Encoded Lytic Transactivator RTA and MARCH8

2016 ◽  
Vol 90 (18) ◽  
pp. 8047-8058 ◽  
Author(s):  
Zhiguo Sun ◽  
Hem Chandra Jha ◽  
Yong-gang Pei ◽  
Erle S. Robertson
Keyword(s):  
Major Histocompatibility Complex ◽  
Immune System ◽  
Kaposi's Sarcoma ◽  
Kaposi’S Sarcoma ◽  
Complex Class ◽  
Transcription Activator ◽  
Mhc Ii ◽  
Histocompatibility Complex ◽  
Kaposi's Sarcoma Associated Herpesvirus ◽  
Kaposi’S Sarcoma Associated Herpesvirus

ABSTRACTKaposi's sarcoma-associated herpesvirus (KSHV) maintains two modes of life cycle, the latent and lytic phases. To evade the attack of the cell host's immune system, KSHV switches from the lytic to the latent phase, a phase in which only a few of viral proteins are expressed. The mechanism by which KSHV evades the attack of the immune system and establishes latency has not been fully understood. Major histocompatibility complex class II (MHC-II) molecules are key components of the immune system defense mechanism against viral infections. Here we report that HLA-DRα, a member of the MHC-II molecules, was downregulated by the replication and transcription activator (RTA) protein encoded by KSHV ORF50, an important regulator of the viral life cycle. RTA not only downregulated HLA-DRα at the protein level through direct binding and degradation through the proteasome pathway but also indirectly downregulated the protein level of HLA-DRα by enhancing the expression of MARCH8, a member of the membrane-associated RING-CH (MARCH) proteins. Our findings indicate that KSHV RTA facilitates evasion of the virus from the immune system through manipulation of HLA-DRα.IMPORTANCEKaposi's sarcoma-associated herpesvirus (KSHV) has a causal role in a number of human cancers, and its persistence in infected cells is controlled by the host's immune system. The mechanism by which KSHV evades an attack by the immune system has not been well understood. This work represents studies which identify a novel mechanism by which the virus can facilitate evasion of an immune system. We now show that RTA, the replication and transcription activator encoded by KSHV (ORF50), can function as an E3 ligase to degrade HLA-DRα. It can directly bind and induce degradation of HLA-DRα through the ubiquitin-proteasome degradation pathway. In addition to the direct regulation of HLA-DRα, RTA can also indirectly downregulate the level of HLA-DRα protein by upregulating transcription of MARCH8. Increased MARCH8 results in the downregulation of HLA-DRα. Furthermore, we also demonstrate that expression of HLA-DRα was impaired in KSHVde novoinfection.

scholarly journals Epstein-Barr Virus Latent Gene Expression in Primary Effusion Lymphomas Containing Kaposi's Sarcoma–Associated Herpesvirus/Human Herpesvirus-8

Blood ◽  
1997 ◽  
Vol 90 (3) ◽  
pp. 1186-1191 ◽  
Author(s):  
Marcelo G. Horenstein ◽  
Roland G. Nador ◽  
Amy Chadburn ◽  
Elizabeth M. Hyjek ◽  
Giorgio Inghirami ◽  
...  
Keyword(s):  
Gene Expression ◽  
Viral Infection ◽  
Kaposi's Sarcoma ◽  
Epstein Barr Virus ◽  
Kaposi’S Sarcoma ◽  
Barr Virus ◽  
Low Levels ◽  
Kaposi's Sarcoma Associated Herpesvirus ◽  
Epstein Barr ◽  
Kaposi’S Sarcoma Associated Herpesvirus

Primary effusion (body cavity–based) lymphoma (PEL) is a recently recognized subtype of malignant lymphoma that exhibits distinctive clinical and biological features, most notably its usual infection with the Kaposi's sarcoma–associated herpesvirus (KSHV). The vast majority of cases also contain Epstein-Barr virus (EBV). This dual viral infection is the first example of a consistent dual herpesviral infection in a human neoplasm and provides a unique model to study viral interactions. We analyzed the pattern of EBV latent gene expression to determine the pathogenic role of this agent in PELs. We examined five PELs coinfected with EBV and KSHV by reverse transcription-polymerase chain reaction (RT-PCR), in situ hybridization, and immunohistochemistry. EBER1 mRNA, a consistent marker of viral latency, was positive in all PEL cases, although at lower levels than in the non-PEL controls due to EBER1 expression by only a variable subset of lymphoma cells. Qp-initiated mRNA, encoding only EBNA1 and characteristic of latencies I and II, was positive in all PEL cases. Wp- and Cp-initiated mRNAs, encoding all EBNAs and characteristic of latency III, were negative in all cases. LMP1 mRNA, expressed in latencies II and III, was present in three cases of PEL, although at very low levels that were not detectable at the protein level by immunohistochemistry. Low levels of LMP2A mRNA were detected in all cases. BZLF1, an early-intermediate lytic phase marker, was weakly positive in four cases, suggesting a productive viral infection in a very small proportion of cells, which was confirmed by ZEBRA antigen expression. Therefore, PELs exhibit a restricted latency pattern, with expression of EBNA1 in all cases, and low LMP1 and LMP2A levels.

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.

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