The latency-associated nuclear antigen of Kaposi's sarcoma-associated herpesvirus interacts preferentially with the terminal repeats of the genome in vivo and this complex is sufficient for episomal DNA replication

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.

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

Journal of Virology ◽

10.1128/jvi.01524-15 ◽

2015 ◽

Vol 89 (20) ◽

pp. 10206-10218 ◽

Cited By ~ 13

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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Kaposi's Sarcoma-Associated Herpesvirus-Encoded Latency-Associated Nuclear Antigen Modulates K1 Expression through Its cis-Acting Elements within the Terminal Repeats

Journal of Virology ◽

10.1128/jvi.80.7.3445-3458.2006 ◽

2006 ◽

Vol 80 (7) ◽

pp. 3445-3458 ◽

Cited By ~ 25

Author(s):

Subhash C. Verma ◽

Ke Lan ◽

Tathagata Choudhuri ◽

Erle S. Robertson

Keyword(s):

Transcription Factor ◽

Binding Sites ◽

Kaposi's Sarcoma ◽

Transcriptional Activity ◽

Kaposi’S Sarcoma ◽

Nuclear Antigen ◽

Reporter Assay ◽

Kaposi's Sarcoma Associated Herpesvirus ◽

Terminal Repeats ◽

Kaposi’S Sarcoma Associated Herpesvirus

ABSTRACT K1 is the first open reading frame encoded by Kaposi's sarcoma-associated herpesvirus (KSHV) and lies positionally to the immediate right of the terminal repeats. K1 is a transmembrane glycoprotein having a functional immunoreceptor tyrosine-based activation motif (ITAM) capable of activating B-cell receptor signaling. K1 is expressed mostly during the lytic cycle of the virus and its promoter lies within the terminal repeat which contains the binding sites for latency-associated nuclear antigen (LANA). The K1 promoter (K1p) having LANA binding sites assayed by reporter assay demonstrated that LANA is capable of down-regulating K1 promoter transcriptional activity. However, the KSHV replication transcription activator RTA up-regulates K1p transcriptional activity. The promoter deleted of LANA binding sites showed loss in LANA-mediated down-regulation but was unaffected for RTA-mediated up-regulation. Increasing amounts of RTA rescued LANA-mediated repression of K1p transcriptional activity in cotransfection experiments. Reporter assay data suggest that LANA binding to its cognate sequence is critical for LANA-mediated repression of K1p as a LANA construct lacking the DNA binding domain was unable to repress K1p transcription. Additionally, KSHV primary infection experiments suggest that K1 is expressed during early infection but is repressed on the establishment of latency and so follows an expression profile similar to that of RTA during infection. Analysis of the promoter sequence revealed the presence of Oct-1 transcription factor binding sites within the −116 to +76 region. Mutational analysis of the Oct-1 sites abolished RTA-mediated transcriptional activation, suggesting that RTA up-regulates K1p transcription through binding to this transcription factor.

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Identification of a virus trans-acting regulatory element on the latent DNA replication of Kaposi's sarcoma-associated herpesvirus

Journal of General Virology ◽

10.1099/vir.0.19510-0 ◽

2004 ◽

Vol 85 (4) ◽

pp. 843-855 ◽

Cited By ~ 17

Author(s):

Chunghun Lim ◽

Taegun Seo ◽

Jun Jung ◽

Joonho Choe

Keyword(s):

Dna Replication ◽

Cell Lines ◽

Kaposi's Sarcoma ◽

Regulatory Element ◽

Kaposi’S Sarcoma ◽

Virus Genome ◽

Nuclear Antigen ◽

N Terminus ◽

Kaposi's Sarcoma Associated Herpesvirus ◽

Kaposi’S Sarcoma Associated Herpesvirus

Latency-associated nuclear antigen 1 (LANA1) of Kaposi's sarcoma-associated herpesvirus (KSHV) plays a pivotal role in the maintenance of the virus genome in latently infected cells. LANA1 links virus genomes to host chromosomes via a C-terminal DNA-binding domain which interacts with the sequences located in terminal repeats (TRs) of the virus genome and via an N-terminal chromosome-binding sequence which associates with the host chromosomes, respectively. Recent data suggest that LANA1 also actively participates in the replication of KSHV TR-containing plasmid in the transient DNA replication assay. In this report, it was found that C33A and COS-1, but not NIH/3T3, cell lines are permissive for the transient replication of KSHV TR-containing plasmid. Using several LANA1-deletion mutants, the minimum domain of LANA1 required for replication activity was also determined. In addition, the N terminus of LANA1 inhibited the transient replication systems of KSHV and Epstein–Barr virus (EBV) in transiently transfected 293 and 293T cells, but the C terminus of LANA1 specifically inhibited the transient replication system of KSHV in other cell lines. Consistent with previous reports, these data further emphasize the functional importance of the N terminus of LANA1 on replication from the KSHV latent origin of DNA replication.

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Kaposi's Sarcoma-Associated Herpesvirus LANA2 Is a B-Cell-Specific Latent Viral Protein That Inhibits p53

Journal of Virology ◽

10.1128/jvi.75.1.429-438.2001 ◽

2001 ◽

Vol 75 (1) ◽

pp. 429-438 ◽

Cited By ~ 213

Author(s):

Carmen Rivas ◽

Ai-En Thlick ◽

Carlo Parravicini ◽

Patrick S. Moore ◽

Yuan Chang

Keyword(s):

Kaposi's Sarcoma ◽

Primary Effusion Lymphoma ◽

Blot Hybridization ◽

Human Herpesvirus ◽

Kaposi’S Sarcoma ◽

Nuclear Antigen ◽

P53 Overexpression ◽

Kaposi's Sarcoma Associated Herpesvirus ◽

Kaposi’S Sarcoma Associated Herpesvirus

ABSTRACT Kaposi's sarcoma-associated herpesvirus (KSHV), or human herpesvirus 8, is associated with three proliferative diseases ranging from viral cytokine-induced hyperplasia to monoclonal neoplasia: multicentric Castleman's disease (CD), Kaposi's sarcoma (KS), and primary effusion lymphoma (PEL). Here we report a new latency-associated 1,704-bp KSHV spliced gene belonging to a cluster of KSHV sequences having homology to the interferon regulatory factor (IRF) family of transcription factors. ORFK10.5 encodes a protein, latency-associated nuclear antigen 2 (LANA2), which is expressed in KSHV-infected hematopoietic tissues, including PEL and CD but not KS lesions. LANA2 is abundantly expressed in the nuclei of cultured KSHV-infected B cells. Transcription of K10.5 in PEL cell cultures is not inhibited by DNA polymerase inhibitors nor significantly induced by phorbol ester treatment. Unlike LANA1, LANA2 does not elicit a serologic response from patients with KS, PEL, or CD as measured by Western blot hybridization. Both KSHV vIRF1 (ORFK9) and LANA2 (ORFK10.5) appear to have arisen through gene duplication of a captured cellular IRF gene. LANA2 is a potent inhibitor of p53-induced transcription in reporter assays. LANA2 antagonizes apoptosis due to p53 overexpression in p53-null SAOS-2 cells and apoptosis due to doxorubicin treatment of wild-type p53 U2OS cells. While LANA2 specifically interacts with amino acids 290 to 393 of p53 in glutathione S-transferase pull-down assays, we were unable to demonstrate LANA2-p53 interaction in vivo by immunoprecipitation. These findings show that KSHV has tissue-specific latent gene expression programs and identify a new latent protein which may contribute to KSHV tumorigenesis in hematopoietic tissues via p53 inhibition.

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A Domain in the C-Terminal Region of Latency-Associated Nuclear Antigen 1 of Kaposi's Sarcoma-Associated Herpesvirus Affects Transcriptional Activation and Binding to Nuclear Heterochromatin

Journal of Virology ◽

10.1128/jvi.77.12.7093-7100.2003 ◽

2003 ◽

Vol 77 (12) ◽

pp. 7093-7100 ◽

Cited By ~ 35

Author(s):

Abel Viejo-Borbolla ◽

Emrah Kati ◽

Julie A. Sheldon ◽

Kavita Nathan ◽

Karin Mattsson ◽

...

Keyword(s):

Amino Acids ◽

Nuclear Matrix ◽

Kaposi's Sarcoma ◽

Kaposi’S Sarcoma ◽

Terminal Region ◽

Nuclear Antigen ◽

Kaposi's Sarcoma Associated Herpesvirus ◽

Carboxy Terminal ◽

Kaposi’S Sarcoma Associated Herpesvirus ◽

Episomal Dna

ABSTRACT The latency-associated nuclear antigen 1 (LANA-1) of Kaposi's sarcoma-associated herpesvirus (KSHV) is required for the maintenance and replication of viral episomal DNA. The binding sites for nuclear heterochromatin and transcriptional repressor complexes are located in an amino-terminal region of LANA-1, whereas those for viral episomal DNA, p53, pRB, and members of the BRD/fsh family of nuclear proteins are located in its carboxy-terminal domain. LANA-1 activates or represses several cellular and viral promoters. In this report we show that a domain of 15 amino acids (amino acids 1129 to 1143), located close to the carboxy-terminal end of LANA-1, is required for the interaction of LANA-1 with nuclear heterochromatin or nuclear matrix, and for the ability of LANA-1 to activate the Epstein-Barr virus Cp promoter. LANA-1 proteins that are tightly associated with nuclear heterochromatin or matrix differ in molecular weight from LANA-1 proteins that can be dissociated from the nuclear matrix by high-salt buffers, suggesting that posttranslational modifications may determine the association of LANA-1 with nuclear heterochromatin or matrix.

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The Kaposi's Sarcoma-Associated Herpesvirus Latency-Associated Nuclear Antigen 1 N Terminus Is Essential for Chromosome Association, DNA Replication, and Episome Persistence

Journal of Virology ◽

10.1128/jvi.78.1.294-301.2004 ◽

2004 ◽

Vol 78 (1) ◽

pp. 294-301 ◽

Cited By ~ 79

Author(s):

Andrew J. Barbera ◽

Mary E. Ballestas ◽

Kenneth M. Kaye

Keyword(s):

Dna Replication ◽

Kaposi's Sarcoma ◽

Kaposi’S Sarcoma ◽

Chromosome Association ◽

Nuclear Antigen ◽

Mitotic Chromosomes ◽

Proliferating Cells ◽

N Terminus ◽

Kaposi's Sarcoma Associated Herpesvirus ◽

Kaposi’S Sarcoma Associated Herpesvirus

ABSTRACT To persist in latently infected, proliferating cells, Kaposi's sarcoma-associated herpesvirus (KSHV) episomes must replicate and efficiently segregate to progeny nuclei. Episome persistence in uninfected cells requires latency-associated nuclear antigen 1 (LANA1) in trans and cis-acting KSHV terminal repeat (TR) DNA. The LANA1 C terminus binds TR DNA, and LANA1 mediates TR-associated DNA replication in transient assays. LANA1 also concentrates at sites of KSHV TR DNA episomes along mitotic chromosomes, consistent with a tethering role to efficiently segregate episomes to progeny nuclei. LANA1 amino acids 5 to 22 constitute a chromosome association region (Piolot et al., J. Virol. 75:3948-3959, 2001). We now investigate LANA1 residues 5 to 22 with scanning alanine substitutions. Mutations targeting LANA1 5GMR7, 8LRS10, and 11GRS13 eliminated chromosome association, DNA replication, and episome persistence. LANA1 mutated at 14TG15 retained the ability to associate with chromosomes but was partially deficient in DNA replication and episome persistence. These results provide genetic support for a key role of the LANA1 N terminus in chromosome association, LANA1-mediated DNA replication, and episome persistence.

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Involvement of SSRP1 in Latent Replication of Kaposi's Sarcoma-Associated Herpesvirus

Journal of Virology ◽

10.1128/jvi.00907-09 ◽

2009 ◽

Vol 83 (21) ◽

pp. 11051-11063 ◽

Cited By ~ 23

Author(s):

Jianhong Hu ◽

Eugene Liu ◽

Rolf Renne

Keyword(s):

Dna Replication ◽

Kaposi's Sarcoma ◽

Latent Infection ◽

Mutational Analysis ◽

Human Herpesvirus ◽

Kaposi’S Sarcoma ◽

Dominant Negative ◽

Nuclear Antigen ◽

Kaposi's Sarcoma Associated Herpesvirus ◽

Kaposi’S Sarcoma Associated Herpesvirus

ABSTRACT Kaposi's sarcoma-associated herpesvirus (also named human herpesvirus 8) is a γ-herpesvirus that undergoes both lytic and latent infection. During latent infection, two viral elements are required: latency-associated nuclear antigen (LANA), which functions as an origin binding protein, and the latent origin, which resides within the terminal repeats (TRs) of the viral genome. Previously, we identified two cis-elements within the TRs which are required for latent DNA replication: two LANA binding sites (LBS1 and LBS2 [LBS1/2]) and a GC-rich replication element (RE) upstream of LBS1/2. To further characterize the RE, we constructed a 71-bp minimal replicon (MR) and performed a detailed mutational analysis. Our data indicate that the first 8 nucleotides within the RE are critical for replication. Moreover, both the position and the distance between the RE and LBS1/2 can affect origin replication activity, suggesting that the RE may function as a loading pad for cellular proteins involved in replication. Using biotinylated DNA fragments of wild-type or mutant MRs as probes, we identified 30 proteins that preferentially bind to the origin. Among these proteins, structure-specific recognition protein 1 (SSRP1), a subunit of the FACT complex, and telomeric repeat binding factor 2 (TRF2) formed complexes with LANA at the MR region. Furthermore, the small interfering RNA-based knockdown of SSRP1, but not the dominant-negative-based knockdown of TRF2, significantly decreased the efficiency of LANA-dependent DNA replication. These results indicate that SSRP1 is a novel cellular protein involved in LANA-dependent DNA replication.

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ORF73 of Herpesvirus Saimiri, a Viral Homolog of Kaposi's Sarcoma-Associated Herpesvirus, Modulates the Two Cellular Tumor Suppressor Proteins p53 and pRb

Journal of Virology ◽

10.1128/jvi.78.19.10336-10347.2004 ◽

2004 ◽

Vol 78 (19) ◽

pp. 10336-10347 ◽

Cited By ~ 31

Author(s):

Sumit Borah ◽

Subhash C. Verma ◽

Erle S. Robertson

Keyword(s):

Cell Cycle ◽

Kaposi's Sarcoma ◽

Kaposi’S Sarcoma ◽

Herpesvirus Saimiri ◽

Nuclear Antigen ◽

Kaposi's Sarcoma Associated Herpesvirus ◽

Kaposi’S Sarcoma Associated Herpesvirus ◽

P53 Inactivation

ABSTRACT All known DNA tumor viruses are known to target and inactivate two main cell cycle regulatory proteins, retinoblastoma protein (pRb) and p53. Inactivation of pRb promotes host cell cycle progression into S phase, and inactivation of p53 promotes cell immortalization. The DNA tumor virus Kaposi's sarcoma associated herpesvirus (KSHV)-encoded latency-associated nuclear antigen (LANA) was shown to target and inactivate pRb as well as p53. In this report we provide evidence that these functions are conserved in the homologous protein encoded by the related gammaherpesvirus herpesvirus saimiri (HVS). ORF73, the HVS homologue of LANA, is shown to bind both p53 and pRb in vitro and in vivo, to colocalize with p53 in human T cells infected with HVS, and in cells overexpressing both ORF73 and p53, as well as to adversely influence pRB/E2F and p53 transcriptional regulation. The C terminus of LANA, the region most highly conserved in ORF73, is shown to be responsible for both pRb and p53 interactions, supporting the hypothesis that these functions are conserved in both homologues. Finally, the region of p53 targeted by LANA (and ORF73) maps to the domain required for tetramerization. However, preliminary cross-linking studies do not detect disruption of p53 tetramerization by either LANA or HVS-encoded ORF73, suggesting that p53 inactivation may be by a mechanism independent of tetramer disruption.

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The Latency-Associated Nuclear Antigen of Kaposi's Sarcoma-Associated Herpesvirus Supports Latent DNA Replication in Dividing Cells

Journal of Virology ◽

10.1128/jvi.76.22.11677-11687.2002 ◽

2002 ◽

Vol 76 (22) ◽

pp. 11677-11687 ◽

Cited By ~ 175

Author(s):

Jianhong Hu ◽

Alexander C. Garber ◽

Rolf Renne

Keyword(s):

Amino Acid ◽

Dna Replication ◽

Dna Binding ◽

Kaposi's Sarcoma ◽

Kaposi’S Sarcoma ◽

Terminal Repeat ◽

Nuclear Antigen ◽

Dividing Cells ◽

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 a multifunctional protein that is consistently expressed in all KSHV-associated malignancies. LANA interacts with a variety of cellular proteins, including the transcriptional cosuppressor complex mSin3 and the tumor suppressors p53 and Rb, thereby regulating viral and cellular gene expression. In addition, LANA is required for maintenance of the episomal viral DNA during latency in dividing cells. Colocalization studies suggest that LANA tethers the viral genome to chromosomes during mitosis. In support of this model, a specific LANA- binding site has recently been identified within the terminal repeat unit, and a chromatin interaction domain was mapped to a short amino acid stretch within the N-terminal domain of LANA. Epstein-Barr virus nuclear antigen 1 (EBNA-1), a functional homologue of LANA, is also required for genome segregation; in addition, EBNA-1 also supports efficient DNA replication of oriP-containing plasmids. By performing short-term replication assays, we demonstrate here for the first time that de novo synthesis of terminal-repeat (TR)-containing plasmids is highly dependent on the presence of LANA. We map the required cis-acting sequences within the TR to a 79-bp region and demonstrate that the DNA-binding domain of LANA is required for this DNA replication activity. Surprisingly, the 233-amino-acid C domain of LANA by itself partially supports replication. Our data show that LANA is a sequence-specific DNA-binding protein that, like EBNA-1, plays an important role in DNA replication and genome segregation. In addition, we show that all necessary cis elements for the origin of replication (ori) function are located within a single TR, suggesting that the putative ori of KSHV is different from those of other gammaherpesviruses, which all contain ori sequences within the unique long sequence outside of their TR. This notion is further strengthened by the unique modular structure of the KSHV TR element.

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