Poly(ADP-Ribose) Polymerase 1 Binds to Kaposi's Sarcoma-Associated Herpesvirus (KSHV) Terminal Repeat Sequence and Modulates KSHV Replication in Latency

Latent DNA replication of Kaposi's sarcoma-associated herpesvirus (KSHV) initiates at the terminal repeat (TR) element and requires trans-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. K aposi's sarcoma-associated herpesvirus (KSHV) or human herpesvirus 8, an oncogenic member of the gammaherpesvirus subfamily, was first identified in 1994 from body cavity-based lymphoma (BCBL) and HIV-infected patients with Kaposi's sarcoma (KS) (1). KSHV is believed to be the etiological agent of several human cancers, including KS (1, 2), primary effusion lymphoma (PEL) or body cavity based lymphoma (3), and multicentric Castleman's disease (MCD) (4, 5). Additionally, there have been reports of KSHV-associated solid lymphomas in HIV-positive and -negative patients as well as KSHV-associated lymphomas in patients with primary immune deficiencies, such as common variable immune deficiency (6, 7). Similar to other members of the gammaherpesvirus family, KSHV establishes a predominantly latent infection in host cells after primary infection (8). During latent infections, the KSHV genome persists as a circular doublestranded DNA (episome) with most viral genes being silenced, except for a small number of latent genes, which include the viral cyclin (v-cyclin), the latency-associated nuclear antigen (LANA), and the viral FLICE inhibitory prote...

Section: Discussionmentioning

confidence: 69%

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

Sun

Jha

Robertson

2015

“…LANA phosphorylation is also achieved by interaction with glycogen synthase kinase 3B (GSK3B), which strengthens the interaction and further inactivates GSK3B (28,29). LANA poly(ADP-ribosyl)ation by poly(ADP-ribose) polymerase 1 (PARP1) negatively regulates latent replication, and LANA phosphorylation by a complex comprising PRKDC (protein kinase, DNA-activated catalytic subunit) and XRCC6/KU70 impairs latent replication (30,31). Moreover, LANA is phosphorylated by PIM1 and PIM3 and acetylated during reactivation (32,33).…”

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confidence: 99%

The Latency-Associated Nuclear Antigen of Kaposi's Sarcoma-Associated Herpesvirus Inhibits Expression of SUMO/Sentrin-Specific Peptidase 6 To Facilitate Establishment of Latency

Lin

Sun

Zhang

et al. 2017

Kaposi's sarcoma-associated herpesvirus (KSHV), which belongs to the Gammaherpesviridae, typically displays two different phases in its life cycle, the latent phase and the lytic phase. Latency-associated nuclear antigen (LANA), the primary viral product during latency, has been reported to bind to a series of cellular gene promoters to modulate gene transcription. To systemically elucidate the cellular genes regulated by LANA, we identified genome-wide LANA binding sites by chromatin immunoprecipitation coupled with sequencing (ChIP-seq). We stratified ChIP-seq data and found that LANA might be involved in the macromolecule catabolic process. Specifically, we found and verified that LANA could directly bind to the promoter of the SUMO/sentrin-specific peptidase 6 (SENP6) gene in vivo and in vitro. LANA could repress SENP6 promoter activity in a dose-dependent manner in a reporter gene assay. LANA expression was sufficient to inhibit endogenous SENP6 expression at both the RNA and protein levels. Moreover, SENP6 overexpression in KSHV-infected cells reduced LANA at the protein level. Mechanistically, we found that SENP6 could interact with LANA and reduce the formation of sumoylated LANA, which relies on the desumoylation ability of SENP6. During de novo infection, SENP6 overexpression would decrease the abundance of LANA and enhance viral gene expression, which would hamper the establishment of latency. Taken together, these data suggest that KSHV-encoded LANA could inhibit SENP6 expression to regulate the abundance of itself, which may play an important role in controlling the establishment of latency.IMPORTANCE LANA, as a key latent protein produced by KSHV, is responsible for episome persistence and regulates viral reactivation. In the present study, our results demonstrated that LANA could bind to the promoter region of the SENP6 gene and inhibit SENP6 expression while the regulated SENP6 could in turn modulate the abundance of LANA through desumoylation. This delicate regulation may provide important insights to explain the abundance of LANA during KSHV latency.

“…LANA binds to the terminal repeats of the KSHV genome (14, 25); links the genomes to the cell chromosomes through interactions with chromatinassociated proteins such as the core histones H2A and H2B, DEK, HP1, Brd4, and MeCP2 (6,28,37,69); and recruits cellular DNA replication machinery to the terminal repeats (45,60,62,64). Expression of LANA in a transgenic mouse generated activated, hyperproliferative B cells, and mice developed lymphoma with a long latency (19).…”

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confidence: 99%

The Kaposi's Sarcoma-Associated Herpesvirus LANA Protein Stabilizes and Activates c-Myc

Liu

Martin

Liao

et al. 2007

115

The Kaposi's sarcoma-associated herpesvirus (KSHV) latency-associated nuclear antigen (LANA) protein is functionally pleiotropic. LANA contributes to KSHV-associated pathogenesis, in part, by increasing entry of cells into S phase through a process that is driven by LANA interaction with the serine-threonine kinase glycogen synthase kinase 3 (GSK-3) and stabilization of ␤-catenin. Kaposi's sarcoma-associated herpesvirus (KSHV) was discovered in lesions of Kaposi's sarcoma using differential display (12) and was subsequently recognized to also be associated with primary effusion lymphoma and multicentric Castleman's disease (10, 18, 52, 59). The KSHV latency-associated nuclear antigen (LANA) is one of a limited number of KSHV genes consistently expressed in latently infected cells and in KSHV-associated malignancies (47). LANA is encoded by KSHV ORF73 and has unique N-terminal and C-terminal domains separated by three sets of repeated sequences that represent approximately half of the total protein sequence. These repeats function similarly to the central repeat region of the Epstein-Barr virus EBNA-1 protein by inhibiting antigen presentation and allowing tumor cells expressing LANA to escape immune surveillance (2,16,70).LANA is a multifunctional protein that is essential for the replication (5, 20, 29, 34) and maintenance (4) of KSHV episomal DNA during latent infection. LANA binds to the terminal repeats of the KSHV genome (14, 25); links the genomes to the cell chromosomes through interactions with chromatinassociated proteins such as the core histones H2A and H2B, DEK, HP1, Brd4, and MeCP2 (6,28,37,69); and recruits cellular DNA replication machinery to the terminal repeats (45,60,62,64). Expression of LANA in a transgenic mouse generated activated, hyperproliferative B cells, and mice developed lymphoma with a long latency (19). LANA has multiple properties that could contribute to tumorigenesis. These include inhibition of p53-mediated apoptosis (9, 21), stimulation of S-phase entry through stabilization of ␤-catenin and upregulation of cyclin D1 (24) and through induction of Rb/ E2F-regulated genes (1, 49), and overcoming G 1 cell cycle arrest mediated by p16 (1) and BRD4 and BRD2 (46). LANA is also responsible for promoting KSHV latency gene expression at the expense of lytic induction and for some of the reprogramming of cell gene expression that occurs in KSHVinfected cells (1,57,65,66). Targeting of LANA to DNA either through the use of Gal4-LANA fusion proteins (38, 53) through binding of LANA to the KSHV terminal repeats (25) or through LANA recruitment to cell (57) or viral promoters (39, 42) leads to transcriptional repression. LANA binds to histone deacetylase-associated corepressors (38) and is also capable of recruiting de novo DNA methyltransferases and the histone methyl transferase SUV39H1 to downregulate targeted cell promoters through CpG methylation (50, 57).LANA has also been reported to increase expression of genes regulated by a variety of transcription factors (40,44,61,63). A source...