A novel KRAB-Zinc finger protein interacts with latency-associated nuclear antigen of Kaposi’s sarcoma-associated herpesvirus and activates transcription via terminal repeat sequences

Kaposi's sarcoma-associated herpesvirus (KSHV) typically displays two different phases in its life cycle, the default latent phase and the lytic phase. There is a short period of lytic gene expression in the early stage of KSHV primary infection. The factors involved in the shutdown process of lytic gene expression are poorly identified. It has been shown that the latency-associated nuclear antigen (LANA) encoded by KSHV plays an important role in the establishment of viral latency. In screening, we identified a host protein, Krüppel-associated box domain-associated protein 1 (KAP1), that bound to LANA. We validated the interaction between LANA and KAP1 in vivo and in vitro, as well as their colocalization in the nucleus. We mapped out that LANA interacted with both the N-and C-terminal domains of KAP1. Based on the interface of LANA-KAP1 interaction determined, we proved that LANA recruited KAP1 to the RTA promoter region of the KSHV genome. We revealed that KAP1 was involved in transcriptional repression by LANA. We found multiple cooccupation sites of LANA and KAP1 on the whole KSHV genome by chromatin immunoprecipitation for sequencing (ChIP-seq) and demonstrated that LANA-recruited KAP1 played a critical role in the shutdown of lytic gene expression during the early stage of KSHV primary infection. Taken together, our data suggest that LANA interacts with KAP1 and represses lytic gene expression to facilitate the establishment of KSHV latency. IMPORTANCEOur study revealed the mechanism of transcriptional repression by LANA during KSHV primary infection, providing new insights into the process of KSHV latency establishment.

Section: Discussionmentioning

confidence: 99%

Kaposi's Sarcoma-Associated Herpesvirus-Encoded LANA Interacts with Host KAP1 To Facilitate Establishment of Viral Latency

Sun

Liang

Gao

et al. 2014

“…Previously, we and others performed yeast two-hybrid screens to identify novel protein-protein interactions with KSHV LANA (34,52,53,79,107). The conventional two-hybrid screen, although very powerful, is based on a transcriptional readout and is therefore unsuited for identifying transcription repressors or proteins whose interaction results in degradation of the bait protein.…”

Section: Discussionmentioning

confidence: 99%

“…LANA contributes to maintenance of latent infection by repressing expression of the KSHV RTA lytic regulator and replication at the lytic origin (56,58,62,85). LANA modifies cell gene expression through interactions with transcription factors (1,9,54,55,60,61,65,69,80,86,101,104,107), by mediating epigenetic silencing (8,91), and by modulating levels of cellular microRNAs (miRNAs) (112). LANA promotes cell cycle progression by binding to pRb (80), by stabilizing and activating c-Myc (8,61), and by increasing ␤-catenin-regulated gene expression (32,33).…”

mentioning

confidence: 99%

“…Information on the mechanisms by which LANA promotes KSHV latency, cell survival, and cell proliferation can be obtained by identifying LANA-interacting partners. Previously, yeast twohybrid screens (34,52,53,79,107), glutathione S-transferase (GST) affinity, and immunoprecipitation assays and chromatography coupled with mass spectroscopy (6,15,48,78) have been applied to the identification of LANA binding proteins. Each approach has strengths and weaknesses, and different proteins tend to be identified by the different screening approaches.…”

mentioning

confidence: 99%

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A Protein Array Screen for Kaposi's Sarcoma-Associated Herpesvirus LANA Interactors Links LANA to TIP60, PP2A Activity, and Telomere Shortening

Shamay

Liu

et al. 2012

The Kaposi's sarcoma-associated herpesvirus (KSHV) LANA protein functions in latently infected cells as an essential participant in KSHV genome replication and as a driver of dysregulated cell growth. To identify novel LANA protein-cell protein interactions that could contribute to these activities, we performed a proteomic screen in which purified, adenovirus-expressed Flag-LANA protein was incubated with an array displaying 4,192 nonredundant human proteins. Sixty-one interacting cell proteins were consistently detected. LANA interactions with high-mobility group AT-hook 1 (HMGA1), HMGB1, telomeric repeat binding factor 1 (TRF1), xeroderma pigmentosum complementation group A (XPA), pygopus homolog 2 (PYGO2), protein phosphatase 2A (PP2A)B subunit, Tat-interactive protein 60 (TIP60), replication protein A1 (RPA1), and RPA2 proteins were confirmed in coimmunoprecipitation assays. LANA-associated TIP60 retained acetyltransferase activity and, unlike human papillomavirus E6 and HIV-1 TAT proteins, LANA did not reduce TIP60 stability. The LANA-bound PP2A B subunit was associated with the PP2A A subunit but not the catalytic C subunit, suggesting a disruption of PP2A phosphatase activity. This is reminiscent of the role of simian virus 40 (SV40) small t antigen. Chromatin immunoprecipitation (ChIP) assays showed binding of RPA1 and RPA2 to the KSHV terminal repeats. Interestingly, LANA expression ablated RPA1 and RPA2 binding to the cell telomeric repeats. In U2OS cells that rely on the alternative mechanism for telomere maintenance, LANA expression had minimal effect on telomere length. However, LANA expression in telomerase immortalized endothelial cells resulted in telomere shortening. In KSHV-infected cells, telomere shortening may be one more mechanism by which LANA contributes to the development of malignancy.

“…However, it is possible that an other cell protein(s) with a role in LANA DNA replication may interact with the internal LANA region. Proteins which have been mapped to regions overlapping with the internal domains fall into several functional categories, including transcription (1,2,8,35,38,39,41,42,44,47,50,51,53,58,60,67,70), chromosome structure or modification (12,29,34,48,61,65,66,72), ubiquitin ligase activity (9), and cell growth control (7, 17-19, 46, 52, 54, 56, 57, 59, 61, 73). It is possible that one or more of these proteins, or an as yet unknown interacting protein, may be responsible for exerting the critical episome maintenance function(s).…”

Section: Discussionmentioning

confidence: 99%

The Internal Kaposi's Sarcoma-Associated Herpesvirus LANA Regions Exert a Critical Role on Episome Persistence

Vázquez

Kaye

2011

Kaposi's sarcoma-associated herpesvirus (KSHV) latency-associated nuclear antigen (LANA) is a 1,162-amino-acid protein that acts on viral terminal repeat (TR) DNA to mediate KSHV episome persistence. The two essential components of episome persistence are DNA replication prior to cell division and episome segregation to daughter nuclei. These functions are located within N-and C-terminal regions of LANA. N-and C-terminal regions of LANA are sufficient for TR DNA replication. In addition, N-and C-terminal regions of LANA tether episomes to mitotic chromosomes to segregate episomes to progeny cell nuclei. To generate a tethering mechanism, N-terminal LANA binds histones H2A/H2B to attach to mitotic chromosomes, and C-terminal LANA binds TR DNA and also associates with mitotic chromosomes. Here, we test the importance of the internal LANA sequence for episome persistence. We generated LANA mutants that contain N-and C-terminal regions of LANA but have most of the internal sequence deleted. As expected, the LANA mutants bound mitotic chromosomes in a wild-type pattern and also bound TR DNA as assayed by electrophoretic mobility shift assays (EMSA). The mutants mediated TR DNA replication, although with reduced efficiency compared with LANA. Despite the ability to replicate DNA and exert the chromosome and DNA binding functions necessary for segregating episomes to daughter nuclei, the mutants were highly deficient for the ability to mediate both short-and long-term episome persistence. These data indicate that internal LANA sequence exerts a critical effect on its ability to maintain episomes, possibly through effects on TR DNA replication.