KSHV LANA—The Master Regulator of KSHV Latency

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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: 68%

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

Sun

Jha

Robertson

2015

Self Cite

“…1Db to d). In contrast, the KSHV latency-associated ORF73/LANA, ORF72/ v-cyclin, ORF71/v-FLIP, and K12/kaposin families of protein-encoding genes (61) were not significantly affected, with only marginal upregulation (Fig. 1Da).…”

Section: Ifi16 Knockdown In Pel Cells Results In Global Increasesmentioning

confidence: 90%

Nuclear Innate Immune DNA Sensor IFI16 Is Degraded during Lytic Reactivation of Kaposi's Sarcoma-Associated Herpesvirus (KSHV): Role of IFI16 in Maintenance of KSHV Latency

Roy

Dutta

Iqbal

et al. 2016

IMPORTANCELike all herpesviruses, latency is an integral part of the life cycle of Kaposi's sarcoma-associated herpesvirus (KSHV), an etiological agent for many human cancers. Herpesviruses utilize viral and host factors to successfully evade the host immune system to maintain latency. Reactivation is a complex event where the latent episomal viral genome springs back to active transcription of lytic cycle genes. Our studies reveal that KSHV has evolved to utilize the innate immune sensor IFI16 to keep lytic cycle transcription in dormancy. We demonstrate that IFI16 binds to the lytic gene promoter, acts as a transcriptional repressor, and thereby helps to maintain latency. We also discovered that during the late stage of lytic replication, KSHV selectively degrades IFI16, thus relieving transcriptional repression. This is the first report to demonstrate the role of IFI16 in latency maintenance of a herpesvirus, and further understanding will lead to the development of strategies to eliminate latent infection. The human gammaherpesvirus Kaposi's sarcoma (KS)-associated herpesvirus (KSHV), also referred to as human herpesvirus 8 (HHV-8), is an oncogenic virus etiologically associated with KS, primary effusion B-cell lymphoma (PEL) or body cavity B-cell lymphoma (BCBL), and plasmablastic multicentric Castleman's disease (pMCD) (1-3). In vivo, KSHV DNA and transcripts have been identified in human B cells, endothelial cells, epithelial cells, macrophages, and keratinocytes (3, 4). Similar to other herpesviruses, KSHV undergoes two distinguishable phases in its life cycle: latent and lytic infection (3, 4). During primary infection, KSHV initially establishes latent infection in specific target cells, during which multiple copies of the viral genome are stably maintained as extrachromosomal episomes (3,5). Only a few viral genes, primarily localized in the major latency locus of the genome, are expressed during this phase. These gene products bestow numerous essential functionalities to the dormant viral genome, such as evading host immune surveillance (6), promoting cellular proliferation (7-9), maintaining the viral episome (10), and tightly suppressing viral lytic gene expression (11). However, both spontaneous reactivation and induced reactivation of the latent genome occur, resulting in the systematic expression of the full repertoire of viral genes, which leads to viral genome replication and virion production (4). Previous studies suggested that lytic reactivation

“…Another interesting promoter is the Orf73Li promoter that drives the transcription of the latency-associated protein LANA. This protein is essential for the establishment, maintenance, and control of viral latency (76)(77)(78)(79)(80). Two other unique transcripts that are more strongly upregulated by RTA isoform 2 than RTA isoform 1 are the Orf47 and Orf49 promoters, which drive the expression of transcription that are antisense to the Orf50 region (Fig.…”

Section: Discussionmentioning

confidence: 99%

Identification of Novel Kaposi's Sarcoma-Associated HerpesvirusOrf50Transcripts: Discovery of New RTA Isoforms with Variable Transactivation Potential

Wakeman

Izumiya

Speck

2017

Kaposi's sarcoma-associated herpesvirus (KSHV) is a gammaherpesvirus that has been associated with primary effusion lymphoma and multicentric Castleman's disease, as well as its namesake Kaposi's sarcoma. As a gammaherpesvirus, KSHV is able to acutely replicate, enter latency, and reactivate from this latent state. A key protein involved in both acute replication and reactivation from latency is the replication and transcriptional activator (RTA) encoded by the gene Orf50. RTA is a known transactivator of multiple viral genes, allowing it to control the switch between latency and virus replication. We report here the identification of six alternatively spliced Orf50 transcripts that are generated from four distinct promoters. These newly identified promoters are shown to be transcriptionally active in 293T (embryonic kidney), Vero (African-green monkey kidney epithelial), 3T12 (mouse fibroblast), and RAW 264.7 (mouse macrophage) cell lines. Notably, the newly identified Orf50 transcripts are predicted to encode four different isoforms of the RTA which differ by 6 to 10 residues at the amino terminus of the protein. We show the global viral transactivation potential of all four RTA isoforms and demonstrate that all isoforms can transcriptionally activate an array of KSHV promoters to various levels. The pattern of transcriptional activation appears to support a transcriptional interference model within the Orf50 region, where silencing of previously expressed isoforms by transcription initiation from upstream Orf50 promoters has the potential to modulate the pattern of viral gene activation.IMPORTANCE Gammaherpesviruses are associated with the development of lymphomas and lymphoproliferative diseases, as well as several other types of cancer. The human gammaherpesvirus, Kaposi's sarcoma-associated herpesvirus (KSHV), is tightly associated with the development of Kaposi's sarcoma and multicentric Castleman's disease, as well as a rare form of B cell lymphoma (primary effusion lymphoma) primarily observed in HIV-infected individuals. RTA is an essential viral gene product involved in the initiation of gammaherpesvirus replication and is conserved among all known gammaherpesviruses. We show here for KSHV that transcription of the gene encoding RTA is complex and leads to the expression of several isoforms of RTA with distinct functions. This observed complexity in KSHV RTA expression and function likely plays a critical role in the regulation of downstream viral and cellular gene expression, leading to the efficient production of mature virions. KEYWORDS Kaposi's sarcoma-associated herpesvirus, RTA isoforms, alternative promoter usage, alternative splicing, transcriptional activation