Site-Specific Association with Host and Viral Chromatin by Kaposi's Sarcoma-Associated Herpesvirus LANA and Its Reversal during Lytic Reactivation

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.

Section: Discussionsupporting

confidence: 86%

supporting

confidence: 81%

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

“…KSHV establishes a latent infection in most infected cells, whereas a small proportion of cells develop lytic infection. The genetic profiles of KSHV-infected populations differ from those of uninfected populations, with host cell transcriptional remodeling observed in latently KSHV-infected cells (4) and global mRNA shutoff noted during lytic replication (5,6).…”

mentioning

confidence: 99%

ORF45-Mediated Prolonged c-Fos Accumulation Accelerates Viral Transcription during the Late Stage of Lytic Replication of Kaposi's Sarcoma-Associated Herpesvirus

Avey

et al. 2015

Kaposi's sarcoma-associated herpesvirus (KSHV) naturally infects humans, and over 95% of healthy persons have no symptoms. KSHV causes three types of malignancies in immunosuppressed patients: Kaposi's sarcoma, body cavity-based lymphoma, and multicentric Castleman's disease (1-3). KSHV establishes a latent infection in most infected cells, whereas a small proportion of cells develop lytic infection. The genetic profiles of KSHV-infected populations differ from those of uninfected populations, with host cell transcriptional remodeling observed in latently KSHV-infected cells (4) and global mRNA shutoff noted during lytic replication (5, 6).A limited number of viral transcripts appear in the latent stage, whereas the viral genome produces all of the viral transcripts during the lytic phase (7). A key viral replication and transcription activator (RTA) switches latent infection to lytic replication (8). Studies have characterized multiple cellular signaling pathways and transcription factors required for RTA expression. Mitogenactivated stimuli or stresses initiate RTA expression through mitogen-activated protein kinase (MAPK) or stress-activated protein kinases (SAPK), respectively (9-14). Thereafter, RTA activates viral lytic transcription and replication. RTA binding sites in the KSHV genome and responsive KSHV promoters have been characterized (15,16); these studies revealed that most viral gene expression is not directly activated by RTA and requires additional cellular transcription factors. Based on the analysis of elements in the RTA, ORF45, and K8 promoters, multiple transcription factors, including c-Fos, c-Jun, Sp1, CREB, C/EBP, c-Myc, and ATF-2, are required for the expression of immediate early (IE) genes (11)(12)(13)(17)(18)(19); most of these transcription factors are the direct or indirect targets of MAPK pathways (20,21). Recently, nonconventional viral DNA elements, viral noncoding RNA, and viral proteins required for KSHV late transcription have been characterized (22)(23)(24)(25)(26). The viral lytic proteins ORF24, ORF31, and ORF34 assemble into a transcriptional activator complex (25), and ORF24 recruits RNA polymerase II to viral late promot-

“…LANA mediates transcriptional upregulation and downregulation (32). Chromatin immunoprecipitation (ChIP) analyses have found LANA associated with the same sequences in the host genome as were characterized in the viral terminal repeats, in association with a separate characterized binding motif and binding indirectly through protein-protein interactions (33)(34)(35). LANA participates in epigenetic silencing (36)(37)(38)(39), increased transcription factor activity (40)(41)(42)(43)(44)(45)(46), and modulation of interferon signaling (33,47), the cell cycle (42,48,49) and kinase activity (50)(51)(52)(53)(54).…”

mentioning

confidence: 99%

A Screen for Extracellular Signal-Regulated Kinase-Primed Glycogen Synthase Kinase 3 Substrates Identifies the p53 Inhibitor iASPP

Woodard

Liao

Goodwin

et al. 2015

The Kaposi's sarcoma-associated herpesvirus (KSHV) LANA protein is essential for the replication and maintenance of virus genomes in latently KSHV-infected cells. LANA also drives dysregulated cell growth through a multiplicity of mechanisms that include altering the activity of the cellular kinases extracellular signal-regulated kinase (ERK) and glycogen synthase kinase 3 (GSK-3). To investigate the potential impact of these changes in enzyme activity, we used protein microarrays to identify cell proteins that were phosphorylated by the combination of ERK and GSK-3. The assays identified 58 potential ERK-primed GSK-3 substrates, of which 23 had evidence for in vivo phosphorylation in mass spectrometry databases. Two of these, SMAD4 and iASPP, were selected for further analysis and were confirmed as ERK-primed GSK-3 substrates. Cotransfection experiments revealed that iASPP, but not SMAD4, was targeted for degradation in the presence of GSK-3. iASPP interferes with apoptosis induced by p53 family members. To determine the importance of iASPP to KSHV-infected-cell growth, primary effusion lymphoma (PEL) cells were treated with an iASPP inhibitor in the presence or absence of the MDM2 inhibitor Nutlin-3. Drug inhibition of iASPP activity induced apoptosis in BC3 and BCBL1 PEL cells but did not induce poly(ADP-ribose) polymerase (PARP) cleavage in virus-negative BJAB cells. The effect of iASPP inhibition was additive with that of Nutlin-3. Interfering with iASPP function is therefore another mechanism that can sensitize KSHV-positive PEL cells to cell death. IMPORTANCEKSHV is associated with several malignancies, including primary effusion lymphoma (PEL). The KSHV-encoded LANA protein is multifunctional and promotes both cell growth and resistance to cell death. LANA is known to activate ERK and limit the activity of another kinase, GSK-3. To discover ways in which LANA manipulation of these two kinases might impact PEL cell survival, we screened a human protein microarray for ERK-primed GSK-3 substrates. One of the proteins identified, iASPP, showed reduced levels in the presence of GSK-3. Further, blocking iASPP activity increased cell death, particularly in p53 wild-type BC3 PEL cells.T he Kaposi's sarcoma-associated herpesvirus (KSHV) latencyassociated nuclear antigen (LANA) is expressed in all KSHVinfected cells, including the tumor cells of the KSHV-associated malignancies Kaposi's sarcoma, primary effusion lymphoma (PEL), and muticentric Castleman disease. LANA functions in the replication and maintenance of latent, episomal KSHV genomes (1, 2) by binding to the KSHV origin of replication in the terminal repeats (3, 4), recruiting cellular replication proteins (5-11), and tethering the KSHV episomal genomes to host chromosomes (12-18). Sequences in the KSHV terminal repeats are bound by the C terminus of LANA, and the X-ray crystal structures of the human and murine gammaherpesvirus 68 (MHV68) LANA DNA-binding domains have been solved (19)(20)(21).KSHV infection leads to a global reprogramming of cel...