scholarly journals Evidence for Multiple Subpopulations of Herpesvirus-Latently Infected Cells

mBio ◽  
2022 ◽  
Author(s):  
Justin T. Landis ◽  
Ryan Tuck ◽  
Yue Pan ◽  
Carson N. Mosso ◽  
Anthony B. Eason ◽  
...  
Keyword(s):  
Kaposi's Sarcoma ◽  
Kaposi’S Sarcoma ◽  
Resistance To Therapy ◽  
Latently Infected ◽  
Infected Cells ◽  
Kaposi's Sarcoma Associated Herpesvirus ◽  
Kaposi’S Sarcoma Associated Herpesvirus ◽  
Primary Effusion Lymphomas

Latency is the defining characteristic of the Herpesviridae and central to the tumorigenesis phenotype of Kaposi’s sarcoma-associated herpesvirus (KSHV). KSHV-driven primary effusion lymphomas (PEL) rapidly develop resistance to therapy, suggesting tumor instability and plasticity.

scholarly journals ORC, MCM, and Histone Hyperacetylation at the Kaposi's Sarcoma-Associated Herpesvirus Latent Replication Origin

2004 ◽  
Vol 78 (22) ◽  
pp. 12566-12575 ◽  
Author(s):  
William Stedman ◽  
Zhong Deng ◽  
Fang Lu ◽  
Paul M. Lieberman
Keyword(s):  
Dna Replication ◽  
Replication Origin ◽  
Kaposi's Sarcoma ◽  
Histone Acetyltransferase ◽  
Histone H3 ◽  
Kaposi’S Sarcoma ◽  
Latently Infected ◽  
Infected Cells ◽  
Kaposi's Sarcoma Associated Herpesvirus ◽  
Kaposi’S Sarcoma Associated Herpesvirus

ABSTRACT The viral genome of Kaposi's sarcoma-associated herpesvirus (KSHV) persists as an extrachromosomal plasmid in latently infected cells. The KSHV latency-associated nuclear antigen (LANA) stimulates plasmid maintenance and DNA replication by binding to an ∼150-bp region within the viral terminal repeats (TR). We have used chromatin immunoprecipitation assays to demonstrate that LANA binds specifically to the replication origin sequence within the KSHV TR in latently infected cells. The latent replication origin within the TR was also bound by LANA-associated proteins CBP, double-bromodomain-containing protein 2 (BRD2), and the origin recognition complex 2 protein (ORC2) and was enriched in hyperacetylated histones H3 and H4 relative to other regions of the latent genome. Cell cycle analysis indicated that the minichromosome maintenance complex protein, MCM3, bound TR in late-G1/S-arrested cells, which coincided with the loss of histone H3 K4 methylation. Micrococcal nuclease studies revealed that TRs are embedded in a highly ordered nucleosome array that becomes disorganized in late G1/S phase. ORC binding to TR was LANA dependent when reconstituted in transfected plasmids. DNA affinity purification confirmed that LANA, CBP, BRD2, and ORC2 bound TR specifically and identified the histone acetyltransferase HBO1 (histone acetyltransferase binding to ORC1) as a potential TR binding protein. Disruption of ORC2, MCM5, and HBO1 expression by small interfering RNA reduced LANA-dependent DNA replication of TR-containing plasmids. These findings are the first demonstration that cellular replication and origin licensing factors are required for KSHV latent cycle replication. These results also suggest that the KSHV latent origin of replication is a unique chromatin environment containing histone H3 hyperacetylation within heterochromatic tandem repeats.

scholarly journals STAT3 Regulates Lytic Activation of Kaposi's Sarcoma-Associated Herpesvirus

2015 ◽  
Vol 89 (22) ◽  
pp. 11347-11355 ◽  
Author(s):  
Christine A. King ◽  
Xiaofan Li ◽  
Arturo Barbachano-Guerrero ◽  
Sumita Bhaduri-McIntosh
Keyword(s):  
Life Cycle ◽  
Molecular Basis ◽  
Kaposi's Sarcoma ◽  
Kaposi’S Sarcoma ◽  
Latently Infected ◽  
Refractory State ◽  
Infected Cells ◽  
Kaposi's Sarcoma Associated Herpesvirus ◽  
Kaposi’S Sarcoma Associated Herpesvirus ◽  
Hsv 1

ABSTRACTLytic activation of Kaposi's sarcoma-associated herpesvirus (KSHV) from latency is a critical contributor to pathogenesis and progression of KSHV-mediated disease. Development of targeted treatment strategies and improvement of lytic-phase-directed oncolytic therapies, therefore, hinge on gaining a better understanding of latency-to-lytic-phase transition. A key observation in that regard, also common to other herpesviruses, is the partial permissiveness of latently infected cells to lytic-cycle-inducing agents. Here, we address the molecular basis of why only some KSHV-infected cells respond to lytic stimuli. Since cellular signal transducer and activator of transcription 3 (STAT3) is constitutively active in KSHV-associated cancers, KSHV activates STAT3, and STAT3 has been found to regulate lytic activation of Epstein-Barr virus (EBV)-infected cells, we asked if STAT3 contributes similarly to the life cycle of KSHV. We found that high levels of STAT3 correlate with the refractory state at the single-cell level under conditions of both spontaneous and induced lytic activation; importantly, STAT3 also regulates lytic susceptibility. Further, knockdown of STAT3 suppresses the cellular transcriptional corepressor Krüppel-associated box domain-associated protein 1 (KAP1; also known as TRIM28), and suppression of KAP1 activates lytic genes, including the viral lytic switch RTA, thereby linking STAT3 via KAP1 to regulation of the balance between lytic and latent cells. These findings, taken together with those from EBV-infected and, more recently, herpes simplex virus 1 (HSV-1)-infected cells, cement the contribution of host STAT3 to persistence of herpesviruses and simultaneously reveal an important lead to devise strategies to improve lytic-phase-directed therapies for herpesviruses.IMPORTANCELytic activation of the cancer-causing Kaposi's sarcoma-associated herpesvirus (KSHV) is vital to its life cycle and causation of disease. Like other herpesviruses, however, a substantial fraction of latently infected cells are resistant to lytic-phase-inducing stimuli. Investigating the molecular basis for this refractory state is essential for understanding how the virus persists and how it causes disease and to guide efforts to improve treatment of KSHV-mediated diseases. We found that, like two other herpesviruses, EBV and HSV-1, KSHV exploits the cellular transcription factor STAT3 to regulate the susceptibility of latently infected cells to lytic triggers. These findings highlight a common STAT3-centered strategy used by herpesviruses to maintain persistence in their hosts while also revealing a key molecule to pursue while devising methods to improve herpesvirus lytic-phase-directed therapies.

scholarly journals Targeting the Kaposi’s sarcoma-associated herpesvirus genome with the CRISPR-Cas9 platform in latently infected cells

2021 ◽  
Vol 18 (1) ◽  
Author(s):  
Coral Orel Haddad ◽  
Inna Kalt ◽  
Yehuda Shovman ◽  
Lei Xia ◽  
Yehuda Schlesinger ◽  
...  
Keyword(s):  
Kaposi's Sarcoma ◽  
Marker Gene ◽  
Kaposi’S Sarcoma ◽  
Infection Process ◽  
Viral Genomes ◽  
Latently Infected ◽  
Infected Cells ◽  
Kaposi's Sarcoma Associated Herpesvirus ◽  
Novel Strategy ◽  
Kaposi’S Sarcoma Associated Herpesvirus

Abstract Background Kaposi’s sarcoma-associated herpesvirus (KSHV) is a transforming gammaherpesvirus. Like other herpesviruses, KSHV infection is for life long and there is no treatment that can cure patients from the virus. In addition, there is an urgent need to target viral genes to study their role during the infection cycle. The CRISPR-Cas9 technology offers a means to target viral genomes and thus may offer a novel strategy for viral cure as well as for better understanding of the infection process. We evaluated the suitability of this platform for the targeting of KSHV. Methods We have used the recombinat KSHV BAC16 genome, which contains an expression cassette encoding hygromycin-resistance and a GFP marker gene. Three genes were targeted: gfp, which serves as a marker for infection; orf45 encoding a lytic viral protein; and orf73, encoding LANA which is crucial for latent infection. The fraction of cells expressing GFP, viral DNA levels and LANA expression were monitored and viral genomes were sequenced. Results We found that KSHV episomes can be targeted by CRISPR-Cas9. Interestingly, the quantity of KSHV DNA declined, even when target sites were not functionally important for latency. In addition, we show that antibiotic selection, used to maintain infection, interferes with the outcome of targeting. Conclusions Our study provides insights into the use of this fundamental approach for the study and manipulation of KSHV. It provides guidelines for the targeting CRISPR-Cas9 to the viral genome and for outcomes interpretation.

scholarly journals Transcriptional Origin of Kaposi's Sarcoma-Associated Herpesvirus MicroRNAs

2006 ◽  
Vol 80 (5) ◽  
pp. 2234-2242 ◽  
Author(s):  
Xuezhong Cai ◽  
Bryan R. Cullen
Keyword(s):  
Kaposi's Sarcoma ◽  
Kaposi’S Sarcoma ◽  
Lytic Replication ◽  
Open Reading Frames ◽  
Polyadenylation Signal ◽  
Latently Infected ◽  
Infected Cells ◽  
Kaposi's Sarcoma Associated Herpesvirus ◽  
The One ◽  
Kaposi’S Sarcoma Associated Herpesvirus

ABSTRACT Kaposi's sarcoma-associated herpesvirus (KSHV) encodes 11 distinct microRNAs, all of which are found clustered within the major latency-associated region of the KSHV genome in the same transcriptional orientation. Because the KSHV microRNAs are all expressed in latently infected cells and are largely unaffected by induction of lytic replication, it appeared probable that they would be processed out of KSHV transcripts that are derived from a latent promoter(s) present in this region. Here, we define three latent transcripts, derived from two distinct KSHV latent promoters, that function as both KSHV primary microRNA precursors and as kaposin pre-mRNAs. These activities require the readthrough of a leaky viral polyadenylation signal located at nucleotide 122070 in the KSHV genome. In contrast, recognition of this polyadenylation signal gives rise to previously identified mRNAs that encode the KSHV open reading frames (ORFs) 71, 72 and 73 proteins as well as a novel unspliced KSHV mRNA that encodes only ORF72 and ORF71. Thus, transcripts initiating at the two latent promoters present in the KSHV latency-associated region can undergo two entirely distinct fates, i.e., processing to give a kaposin mRNA and viral microRNAs on the one hand or expression as KSHV ORF71, ORF72, or ORF73 mRNAs on the other, depending on whether the viral polyadenylation site located at position 122070 is ignored or recognized, respectively.

scholarly journals Targeting the Kaposi’s Sarcoma-associated Herpesvirus Genome With the CRISPR-Cas9 Platform in Latently Infected Cells

2020 ◽  
Author(s):  
Coral Orel Haddad ◽  
Inna Kalt ◽  
Yehuda Shovman ◽  
Lei Xia ◽  
Yehuda Schlesinger ◽  
...  
Keyword(s):  
Kaposi's Sarcoma ◽  
Marker Gene ◽  
Kaposi’S Sarcoma ◽  
Infection Process ◽  
Viral Genomes ◽  
Latently Infected ◽  
Infected Cells ◽  
Kaposi's Sarcoma Associated Herpesvirus ◽  
Novel Strategy ◽  
Kaposi’S Sarcoma Associated Herpesvirus

Abstract Background: Kaposi’s sarcoma-associated herpesvirus (KSHV) is a transforming gammaherpes. Like other herpesviruses, KSHV infection is for life long and there is no treatment that can cure of patients from the virus. In addition, there is urgent need to target viral genes to study their role during the infection cycle. The CRISPR-Cas9 technology offers a means to target viral genomes and thus may offer a novel strategy for viral cure as well for better understanding of the infection process. We evaluated the suitability of this platform for the targeting of KSHV. Methods: We have used BAC16 genome, which contains an expression cassette encoding hygromycin-resistance and a GFP marker gene. Three genes were targeted: gfp which serves as a marker for infection; orf45 encoding a lytic viral protein; and orf73, encoding LANA which is crucial for latent infection. The fraction of cells expressing GFP as well as viral DNA levels and LANA expression were monitored and viral genomes were sequenced. Results: We found that KSHV episomes can be targeted by CRISPR-Cas9. Interestingly, the quantity of KSHV DNA declined, even when target sites were not functionally important for latency. In addition, we show that antibiotic selection, used to maintain infection, interferes with the outcome of targeting.Conclusions: Our study provides insights to the use of this fundamental approach for the study and manipulation of KSHV. It provides guidelines for the targeting CRISPR-Cas9 to the viral genome and for outcomes interpretation.

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

2015 ◽  
Vol 89 (20) ◽  
pp. 10206-10218 ◽  
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.

scholarly journals Latency-Associated Nuclear Antigen of Kaposi's Sarcoma-Associated Herpesvirus (KSHV) Upregulates Survivin Expression in KSHV-Associated B-Lymphoma Cells and Contributes to Their Proliferation

2009 ◽  
Vol 83 (14) ◽  
pp. 7129-7141 ◽  
Author(s):  
Jie Lu ◽  
Subhash C. Verma ◽  
Masanao Murakami ◽  
Qiliang Cai ◽  
Pankaj Kumar ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Kaposi's Sarcoma ◽  
Survivin Expression ◽  
Kaposi’S Sarcoma ◽  
Nuclear Antigen ◽  
Mrna Levels ◽  
Survivin Promoter ◽  
Infected Cells ◽  
Kaposi's Sarcoma Associated Herpesvirus ◽  
Kaposi’S Sarcoma Associated Herpesvirus

ABSTRACT Survivin is a master regulator of cell proliferation and cell viability and is highly expressed in most human tumors. The molecular network linked to survivin expression in tumors has not been completely elucidated. In this study, we show that latency-associated nuclear antigen (LANA), a multifunctional protein of Kaposi's sarcoma-associated herpesvirus (KSHV) that is found in Kaposi's sarcoma tumors, upregulates survivin expression and increases the proliferation of KSHV-infected B cells. Analysis of pathway-specific gene arrays showed that survivin expression was highly upregulated in BJAB cells expressing LANA. The mRNA levels of survivin were also upregulated in HEK 293 and BJAB cells expressing LANA. Similarly, protein levels of survivin were significantly higher in LANA-expressing, as well as KSHV-infected, cells. Survivin promoter activity assays identified GC/Sp1 and p53 cis-acting elements within the core promoter region as being important for LANA activity. Gel mobility shift assays revealed that LANA forms a complex with Sp1 or Sp1-like proteins bound to the GC/Sp1 box of the survivin promoter. In addition, a LANA/p53 complex bound to the p53 cis-acting element within the survivin promoter, indicating that upregulation of survivin expression can also occur through suppression of p53 function. Furthermore, immunohistochemistry analyses revealed that survivin expression was upregulated in KSHV-associated Kaposi's sarcoma tissue, suggesting that LANA plays an important role in the upregulation of survivin expression in KSHV-infected endothelial cells. Knockdown of survivin expression by lentivirus-delivered small hairpin RNA resulted in loss of cell proliferation in KSHV-infected cells. Therefore, upregulation of survivin expression in KSHV-associated human cells contributes to their proliferation.

scholarly journals Transcriptome Analysis of Kaposi's Sarcoma-Associated Herpesvirus duringDe NovoPrimary Infection of Human B and Endothelial Cells

2014 ◽  
Vol 89 (6) ◽  
pp. 3093-3111 ◽  
Author(s):  
Pravinkumar Purushothaman ◽  
Suhani Thakker ◽  
Subhash C. Verma
Keyword(s):  
Primary Infection ◽  
Kaposi's Sarcoma ◽  
De Novo ◽  
Kaposi’S Sarcoma ◽  
Viral Gene ◽  
Target Cells ◽  
Viral Genes ◽  
Infected Cells ◽  
Kaposi's Sarcoma Associated Herpesvirus ◽  
Kaposi’S Sarcoma Associated Herpesvirus

ABSTRACTKaposi's sarcoma-associated herpesvirus (KSHV) infects many target cells (e.g., endothelial, epithelial, and B cells, keratinocytes, and monocytes) to establish lifelong latent infections. Viral latent-protein expression is critical in inducing and maintaining KSHV latency. Infected cells are programmed to retain the incoming viral genomes during primary infection. Immediately after infection, KSHV transcribes many lytic genes that modulate various cellular pathways to establish successful infection. Analysis of the virion particle showed that the virions contain viral mRNAs, microRNAs, and other noncoding RNAs that are transduced into the target cells during infection, but their biological functions are largely unknown. We performed a comprehensive analysis of the KSHV virion packaged transcripts and the profiles of viral genes transcribed afterde novoinfections of various cell types (human peripheral blood mononuclear cells [PBMCs], CD14+monocytes, and telomerase-immortalized vascular endothelial [TIVE] cells), from viral entry until latency establishment. A next-generation sequence analysis of the total transcriptome showed that several viral RNAs (polyadenylated nuclear RNA, open reading frame 58 [ORF58], ORF59, T0.7, and ORF17) were abundantly present in the KSHV virions and effectively transduced into the target cells. Analysis of the transcription profiles of each viral gene showed specific expression patterns in different cell lines, with the majority of the genes, other than latent genes, silencing after 24 h postinfection. We differentiated the actively transcribing genes from the virion-transduced transcripts using a nascent RNA capture approach (Click-iT chemistry), which identified transcription of a number of viral genes during primary infection. Treating the infected cells with phosphonoacetic acid (PAA) to block the activity of viral DNA polymerase confirmed the involvement of lytic DNA replication during primary infection. To further understand the role of DNA replication during primary infection, we performedde novoPBMC infections with a recombinant ORF59-deleted KSHV virus, which showed significantly reduced numbers of viral copies in the latently infected cells. In summary, the transduced KSHV RNAs as well as the actively transcribed genes control critical processes of early infection to establish KSHV latency.IMPORTANCEKaposi's sarcoma-associated herpesvirus (KSHV) is the causative agent of multiple human malignancies in immunocompromised individuals. KSHV establishes a lifelong latency in the infected host, during which only a limited number of viral genes are expressed. However, a fraction of latently infected cells undergo spontaneous reactivation to produce virions that infect the surrounding cells. These newly infected cells are primed early to retain the incoming viral genome and induce cell growth. KSHV transcribes a variety of lytic proteins duringde novoinfections that modulate various cellular pathways to establish the latent infection. Interestingly, a large number of viral proteins and RNA are encapsidated in the infectious virions and transduced into the infected cells during ade novoinfection. This study determined the kinetics of the viral gene expression duringde novoKSHV infections and the functional role of the incoming viral transcripts in establishing latency.

Sign in / Sign up

Export Citation Format

Share Document