The Kaposi's sarcoma-associated herpesvirus (KSHV) non-structural membrane protein K15 is required for viral lytic replication and may represent a therapeutic target

Abere, Bizunesh; Mamo, Tamrat M.; Hartmann, Silke; Samarina, Naira; Hage, Elias; Rückert, Jessica; Hotop, Sven-Kevin; Büsche, Guntram; Schulz, Thomas F.

doi:10.1371/journal.ppat.1006639

Cited by 24 publications

(52 citation statements)

References 104 publications

(157 reference statements)

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“…Virus production and Histodenz gradient purification. The rKSHV.219 virus was produced as previously described (69,82). Briefly, to induce the KSHV lytic cycle, BJAB-rKSHV.219 cells were inoculated at a density of 6 ϫ 10 5 cells/ml in a total volume of 800 ml culture in the presence of 3 g/ml of anti-human IgM antibody (catalog no.…”

Section: Methodsmentioning

confidence: 99%

Kaposi’s Sarcoma-Associated Herpesvirus-Encoded circRNAs Are Expressed in Infected Tumor Tissues and Are Incorporated into Virions

Abere

Zhou

et al. 2020

mBio

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Kaposi's sarcoma-associated herpesvirus (KSHV) has recently been found to generate circular RNAs (circRNAs) from several KSHV genes, most abundantly from K10 (viral interferon regulatory factor 4 [vIRF4]), K7.3, and polyadenylated nuclear (PAN) RNA. To define expression of these circRNAs, KSHV-infected cell lines, patient tissues, and purified virions were examined. KSHV circRNA expression was universally detected in tests of six primary effusion lymphoma (PEL) cell lines but ranged from low-level expression in BC-1 cells dually infected with tightly latent KSHV and Epstein-Barr virus to abundant expression in KSHV-only BCBL-1 cells with spontaneous virus production. Generally, the PAN/K7.3 locus broadly and bidirectionally generated circRNA levels that paralleled the corresponding linear RNA levels. However, RNA corresponding to a particular KSHV circularization site (circ-vIRF4) was minimally induced, despite linear vIRF4 RNA being activated by virus induction. In situ hybridization showed abundant circ-vIRF4 in noninduced PEL cells. All three KSHV circRNAs were isolated as nuclease-protected forms from gradient-purified virions collected from BrK.219 cells infected with a KSHV molecular clone. For circ-vIRF4, the fully processed form that is exported to the cytoplasm was incorporated into virus particles but the nuclear, intron-retaining form was not. The half-life of circ-vIRF4 was twice as long as that of its linear counterpart. The KSHV circRNAs could be detected at a higher rate than their corresponding linear counterparts by in situ hybridization in archival tissues and by reverse transcription-PCR (RT-PCR) in sera stored for over 25 years. In summary, KSHV circRNAs are expressed in infectionassociated diseases, can be regulated depending on virus life cycle, and are incorporated into viral particles for preformed delivery, suggesting a potential function in early infection. IMPORTANCE KSHV has recently been found to encode circRNAs. circRNAs result from back-splicing of an upstream pre-mRNA splice donor exon-intron junction to an acceptor site, generating a covalently closed circle. This study revealed that for one KSHV region, the PAN/K7.3 locus, broadly and bidirectionally generated circRNA levels parallel corresponding linear RNA levels. Another KSHV circularization site (circ-vIRF4), however, showed expression that differed from that of the corresponding linear RNA. All KSHV circRNAs are incorporated into KSHV virions and are potentially expressed as immediate early products in newly infected cells.

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Section: Methodsmentioning

confidence: 99%

Kaposi’s Sarcoma-Associated Herpesvirus-Encoded circRNAs Are Expressed in Infected Tumor Tissues and Are Incorporated into Virions

Abere

Zhou

et al. 2020

mBio

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“…We further show that the late lytic protein K8.1 is expressed in the majority of the studied KS biopsies, suggesting that, similar to KLECs in culture, lytic protein expression is likely a common feature of KS tumours. This is supported by detection of the lytic proteins K5 and K15 in KS biopsies 37, 38 and a recent RNA-seq study on African-KS lesions where KSHV lytic transcripts were detected in the tumours analysed 15 . It is not known whether KS-SCs, like the KLEC cultures, constitutively produce and release infectious virus.…”

Section: Discussionmentioning

confidence: 64%

Oncogenic herpesvirus engages the endothelial transcription factors SOX18 and PROX1 to increase viral genome copies and virus production

Gramolelli

Elbasani

Nurminen

et al. 2019

Preprint

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27Kaposi sarcoma (KS) is a tumour of endothelial origin caused by KS herpesvirus (KSHV) infection 28 and suggested to originate from lymphatic endothelial cells (LECs). While KSHV establishes latency 29 in virtually all susceptible cell types, LECs support a spontaneous lytic gene expression program with 30 high viral genome copies and release of infectious virus. Here, we investigated the role of PROX1, 31 SOX18 and COUPTF2, drivers of lymphatic endothelial fate during embryogenesis, in this unique 32 KSHV infection program. We found that these factors were co-expressed in KS tumours with the 33 viral lytic marker K8.1, and that SOX18 and PROX1 regulate KSHV infection via two independent 34 mechanisms. SOX18 binds to the viral origins of replication and its depletion or chemical inhibition 35 significantly reduced the KSHV genome copies in LECs. PROX1 interacts with ORF50, the initiator 36 of the lytic cascade, increases lytic gene expression and virus production and its depletion reduces 37 KSHV spontaneous lytic reactivation. Upon lytic replication, PROX1 binds to the KSHV genome in 38 the promoter region of ORF50 and enhances its transactivation activity. These results demonstrate 39 the importance of two endothelial transcription factors in the regulation of the KSHV life cycle and 40 introduce SOX18 inhibition as a potential, novel therapeutic modality for KS. 41 42 43 KEYWORDS 44 Kaposi sarcoma, KSHV, herpesvirus, K8.1, transcription factors, PROX1, SOX18, lymphatic 45 endothelial cells, viral genome replication, viral reactivation. 46 47 3 MAIN 48Kaposi sarcoma (KS) is an angiogenic endothelial tumour caused by KS herpesvirus (KSHV). KS is 49 the most common cancer among AIDS patients and a frequent malignancy in Sub-Saharan Africa 1 . 50 KS presents with skin lesions that progress from patch to plaque and ultimately to nodular tumours; 51 in advanced disease visceral involvement is also seen 2,3 . The histopathological hallmark of KS is the 52 presence of KSHV-positive spindle cells (SC), the tumour cells of KS 2,3 . The cell of origin of SC has 53 been debated for two decades 3 . The prevailing hypothesis suggests lymphatic endothelial origin, 54 although blood endothelial cells or mesenchymal cells are also candidates 1,4 . In vitro, latency is the 55 default replication program in KSHV-infected cells with undetectable levels of lytic genes expressed. 56 However, KSHV infection of lymphatic, but not blood, endothelial cells (LEC and BEC) leads to a 57 unique infection program characterized by high KSHV genome copies, spontaneous lytic gene 58 expression and release of infectious virus 5-8 . 59 60 During embryonic development, LEC precursors originate from COUPTF2/SOX18 double-positive 61 BECs that physically separate from the cardinal vein to establish a primary lymphatic vascular plexus. 62 In this process, COUPTF2 and SOX18 drive the expression of PROX1 thereby orchestrating LEC 63 differentiation 9,10 . Here, we explored the role of these transcription factors (TF), instrumental for 64 establishment...

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“…By using a monoclonal antibody (mAb) to the K15 C-terminal cytoplasmic tail, pK15 protein expression has been immunohistochemically detected in latently infected PEL cells and in KSHV latency-associated nuclear antigen (LANA)-positive plasmablasts in MCD (35). Similarly, we have recently demonstrated the expression of the K15 protein in KS tumors obtained from AIDS-KS patients (19).…”

Section: Importancementioning

confidence: 89%

“…Among these are the viral G-protein-coupled receptor (vGPCR), K1, viral interleukin-6 (vIL-6), homologues of cellular CC chemokines, and K15. Cellular signaling subsequently leads to the expression of inflammatory and angiogenic molecules that are known to play an important role in KSHV lytic replication and disease progression (13)(14)(15)(16)(17)(18)(19)(20).…”

Section: Importancementioning

confidence: 99%

“…The expression of K15 mRNA and protein can be detected in latently infected (unstimulated) cells, while their expression increases upon the induction of the KSHV lytic cycle (16,17,19,21,(24)(25)(26). Consistent with its classical description as an early lytic gene, the promoter of the K15 gene can be trans-activated by regulator of transcription activation (RTA) in the PEL cell line BCBL-1 (27), and it has been shown to be expressed predominantly during the KSHV lytic cycle in PEL cell lines (28)(29)(30).…”

Section: Importancementioning

confidence: 99%

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Kaposi's Sarcoma-Associated Herpesvirus Nonstructural Membrane Protein pK15 Recruits the Class II Phosphatidylinositol 3-Kinase PI3K-C2α To Activate Productive Viral Replication

Abere

Samarina

Gramolelli

et al. 2018

J Virol

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Kaposi's sarcoma (KS)-associated herpesvirus (KSHV)/human herpesvirus 8 (HHV-8) causes the angiogenic tumor KS and two B-cell malignancies. The KSHV nonstructural membrane protein encoded by the open reading frame (ORF) K15 recruits and activates several cellular proteins, including phospholipase Cγ1 (PLCγ1), components of the NF-κB pathway, as well as members of the Src family of nonreceptor tyrosine kinases, and thereby plays an important role in the activation of angiogenic and inflammatory pathways that contribute to the pathogenesis of KS as well as KSHV productive (lytic) replication. In order to identify novel cellular components involved in the biology of pK15, we immunoprecipitated pK15 from KSHV-infected endothelial cells and identified associated proteins by label-free quantitative mass spectrometry. Cellular proteins interacting with pK15 point to previously unappreciated cellular processes, such as the endocytic pathway, that could be involved in the function of pK15. We found that the class II phosphatidylinositol 3-kinase (PI3K) PI3K-C2α, which is involved in the endocytosis of activated receptor tyrosine kinases and their signaling from intracellular organelles, interacts and colocalizes with pK15 in vesicular structures abundant in the perinuclear area. Further functional analysis revealed that PI3K-C2α contributes to the pK15-dependent phosphorylation of PLCγ1 and Erk1/2. PI3K-C2α also plays a role in KSHV lytic replication, as evidenced by the reduced expression of the viral lytic genes K-bZIP and ORF45 as well as the reduced release of infectious virus in PI3K-C2α-depleted KSHV-infected endothelial cells. Taken together, our results suggest a role of the cellular PI3K-C2α protein in the functional properties of the KSHV pK15 protein. The nonstructural membrane protein encoded by open reading frame K15 of Kaposi's sarcoma-associated herpesvirus (KSHV) (HHV8) activates several intracellular signaling pathways that contribute to the angiogenic properties of KSHV in endothelial cells and to its reactivation from latency. A detailed understanding of how pK15 activates these intracellular signaling pathways is a prerequisite for targeting these processes specifically in KSHV-infected cells. By identifying pK15-associated cellular proteins using a combination of immunoprecipitation and mass spectrometry, we provide evidence that pK15-dependent signaling may occur from intracellular vesicles and rely on the endocytotic machinery. Specifically, a class II PI3K, PI3K-C2α, is recruited by pK15 and involved in pK15-dependent intracellular signaling and viral reactivation from latency. These findings are of importance for future intervention strategies that aim to disrupt the activation of intracellular signaling by pK15 in order to antagonize KSHV productive replication and tumorigenesis.

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The Kaposi's sarcoma-associated herpesvirus (KSHV) non-structural membrane protein K15 is required for viral lytic replication and may represent a therapeutic target

Cited by 24 publications

References 104 publications

Kaposi’s Sarcoma-Associated Herpesvirus-Encoded circRNAs Are Expressed in Infected Tumor Tissues and Are Incorporated into Virions

Kaposi’s Sarcoma-Associated Herpesvirus-Encoded circRNAs Are Expressed in Infected Tumor Tissues and Are Incorporated into Virions

Oncogenic herpesvirus engages the endothelial transcription factors SOX18 and PROX1 to increase viral genome copies and virus production

Kaposi's Sarcoma-Associated Herpesvirus Nonstructural Membrane Protein pK15 Recruits the Class II Phosphatidylinositol 3-Kinase PI3K-C2α To Activate Productive Viral Replication

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