The Inflammatory Kinase MAP4K4 Promotes Reactivation of Kaposi's Sarcoma Herpesvirus and Enhances the Invasiveness of Infected Endothelial Cells

Kaposi's sarcoma (KS) is a mesenchymal tumour, which is caused by Kaposi's sarcoma herpesvirus (KSHV) and develops under inflammatory conditions. KSHV-infected endothelial spindle cells, the neoplastic cells in KS, show increased invasiveness, attributed to the elevated expression of metalloproteinases (MMPs) and cyclooxygenase-2 (COX-2). The majority of these spindle cells harbour latent KSHV genomes, while a minority undergoes lytic reactivation with subsequent production of new virions and viral or cellular chemo- and cytokines, which may promote tumour invasion and dissemination. In order to better understand KSHV pathogenesis, we investigated cellular mechanisms underlying the lytic reactivation of KSHV. Using a combination of small molecule library screening and siRNA silencing we found a STE20 kinase family member, MAP4K4, to be involved in KSHV reactivation from latency and to contribute to the invasive phenotype of KSHV-infected endothelial cells by regulating COX-2, MMP-7, and MMP-13 expression. This kinase is also highly expressed in KS spindle cells in vivo. These findings suggest that MAP4K4, a known mediator of inflammation, is involved in KS aetiology by regulating KSHV lytic reactivation, expression of MMPs and COX-2, and, thereby modulating invasiveness of KSHV-infected endothelial cells. © 2013 Haas et al

The ubiquitin-specific protease USP7 Modulates the replication of kaposi's sarcoma-associated herpesvirus latent episomal DNA

Kaposi's sarcoma herpesvirus (KSHV) belongs to the gamma-2 Herpesviridae and is associated with three neoplastic disorders: Kaposi's sarcoma (KS), primary effusion lymphoma (PEL), and multicentric Castleman's disease (MCD). The viral latency-associated nuclear antigen 1 (LANA) is expressed in all latently KSHV-infected cells and is involved in viral latent replication and maintenance of the viral genome. We show that LANA interacts with the ubiquitin-specific protease USP7 through its N-terminal TRAF (tumor necrosis factor [TNF] receptor-associated factor) domain. This interaction involves a short sequence (amino acids [aa] 971 to 986) within the C-terminal domain of LANA with strong similarities to the USP7 binding site of the Epstein-Barr virus (EBV) EBNA-1 protein. A LANA mutant with a deletion of the identified USP7 binding site showed an enhanced ability to replicate a plasmid containing the KSHV latent origin of replication but was comparable to the wild-type LANA (LANA WT) with regard to the regulation of viral and cellular promoters. Furthermore, the LANA homologues of two other gamma-2 herpesviruses, MHV68 and RRV, also recruit USP7. Our findings suggest that recruitment of USP7 to LANA could play a role in the regulation of viral latent replication. The recruitment of USP7, and its role in herpesvirus latent replication, previously described for the latent EBNA-1 protein of the gamma-1 herpesvirus (lymphocryptovirus) EBV (M. N. Holowaty et al., J. Biol. Chem. 278:29987-29994, 2003), may thereby be a conserved feature among gammaherpesvirus latent origin binding proteins.EU Integrated Project INCA (LSHC-CT-18730); DFG Priority program SPP1130; Collaborative Research Centre DFG SFB900Peer Reviewe

Kaposi Sarcoma Herpesvirus (KSHV) Latency-Associated Nuclear Antigen (LANA) recruits components of the MRN (Mre11-Rad50-NBS1) repair complex to modulate an innate immune signaling pathway and viral latency.

Kaposi Sarcoma Herpesvirus (KSHV), a γ2-herpesvirus and class 1 carcinogen, is responsible for at least three human malignancies: Kaposi Sarcoma (KS), Primary Effusion Lymphoma (PEL) and Multicentric Castleman's Disease (MCD). Its major nuclear latency protein, LANA, is indispensable for the maintenance and replication of latent viral DNA in infected cells. Although LANA is mainly a nuclear protein, cytoplasmic isoforms of LANA exist and can act as antagonists of the cytoplasmic DNA sensor, cGAS. Here, we show that cytosolic LANA also recruits members of the MRN (Mre11-Rad50-NBS1) repair complex in the cytosol and thereby inhibits their recently reported role in the sensing of cytoplasmic DNA and activation of the NF-κB pathway. Inhibition of NF-κB activation by cytoplasmic LANA is accompanied by increased lytic replication in KSHV-infected cells, suggesting that MRN-dependent NF-κB activation contributes to KSHV latency. Cytoplasmic LANA may therefore support the activation of KSHV lytic replication in part by counteracting the activation of NF-κB in response to cytoplasmic DNA. This would complement the recently described role of cytoplasmic LANA in blocking an interferon response triggered by cGAS and thereby promoting lytic reactivation. Our findings highlight a second point at which cytoplasmic LANA interferes with the innate immune response, as well as the importance of the recently discovered role of cytoplasmic MRN complex members as innate sensors of cytoplasmic DNA for the control of KSHV replication

An endothelial cell line infected by Kaposi's sarcoma-associated herpes virus (KSHV) allows the investigation of Kaposi's sarcoma and the validation of novel viral inhibitors in vitro and in vivo.

Kaposi's sarcoma-associated herpesvirus (KSHV) is the etiological agent of Kaposi's sarcoma (KS), a tumor of endothelial origin predominantly affecting immunosuppressed individuals. Up to date, vaccines and targeted therapies are not available. Screening and identification of anti-viral compounds are compromised by the lack of scalable cell culture systems reflecting properties of virus-transformed cells in patients. Further, the strict specificity of the virus for humans limits the development of in vivo models. In this study, we exploited a conditionally immortalized human endothelial cell line for establishment of in vitro 2D and 3D KSHV latency models and the generation of KS-like xenograft tumors in mice. Importantly, the invasive properties and tumor formation could be completely reverted by purging KSHV from the cells, confirming that tumor formation is dependent on the continued presence of KSHV, rather than being a consequence of irreversible transformation of the infected cells. Upon testing a library of 260 natural metabolites, we selected the compounds that induced viral loss or reduced the invasiveness of infected cells in 2D and 3D endothelial cell culture systems. The efficacy of selected compounds against KSHV-induced tumor formation was verified in the xenograft model. Together, this study shows that the combined use of anti-viral and anti-tumor assays based on the same cell line is predictive for tumor reduction in vivo and therefore allows faithful selection of novel drug candidates against Kaposi's sarcoma. KEY MESSAGES: Novel 2D, 3D, and xenograft mouse models mimic the consequences of KSHV infection. KSHV-induced tumorigenesis can be reverted upon purging the cells from the virus. A 3D invasiveness assay is predictive for tumor reduction in vivo. Chondramid B, epothilone B, and pretubulysin D diminish KS-like lesions in vivo

KSHV LANA recruits MRN (Mre11-Rad50-NBS1) complex.

<p>(<b>A</b>) Co-immunoprecipitation of endogenous LANA and MRN proteins in BC3 cells. Cells were lysed using TBS-T buffer and the cell lysate was incubated with benzonase. After centrifugation, supernatant was incubated overnight with anti-LANA or IgG-control beads. The precipitated complexes were analyzed for the presence of endogenous Rad50, Mre11 and NBS1 by SDS-PAGE and immunoblotting. For the input, see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006335#sec008" target="_blank">Materials and methods</a>. (<b>B</b>) Co-immunoprecipitation of endogenous LANA and Rad50 in BC3 cells. Co-immunoprecipitation of endogenous Rad50 was performed and analysed as in (A), but with anti-Rad50-antibody-coated-beads (left) or anti-LANA coated beads (right). The arrowhead indicates the smaller LANA forms co-immunoprecipitating with Rad50 (see text). (<b>C</b>) Schematic representation of LANA domain structure. NLS: Nuclear Localization Signal; TR: KSHV Terminal Repeats. (<b>D</b>) Pull-down assay with GST-fused LANA-C (aa 931–1162) and LANA-N (aa 1–312) proteins and HEK293T cell lysates. HEK293T were lysed with TBS-T buffer and incubated 4 hours with GST-fused proteins or GST alone, as negative control. <i>Top</i>: immunoblot for endogenous Rad50, Mre11 and NBS1 bound to GST-fused LANA fragments. <i>Bottom</i>: Ponceau staining to detect GST-fused proteins. (M) for marker.</p

LANA Δ161 modulates the activation of NF-kB cascade triggered by cytosolic DNA.

<p>HeLa.CNX cells were transfected with the LANA Δ161 plasmid (or empty vector, EV) for about 48 hours. Cells were then stimulated with ISD (4μg/well) using Lipofectamine2000 following the manufacturer‘s instructions for 6 hours. Afterwards cells were lysed with TBS-T buffer and phosphorylation levels of p65 were analyzed by immunoblotting.</p

Inhibition of canonical NF-κB cascade and KSHV lytic reactivation upon LANA Δ161 overexpression.

<p>(<b>A-B</b>) HeLa.CNX.rKSHV and the parental HeLa.CNX cells were transfected with LANA Δ161 plasmid or empty vector for 48 hours and (<b>B</b>) treated with 5% RTA (vol/vol, see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006335#sec008" target="_blank">Materials and methods</a>) for about 24 hours. Cells were lysed with TBS-T buffer and lysates resolved by immunoblotting. Phospho-p65 levels were digitally quantified (see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006335#sec008" target="_blank">Materials and methods</a>). (<b>C</b>) HEK293 cells were transfected with the plasmid expressing full-length LANA, the truncated LANA isoforms or the empty vector (2 μg/well), together with an NF-κB reporter vector (200 ng/well). The luciferase activity was measured 48 hours later in duplicates and the statistical analysis was performed with two-tailed student’s t-test. Statistical significance of the difference between control (pcDNA3.1) and LANA (FL or Δ161 or Δ282) transfected samples is shown: (***) for p<0.005 and (ns) for not significant. (<b>D</b>) HeLa.CNX cells were transfected with the plasmid expressing vFLIP or the empty vector (1μg) and additionally with the plasmid expressing full-length LANA (1μg) or the truncated isoform (Δ161, 0.5–1μg) or the empty vector (2μg of plasmid DNA in total per condition), together with NF-κB reporter vector (200 ng/well). The luciferase activity was measured 48 hours later in duplicates and the statistical analysis was performed with two-tailed student’s t-test. Statistical significance of the difference between vFLIP alone and vFLIP-LANA (FL or Δ161) transfected samples is shown: (*) for p<0,05 and (ns) for not significant.</p

Model of LANA antagonizing cytoplasmic DNA sensors.

<p>Cytoplasmic KSHV LANA isoforms recruit and antagonize cellular DNA sensor proteins cGAS as well as the Rad50-Mre11-CARD9 complex to inhibit innate immunity responses (IFN-β and NF-κB) and support KSHV lytic reactivation from latency. During KSHV lytic reactivation, free viral DNA in the cytosol is detected by host DNA sensors, such as cGAS and the Rad50/Mre11/CARD9 complex. The cGAS-STING cascade leads to IFN-β production, whereas the Rad50/Mre11/CARD9 complex is responsible for NF-κB cascade activation. A cross-talk between these two pathways may also be possible as indicated by the dashed arrows (Fig 6). Triggering of the Rad50/Mre11/CARD9 complex leads to the activation and nuclear accumulation of NF-κB p65 and the subsequent production of chemokines and cytokines. These events would interfere with the efficient KSHV lytic replication and therefore KSHV LANA ΔN isoforms block these signalling cascades by recruiting and inhibiting the upstream activators (cGAS as well as Rad50/Mre11).</p

KSHV LANA recruits Rad50 and Mre11 in the cytosol.

<p>(<b>A</b>) Co-immunoprecipitation of endogenous LANA, Rad50, Mre11 and Brd4 in BCBL-1 cells upon cytosolic-nuclear fractionation. Cells were lysed and cytoplasmic extracts (Cyto) and nuclear extracts (Nu) were prepared using the Thermo-Fischer Nu-Cyto fractionation kit following the manufacturer‘s instructions. Cytoplasmic and nuclear fractions were incubated overnight with sepharose beads coated with LANA-antibody or IgG-control. Left (INPUT, see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006335#sec008" target="_blank">Materials and methods</a>): Brd4, Lamin A/C and GAPDH immunoblots were analyzed to confirm the efficiency of the fractionation. Right (IP): immunoprecipitation with LANA-antibody or IgG-control coated-beads and immunoblot for endogenous Rad50, Mre11 and Brd4. (<b>B</b>) Co-immunoprecipitation of endogenous Rad50 and full-length LANA or ΔN mutants (Δ161 and Δ282) transfected into HEK293 cells. HEK293 cells were transfected with LANA constructs (or empty vector). 48 hours later cells were lysed and incubated with benzonase. After centrifugation, cells were incubated overnight with beads coated with LANA-antibody. Left (INPUT): immunoblot to check the expression of LANA constructs and the endogenous Rad50 in the cells. Right (IP from LANA-antibody-coated-beads): immunoblot for endogenous Rad50 co-immunoprecipitation. (<b>C</b>) Co-immunoprecipitation of endogenous LANA and Rad50, Mre11 and CARD9 in latently KSHV-infected THP-1 cells (TrK.219 cells, see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006335#sec008" target="_blank">Materials and methods</a>). Cells were lysed and incubated with benzonase. After centrifugation, whole cell lysates were incubated overnight with beads coated with anti-LANA or IgG-control antibody. Precipitated complexes were analysed by SDS-PAGE and immunoblotting with the indicated antibodies.</p