an-vitro-model-for-studying-cellular-transformation-kaposi-sarcoma

An <em>In Vitro </em>Model for Studying Cellular Transformation by Kaposi Sarcoma Herpesvirus

Kaposi sarcoma (KS) is an unusual tumor composed of proliferating spindle cells that is initiated by infection of endothelial cells (EC) with KSHV, and ...

Angiogenesis is a critical factor in the growth and dissemination of solid tumors. Indeed, tumor vasculature is abnormal and contributes to the development and spread of malignancies by creating a hostile microenvironment. The alternative SDF-1/CXCL12 receptor, CXCR7, is frequently and specifically expressed in tumor-associated vessels. In this study, we examine the role of endothelium-expressed CXCR7 in tumor vascular dysfunction by specifically examining the contribution of CXCR7 to endothelial cell (EC) proliferation. We demonstrate that CXCR7 expression is sufficient to drive post-confluent growth in EC cultures. Further, we provide a novel mechanism for CXCR7-mediated proliferation via proteasomal degradation of the tumor suppressor protein Rb. These findings identify a heretofore unappreciated role for CXCR7 in vascular dysfunction and confirm this receptor as a plausible target for anti-tumor therapy.

Aberrant proliferation in CXCR7+ endothelial cells via degradation of the retinoblastoma protein.

Persistent human cytomegalovirus (HCMV) infection has been linked to several diseases, including atherosclerosis, transplant vascular sclerosis (TVS), restenosis, and glioblastoma. We have previously shown that factors secreted from HCMV-infected cells induce angiogenesis and that this process is due, at least in part, to increased secretion of interleukin-6 (IL-6). In order to identify the HCMV gene(s) responsible for angiogenesis promotion, we constructed a large panel of replication-competent HCMV recombinants. One HCMV recombinant deleted for UL1 to UL10 was unable to induce secretion of factors necessary for angiogenesis. Fine mapping using additional HCMV recombinants identified UL7 as a viral gene required for production of angiogenic factors from HCMV-infected cells. Transient expression of pUL7 induced phosphorylation of STAT3 and ERK1/2 MAP kinases and production of proangiogenic factors, including IL-6. Addition of recombinant pUL7 to cells was sufficient for angiogenesis and was again associated with increased IL-6 expression. Analysis of the UL7 structure revealed a conserved domain similar to the immunoglobulin superfamily domain and related to the N-terminal V-like domain of carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1). Our report therefore identifies UL7 as a novel HCMV-encoded molecule that is both structurally and functionally related to cellular CEACAM1, a proangiogenic factor highly expressed during vasculogenesis.

Human cytomegalovirus-encoded pUL7 is a novel CEACAM1-like molecule responsible for promotion of angiogenesis.

The lower respiratory tract is a latency site of Epstein-Barr virus (EBV); however, its pathogenic role is poorly known, particularly in transplant patients. The aim of this study was to evaluate the prevalence and role of EBV in bronchoalveolar lavages (BAL) from transplant recipients (TR) in comparison with nontransplant (NT) patients.

Quantitative detection of Epstein-Barr virus in bronchoalveolar lavage from transplant and nontransplant patients.

Human rhinoviruses (HRV) usually cause upper airway infections. However, viral replication in the tracheobronchial tree has been disclosed, although its clinical role is poorly known. We evaluated the prevalence of HRV in 159 bronchoalveolar lavages from 88 patients and describe a lung transplant recipient with a high HRV load in association with acute rejection. HRV was detected in 22/88 patients (25.0%): 7/18 lung transplant recipients, 11/41 immunocompetent, and 4/29 immunocompromised (p = n.s.). No lung disease was significantly associated with HRV positivity. It should be recommended to include HRV in the virological diagnostic work-up of lower respiratory specimens to elucidate their role.

What role for human rhinoviruses in the lower respiratory tract?

The molecular epidemiology and tissue distribution of Human Parvovirus 4 (PARV4) and its variant PARV5 (Parvoviridae family) are poorly known. The aim of this study was to investigate the epidemiological role and prevalence of PARV4/5 by a nested-PCR on different clinical specimens, including blood samples from healthy donors, healthy and pathological skin samples, and bronchoalveolar lavages (BAL). Among blood specimens, 2/53 were positive; 3/37 and 23/105 of healthy and pathological skin specimens resulted positive, respectively, whereas no BAL was positive. PARV4/5 may be present in different healthy and pathological samples, suggesting the need for further investigating its tissue distribution.

Detection of PARV4, genotypes 1 and 2, in healthy and pathological clinical specimens.

Human cytomegalovirus (HCMV) is linked to the acceleration of vascular diseases such as atherosclerosis and transplant vasculopathy. One of the hallmarks of these diseases is angiogenesis (AG) and neovessel formation. Endothelial cells (ECs) are an integral part of AG and are sites of HCMV persistence. AG requires multiple synchronous processes that include EC proliferation, migration, and vessel stabilization. Virus-free supernatant (secretome) from HCMV-infected ECs induces AG. To identify factor(s) involved in this process, we performed a human cytokine array. Several cytokines were significantly induced in the HCMV secretomes including interleukin-6 (IL-6), granulocyte macrophage colony-stimulating factor, and IL-8/CXCL8. Using in vitro AG assays, neutralization of IL-6 significantly reduced neovessel formation. Addition of the HCMV secretome to preformed vessels extended neovessel survival, but this effect was blocked by neutralization of IL-6. In these cells, IL-6 prevented apoptosis by blocking caspase-3 and -7 activation through the induction of survivin. Neutralization of IL-6 receptor on ECs abolished the ability of HCMV secretome to increase survivin expression and activated effector caspases. Moreover, survivin shRNA expression induced rapid regression of tubule capillary networks in ECs stimulated with HCMV secretome and activated effector caspases. These observations may explain how CMV accelerates vascular disease despite limited infection in tissues.

IL-6 in human cytomegalovirus secretome promotes angiogenesis and survival of endothelial cells through the stimulation of survivin.

Human outbreaks of Ebola virus (EBOV) are a serious human health concern in Central Africa. Great apes (gorillas/chimpanzees) are an important source of EBOV transmission to humans due to increased hunting of wildlife including the 'bush-meat' trade. Cytomegalovirus (CMV) is an highly immunogenic virus that has shown recent utility as a vaccine platform. CMV-based vaccines also have the unique potential to re-infect and disseminate through target populations regardless of prior CMV immunity, which may be ideal for achieving high vaccine coverage in inaccessible populations such as great apes.

A replicating cytomegalovirus-based vaccine encoding a single Ebola virus nucleoprotein CTL epitope confers protection against Ebola virus.

Cytomegalovirus (CMV) is a highly immunogenic virus that results in a persistent, life-long infection in the host typically with no ill effects. Certain unique features of CMV, including its capacity to actively replicate in the presence of strong host CMV-specific immunity, may give CMV an advantage compared with other virus-based vaccine delivery platforms. In the present study, we tested the utility of mouse CMV (mCMV)-based vaccines expressing human prostate-specific antigen (PSA) for prostate cancer immunotherapy in double-transgenic mice expressing PSA and HLA-DRB1*1501 (DR2bxPSA F1 mice). We assessed the capacity of 2 mCMV-based vectors to induce PSA-specific CD8 T-cell responses and affect the growth of PSA-expressing Transgenic Adenocarcinoma of the Mouse Prostate tumors (TRAMP-PSA). In the absence of tumor challenge, immunization with mCMV vectors expressing either a H2-D(b)-restricted epitope PSA(65-73) (mCMV/PSA(65-73)) or the full-length gene for PSA (mCMV/PSA(FL)) induced comparable levels of CD8 T-cell responses that increased (inflated) with time. Upon challenge with TRAMP-PSA tumor cells, animals immunized with mCMV/PSA(65-73) had delay of tumor growth and increased PSA-specific CD8 T-cell responses, whereas animals immunized with mCMV/PSA(FL) showed progressive tumor growth and no increase in number of splenic PSA(65-73)-specific T cells. The data show that a prototype CMV-based prostate cancer vaccine can induce an effective antitumor immune response in a "humanized" double-transgenic mouse model. The observation that mCMV/PSA(FL) is not effective against TRAMP-PSA is consistent with our previous findings that HLA-DRB1*1501-restricted immune responses to PSA are associated with suppression of effective CD8 T-cell responses to TRAMP-PSA tumors.

A cytomegalovirus-based vaccine expressing a single tumor-specific CD8+ T-cell epitope delays tumor growth in a murine model of prostate cancer.

Ebola virus (Zaire ebolavirus; EBOV) is a highly lethal hemorrhagic disease virus that most recently was responsible for two independent 2014 outbreaks in multiple countries in Western Africa, and the Democratic Republic of the Congo, respectively. Herein, we show that a cytomegalovirus (CMV)-based vaccine provides durable protective immunity from Ebola virus following a single vaccine dose. This study has implications for human vaccination against ebolaviruses, as well as for development of a 'disseminating' vaccine to target these viruses in wild African great apes.

A cytomegalovirus-based vaccine provides long-lasting protection against lethal Ebola virus challenge after a single dose.

Kaposi sarcoma (KS) herpesvirus (KSHV) infection of endothelial cells (EC) is associated with strong induction of heme oxygenase-1 (HO-1), a stress-inducible host gene that encodes the rate-limiting enzyme responsible for heme catabolism. KS is an angioproliferative tumor characterized by the proliferation of KSHV-infected spindle cells, and HO-1 is highly expressed in such cells. HO-1 converts the pro-oxidant, proinflammatory heme molecule into metabolites with antioxidant, anti-inflammatory, and proliferative activities. Previously published work has shown that KSHV-infected EC in vitro proliferate in response to free heme in a HO-1-dependent manner, thus implicating virus-enhanced HO-1 activity in KS tumorigenesis. The present study investigated the molecular mechanisms underlying KSHV induction of HO-1 in lymphatic EC (LEC), which are the likely spindle cell precursors. In a time course analysis of KSHV-infected cells, HO-1 expression displays biphasic kinetics characterized by an early transient induction that is followed by a more sustained upregulation coincident with the establishment of viral latency. A viral microRNA miR-K12-11 deletion mutant of KSHV was found to be defective for induction of HO-1 during latency. A potential mechanism for this phenotype was provided by BACH1, a cellular HO-1 transcriptional repressor targeted by miR-K12-11. In fact, in KSHV-infected LEC, the BACH1 message level is reduced, BACH1 subcellular localization is altered, and miR-K12-11 mediates the inverse regulation of HO-1 and BACH1 during viral latency. Interestingly, the data indicate that neither miR-K12-11 nor de novo KSHV gene expression is required for the burst of HO-1 expression observed at early times postinfection, which suggests that additional virion components promote this phenotype.

Kaposi Sarcoma Herpesvirus Induces HO-1 during De Novo Infection of Endothelial Cells via Viral miRNA-Dependent and -Independent Mechanisms.

Kaposi sarcoma herpesvirus (KSHV) is the etiologic agent of Kaposi sarcoma (KS) and certain rare B cell lymphoproliferative disorders. KSHV infection of endothelial cells (EC) in vitro increases expression of the inducible host-encoded enzyme heme oxygenase-1 (HO-1), which is also strongly expressed in KS tumors. HO-1 catalyzes the rate-limiting step in the conversion of heme into iron, biliverdin and the gasotransmitter carbon monoxide (CO), all of which share anti-apoptotic, anti-inflammatory, pro-survival, and tumorigenic activities. Our previous work has shown that HO-1 expression in KSHV-infected EC is characterized by a rapid yet transient induction at early times post-infection, followed by a sustained upregulation co-incident with establishment of viral latency. These two phases of expression are independently regulated, suggesting distinct roles for HO-1 in the virus life cycle. Here, we investigated the role of HO-1 during acute infection, prior to the onset of viral gene expression. The early infection phase involves a series of events that culminate in delivery of the viral genome to the nucleus. Primary infection also leads to activation of host innate immune effectors, including the pattern recognition receptor TLR4, to induce an antiviral response. It has been shown that TLR4-deficient EC are more susceptible to KSHV infection than wild-type controls, suggesting an important inhibitory role for TLR4 in the KSHV life cycle. TLR4 signaling is in turn subject to regulation by several virus-encoded immune evasion factors. In this report we identify HO-1 as a host protein co-opted by KSHV as part of a rapid immune evasion strategy. Specifically, we show that early HO-1 induction by KSHV results in increased levels of endogenous CO, which functions as a TLR4 signaling inhibitor. In addition, we show that CO-mediated inhibition of TLR4 signaling leads to reduced expression of TLR4-induced antiviral genes, thus dampening the host antiviral response and facilitating KSHV infection. Conversely, inhibition of HO-1 activity decreases intracellular CO, enhances the host antiviral response and inhibits KSHV infection. In conclusion, this study identifies HO-1 as a novel innate immune evasion factor in the context of KSHV infection and supports HO-1 inhibition as a viable therapeutic strategy for KS.

Article	Year
Aberrant proliferation in CXCR7+ endothelial cells via degradation of the retinoblastoma protein. PloS one\| PubMed ID: 23894550	2013
Human cytomegalovirus-encoded pUL7 is a novel CEACAM1-like molecule responsible for promotion of angiogenesis. mBio\| PubMed ID: 25352622	2014
Quantitative detection of Epstein-Barr virus in bronchoalveolar lavage from transplant and nontransplant patients. Transplantation\| PubMed ID: 19034008	2008
What role for human rhinoviruses in the lower respiratory tract? The new microbiologica\| PubMed ID: 19382677	2009
Detection of PARV4, genotypes 1 and 2, in healthy and pathological clinical specimens. The new microbiologica\| PubMed ID: 19579698	2009
IL-6 in human cytomegalovirus secretome promotes angiogenesis and survival of endothelial cells through the stimulation of survivin. Blood\| PubMed ID: 20930069	2011
A replicating cytomegalovirus-based vaccine encoding a single Ebola virus nucleoprotein CTL epitope confers protection against Ebola virus. PLoS neglected tropical diseases\| PubMed ID: 21858240	2011
A cytomegalovirus-based vaccine expressing a single tumor-specific CD8+ T-cell epitope delays tumor growth in a murine model of prostate cancer. Journal of immunotherapy (Hagerstown, Md. : 1997)\| PubMed ID: 22576344	2012
A cytomegalovirus-based vaccine provides long-lasting protection against lethal Ebola virus challenge after a single dose. Vaccine\| PubMed ID: 25820063	2015
Kaposi Sarcoma Herpesvirus Induces HO-1 during De Novo Infection of Endothelial Cells via Viral miRNA-Dependent and -Independent Mechanisms. mBio\| PubMed ID: 26045540	2015
The Heme Metabolite Carbon Monoxide Facilitates KSHV Infection by Inhibiting TLR4 Signaling in Endothelial Cells. Frontiers in microbiology\| PubMed ID: 28421060	2017

Sara Botto

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