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Kaposi's sarcoma-associated herpesvirus K3 and K5 proteins block distinct steps in transendothelial migration of effector memory CD4+ T cells by targeting different endothelial proteins.

ABSTRACT: ORFK3 (K3) and ORFK5 (K5) are Kaposi's sarcoma-associated herpesvirus-encoded E3 ubiquitin ligases that differentially reduce surface expression of various proteins in infected cells. In this study, we describe their effects on human dermal microvascular endothelial cells (ECs), a natural target of Kaposi's sarcoma-associated herpesvirus infection. TNF-treated human dermal microvascular ECs transduced to express K5 show reduced capacity to capture effector memory (EM) CD4+ T cells under conditions of venular shear stress. K5 but not K3 transduction significantly reduces ICAM-1 expression and the inhibition of T cell capture was phenocopied by small interfering RNA knockdown of ICAM-1 and by anti-ICAM-1 Ab blocking. Cotransduction with an ICAM-1 truncated construct not subject to K5 ubiquitylation restored EM CD4+ T cell capture. K3 transductants effectively capture EM CD4+ T cells, but fail to support their transendothelial migration (TEM) in response to TCR engagement by superantigen presented by the ECs, leaving intact chemokine-dependent TEM. K3 but not K5 transduction significantly reduces PECAM-1 expression, and the effect on TCR-induced TEM is phenocopied by small interfering RNA knockdown of PECAM-1 and by anti-PECAM-1 Ab blocking. TCR-dependent TEM was restored in K3 transductants cotransduced to express a mutant of PECAM-1 not subject to K3-induced ubiquitylation. EM CD4+ T cells lack any known PECAM-1 counter receptor, but heterophilic engagement of PECAM-1 can involve glycosaminoglycans. In addition, TCR-induced TEM, but not chemokine-induced TEM, appears to involve a heparan- or chondroitin-like molecule on T cells. These results both identify specific roles of K5 and K3 in immune evasion and further differentiate the processes of inflammatory chemokine- versus TCR-dependent recruitment of human EM CD4+ T cells.

SUBMITTER: Manes TD

PROVIDER: S-EPMC2877909 | biostudies-literature | 2010 May

REPOSITORIES: biostudies-literature

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Kaposi's sarcoma-associated herpesvirus K3 and K5 proteins block distinct steps in transendothelial migration of effector memory CD4+ T cells by targeting different endothelial proteins.

Manes Thomas D TD Hoer Simon S Muller William A WA Lehner Paul J PJ Pober Jordan S JS

Journal of immunology (Baltimore, Md. : 1950) 20100331 9

ORFK3 (K3) and ORFK5 (K5) are Kaposi's sarcoma-associated herpesvirus-encoded E3 ubiquitin ligases that differentially reduce surface expression of various proteins in infected cells. In this study, we describe their effects on human dermal microvascular endothelial cells (ECs), a natural target of Kaposi's sarcoma-associated herpesvirus infection. TNF-treated human dermal microvascular ECs transduced to express K5 show reduced capacity to capture effector memory (EM) CD4+ T cells under conditio ...[more]

PMID: 20357254

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Project description:The downregulation of immune synapse components such as major histocompatibility complex class I (MHC-I) and ICAM-1 is a common viral immune evasion strategy that protects infected cells from targeted elimination by cytolytic effector functions of the immune system. Kaposi's sarcoma-associated herpesvirus (KSHV) encodes two membrane-bound ubiquitin E3 ligases, called K3 and K5, which share the ability to induce internalization and degradation of MHC-I molecules. Although individual functions of K3 and K5 outside the viral genome are well characterized, their roles during the KSHV life cycle are still unclear. In this study, we individually introduced the amino acid-coding sequences of K3 or K5 into a ?K3 ?K5 recombinant virus, at either original or interchanged genomic positions. Recombinants harboring coding sequences within the K5 locus showed higher K3 and K5 protein expression levels and more rapid surface receptor downregulation than cognate recombinants in which coding sequences were introduced into the K3 locus. To identify infected cells undergoing K3-mediated downregulation of MHC-I, we employed a novel reporter virus, called red-green-blue-BAC16 (RGB-BAC16), which was engineered to harbor three fluorescent protein expression cassettes: EF1?-monomeric red fluorescent protein 1 (mRFP1), polyadenylated nuclear RNA promoter (pPAN)-enhanced green fluorescent protein (EGFP), and pK8.1-monomeric blue fluorescent protein (tagBFP), marking latent, immediate early, and late viral gene expression, respectively. Analysis of RGB-derived K3 and K5 deletion mutants showed that while the K5-mediated downregulation of MHC-I was concomitant with pPAN induction, the reduction of MHC-I surface expression by K3 was evident in cells that were enriched for pPAN-driven EGFP(high) and pK8.1-driven blue fluorescent protein-positive (BFP(+)) populations. These data support the notion that immunoreceptor downregulation occurs by a sequential process wherein K5 is critical during the immediately early phase and K3 plays a significant role during later stages.Although the roles of K3 and K5 outside the viral genome are well characterized, the function of these proteins in the context of the KSHV life cycle has remained unclear, particularly in the case of K3. This study examined the relative contributions of K3 and K5 to the downregulation of MHC-I during the lytic replication of KSHV. We show that while K5 acts immediately upon entry into the lytic phase, K3-mediated downregulation of MHC-I was evident during later stages of lytic replication. The identification of distinctly timed K3 and K5 activities significantly advances our understanding of KSHV-mediated immune evasion. Crucial to this study was the development of a novel recombinant KSHV, called RGB-BAC16, which facilitated the delineation of stage-specific phenotypes.

Project description:Primary effusion lymphoma (PEL) is a lymphogenic disorder associated with Kaposi's sarcoma-associated herpesvirus (KSHV) infection. Key to the survival and proliferation of PEL is the canonical NF-?B pathway, which becomes constitutively activated following overexpression of the viral oncoprotein KSHV vFLIP (ks-vFLIP). This arises from its capacity to form a complex with the modulatory subunit of the I?B kinase (IKK) kinase, IKK? (or NEMO), resulting in the overproduction of proteins that promote cellular survival and prevent apoptosis, both of which are important drivers of tumorigenesis. Using a combination of cell-based and biophysical assays together with structural techniques, we showed that the observed resistance to cell death is largely independent of autophagy or major death receptor signaling pathways and demonstrated that direct targeting of the ks-vFLIP-IKK? interaction both in cells and in vitro can be achieved using IKK?-mimetic peptides. Our results further reveal that these peptides not only induce cell killing but also potently sensitize PEL to the proapoptotic agents tumor necrosis factor alpha and etoposide and are the first to confirm ks-vFLIP as a tractable target for the treatment of PEL and related disorders.IMPORTANCE KSHV vFLIP (ks-vFLIP) has been shown to have a crucial role in cellular transformation, in which it is vital for the survival and proliferation of primary effusion lymphoma (PEL), an aggressive malignancy associated with infection that is resistant to the majority of chemotherapeutic drugs. It operates via subversion of the canonical NF-?B pathway, which requires a physical interaction between ks-vFLIP and the IKK kinase modulatory subunit IKK?. While this interaction has been directly linked to protection against apoptosis, it is unclear whether the suppression of other cell death pathways implicated in ks-vFLIP pathogenesis is an additional contributor. We demonstrate that the interaction between ks-vFLIP and IKK? is pivotal in conferring resistance to apoptosis. Additionally, we show that the ks-vFLIP-IKK? complex can be disrupted using peptides leading to direct killing and the sensitization of PEL cells to proapoptotic agents. Our studies thus provide a framework for future therapeutic interventions.

Dataset Information

Kaposi's sarcoma-associated herpesvirus K3 and K5 proteins block distinct steps in transendothelial migration of effector memory CD4+ T cells by targeting different endothelial proteins.

Publications

Kaposi's sarcoma-associated herpesvirus K3 and K5 proteins block distinct steps in transendothelial migration of effector memory CD4+ T cells by targeting different endothelial proteins.

Similar Datasets

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