Kaposi's Sarcoma-associated Herpesvirus Activation of Vascular Endothelial Growth Factor Receptor 3 Alters Endothelial Function and Enhances Infection

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140

Kaposi's sarcoma-associated herpesvirus (KSHV) interacts with cell surface heparan sulfate (HS) and ␣3␤1 integrin during the early stages of infection of human dermal microvascular endothelial cells (HMVEC-d) and human foreskin fibroblasts (HFF), and these interactions are followed by virus entry overlapping with the induction of preexisting host cell signal pathways. KSHV also utilizes the amino acid transporter protein xCT for infection of adherent cells, and the xCT molecule is part of the cell surface heterodimeric membrane glycoprotein CD98 (4F2 antigen) complex known to interact with ␣3␤1 and ␣V␤3 integrins. KSHV gB mediates adhesion of HMVEC-d, CV-1, and HT-1080 cells and HFF via its RGD sequence. Anti-␣V and -␤1 integrin antibodies inhibited the cell adhesion mediated by KSHV-gB. Variable levels of neutralization of HMVEC-d and HFF infection were observed with antibodies against ␣V␤3 and ␣V␤5 integrins. Similarly, variable levels of inhibition of virus entry into adherent HMVEC-d, 293 and Vero cells, and HFF was observed by preincubating virus with soluble ␣3␤1, ␣V␤3, and ␣V␤5 integrins, and cumulative inhibition was observed with a combination of integrins. We were unable to infect HT1080 cells. Virus binding and DNA internalization studies suggest that ␣V␤3 and ␣V␤5 integrins also play roles in KSHV entry. We observed time-dependent temporal KSHV interactions with HMVEC-d integrins and CD98/xCT with three different patterns of association and dissociation. Integrin ␣V␤5 interaction with CD98/xCT predominantly occurred by 1 min postinfection (p.i.) and dissociated at 10 min p.i., whereas ␣3␤1-CD98/xCT interaction was maximal at 10 min p.i. and dissociated at 30 min p.i., and ␣V␤3-CD98/xCT interaction was maximal at 10 min p.i. and remained at the observed 30 min p.i. Fluorescence microscopy also showed a similar time-dependent interaction of ␣V␤5-CD98. Confocal-microscopy studies confirmed the association of CD98/xCT with ␣3␤1 and KSHV. Preincubation of KSHV with soluble heparin and ␣3␤1 significantly inhibited this association, suggesting that the first contact with HS and integrin is an essential element in subsequent CD98-xCT interactions. Anti-CD98 and xCT antibodies did not block virus binding and entry and nuclear delivery of viral DNA; however, viral-gene expression was significantly inhibited, suggesting that CD98-xCT play roles in the post-entry stage of infection, possibly in mediating signal cascades essential for viral-gene expression. Together, these studies suggest that KSHV interacts with functionally related integrins (␣V␤3, ␣3␤1, and ␣V␤5) and CD98/xCT molecules in a temporal fashion to form a multimolecular complex during the early stages of endothelial cell infection, probably mediating multiple roles in entry, signal transduction, and viral-gene expression.The ␥-2 herpesvirus Kaposi's sarcoma (KS)-associated herpesvirus (KSHV), or human herpesvirus 8, is etiologically associated with KS; primary effusion lymphoma, or body cavitybased B-cell lymphoma (BCBL); and multicentric Castleman...

Section: Discussionsupporting

confidence: 52%

Kaposi's Sarcoma-Associated Herpesvirus Forms a Multimolecular Complex of Integrins (αVβ5, αVβ3, and α3β1) and CD98-xCT during Infection of Human Dermal Microvascular Endothelial Cells, and CD98-xCT Is Essential for the Postentry Stage of Infection

Veettil

Sadagopan

Sharma-Walia

et al. 2008

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140

“…To support these transcriptional studies, ELISAs were performed to determine the levels of VEGF, a downstream target of HIF-1␣, which was previously demonstrated to be induced by KSHV infection (21,47). The data obtained from cells transiently expressing LANA showed that the levels of VEGF produced were also significantly increased in HEK293T cells transfected with LANA compared to only HIF-1␣ expression (Fig.…”

Section: Resultsmentioning

confidence: 99%

A Potential α-Helix Motif in the Amino Terminus of LANA Encoded by Kaposi's Sarcoma-Associated Herpesvirus Is Critical for Nuclear Accumulation of HIF-1α in Normoxia

Cai

Murakami

et al. 2007

Hypoxia-inducible factor 1 (HIF-1) is a ubiquitously expressed transcriptional regulator involved in induction of numerous genes associated with angiogenesis and tumor growth. Kaposi's sarcoma, associated with increased angiogenesis, is a highly vascularized, endothelial cell-derived tumor. Previously, we have shown that the latency-associated nuclear antigen (LANA) encoded by Kaposi's sarcoma-associated herpesvirus (KSHV) targets the HIF-1␣ suppressors von Hippel-Lindau protein and p53 for degradation via its suppressor of cytokine signaling-box motif, which recruits the EC 5 S ubiquitin complex. Here we further show that HIF-1␣ was aberrantly accumulated in KSHV latently infected primary effusion lymphoma (PEL) cells, as well as HEK293 cells infected with KSHV, and also show that a potential ␣-helical amino-terminal domain of LANA was important for HIF-1␣ nuclear accumulation in normoxic conditions. Moreover, we have now determined that this association was dependent on the residues 46 to 89 of LANA and the oxygen-dependent degradation domain of HIF-1␣. Introduction of specific small interfering RNA against LANA into PEL cells also resulted in a diminished nuclear accumulation of HIF-1␣. Therefore, these data show that LANA can function not only as an inhibitor of HIF-1␣ suppressor proteins but can also induce nuclear accumulation of HIF-1␣ during KSHV latent infection.

“…Cells express- ing VEGFR-3 are slightly more infectible by KSHV, suggesting that the upregulation of lymphatic markers could enhance viral infection or reinfection, which may be required for the maintenance of KSHV latency (58,63). However, VEGFR-3 is neither necessary nor sufficient for viral entry.…”

Section: Discussionmentioning

confidence: 99%

Activation of Akt through gp130 Receptor Signaling Is Required for Kaposi's Sarcoma-Associated Herpesvirus-Induced Lymphatic Reprogramming of Endothelial Cells

Morris

Punjabi

Lagunoff

2008

Kaposi's sarcoma (KS) is the most common tumor of AIDS patients worldwide. KS-associated herpesvirus (KSHV) is the infectious cause of this highly vascularized skin tumor. The main cell type found within a KS lesion, the spindle cell, is latently infected with KSHV and has markers of both blood and lymphatic endothelial cells. During development, lymphatic endothelial cells differentiate from preexisting blood endothelial cells. Interestingly, KSHV infection of blood endothelial cells induces lymphatic endothelial cell differentiation. Here, we show that KSHV gene expression is necessary to maintain the expression of the lymphatic markers vascular endothelial growth factor receptor 3 (VEGFR-3) and podoplanin. KSHV infection activates many cell signaling pathways in endothelial cells and persistently activates STAT3 through the gp130 receptor, the common receptor of the interleukin 6 family of cytokines. We find that KSHV infection also activates the phosphatidylinositol 3-OH-kinase (PI3K)/Akt cell signaling pathway in latently infected endothelial cells and that gp130 receptor signaling is necessary for Akt activation. Using both pharmacological inhibitors and small interfering RNA knockdown, we show that the gp130 receptor-mediated activation of both the JAK2/STAT3 and PI3K/Akt cell signaling pathways is necessary for KSHV-induced lymphatic reprogramming of endothelial cells. The induction of the lymphatic endothelial cell-specific transcription factor Prox1 is also involved in KSHV-induced lymphatic reprogramming. The activation of gp130 receptor signaling is a novel mechanism for the differentiation of blood endothelial cells into lymphatic endothelial cells and may be relevant to the developmental or pathological differentiation of lymphatic endothelial cells as well as to KSHV pathogenesis.