Abstract
KSHV envelope glycoproteins interact with cell surface heparan sulfate and integrins, and activate FAK, Src, PI3-K, c-Cbl, and Rho-GTPase signal molecules in human microvascular dermal endothelial (HMVEC-d) cells. c-Cbl mediates the translocation of virus bound α3β1 and αVβ3 integrins into lipid rafts (LRs), where KSHV interacts and activates EphrinA2 (EphA2). EphA2 associates with c-Cbl-myosin IIA and augmented KSHV-induced Src and PI3-K signals in LRs, leading to bleb formation and macropinocytosis of KSHV. To identify the factor(s) coordinating the EphA2-signal complex, the role of CIB1 (calcium and integrin binding protein-1) associated with integrin signaling was analyzed. CIB1 knockdown did not affect KSHV binding to HMVEC-d cells but significantly reduced its entry and gene expression. In contrast, CIB1 overexpression increased KSHV entry in 293 cells. Single virus particle infection and trafficking during HMVEC-d cell entry was examined by utilizing DiI (envelope) and BrdU (viral DNA) labeled virus. CIB1 was associated with KSHV in membrane blebs and in Rab5 positive macropinocytic vesicles. CIB1 knockdown abrogated virus induced blebs, macropinocytosis and virus association with the Rab5 macropinosome. Infection increased the association of CIB1 with LRs, and CIB1 was associated with EphA2 and KSHV entry associated signal molecules such as Src, PI3-K, and c-Cbl. CIB1 knockdown significantly reduced the infection induced EphA2, Src and Erk1/2 activation. Mass spectrometry revealed the simultaneous association of CIB1 and EphA2 with the actin cytoskeleton modulating myosin IIA and alpha-actinin 4 molecules, and CIB1 knockdown reduced EphA2's association with myosin IIA and alpha-actinin 4. Collectively, these studies revealed for the first time that CIB1 plays a role in virus entry and macropinocytosis, and suggested that KSHV utilizes CIB1 as one of the key molecule(s) to coordinate and sustain the EphA2 mediated signaling involved in its entry, and CIB1 is an attractive therapeutic target to block KSHV infection.
Highlights
Kaposi’s sarcoma-associated herpes virus or human herpes virus 8 (HHV-8), a member of the lymphotrophic (c2) herpesvirus subfamily, is etiologically linked to endothelial cell neoplasm Kaposi’s sarcoma (KS), and B-cell neoplasms primary effusion lymphoma (PEL) or body cavity based B-cell lymphoma (BCBL), and multicentric Castleman’s disease (MCD) [1,2,3]
The de novo KSHV infection of endothelial HMVEC-d cells is initiated by its attachment to cell surface integrins, activation of cellular signals, and interaction with the receptor tyrosine kinase EphrinA2
This results in plasma membrane protrusions in the lipid raft regions that engulf and internalize the virus, a process known as macropinocytosis
Summary
Kaposi’s sarcoma-associated herpes virus or human herpes virus 8 (HHV-8), a member of the lymphotrophic (c2) herpesvirus subfamily, is etiologically linked to endothelial cell neoplasm Kaposi’s sarcoma (KS), and B-cell neoplasms primary effusion lymphoma (PEL) or body cavity based B-cell lymphoma (BCBL), and multicentric Castleman’s disease (MCD) [1,2,3]. Other downstream molecules activated by KSHV such as PKC-f, MEK, ERK1/2, and NFkB are essential for the initiation of viral and host gene expression early during infection [18,19]. These studies revealed a novel paradigm that by interacting with integrins and a family of functionally related molecules at the cell surfaces early during infection, KSHV utilizes ligand mimicry as an opportunistic mechanism to subvert host signal molecules for its entry and successful infection. Further understanding of signal induction and host cell molecules modulated by KSHV are essential to develop
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