p130Cas Scaffolds the Signalosome To Direct Adaptor-Effector Cross Talk during Kaposi's Sarcoma-Associated Herpesvirus Trafficking in Human Microvascular Dermal Endothelial Cells

Abstract

Kaposi's sarcoma-associated herpesvirus (KSHV) interacts with cell surface receptors, such as heparan sulfate, integrins (␣3␤1, ␣V␤3, and ␣V␤5), and EphrinA2 (EphA2), and activates focal adhesion kinase (FAK), Src, phosphoinositol 3-kinase (PI3-K), c-Cbl, and RhoA GTPase signal molecules early during lipid raft (LR)-dependent productive macropinocytic entry into human dermal microvascular endothelial cells. Our recent studies have identified CIB1 as a signal amplifier facilitating EphA2 phosphorylation and subsequent cytoskeletal cross talk during KSHV macropinocytosis. Although CIB1 lacks an enzymatic activity and traditional adaptor domain or known interacting sequence, it associated with the KSHV entry signal complex and the CIB1-KSHV association was sustained over 30 min postinfection. To identify factors scaffolding the EphA2-CIB1 signal axis, the role of major cellular scaffold protein p130Cas (Crk-associated substrate of Src) was investigated. Inhibitor and small interfering RNA (siRNA) studies demonstrated that KSHV induced p130Cas in an EphA2-, CIB1-, and Src-dependent manner. p130Cas and Crk were associated with KSHV, LRs, EphA2, and CIB1 early during infection. Live-cell microscopy and biochemical studies demonstrated that p130Cas knockdown did not affect KSHV entry but significantly reduced productive nuclear trafficking of viral DNA and routed KSHV to lysosomal degradation. p130Cas aided in scaffolding adaptor Crk to downstream guanine nucleotide exchange factor phospho-C3G possibly to coordinate GTPase signaling during KSHV trafficking. Collectively, these studies demonstrate that p130Cas acts as a bridging molecule between the KSHV-induced entry signal complex and the downstream trafficking signalosome in endothelial cells and suggest that simultaneous targeting of KSHV entry receptors with p130Cas would be an attractive potential avenue for therapeutic intervention in KSHV infection. IMPORTANCEEukaryotic cell adaptor molecules, without any intrinsic enzymatic activity, are well known to allow a great diversity of specific and coordinated protein-protein interactions imparting signal amplification to different networks for physiological and pathological signaling. They are involved in integrating signals from growth factors, extracellular matrix molecules, bacterial pathogens, and apoptotic cells. The present study identifies human microvascular dermal endothelial (HMVEC-d) cellular scaffold protein p130Cas (Crk-associated substrate) as a platform to promote Kaposi's sarcoma-associated herpesvirus (KSHV) trafficking. Early during KSHV de novo infection, p130Cas associates with lipid rafts and scaffolds EphrinA2 (EphA2)-associated critical adaptor members to downstream effector molecules, promoting successful nuclear delivery of the KSHV genome. Hence, simultaneous targeting of the receptor EphA2 and scaffolding action of p130Cas can potentially uncouple the signal cross talk of the KSHV entry-associated upstream signal complex from the immediate downstream trafficking-associated signalosom...

KSHV Entry and Trafficking in Target Cells—Hijacking of Cell Signal Pathways, Actin and Membrane Dynamics

“…During adeno virus infection, αVβ5 integrin-PI3-K-p130cas has been shown to facilitate viral endocytosis [97]. Studies demonstrate that p130Cas plays a critical role during KSHV infection in HMVEC-d cells [65]. KSHV integrin α3β1 and αVβ3 interaction induced signal molecules promote the recruitment of CIB1, p130Cas, and Crk molecules to the integrin-KSHV interacting sites on the NLR regions of the plasma membrane.…”

“…Virus interaction with EphA2R in the LR region induces the association of EphA2R with CIB1 resulting in the phosphorylation of p130Cas, assembling of the EphA2R-CIB1-c-Cbl-Crk signalosome which in turn activates the GEF-C3G (guanine nucleotide exchange factor phospho-C3G) molecule which probably directs the GTPase signaling to accelerate the KSHV containing macropinosome trafficking (Figure 1). Live cell imaging of KSHV infection of control and p130Cas knockdown HMVEC-d cells show that in the absence of p130Cas, KSHV is directed toward lysosomal degradation and thus demonstrating the critical role of p130Cas in KSHV infection [65]. …”

ESCRT-0 Component Hrs Promotes Macropinocytosis of Kaposi's Sarcoma-Associated Herpesvirus in Human Dermal Microvascular Endothelial Cells

“…Proximity ligation assay (PLA) was performed using the Duolink in situ detection kit (Sigma) as per the manufacturer's instructions (33,34). Briefly, uninfected and infected cells were fixed with 4% paraformaldehyde solution for 15 min, permeabilized with Triton X-100 for 5 min, washed with phosphate-buffered saline (PBS), and incubated for 1 h with the Duolink blocking buffer.…”

A Kaposi's Sarcoma-Associated Herpesvirus Infection Mechanism Is Independent of Integrins α3β1, αVβ3, and αVβ5

“…However, in these two well-characterized infection models, the interaction between KSHV gB and the canonical integrin receptors initiates a signaling cascade of focal adhesion kinase (FAK), Src, and phosphatidylinositol 3-kinase (PI-3K) (29-31; reviewed in reference 4). KSHV gH/gL binds EphA2, which amplifies this cascade and coordinates endocytosis effectors together with c-Cbl and myosin IIA (32)(33)(34)(35)(36)(37). Still, there are important differences in the entry mechanisms used during infection of HFF and primary microvascular endothelial cells, such as the form of endocytosis used to ultimately internalize the virion, hinting that KSHV initiates different entry processes in different types of cells while using the same receptors.…”