KSHV is etiologically associated with
Kaposi's sarcoma (KS), an angioproliferative endothelial cell
malignancy. Macropinocytosis is the predominant mode of in vitro entry of KSHV into its natural target cells, human dermal microvascular endothelial (HMVEC-d) cells. Although macropinocytosis is known to be a major route of entry for many viruses, the molecule(s) involved in the recruitment and integration of signaling early during macropinosome formation is less well studied. Here we demonstrate that
tyrosine phosphorylation of the adaptor
protein c-Cbl is required for KSHV induced membrane blebbing and macropinocytosis. KSHV induced the
tyrosine phosphorylation of c-Cbl as early as 1 min post-
infection and was recruited to the sites of
bleb formation.
Infection also led to an increase in the interaction of c-Cbl with PI3-K p85 in a time dependent manner. c-Cbl
shRNA decreased the formation of KSHV induced membrane
blebs and macropinocytosis as well as virus entry. Immunoprecipitation of c-Cbl followed by mass spectrometry identified the interaction of c-Cbl with a novel molecular partner, non-muscle
myosin heavy chain IIA (
myosin IIA), in
bleb associated macropinocytosis. Phosphorylated c-Cbl colocalized with phospho-
myosin light chain II in the interior of
blebs of infected cells and this interaction was abolished by c-Cbl
shRNA. Studies with the
myosin II inhibitor
blebbistatin demonstrated that
myosin IIA is a biologically significant component of the c-Cbl signaling pathway and c-Cbl plays a new role in the recruitment of
myosin IIA to the
blebs during KSHV
infection.
Myosin II associates with actin in KSHV induced
blebs and the absence of actin and
myosin ubiquitination in c-Cbl
ShRNA cells suggested that c-Cbl is also responsible for the ubiquitination of these
proteins in the infected cells. This is the first study demonstrating the role of c-Cbl in viral entry as well as macropinocytosis, and provides the evidence that a signaling complex containing c-Cbl and
myosin IIA plays a crucial role in blebbing and macropinocytosis during
viral infection and suggests that targeting c-Cbl could lead to a block in KSHV
infection.