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The 11 th International Workshop on KSHV & Related Agents, Birmingham, UK
Virus-Cell Interactions I Abstract 28
KAPOSI’S SARCOMA ASSOCIATED HERPESVIRUS (KSHV/HHV-8) UTILIZES
MACROPINOCYTIC PATHWAY TO ENTER HUMAN DERMAL MICROVASCULAR
ENDOTHELIAL (HMVEC-D) AND HUMAN UMBILICAL VEIN ENDOTHELIAL
(HUVEC) CELLS
Hari Raghu, Neelam Sharma Walia, Mohanan Valiya Veettil, Sathish Sadagopan and Bala
Chandran
H.M. Bligh cancer research laboratories, Department of Microbiology and Immunology,
H.M. Bligh Cancer Research Laboratories, Chicago Medical School, Rosalind Franklin
University of Medicine and Science, 3333 Green Bay Road, North Chicago, IL. USA
Abstract
KSHV utilizes the clathrin mediated endocytic pathway for its infectious entry into human
foreskin fibroblast (HFF) cells (J.Virology. 2003. 77: 7978-7990). Here, we characterized
KSHV entry in primary HMVEC-d and HUVEC cells. Similar to HMVEC-d cells, KSHV
infection of HUVEC cells also resulted in the initial high level lytic ORF50 and K8 gene
expression and subsequent decline, while the latent gene expression persisted. In
contrast to HFF cells, cytochalasin D affecting actin polymerization significantly blocked
virus entry and gene expression in both endothelial cell types tested. Chlorpromazine, a
clathrin mediated endocytosis inhibitor which inhibited KSHV entry in HFF cells did not
have any effect on KSHV binding, internalization and gene expression in HMVEC-d and
HUVEC cells. No significant inhibition was observed in both the endothelial cell types with
filipin, a caveolar endocytosis inhibitor. In contrast, internalization and gene expression
was significantly inhibited in both the endothelial cell types by macropinocytosis
inhibitors EIPA and Rottlerin. Internalized virus particles enclosed in large vesicles were
seen by electron microscopy. Confocal microscopy localized the viral capsid with KSHV
envelope glycoprotein gpK8.1A at 5’ and 10’ post infection. Inhibition of macropinocytosis
resulted in the distribution of viral capsids at the cell periphery and very little association
with microtubules. Internalized viral glycoprotein gpK8.1A was associated with dextran, a
marker for macropinocytosis, and KSHV entry in HMVEC-d cells was dynamin
independent. Although KSHV was not associated with the early endosome marker EEA-1,
internalized KSHV was associated with Rab5 and Rab34 GTPases that are known to
regulate macropinocytosis. Taken together, these findings suggest that for its infectious
entry in HMVEC-d and HUVEC cells, KSHV utilizes a macropinocytic pathway.
Presenting author Email: bala.chandran@rosalindfranklin.edu
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