Cargando…

ESCRT-I Protein Tsg101 Plays a Role in the Post-macropinocytic Trafficking and Infection of Endothelial Cells by Kaposi’s Sarcoma-Associated Herpesvirus

Kaposi’s sarcoma-associated herpesvirus (KSHV) binding to the endothelial cell surface heparan sulfate is followed by sequential interactions with α3β1, αVβ3 and αVβ5 integrins and Ephrin A2 receptor tyrosine kinase (EphA2R). These interactions activate host cell pre-existing FAK, Src, PI3-K and Rho...

Descripción completa

Detalles Bibliográficos
Autores principales: Kumar, Binod, Dutta, Dipanjan, Iqbal, Jawed, Ansari, Mairaj Ahmed, Roy, Arunava, Chikoti, Leela, Pisano, Gina, Veettil, Mohanan Valiya, Chandran, Bala
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Public Library of Science 2016
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5072609/
https://www.ncbi.nlm.nih.gov/pubmed/27764233
http://dx.doi.org/10.1371/journal.ppat.1005960
_version_ 1782461423327117312
author Kumar, Binod
Dutta, Dipanjan
Iqbal, Jawed
Ansari, Mairaj Ahmed
Roy, Arunava
Chikoti, Leela
Pisano, Gina
Veettil, Mohanan Valiya
Chandran, Bala
author_facet Kumar, Binod
Dutta, Dipanjan
Iqbal, Jawed
Ansari, Mairaj Ahmed
Roy, Arunava
Chikoti, Leela
Pisano, Gina
Veettil, Mohanan Valiya
Chandran, Bala
author_sort Kumar, Binod
collection PubMed
description Kaposi’s sarcoma-associated herpesvirus (KSHV) binding to the endothelial cell surface heparan sulfate is followed by sequential interactions with α3β1, αVβ3 and αVβ5 integrins and Ephrin A2 receptor tyrosine kinase (EphA2R). These interactions activate host cell pre-existing FAK, Src, PI3-K and RhoGTPase signaling cascades, c-Cbl mediated ubiquitination of receptors, recruitment of CIB1, p130Cas and Crk adaptor molecules, and membrane bleb formation leading to lipid raft dependent macropinocytosis of KSHV into human microvascular dermal endothelial (HMVEC-d) cells. The Endosomal Sorting Complexes Required for Transport (ESCRT) proteins, ESCRT-0, -I, -II, and–III, play a central role in clathrin-mediated internalized ubiquitinated receptor endosomal trafficking and sorting. ESCRT proteins have also been shown to play roles in viral egress. We have recently shown that ESCRT-0 component Hrs protein associates with the plasma membrane during macropinocytosis and mediates KSHV entry via ROCK1 mediated phosphorylation of NHE1 and local membrane pH change. Here, we demonstrate that the ESCRT-I complex Tsg101 protein also participates in the macropinocytosis of KSHV and plays a role in KSHV trafficking. Knockdown of Tsg101 did not affect virus entry in HMVEC-d and human umbilical vein endothelial (HUVEC) cells but significantly inhibited the KSHV genome entry into the nucleus and consequently viral gene expression in these cells. Double and triple immunofluorescence, proximity ligation immunofluorescence and co-immuoprecipitation studies revealed the association of Tsg101 with the KSHV containing macropinosomes, and increased levels of Tsg101 association/interactions with EphA2R, c-Cbl, p130Cas and Crk signal molecules, as well as with upstream and downstream ESCRT components such as Hrs (ESCRT-0), EAP45 (ESCRT-II), CHMP6 (ESCRT-III) and CHMP5 (ESCRT-III) in the KSHV infected cells. Tsg101 was also associated with early (Rab5) and late endosomal (Rab7) stages of KSHV intracellular trafficking, and CHMP5 (ESCRT-III) was also associated with Rab 5 and Rab 7. Knockdown of Tsg101 significantly inhibited the transition of virus from early to late endosomes. Collectively, our studies reveal that Tsg101 plays a role in the trafficking of macropinocytosed KSHV in the endothelial cells which is essential for the successful viral genome delivery into the nucleus, viral gene expression and infection. Thus, ESCRT molecules could serve as therapeutic targets to combat KSHV infection.
format Online
Article
Text
id pubmed-5072609
institution National Center for Biotechnology Information
language English
publishDate 2016
publisher Public Library of Science
record_format MEDLINE/PubMed
spelling pubmed-50726092016-10-27 ESCRT-I Protein Tsg101 Plays a Role in the Post-macropinocytic Trafficking and Infection of Endothelial Cells by Kaposi’s Sarcoma-Associated Herpesvirus Kumar, Binod Dutta, Dipanjan Iqbal, Jawed Ansari, Mairaj Ahmed Roy, Arunava Chikoti, Leela Pisano, Gina Veettil, Mohanan Valiya Chandran, Bala PLoS Pathog Research Article Kaposi’s sarcoma-associated herpesvirus (KSHV) binding to the endothelial cell surface heparan sulfate is followed by sequential interactions with α3β1, αVβ3 and αVβ5 integrins and Ephrin A2 receptor tyrosine kinase (EphA2R). These interactions activate host cell pre-existing FAK, Src, PI3-K and RhoGTPase signaling cascades, c-Cbl mediated ubiquitination of receptors, recruitment of CIB1, p130Cas and Crk adaptor molecules, and membrane bleb formation leading to lipid raft dependent macropinocytosis of KSHV into human microvascular dermal endothelial (HMVEC-d) cells. The Endosomal Sorting Complexes Required for Transport (ESCRT) proteins, ESCRT-0, -I, -II, and–III, play a central role in clathrin-mediated internalized ubiquitinated receptor endosomal trafficking and sorting. ESCRT proteins have also been shown to play roles in viral egress. We have recently shown that ESCRT-0 component Hrs protein associates with the plasma membrane during macropinocytosis and mediates KSHV entry via ROCK1 mediated phosphorylation of NHE1 and local membrane pH change. Here, we demonstrate that the ESCRT-I complex Tsg101 protein also participates in the macropinocytosis of KSHV and plays a role in KSHV trafficking. Knockdown of Tsg101 did not affect virus entry in HMVEC-d and human umbilical vein endothelial (HUVEC) cells but significantly inhibited the KSHV genome entry into the nucleus and consequently viral gene expression in these cells. Double and triple immunofluorescence, proximity ligation immunofluorescence and co-immuoprecipitation studies revealed the association of Tsg101 with the KSHV containing macropinosomes, and increased levels of Tsg101 association/interactions with EphA2R, c-Cbl, p130Cas and Crk signal molecules, as well as with upstream and downstream ESCRT components such as Hrs (ESCRT-0), EAP45 (ESCRT-II), CHMP6 (ESCRT-III) and CHMP5 (ESCRT-III) in the KSHV infected cells. Tsg101 was also associated with early (Rab5) and late endosomal (Rab7) stages of KSHV intracellular trafficking, and CHMP5 (ESCRT-III) was also associated with Rab 5 and Rab 7. Knockdown of Tsg101 significantly inhibited the transition of virus from early to late endosomes. Collectively, our studies reveal that Tsg101 plays a role in the trafficking of macropinocytosed KSHV in the endothelial cells which is essential for the successful viral genome delivery into the nucleus, viral gene expression and infection. Thus, ESCRT molecules could serve as therapeutic targets to combat KSHV infection. Public Library of Science 2016-10-20 /pmc/articles/PMC5072609/ /pubmed/27764233 http://dx.doi.org/10.1371/journal.ppat.1005960 Text en © 2016 Kumar et al http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.
spellingShingle Research Article
Kumar, Binod
Dutta, Dipanjan
Iqbal, Jawed
Ansari, Mairaj Ahmed
Roy, Arunava
Chikoti, Leela
Pisano, Gina
Veettil, Mohanan Valiya
Chandran, Bala
ESCRT-I Protein Tsg101 Plays a Role in the Post-macropinocytic Trafficking and Infection of Endothelial Cells by Kaposi’s Sarcoma-Associated Herpesvirus
title ESCRT-I Protein Tsg101 Plays a Role in the Post-macropinocytic Trafficking and Infection of Endothelial Cells by Kaposi’s Sarcoma-Associated Herpesvirus
title_full ESCRT-I Protein Tsg101 Plays a Role in the Post-macropinocytic Trafficking and Infection of Endothelial Cells by Kaposi’s Sarcoma-Associated Herpesvirus
title_fullStr ESCRT-I Protein Tsg101 Plays a Role in the Post-macropinocytic Trafficking and Infection of Endothelial Cells by Kaposi’s Sarcoma-Associated Herpesvirus
title_full_unstemmed ESCRT-I Protein Tsg101 Plays a Role in the Post-macropinocytic Trafficking and Infection of Endothelial Cells by Kaposi’s Sarcoma-Associated Herpesvirus
title_short ESCRT-I Protein Tsg101 Plays a Role in the Post-macropinocytic Trafficking and Infection of Endothelial Cells by Kaposi’s Sarcoma-Associated Herpesvirus
title_sort escrt-i protein tsg101 plays a role in the post-macropinocytic trafficking and infection of endothelial cells by kaposi’s sarcoma-associated herpesvirus
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5072609/
https://www.ncbi.nlm.nih.gov/pubmed/27764233
http://dx.doi.org/10.1371/journal.ppat.1005960
work_keys_str_mv AT kumarbinod escrtiproteintsg101playsaroleinthepostmacropinocytictraffickingandinfectionofendothelialcellsbykaposissarcomaassociatedherpesvirus
AT duttadipanjan escrtiproteintsg101playsaroleinthepostmacropinocytictraffickingandinfectionofendothelialcellsbykaposissarcomaassociatedherpesvirus
AT iqbaljawed escrtiproteintsg101playsaroleinthepostmacropinocytictraffickingandinfectionofendothelialcellsbykaposissarcomaassociatedherpesvirus
AT ansarimairajahmed escrtiproteintsg101playsaroleinthepostmacropinocytictraffickingandinfectionofendothelialcellsbykaposissarcomaassociatedherpesvirus
AT royarunava escrtiproteintsg101playsaroleinthepostmacropinocytictraffickingandinfectionofendothelialcellsbykaposissarcomaassociatedherpesvirus
AT chikotileela escrtiproteintsg101playsaroleinthepostmacropinocytictraffickingandinfectionofendothelialcellsbykaposissarcomaassociatedherpesvirus
AT pisanogina escrtiproteintsg101playsaroleinthepostmacropinocytictraffickingandinfectionofendothelialcellsbykaposissarcomaassociatedherpesvirus
AT veettilmohananvaliya escrtiproteintsg101playsaroleinthepostmacropinocytictraffickingandinfectionofendothelialcellsbykaposissarcomaassociatedherpesvirus
AT chandranbala escrtiproteintsg101playsaroleinthepostmacropinocytictraffickingandinfectionofendothelialcellsbykaposissarcomaassociatedherpesvirus