title: ESCRT-0 Component Hrs Promotes Macropinocytosis of Kaposi's Sarcoma-Associated Herpesvirus in Human Dermal Microvascular Endothelial Cells.
abstract: Kaposi's sarcoma-associated herpesvirus (KSHV) enters human dermal microvascular endothelial cells (HMVEC-d), its naturalin vivotarget cells, by lipid raft-dependent macropinocytosis. The internalized viral envelope fuses with the macropinocytic membrane, and released capsid is transported to the nuclear vicinity, resulting in the nuclear entry of viral DNA. The endosomal sorting complexes required for transport (ESCRT) proteins, which include ESCRT-0, -I, -II, and -III, play a central role in endosomal trafficking and sorting of internalized and ubiquitinated receptors. Here, we examined the role of ESCRT-0 component Hrs (hepatocyte growth factor-regulated tyrosine kinase substrate) in KSHV entry into HMVEC-d by macropinocytosis. Knockdown of Hrs by short hairpin RNA (shRNA) transduction resulted in significant decreases in KSHV entry and viral gene expression. Immunofluorescence analysis (IFA) and plasma membrane isolation and proximity ligation assay (PLA) demonstrated the translocation of Hrs from the cytosol to the plasma membrane of infected cells and association with α-actinin-4. In addition, infection induced the plasma membrane translocation and activation of the serine/threonine kinase ROCK1, a downstream target of the RhoA GTPase. Hrs knockdown reduced these associations, suggesting that the recruitment of ROCK1 is an Hrs-mediated event. Interaction between Hrs and ROCK1 is essential for the ROCK1-induced phosphorylation of NHE1 (Na(+)/H(+)exchanger 1), which is involved in the regulation of intracellular pH. Thus, our studies demonstrate the plasma membrane association of ESCRT protein Hrs during macropinocytosis and suggest that KSHV entry requires both Hrs- and ROCK1-dependent mechanisms and that ROCK1-mediated phosphorylation of NHE1 and pH change is an essential event required for the macropinocytosis of KSHV.
  
  
  OBJECTIVE
  Macropinocytosis is the major entry pathway of KSHV in human dermal microvascular endothelial cells, the natural target cells of KSHV. Although the role of ESCRT protein Hrs has been extensively studied with respect to endosomal movement and sorting of ubiquitinated proteins into lysosomes, its function in macropinocytosis is not known. In the present study, we demonstrate for the first time that upon KSHV infection, the endogenous Hrs localizes to the plasma membrane and the membrane-associated Hrs facilitates assembly of signaling molecules, macropinocytosis, and virus entry. Hrs recruits ROCK1 to the membrane, which is required for the activation of NHE1 and an increase in submembranous intracellular pH occurring during macropinocytosis. These studies demonstrate that the localization of Hrs from the cytosol to the plasma membrane is important for coupling membrane dynamics to the cytosolic signaling events during macropinocytosis of KSHV.
authors.affiliation: H. M. Bligh Cancer Research Laboratories, Department of Microbiology and Immunology, Chicago Medical School, Rosalind Franklin University of Medicine and Science, North Chicago, Illinois, USA mohanan.veettil@rosalindfranklin.edu.
authors.forename: Mohanan Valiya
authors.initials: MV
authors.lastname: Veettil
authors.affiliation: H. M. Bligh Cancer Research Laboratories, Department of Microbiology and Immunology, Chicago Medical School, Rosalind Franklin University of Medicine and Science, North Chicago, Illinois, USA.
authors.forename: Binod
authors.initials: B
authors.lastname: Kumar
authors.affiliation: H. M. Bligh Cancer Research Laboratories, Department of Microbiology and Immunology, Chicago Medical School, Rosalind Franklin University of Medicine and Science, North Chicago, Illinois, USA.
authors.forename: Mairaj Ahmed
authors.initials: MA
authors.lastname: Ansari
authors.affiliation: H. M. Bligh Cancer Research Laboratories, Department of Microbiology and Immunology, Chicago Medical School, Rosalind Franklin University of Medicine and Science, North Chicago, Illinois, USA.
authors.forename: Dipanjan
authors.initials: D
authors.lastname: Dutta
authors.affiliation: H. M. Bligh Cancer Research Laboratories, Department of Microbiology and Immunology, Chicago Medical School, Rosalind Franklin University of Medicine and Science, North Chicago, Illinois, USA.
authors.forename: Jawed
authors.initials: J
authors.lastname: Iqbal
authors.affiliation: H. M. Bligh Cancer Research Laboratories, Department of Microbiology and Immunology, Chicago Medical School, Rosalind Franklin University of Medicine and Science, North Chicago, Illinois, USA.
authors.forename: Olsi
authors.initials: O
authors.lastname: Gjyshi
authors.affiliation: H. M. Bligh Cancer Research Laboratories, Department of Microbiology and Immunology, Chicago Medical School, Rosalind Franklin University of Medicine and Science, North Chicago, Illinois, USA.
authors.forename: Virginie
authors.initials: V
authors.lastname: Bottero
authors.affiliation: H. M. Bligh Cancer Research Laboratories, Department of Microbiology and Immunology, Chicago Medical School, Rosalind Franklin University of Medicine and Science, North Chicago, Illinois, USA.
authors.forename: Bala
authors.initials: B
authors.lastname: Chandran
mesh_terms: D000185:Actinin / Q000378:metabolism; D002460:Cell Line; D002462:Cell Membrane / Q000821:virology; D020405:Dermis / Q000098:blood supply / Q000821:virology; D056827:Endosomal Sorting Complexes Required for Transport / Q000235:genetics / Q000502:physiology*; D004730:Endothelium, Vascular / Q000821:virology*; D015870:Gene Expression; D055785:Gene Knockdown Techniques; D019288:Herpesvirus 8, Human / Q000502:physiology*; D006801:Humans; D055806:Microvessels / Q000166:cytology / Q000821:virology; D010750:Phosphoproteins / Q000235:genetics / Q000502:physiology*; D010873:Pinocytosis; D053586:Virus Internalization; D054460:rho-Associated Kinases / Q000378:metabolism
chemical_list: D056827:Endosomal Sorting Complexes Required for Transport; D010750:Phosphoproteins; C096164:hepatocyte growth factor-regulated tyrosine kinase substrate; D000185:Actinin; C515851:ROCK1 protein, human; D054460:rho-Associated Kinases
references.citation: Nat Med. 2012 Jun;18(6):961-6
references.pmid: 22635007
references.citation: Viruses. 2014 Oct;6(10):4024-46
references.pmid: 25341665
references.citation: J Virol. 2009 May;83(10):4895-911
references.pmid: 19279100
references.citation: Front Microbiol. 2012 Jan 20;3:6
references.pmid: 22319516
references.citation: PLoS Pathog. 2010;6(12):e1001238
references.pmid: 21203488
references.citation: J Gen Virol. 2014 Aug;95(Pt 8):1770-82
references.pmid: 24814923
references.citation: J Virol. 2003 Jul;77(14):8147-52
references.pmid: 12829853
references.citation: J Virol. 2004 Apr;78(8):4207-23
references.pmid: 15047836
references.citation: J Cell Sci. 2000 Sep;113 ( Pt 18):3329-40
references.pmid: 10954430
references.citation: Curr Biol. 2012 Feb 21;22(4):R116-20
references.pmid: 22361144
references.citation: J Virol. 2006 Dec;80(23):11432-46
references.pmid: 17005646
references.citation: Biochem J. 1994 Sep 1;302 ( Pt 2):551-7
references.pmid: 8093010
references.citation: Dev Cell. 2011 Jul 19;21(1):77-91
references.pmid: 21763610
references.citation: Mol Biol Cell. 2005 May;16(5):2470-82
references.pmid: 15772161
references.citation: Nat Cell Biol. 2001 Apr;3(4):346-52
references.pmid: 11283607
references.citation: Cell Struct Funct. 2002 Dec;27(6):403-8
references.pmid: 12576633
references.citation: J Virol. 2003 Mar;77(5):3131-47
references.pmid: 12584338
references.citation: Nat Rev Mol Cell Biol. 2007 May;8(5):355-68
references.pmid: 17450176
references.citation: J Virol. 2008 Feb;82(3):1570-80
references.pmid: 18045938
references.citation: PLoS Pathog. 2014 Oct;10(10):e1004389
references.pmid: 25299066
references.citation: Prog Mol Subcell Biol. 1999;22:23-38
references.pmid: 10081063
references.citation: Science. 1996 Jul 12;273(5272):245-8
references.pmid: 8662509
references.citation: Nat Cell Biol. 2009 May;11(5):510-20
references.pmid: 19404330
references.citation: Genes Dev. 1999 Jun 1;13(11):1475-85
references.pmid: 10364163
references.citation: Biochem Soc Trans. 2001 Aug;29(Pt 4):472-5
references.pmid: 11498011
references.citation: J Virol. 2011 Dec;85(23):12410-30
references.pmid: 21937638
references.citation: J Virol. 2004 Apr;78(7):3601-20
references.pmid: 15016882
references.citation: Traffic. 2006 Nov;7(11):1551-66
references.pmid: 17014699
references.citation: J Biol Chem. 1996 Mar 15;271(11):6152-8
references.pmid: 8626403
references.citation: Biochem J. 2000 Jun 1;348 Pt 2:241-55
references.pmid: 10816416
references.citation: J Virol. 2003 Jul;77(14):7978-90
references.pmid: 12829837
references.citation: Proc Natl Acad Sci U S A. 1991 Sep 1;88(17):7849-53
references.pmid: 1652767
references.citation: J Virol. 2005 Jan;79(2):1191-206
references.pmid: 15613346
references.citation: Genes Dev. 1998 Dec 1;12(23):3663-74
references.pmid: 9851973
references.citation: EMBO J. 1999 Jul 1;18(13):3616-28
references.pmid: 10393178
references.citation: Virology. 2001 Apr 10;282(2):245-55
references.pmid: 11289807
references.citation: J Virol. 2008 Dec;82(24):12126-44
references.pmid: 18829766
references.citation: J Virol. 2010 Mar;84(5):2188-99
references.pmid: 19923183
references.citation: Nat Protoc. 2006;1(1):241-5
references.pmid: 17406239
references.citation: Virology. 1999 Sep 15;262(1):237-49
references.pmid: 10489357
references.citation: PLoS Pathog. 2015 Jul;11(7):e1005019
references.pmid: 26134128
references.citation: J Cell Biol. 2010 Feb 22;188(4):547-63
references.pmid: 20156964
references.citation: J Virol. 2007 Oct;81(20):10879-89
references.pmid: 17670830
references.citation: Virology. 2014 Sep;464-465:118-33
references.pmid: 25063885
references.citation: PLoS Pathog. 2009 Jul;5(7):e1000512
references.pmid: 19593382
references.citation: Proc Natl Acad Sci U S A. 2012 May 8;109(19):E1163-72
references.pmid: 22509030
references.citation: J Virol. 2014 Dec;88(23):13858-78
references.pmid: 25253349
references.citation: Mol Biol Cell. 2003 Jul;14(7):2655-64
references.pmid: 12857854
references.citation: J Virol. 2008 May;82(10):4793-806
references.pmid: 18337571
references.citation: Cell. 2002 Feb 8;108(3):407-19
references.pmid: 11853674
references.citation: J Cell Biol. 2003 Aug 4;162(3):435-42
references.pmid: 12900395
references.citation: J Cell Biol. 2005 Aug 1;170(3):443-53
references.pmid: 16043513
references.citation: PLoS Pathog. 2014 Feb;10(2):e1003941
references.pmid: 24550731
references.citation: Mol Biol Cell. 1998 Aug;9(8):2287-303
references.pmid: 9693382
references.citation: Nat Cell Biol. 2002 May;4(5):394-8
references.pmid: 11988743