ESCRT-0 Component Hrs Promotes Macropinocytosis of Kaposi's Sarcoma

45 Pages·2016·6.57 MB·English

JVI Accepted Manuscript Posted Online 27 January 2016
J. Virol. doi:10.1128/JVI.02704-15
Copyright © 2016, American Society for Microbiology. All Rights Reserved.
1 ESCRT-0 Component Hrs Promotes Macropinocytosis of Kaposi’s Sarcoma-
2 Associated Herpesvirus in Human Dermal Microvascular Endothelial Cells
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4 Mohanan Valiya Veettil#, Binod Kumar, Mairaj Ahmed Ansari, Dipanjan Dutta, Jawed
5 Iqbal, Olsi Gjyshi, Virginie Bottero and Bala Chandran
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7 H. M. Bligh Cancer Research Laboratories, Department of Microbiology and e
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8 Immunology, Chicago Medical School, Rosalind Franklin University of Medicine and o
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9 Science, North Chicago, Illinois 60064, USA. t
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11 Running title: Role of Hrs in macropinocytosis of KSHV .o
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13 # Corresponding author. A
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15 Department of Microbiology and Immunology, Chicago Medical School, 0
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16 Rosalind Franklin University of Medicine and Science, b
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17 3333 Green Bay Road, North Chicago, IL 60064. u
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18 Phone [phone]x7716; Fax [phone].
19 E-mail: [email protected]
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23 ABSTRACT
24 Kaposi’s sarcoma-associated herpesvirus (KSHV) enters human dermal microvascular
25 endothelial cells (HMVEC-d), its natural in vivo target cells, by lipid raft dependent
26 macropinocytosis. The internalized viral envelope fuses with the macropinocytic membrane and
27 released capsid is transported to the nuclear vicinity resulting in the nuclear entry of viral DNA.
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28 The Endosomal Sorting Complexes Required for Transport (ESCRT) proteins which include o
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29 ESCRT-0, -I, -II, and -III play a central role in endosomal trafficking and sorting of internalized lo
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30 and ubiquitinated receptors. Here, we examined the role of ESCRT-0 component Hrs e
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31 (Hepatocyte growth factor regulated tyrosine kinase substrate) in KSHV entry into HMVEC-d o
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32 cells by macropinocytosis. Knockdown of Hrs by shRNA transduction results in significant t
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33 decrease in KSHV entry and viral gene expression. Immunofluorescence analysis (IFA) and //jv
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34 plasma membrane isolation and proximity ligation assay (PLA) demonstrate the translocation of s
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35 Hrs from the cytosol to the plasma membranes of infected cells and association with α-actinin-4. o
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36 In addition, infection induces the plasma membrane translocation and activation of the o
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37 serine/threonine kinase ROCK1, a downstream target of the RhoA GTPase. Hrs knockdown p
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38 reduces these associations suggesting that the recruitment of ROCK1 is an Hrs mediated event. 6,

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39 Interaction between Hrs and ROCK1 is essential for the ROCK1 induced phosphorylation of 1
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40 NHE1 (Na+/H+ exchanger 1) involved in the regulation of intracellular pH. Thus, our studies y

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41 demonstrate the plasma membrane association of ESCRT protein Hrs during macropinocytosis e
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42 and suggest that KSHV entry requires both Hrs and ROCK1 dependent mechanisms, and
43 ROCK1 mediated phosphorylation of NHE1 and pH change is an essential event required for the
44 macropinocytosis of KSHV.
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46 IMPORTANCE
47 Macropinocytosis is the major entry pathway of KSHV in human dermal microvascular
48 endothelial cells, the natural target cells of KSHV. Although the role of ESCRT protein Hrs has
49 been extensively studied with respect to endosomal movement and sorting of ubiquitinated
50 proteins into lysosomes, its function in macropinocytosis is not known. In the present study, we
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51 demonstrate for the first time that upon KSHV infection the endogenous Hrs localizes to the w
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52 plasma membrane and the membrane associated Hrs facilitates assembly of signaling molecules, a
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53 macropinocytosis and virus entry. Hrs recruits ROCK1 to the membrane which is required for d

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54 the activation of NHE1 and an increase in submembraneous intracellular pH occurring during m

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55 macropinocytosis. These studies demonstrate that the localization of Hrs from the cytosol to the tp
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56 plasma membrane is important for coupling membrane dynamics to the cytosolic signaling jv
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57 events during macropinocytosis of KSHV. m
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69 INTRODUCTION
70 KSHV entry into its in vitro adherent target cells is a multistage process which involves
71 binding of viral glycoproteins to cell surface heparan sulfate receptors, followed by interaction
72 with specific entry receptors, induction of cell signaling pathways and endocytosis. KSHV
73 exploits multiple host cell surface receptors including integrins-α3β1, αVβ3, αVβ5, α9β1, and
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74 non-integrins CD98/xCT, and EphA2 to enter the adherent target cells such as HMVEC-d o
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75 (Human dermal microvascular endothelial) and HFF (Human foreskin fibroblasts) cells (1-6). lo
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76 The interaction of KSHV with its specific entry receptors leads to the formation of a e
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77 multimolecular receptor complex consisting of integrins, xCT and EphA2 (2, 4, 7). Receptor o
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78 engagement and multimolecular receptor complex formation results in auto phosphorylation of t
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79 FAK, and the activation of Src, PI3-K and Rho GTPases, and all these molecules are targeted to //jv
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80 specific entry sites on the plasma membrane (8-10). Our previous studies have demonstrated that s
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81 the signal transduction pathways induced by KSHV and the consequent activation of their o
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82 downstream molecules plays a central role in coordinating the actin dynamics and the membrane o
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83 protein assembly required for the successful entry of the virus into the cytoplasm (8-10). The p
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84 endocytosed viral particles are then transported towards the nuclear periphery along the 6,

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85 microtubules by utilizing dynein motor proteins to deliver its DNA content into the nucleus (11). 1
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86 KSHV utilizes different endocytic pathways to enter different cell types (12-17). In HFF y

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87 cells, primary B cells, and 293 cells, clathrin dependent endocytosis is the predominant pathway e
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88 of entry (12-14), whereas in HMVEC-d cells entry occurs by bleb associated macropinocytosis
89 (15, 16, 18). Bleb associated macropinocytosis begins with a remarkable set of events including
90 the formation of blebs, actomyosin contraction, bleb retraction, macropinosome formation and
91 eventually virus entry (18). Our studies have established that the adaptor protein c-Cbl and its
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92 interaction with myosin IIA light chain (MLC) plays a significant role in blebbing and that
93 myosin IIA is required for both actomyosin contraction and retraction of the bleb (18). Our
94 subsequent studies proved that c-Cbl is also required for both translocation of the receptors into
95 the lipid raft and ubiquitination of the α3β1 and αVβ3 receptors which are critical determinants
96 of the macropinocytic entry, trafficking, and productive infection of KSHV (19).
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97 Ubiquitination of receptors and the adaptor proteins such as c-Cbl serves to facilitate the o
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98 endocytosis of receptors and their sorting from membrane to lysosomes and subsequent lo
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99 degradation (20, 21). In mammalian cells, the Endosomal Sorting Complexes Required for e
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100 Transport (ESCRT) helps in sorting ubiquitinated proteins and their delivery into lysosomes. The o
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101 ESCRT machinery consists a family of four complexes, ESCRT-0, -I, -II, and –III, which t
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102 sequentially assemble on the endosomal membrane. The ESCRT-0 component Hrs (Hepatocyte //jv
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103 growth factor (HGF)-regulated tyrosine kinase substrate) acts upstream to ESCRT complexes s
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104 and can recruit ESCRT-I to the endosomal membranes. This complex subsequently recruits o
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105 ESCRT-II and ESCRT-III complexes, two important complexes required for the sorting of o
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106 ubiquitinated proteins into late endosomes (22-25). p
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107 Hrs protein contains several domains: an N-terminal VHS-domain (Vps27p, Hrs, STAM), 6,

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108 FYVE domain, an ubiquitin interacting motif, a proline-rich domain, two coiled-coil domains, 1
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109 and a C-terminal clathrin binding domain (26). The functional role of the different domains of y

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110 Hrs includes protein-protein interaction, cellular localization, cargo sorting, and endosome e
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111 motility (24, 25, 27, 28). Although the function of Hrs in intracellular trafficking and signal
112 transduction is well established, its role in macropinocytosis remains unclear. In the present
113 study, we provide evidence that Hrs is recruited to the plasma membrane early during KSHV
114 infection which is required for the virus entry by macropinocytosis. Interestingly, we found that
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115 Hrs interacts with the Rho-associated protein kinase ROCK1, which in turn activates MLC and
116 the intracellular pH-regulating protein NHE1 to promote actomyosin contraction and
117 intracellular pH changes required for macropinocytosis.
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138 MATERIALS AND METHODS
139 Cells and virus: Primary human dermal microvascular enodthelial cells (HMVEC-d CC-2543;
140 Clonetics, Walkersville, MD) were grown in EBM2 medium with growth factors (Cambrex,
141 Walkersville, MD). BCBL-1 cell culture, supernatant collection after TPA induction, and virus
142 purification procedures were performed as described previously (1). The isolation of KSHV
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143 DNA from the virus, and the determination of viral copy numbers by real-time DNA PCR using o
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144 KSHV ORF73 gene-specific primers were performed as previously described (29). All infections lo
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145 were carried out with 20 DNA copies/cell (multiplicity of infection-MOI) of KSHV, unless e
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146 stated otherwise. o
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147 Antibodies and reagents: Antibodies and reagents used for this study include: Anti-ROCK1 t
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148 rabbit monoclonal antibody, anti-phospho MLC rabbit polyclonal antibody, anti-Na, K-ATPase //jv
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149 rabbit polyclonal antibody (Cell Signaling Technology); anti-Hrs rabbit polyclonal antibody s
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150 (Sigma); anti-NHE1 mouse monoclonal antibody (Santa Cruz); anti-calnexin rabbit monoclonal o
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151 antibody, mouse anti-phospho serine antibody (Abcam); anti-α-actinin-4 rabbit monoclonal o
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152 antibody (Millipore); rabbit anti-gB and mouse anti-gpK8.1A antibodies were created in Dr. p
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153 Chandran’s laboratory (30, 31); anti-rabbit and anti-mouse antibodies linked to horseradish 6,

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154 peroxidase (KPL Inc., Gaithersburg, Md.); Texas Red conjugated dextran, Alexa 594 conjugated 1
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155 phalloidin and anti-rabbit and anti-mouse secondary antibodies conjugated to Alexa 488 or Alexa y

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156 594 (Invitrogen); protein A and G–Sepharose CL-4B beads (Amersham Pharmacia Biotech, e
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157 Piscataway, NJ).
158 Hrs shRNA knockdown: For shRNA knockdown of Hrs, five sets of bacterial glycerol stock
159 Hrs shRNA plasmid vectors (TRCN00000037894, -37895, -37896, -37897, and -37898) were
160 purchased from Thermoscientific. The Hrs shRNA lentiviral plasmids were purified from the
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161 bacterial cultures (Invitrogen) and the lentivial particles were produced by transfection with a
162 four-plasmid system, as previously described (32). Briefly, HEK 293T cells were transiently
163 transfected with shRNA lentiviral constructs and the plasmid packaging system (Gag-Pol, Rev
164 and VSV-G), the supernatants were collected, and filtered. The lentiviral particles produced from
165 each plasmid vector were tested for knockdown efficiency by Western blot analysis. We found
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166 that TRCN00000037897 and TRCN00000037898 had the highest knockdown efficiency among o
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167 the five shRNAs. Therefore, we used Hrs shRNAs created with these two plasmids (Hrs- lo
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168 shRNA1 and Hrs-shRNA2) for the experiments in this manuscript. We used Hrs shRNA1 for all e
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169 experiments except figure 1A and 1B where we used both Hrs shRNA1 and 2. For negative o
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170 control, a nontargeting shRNA lentiviral pool was used. t
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171 Western blotting: Cells lysates were prepared in RIPA lysis buffer (15 mM NaCl, 1 mM //jv
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172 MgCl2, 1 mM MnCl2, 2 mM CaCl2, 2 mM phenylmethylsulfonyl fluoride, and protease s
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173 inhibitor mixture (Sigma). The lysates were centrifuged at 12,000 rpm for 15 min at 4°C and the o
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174 protein concentration was measured with BCA reagent. Equal amounts of proteins from each o
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175 sample were separated on SDS-PAGE and transferred to nitrocellulose membranes. Membranes p
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176 were probed with the indicated primary antibodies and detected by incubation with species- 6,

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177 specific HRP-conjugated secondary antibodies. Immunoreactive protein bands were visualized 1
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178 and imaged by enhanced chemiluminescent HRP substrate kit (Pierce, Rockford, IL). The bands y

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179 were scanned and densitometric quantification was performed using the FluorChem FC2 and e
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180 Alpha-Imager Systems (Alpha Innotech Corporation, San Leonardo, CA).
181 Immunoprecipitation: Cell lysates were immunoprecipitated for 2h with appropriate antibodies
182 and the immune complexes were captured by protein A or G-Sepharose. The immune complexes
183 were then eluted in sample buffer for SDS-PAGE and run on 10% gels and transferred to
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184 membranes. The membranes were tested by Western blot with specific primary and secondary
185 antibodies.
186 Immunofluorescence microscopy: Cells grown in 8-well chamber slides were fixed with 4%
187 paraformaldehyde after infection and permeabilized with 0.2% Triton X-100. The cells were
188 blocked with Image-iT FX signal enhancer (Invitrogen) for 20 min, and then incubated with
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189 primary antibodies against the specific proteins and subsequently stained with secondary o
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190 antibodies conjugated to Alexa 488 or 594. The stained cells were mounted in mounting medium lo
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191 with DAPI (4′,6′-diamidino-2-phenylindole) (Invitrogen) and visualized with a Nikon 80i e
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192 fluorescent microscope equipped with Metamorph digital imaging software. o
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193 Proximity Ligation Assay: Proximity Ligation Assay (PLA) was performed using the Duolink t
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194 in situ detection kit (Sigma) as per the manufacturer’s instructions (33, 34). Briefly, uninfected //jv
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195 and infected cells fixed with 4% paraformaldehyde solution for 15 min, permeabilized with s
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196 Triton X- 100 for 5 min and washed with PBS, and incubated for 1h with the Duolink blocking o
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197 buffer. The cells were then incubated with pairs of primary antibodies in Duolink antibody o
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198 diluent solution and then labeled with Duolink PLA PLUS and MINUS probes for 1h at 37°C. p
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199 This was followed by hybridization, ligation, amplification, and detection using a red fluorescent 6,

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200 probe. The red fluorescent fluorophore-tagged oligonucleotides were visualized using a Nikon 1
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201 Eclipse 80i microscope equipped with Metamorph digital imaging software. y

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202 Determination of KSHV entry by real-time DNA PCR: Target cells were infected with 20 e
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203 MOI of KSHV at 37°C for 2h. After infection, the cells were washed with HBSS, and treated
204 with 0.25% trypsin-EDTA for 5 min at 37°C to remove the bound but non-internalized virus.
205 Total DNA was isolated from the uninfected and infected cells using DNeasy kit (QIAGEN,
206 Valencia, CA) and real time DNA PCR was carried out using primer sets described previously
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207 (29). To calculate the percent inhibition of KSHV entry, internalized KSHV DNA was
208 quantitated by amplification of the ORF73 gene by real-time DNA PCR. The KSHV ORF73
209 gene cloned in the pGEM-T vector (Promega) was used for the external standard.
210 Determination of KSHV gene expression by real-time RT-PCR: Isolation of total RNA from
211 cells, reverse transcription, and qPCR analysis were carried out using primer sets described
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212 previously. Briefly, total RNA was isolated from the lysate using RNeasy kit (QIAGEN), o
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213 quantified, and the ORF73 expression was detected by real-time reverse transcription PCR using lo
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214 specific primers and TaqMan probes (29). GAPDH (glyceraldehyde-3-phosphate e
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215 dehydrogenase) gene expression was used to normalize the expression levels of ORF73. o
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216 Isolation of membrane fraction: Subcellular fractionation and cell membrane preparation were t
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217 performed as described previously (18, 35). Briefly, cells were homogenized with a Dounce- //jv
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218 homogenizer in homogenization buffer (250 mM sucrose, 20 mM HEPES, 10 mM KCl, 1 mM s
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219 EDTA, 1 mM EGTA and protease inhibitors). The homogenate was centrifuged at 3,000 rpm for o
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220 5 min, and the post-nuclear supernatant was centrifuged at 8,000 rpm for 5 min at 4ºC. The o
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221 supernatant was again centrifuged at 40,000 rpm for 1h at 4ºC, and the cytosolic (supernatant) p
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222 and membrane (pellet) fractions were separated. The membrane fractions were lysed in RIPA 6,

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223 buffer and used for Western blot. 1
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224 Analysis of dextran uptake: For the dextran uptake study, HMVEC-d cells were incubated with y

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225 Texas Red labeled dextran (40 kDa, 0.5 mg/ml; Invitrogen) and KSHV for 30 min. The cells e
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226 were then washed twice in HBSS, fixed with 4% paraformaldehyde for 15 min, and washed three
227 times with PBS. Internalized dextran was observed under immunofluorescence microscope.
228 To perform quantitative analysis of dextran uptake, the dextran positive cells were counted
229 under immunofluorescence microscope. At least 10 different microscopic fields of 25-30 cells
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