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-
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Associated Herpesvirus in Human Dermal Microvascular Endothelial Cells
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Mohanan Valiya Veettil#, Binod Kumar, Mairaj Ahmed Ansari, Dipanjan Dutta, Jawed
5
Iqbal, Olsi Gjyshi, Virginie Bottero and Bala Chandran
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H. M. Bligh Cancer Research Laboratories, Department of Microbiology and
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Immunology, Chicago Medical School, Rosalind Franklin University of Medicine and
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Science, North Chicago, Illinois 60064, USA.
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Running title: Role of Hrs in macropinocytosis of KSHV
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# Corresponding author.
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Mailing Address:
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Department of Microbiology and Immunology, Chicago Medical School,
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Rosalind Franklin University of Medicine and Science,
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3333 Green Bay Road, North Chicago, IL 60064.
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Phone (847) 578-8700x7716; Fax (847) 578-3349.
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E-mail: [email protected]
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ABSTRACT
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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.
28
The Endosomal Sorting Complexes Required for Transport (ESCRT) proteins which include
29
ESCRT-0, -I, -II, and -III play a central role in endosomal trafficking and sorting of internalized
30
and ubiquitinated receptors. Here, we examined the role of ESCRT-0 component Hrs
31
(Hepatocyte growth factor regulated tyrosine kinase substrate) in KSHV entry into HMVEC-d
32
cells by macropinocytosis. Knockdown of Hrs by shRNA transduction results in significant
33
decrease in KSHV entry and viral gene expression. Immunofluorescence analysis (IFA) and
34
plasma membrane isolation and proximity ligation assay (PLA) demonstrate the translocation of
35
Hrs from the cytosol to the plasma membranes of infected cells and association with α-actinin-4.
36
In addition, infection induces the plasma membrane translocation and activation of the
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serine/threonine kinase ROCK1, a downstream target of the RhoA GTPase. Hrs knockdown
38
reduces these associations suggesting that the recruitment of ROCK1 is an Hrs mediated event.
39
Interaction between Hrs and ROCK1 is essential for the ROCK1 induced phosphorylation of
40
NHE1 (Na+/H+ exchanger 1) involved in the regulation of intracellular pH. Thus, our studies
41
demonstrate the plasma membrane association of ESCRT protein Hrs during macropinocytosis
42
and suggest that KSHV entry requires both Hrs and ROCK1 dependent mechanisms, and
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ROCK1 mediated phosphorylation of NHE1 and pH change is an essential event required for the
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macropinocytosis of KSHV.
45
2
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
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been extensively studied with respect to endosomal movement and sorting of ubiquitinated
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proteins into lysosomes, its function in macropinocytosis is not known. In the present study, we
51
demonstrate for the first time that upon KSHV infection the endogenous Hrs localizes to the
52
plasma membrane and the membrane associated Hrs facilitates assembly of signaling molecules,
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macropinocytosis and virus entry. Hrs recruits ROCK1 to the membrane which is required for
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the activation of NHE1 and an increase in submembraneous intracellular pH occurring during
55
macropinocytosis. These studies demonstrate that the localization of Hrs from the cytosol to the
56
plasma membrane is important for coupling membrane dynamics to the cytosolic signaling
57
events during macropinocytosis of KSHV.
58 59 60 61 62 63 64 65 66 67 68 3
69
INTRODUCTION
70
KSHV entry into its in vitro adherent target cells is a multistage process which involves
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binding of viral glycoproteins to cell surface heparan sulfate receptors, followed by interaction
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with specific entry receptors, induction of cell signaling pathways and endocytosis. KSHV
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exploits multiple host cell surface receptors including integrins-α3β1, αVβ3, αVβ5, α9β1, and
74
non-integrins CD98/xCT, and EphA2 to enter the adherent target cells such as HMVEC-d
75
(Human dermal microvascular endothelial) and HFF (Human foreskin fibroblasts) cells (1-6).
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The interaction of KSHV with its specific entry receptors leads to the formation of a
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multimolecular receptor complex consisting of integrins, xCT and EphA2 (2, 4, 7). Receptor
78
engagement and multimolecular receptor complex formation results in auto phosphorylation of
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FAK, and the activation of Src, PI3-K and Rho GTPases, and all these molecules are targeted to
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specific entry sites on the plasma membrane (8-10). Our previous studies have demonstrated that
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the signal transduction pathways induced by KSHV and the consequent activation of their
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downstream molecules plays a central role in coordinating the actin dynamics and the membrane
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protein assembly required for the successful entry of the virus into the cytoplasm (8-10). The
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endocytosed viral particles are then transported towards the nuclear periphery along the
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microtubules by utilizing dynein motor proteins to deliver its DNA content into the nucleus (11).
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KSHV utilizes different endocytic pathways to enter different cell types (12-17). In HFF
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cells, primary B cells, and 293 cells, clathrin dependent endocytosis is the predominant pathway
88
of entry (12-14), whereas in HMVEC-d cells entry occurs by bleb associated macropinocytosis
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(15, 16, 18). Bleb associated macropinocytosis begins with a remarkable set of events including
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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
4
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interaction with myosin IIA light chain (MLC) plays a significant role in blebbing and that
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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
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the lipid raft and ubiquitination of the α3β1 and αVβ3 receptors which are critical determinants
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of the macropinocytic entry, trafficking, and productive infection of KSHV (19).
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Ubiquitination of receptors and the adaptor proteins such as c-Cbl serves to facilitate the
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endocytosis of receptors and their sorting from membrane to lysosomes and subsequent
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degradation (20, 21). In mammalian cells, the Endosomal Sorting Complexes Required for
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Transport (ESCRT) helps in sorting ubiquitinated proteins and their delivery into lysosomes. The
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ESCRT machinery consists a family of four complexes, ESCRT-0, -I, -II, and –III, which
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sequentially assemble on the endosomal membrane. The ESCRT-0 component Hrs (Hepatocyte
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growth factor (HGF)-regulated tyrosine kinase substrate) acts upstream to ESCRT complexes
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and can recruit ESCRT-I to the endosomal membranes. This complex subsequently recruits
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ESCRT-II and ESCRT-III complexes, two important complexes required for the sorting of
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ubiquitinated proteins into late endosomes (22-25).
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Hrs protein contains several domains: an N-terminal VHS-domain (Vps27p, Hrs, STAM),
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FYVE domain, an ubiquitin interacting motif, a proline-rich domain, two coiled-coil domains,
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and a C-terminal clathrin binding domain (26). The functional role of the different domains of
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Hrs includes protein-protein interaction, cellular localization, cargo sorting, and endosome
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motility (24, 25, 27, 28). Although the function of Hrs in intracellular trafficking and signal
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transduction is well established, its role in macropinocytosis remains unclear. In the present
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study, we provide evidence that Hrs is recruited to the plasma membrane early during KSHV
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infection which is required for the virus entry by macropinocytosis. Interestingly, we found that
5
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Hrs interacts with the Rho-associated protein kinase ROCK1, which in turn activates MLC and
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the intracellular pH-regulating protein NHE1 to promote actomyosin contraction and
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intracellular pH changes required for macropinocytosis.
118 119 120 121 122 123 124 125 126 127 128 129 130 131 132 133 134 135 136 137
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MATERIALS AND METHODS
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Cells and virus: Primary human dermal microvascular enodthelial cells (HMVEC-d CC-2543;
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Clonetics, Walkersville, MD) were grown in EBM2 medium with growth factors (Cambrex,
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Walkersville, MD). BCBL-1 cell culture, supernatant collection after TPA induction, and virus
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purification procedures were performed as described previously (1). The isolation of KSHV
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DNA from the virus, and the determination of viral copy numbers by real-time DNA PCR using
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KSHV ORF73 gene-specific primers were performed as previously described (29). All infections
145
were carried out with 20 DNA copies/cell (multiplicity of infection-MOI) of KSHV, unless
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stated otherwise.
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Antibodies and reagents: Antibodies and reagents used for this study include: Anti-ROCK1
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rabbit monoclonal antibody, anti-phospho MLC rabbit polyclonal antibody, anti-Na, K-ATPase
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rabbit polyclonal antibody (Cell Signaling Technology); anti-Hrs rabbit polyclonal antibody
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(Sigma); anti-NHE1 mouse monoclonal antibody (Santa Cruz); anti-calnexin rabbit monoclonal
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antibody, mouse anti-phospho serine antibody (Abcam); anti-α-actinin-4 rabbit monoclonal
152
antibody (Millipore); rabbit anti-gB and mouse anti-gpK8.1A antibodies were created in Dr.
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Chandran’s laboratory (30, 31); anti-rabbit and anti-mouse antibodies linked to horseradish
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peroxidase (KPL Inc., Gaithersburg, Md.); Texas Red conjugated dextran, Alexa 594 conjugated
155
phalloidin and anti-rabbit and anti-mouse secondary antibodies conjugated to Alexa 488 or Alexa
156
594 (Invitrogen); protein A and G–Sepharose CL-4B beads (Amersham Pharmacia Biotech,
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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
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purchased from Thermoscientific. The Hrs shRNA lentiviral plasmids were purified from the
7
161
bacterial cultures (Invitrogen) and the lentivial particles were produced by transfection with a
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four-plasmid system, as previously described (32). Briefly, HEK 293T cells were transiently
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transfected with shRNA lentiviral constructs and the plasmid packaging system (Gag-Pol, Rev
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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
166
that TRCN00000037897 and TRCN00000037898 had the highest knockdown efficiency among
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the five shRNAs. Therefore, we used Hrs shRNAs created with these two plasmids (Hrs-
168
shRNA1 and Hrs-shRNA2) for the experiments in this manuscript. We used Hrs shRNA1 for all
169
experiments except figure 1A and 1B where we used both Hrs shRNA1 and 2. For negative
170
control, a nontargeting shRNA lentiviral pool was used.
171
Western blotting: Cells lysates were prepared in RIPA lysis buffer (15 mM NaCl, 1 mM
172
MgCl2, 1 mM MnCl2, 2 mM CaCl2, 2 mM phenylmethylsulfonyl fluoride, and protease
173
inhibitor mixture (Sigma). The lysates were centrifuged at 12,000 rpm for 15 min at 4°C and the
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protein concentration was measured with BCA reagent. Equal amounts of proteins from each
175
sample were separated on SDS-PAGE and transferred to nitrocellulose membranes. Membranes
176
were probed with the indicated primary antibodies and detected by incubation with species-
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specific HRP-conjugated secondary antibodies. Immunoreactive protein bands were visualized
178
and imaged by enhanced chemiluminescent HRP substrate kit (Pierce, Rockford, IL). The bands
179
were scanned and densitometric quantification was performed using the FluorChem FC2 and
180
Alpha-Imager Systems (Alpha Innotech Corporation, San Leonardo, CA).
181
Immunoprecipitation: Cell lysates were immunoprecipitated for 2h with appropriate antibodies
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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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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%
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paraformaldehyde after infection and permeabilized with 0.2% Triton X-100. The cells were
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blocked with Image-iT FX signal enhancer (Invitrogen) for 20 min, and then incubated with
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primary antibodies against the specific proteins and subsequently stained with secondary
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antibodies conjugated to Alexa 488 or 594. The stained cells were mounted in mounting medium
191
with DAPI (4′,6′-diamidino-2-phenylindole) (Invitrogen) and visualized with a Nikon 80i
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fluorescent microscope equipped with Metamorph digital imaging software.
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Proximity Ligation Assay: Proximity Ligation Assay (PLA) was performed using the Duolink
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in situ detection kit (Sigma) as per the manufacturer’s instructions (33, 34). Briefly, uninfected
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and infected cells fixed with 4% paraformaldehyde solution for 15 min, permeabilized with
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Triton X- 100 for 5 min and washed with PBS, and incubated for 1h with the Duolink blocking
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buffer. The cells were then incubated with pairs of primary antibodies in Duolink antibody
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diluent solution and then labeled with Duolink PLA PLUS and MINUS probes for 1h at 37°C.
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This was followed by hybridization, ligation, amplification, and detection using a red fluorescent
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probe. The red fluorescent fluorophore-tagged oligonucleotides were visualized using a Nikon
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Eclipse 80i microscope equipped with Metamorph digital imaging software.
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Determination of KSHV entry by real-time DNA PCR: Target cells were infected with 20
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MOI of KSHV at 37°C for 2h. After infection, the cells were washed with HBSS, and treated
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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,
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Valencia, CA) and real time DNA PCR was carried out using primer sets described previously
9
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
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gene cloned in the pGEM-T vector (Promega) was used for the external standard.
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Determination of KSHV gene expression by real-time RT-PCR: Isolation of total RNA from
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cells, reverse transcription, and qPCR analysis were carried out using primer sets described
212
previously. Briefly, total RNA was isolated from the lysate using RNeasy kit (QIAGEN),
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quantified, and the ORF73 expression was detected by real-time reverse transcription PCR using
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specific
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dehydrogenase) gene expression was used to normalize the expression levels of ORF73.
216
Isolation of membrane fraction: Subcellular fractionation and cell membrane preparation were
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performed as described previously (18, 35). Briefly, cells were homogenized with a Dounce-
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homogenizer in homogenization buffer (250 mM sucrose, 20 mM HEPES, 10 mM KCl, 1 mM
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EDTA, 1 mM EGTA and protease inhibitors). The homogenate was centrifuged at 3,000 rpm for
220
5 min, and the post-nuclear supernatant was centrifuged at 8,000 rpm for 5 min at 4ºC. The
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supernatant was again centrifuged at 40,000 rpm for 1h at 4ºC, and the cytosolic (supernatant)
222
and membrane (pellet) fractions were separated. The membrane fractions were lysed in RIPA
223
buffer and used for Western blot.
224
Analysis of dextran uptake: For the dextran uptake study, HMVEC-d cells were incubated with
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Texas Red labeled dextran (40 kDa, 0.5 mg/ml; Invitrogen) and KSHV for 30 min. The cells
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.
primers
and
TaqMan
probes
(29).
GAPDH
(glyceraldehyde-3-phosphate
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
10
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each were counted for each experiment and the results displayed as percentage of dextran
231
positive cells.
232
Intracellular pH measurement: An equal number of cells in 96 well plates were loaded with
233
BCECF-AM (bis-(2-carboxy-ethyl)-5-(and-6)-carboxyfluorescein, acetoxy-methyl, 10 μM) in
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HBSS for 30 min. The cells were washed with HBSS to remove the free dye, and then infected
235
with KSHV for 2, 5, and 10 min at 37°C. Immediately after infection, BCECF fluorescence
236
excited with a 485/20 filter and the emission was measured with a 528/20 filter on a BioTek
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Synergy2 HT microplate reader. The average readings for each time point were calculated and
238
the changes in pH expressed as relative fluorescent units.
239
Statistical analyses.
240
Statistical significance was calculated using a two tailed Student’s t-test. P < 0.05 was
241
considered significant.
242 243 244 245 246 247 248 249 250 251 252
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RESULTS
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Hrs knockdown inhibits KSHV entry and gene expression in HMVEC-d cells.
255
To explore the role of Hrs in KSHV infection, we performed Hrs knockdown in
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HMVEC-d cells using two different lentiviral Hrs shRNAs (Hrs shRNA1 and 2). HMVEC-d
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cells were transduced with control and Hrs shRNAs and the efficiency of knockdown was
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determined 48h after transduction by Western blotting with anti-Hrs antibody. As shown in
259
figure 1A, both the Hrs shRNA1 and Hrs shRNA2 completely knockdown endogenous Hrs
260
expression. To address whether Hrs inhibition affects the entry of KSHV, control and Hrs
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shRNA transduced HMVEC-d cells were infected with KSHV for 2h, DNA was isolated and the
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internalized viral DNA copy numbers were determined by measuring viral ORF73 DNA copy
263
numbers by real-time DNA PCR. Compared to the control shRNA transduced cells (copy
264
number 18009 ± 938), the entry of KSHV was significantly reduced (~65-70 %) in both Hrs
265
shRNA1 (4427 ± 49) and Hrs shRNA2 (5330± 214) knockdown cells. Heparin-treated virus,
266
which cannot bind and enter the cells, was used as a control (Fig. 1B). We next investigated
267
whether Hrs knockdown also results in decreased viral gene expression. Control and Hrs shRNA
268
transduced HMVEC-d cells were infected with KSHV for 24h, and viral gene expression of the
269
latency associated ORF73 gene was determined by real-time RT-PCR analysis. Similar to the
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entry results, the expression of ORF73 gene in both Hrs shRNA1 (copy number 7742 ± 2180)
271
and shRNA2 (8364 ± 963) was reduced compared to control cells (41135 ± 680). Hrs shRNA
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cells showed 70–80% inhibition of ORF73 gene expression (Fig. 1C), suggesting that the
273
decreased viral entry results in decreased gene expression in Hrs knockdown cells.
274
Since macropinocytosis is the major pathway of KSHV entry leading to a productive
275
infection in HMVEC-d cells (16, 18), we asked whether the decreased entry in Hrs shRNA cells
12
276
is correlated with macropinocytosis. The macropinocytosis of KSHV in the control and Hrs
277
shRNA cells was assessed by incubating the cells with the macropinocytosis marker dextran
278
(Texas Red labeled) in the presence or absence of virus for 30 min. Compared to the uninfected
279
cells, KSHV infection resulted in a considerable increase in dextran uptake in the control shRNA
280
transduced cells (Fig. 1D, upper and middle panel). However, Hrs shRNA cells incubated with
281
dextran and KSHV showed a substantial decrease in dextran uptake compared to the control
282
shRNA cells (Fig. 1D, middle and lower panel). Quantitative analysis performed by counting the
283
number of dextran positive cells in the control and Hrs shRNA cells confirmed the decreased
284
dextran uptake in Hrs shRNA cells (Fig. 1E). This result suggested that Hrs plays a role in
285
macropinocytosis mediated entry of KSHV into endothelial cells.
286
In addition to macropinocytic mode of entry in endothelial cells, KSHV is known to enter
287
by clathrin mediated endocytosis in other target cells such as HFF, HEK293 and B-cells. To
288
assess whether Hrs is involved in clathrin mediated endocytosis of KSHV, we examined the
289
effect of Hrs knockdown on the entry of KSHV in HFF cells (Fig.1.F). HFF cells transduced
290
with control or lentiviral Hrs shRNA were infected with KSHV for 2h, and the entry of KSHV
291
was determined by measuring viral ORF73 DNA copy numbers by real-time DNA PCR.
292
Interestingly, real-time PCR analysis demonstrated that Hrs knockdown had no significant effect
293
on the entry of virus in HFF cells (Fig.1.F). These results suggest that Hrs plays a specific role in
294
regulating macropinocytosis-mediated entry of KSHV, but not in clathrin mediated entry.
295
Hrs localizes to the plasma membrane of KSHV infected cells.
296
To ascertain the mechanism of Hrs function in the entry process of KSHV, we next
297
analyzed the cellular localization of endogenous Hrs protein in the infected cells by
298
immunofluorescent labeling. HMVEC-d cells were infected with KSHV for 5, 10 and 30 min
13
299
and then stained for the KSHV envelope glycoprotein gB and host Hrs protein. As shown in
300
figure 2A, Hrs was distributed throughout the cytoplasm in the uninfected cells. In contrast, at 5
301
and 10 min post-KSHV infection, Hrs localized predominantly in the plasma membranes (Fig.
302
2A, arrow heads), and the KSHV gB staining (Fig. 2A, block arrows) was also observed in
303
similar regions of the infected cell membrane. After 10 and 30 min of infection, in addition to
304
membrane staining, we observed cytoplasmic staining as well as colocalization of Hrs with
305
KSHV-gB in the infected cells (Fig. 2A, yellow spots).
306
To confirm the membrane localization of Hrs in the infected cells, plasma membranes
307
were isolated from uninfected and infected cells and tested for the presence of Hrs using a
308
specific anti-Hrs antibody. As shown in figure 2B, first panel, lanes 1-5, detection of Hrs in the
309
plasma membrane fractions confirmed the membrane localization with a pattern similar to that of
310
the IFA results in figure 2A. However, in the cells infected with heparin treated virus in which
311
KSHV cannot bind and enter the cells (36), we did not observe the induction of membrane
312
translocation (Fig. 2B, lane 6). This indicates that KSHV binding and interaction with receptors
313
are essential for the translocation of Hrs to the membrane. The purity of the membrane fraction
314
was assessed by the detection of membrane positive marker Na, K-ATPase in the purified
315
membrane fractions (Fig. 2B, second panel). Additionally, the absence of ER membrane marker
316
calnexin by Western blot indicated that the fraction was not contaminated with the internal
317
membrane proteins (Fig. 2B, third panel). Western blot of whole cell lysate using Hrs antibody
318
showed no variation in the total Hrs protein level (Fig. 2B, fourth panel). The recruitment of Hrs
319
to the plasma membrane of the infected cells indicated that Hrs may be involved in the initial
320
stages of macropinosome formation and cytoplasmic entry of the virus.
14
321
To further evaluate whether Hrs affects the cytoplasmic entry of KSHV, we examined the
322
localization of viral particles in control and Hrs shRNA cells. Cells infected with KSHV for 30
323
min were stained with rhodamine-phalloidin for filamentous actin and KSHV-gB to visualize the
324
localization of KSHV. In control cells infected with KSHV, virus particles were detected at the
325
nuclear periphery by 30 min post-infection, whereas in Hrs shRNA cells the virus particles failed
326
to enter the cells and were found stuck at the cell surface (Fig. 2C). This result indicated that
327
there is a significant correlation between the membrane recruitment of Hrs and the early stages
328
of KSHV entry by macropinocytosis.
329
Hrs localizes to the membrane by interacting with α-actinin-4 in the infected cells.
330
Macropinocytosis is an actin driven process that depends on actin polymerization and
331
membrane ruffling (37). As membrane associated Hrs affects the macropinocytosis of KSHV, we
332
rationalized that membrane localization may be achieved through the interaction of Hrs with an
333
actin binding protein that is localized to the plasma membrane. It has been shown that Hrs is a
334
binding partner of α-actinin-4 (38), a cytoskeleton scaffolding protein that transiently associates
335
with macropinosomes (39). Moreover, we have reported before that α-actinin-4 formed a
336
complex with the membrane associated proteins EphA2, myosin IIA, and CIB1 in KSHV
337
infected cells (15). Since α-actinin-4 primarily cross-links F-actin and forms α-actinin-4-F-actin
338
network surrounding macropinosomes, we hypothesized that the interaction of α-actinin-4 with
339
Hrs may localize Hrs to the membrane and influence macropinosome formation. To test this
340
hypothesis, we first determined whether KSHV infection results in the recruitment of α-actinin-4
341
to the membrane of infected cells. Immunofluorescence analysis of uninfected and KSHV
342
infected cells with α-actinin-4 and KSHV envelope glycoprotein gpK8.1A antibody
343
demonstrated that the virus particles colocalize with α-actinin-4 at 10 min post infection at the
15
344
membrane of infected cells (Fig. 3A). To evaluate whether α-actinin-4 and Hrs could associate
345
with each other at the membrane, we examined the colocalization of Hrs and α-actinin-4 in
346
KSHV infected cells. Immunofluorescent analysis of uninfected and cells infected with KSHV
347
for 10 min demonstrated that Hrs colocalizes with α-actinin-4 at the membrane of infected cells
348
(Fig. 3B).
349
To visualize that the interaction of α-actinin-4 with Hrs leads to the recruitment of Hrs to
350
the cell membrane of infected cells, we used a proximity ligation assay (PLA) with antibodies
351
against Hrs and α-actinin-4. PLA enables the identification of the association between two
352
proteins which are located in close proximity (
1
ESCRT-0 Component Hrs Promotes Macropinocytosis of Kaposi’s Sarcoma-
2
Associated Herpesvirus in Human Dermal Microvascular Endothelial Cells
3 4
Mohanan Valiya Veettil#, Binod Kumar, Mairaj Ahmed Ansari, Dipanjan Dutta, Jawed
5
Iqbal, Olsi Gjyshi, Virginie Bottero and Bala Chandran
6 7
H. M. Bligh Cancer Research Laboratories, Department of Microbiology and
8
Immunology, Chicago Medical School, Rosalind Franklin University of Medicine and
9
Science, North Chicago, Illinois 60064, USA.
10 11
Running title: Role of Hrs in macropinocytosis of KSHV
12 13
# Corresponding author.
14
Mailing Address:
15
Department of Microbiology and Immunology, Chicago Medical School,
16
Rosalind Franklin University of Medicine and Science,
17
3333 Green Bay Road, North Chicago, IL 60064.
18
Phone (847) 578-8700x7716; Fax (847) 578-3349.
19
E-mail: [email protected]
20 21 22 1
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.
28
The Endosomal Sorting Complexes Required for Transport (ESCRT) proteins which include
29
ESCRT-0, -I, -II, and -III play a central role in endosomal trafficking and sorting of internalized
30
and ubiquitinated receptors. Here, we examined the role of ESCRT-0 component Hrs
31
(Hepatocyte growth factor regulated tyrosine kinase substrate) in KSHV entry into HMVEC-d
32
cells by macropinocytosis. Knockdown of Hrs by shRNA transduction results in significant
33
decrease in KSHV entry and viral gene expression. Immunofluorescence analysis (IFA) and
34
plasma membrane isolation and proximity ligation assay (PLA) demonstrate the translocation of
35
Hrs from the cytosol to the plasma membranes of infected cells and association with α-actinin-4.
36
In addition, infection induces the plasma membrane translocation and activation of the
37
serine/threonine kinase ROCK1, a downstream target of the RhoA GTPase. Hrs knockdown
38
reduces these associations suggesting that the recruitment of ROCK1 is an Hrs mediated event.
39
Interaction between Hrs and ROCK1 is essential for the ROCK1 induced phosphorylation of
40
NHE1 (Na+/H+ exchanger 1) involved in the regulation of intracellular pH. Thus, our studies
41
demonstrate the plasma membrane association of ESCRT protein Hrs during macropinocytosis
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.
45
2
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
51
demonstrate for the first time that upon KSHV infection the endogenous Hrs localizes to the
52
plasma membrane and the membrane associated Hrs facilitates assembly of signaling molecules,
53
macropinocytosis and virus entry. Hrs recruits ROCK1 to the membrane which is required for
54
the activation of NHE1 and an increase in submembraneous intracellular pH occurring during
55
macropinocytosis. These studies demonstrate that the localization of Hrs from the cytosol to the
56
plasma membrane is important for coupling membrane dynamics to the cytosolic signaling
57
events during macropinocytosis of KSHV.
58 59 60 61 62 63 64 65 66 67 68 3
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
74
non-integrins CD98/xCT, and EphA2 to enter the adherent target cells such as HMVEC-d
75
(Human dermal microvascular endothelial) and HFF (Human foreskin fibroblasts) cells (1-6).
76
The interaction of KSHV with its specific entry receptors leads to the formation of a
77
multimolecular receptor complex consisting of integrins, xCT and EphA2 (2, 4, 7). Receptor
78
engagement and multimolecular receptor complex formation results in auto phosphorylation of
79
FAK, and the activation of Src, PI3-K and Rho GTPases, and all these molecules are targeted to
80
specific entry sites on the plasma membrane (8-10). Our previous studies have demonstrated that
81
the signal transduction pathways induced by KSHV and the consequent activation of their
82
downstream molecules plays a central role in coordinating the actin dynamics and the membrane
83
protein assembly required for the successful entry of the virus into the cytoplasm (8-10). The
84
endocytosed viral particles are then transported towards the nuclear periphery along the
85
microtubules by utilizing dynein motor proteins to deliver its DNA content into the nucleus (11).
86
KSHV utilizes different endocytic pathways to enter different cell types (12-17). In HFF
87
cells, primary B cells, and 293 cells, clathrin dependent endocytosis is the predominant pathway
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
4
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).
97
Ubiquitination of receptors and the adaptor proteins such as c-Cbl serves to facilitate the
98
endocytosis of receptors and their sorting from membrane to lysosomes and subsequent
99
degradation (20, 21). In mammalian cells, the Endosomal Sorting Complexes Required for
100
Transport (ESCRT) helps in sorting ubiquitinated proteins and their delivery into lysosomes. The
101
ESCRT machinery consists a family of four complexes, ESCRT-0, -I, -II, and –III, which
102
sequentially assemble on the endosomal membrane. The ESCRT-0 component Hrs (Hepatocyte
103
growth factor (HGF)-regulated tyrosine kinase substrate) acts upstream to ESCRT complexes
104
and can recruit ESCRT-I to the endosomal membranes. This complex subsequently recruits
105
ESCRT-II and ESCRT-III complexes, two important complexes required for the sorting of
106
ubiquitinated proteins into late endosomes (22-25).
107
Hrs protein contains several domains: an N-terminal VHS-domain (Vps27p, Hrs, STAM),
108
FYVE domain, an ubiquitin interacting motif, a proline-rich domain, two coiled-coil domains,
109
and a C-terminal clathrin binding domain (26). The functional role of the different domains of
110
Hrs includes protein-protein interaction, cellular localization, cargo sorting, and endosome
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
5
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.
118 119 120 121 122 123 124 125 126 127 128 129 130 131 132 133 134 135 136 137
6
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
143
DNA from the virus, and the determination of viral copy numbers by real-time DNA PCR using
144
KSHV ORF73 gene-specific primers were performed as previously described (29). All infections
145
were carried out with 20 DNA copies/cell (multiplicity of infection-MOI) of KSHV, unless
146
stated otherwise.
147
Antibodies and reagents: Antibodies and reagents used for this study include: Anti-ROCK1
148
rabbit monoclonal antibody, anti-phospho MLC rabbit polyclonal antibody, anti-Na, K-ATPase
149
rabbit polyclonal antibody (Cell Signaling Technology); anti-Hrs rabbit polyclonal antibody
150
(Sigma); anti-NHE1 mouse monoclonal antibody (Santa Cruz); anti-calnexin rabbit monoclonal
151
antibody, mouse anti-phospho serine antibody (Abcam); anti-α-actinin-4 rabbit monoclonal
152
antibody (Millipore); rabbit anti-gB and mouse anti-gpK8.1A antibodies were created in Dr.
153
Chandran’s laboratory (30, 31); anti-rabbit and anti-mouse antibodies linked to horseradish
154
peroxidase (KPL Inc., Gaithersburg, Md.); Texas Red conjugated dextran, Alexa 594 conjugated
155
phalloidin and anti-rabbit and anti-mouse secondary antibodies conjugated to Alexa 488 or Alexa
156
594 (Invitrogen); protein A and G–Sepharose CL-4B beads (Amersham Pharmacia Biotech,
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
7
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
166
that TRCN00000037897 and TRCN00000037898 had the highest knockdown efficiency among
167
the five shRNAs. Therefore, we used Hrs shRNAs created with these two plasmids (Hrs-
168
shRNA1 and Hrs-shRNA2) for the experiments in this manuscript. We used Hrs shRNA1 for all
169
experiments except figure 1A and 1B where we used both Hrs shRNA1 and 2. For negative
170
control, a nontargeting shRNA lentiviral pool was used.
171
Western blotting: Cells lysates were prepared in RIPA lysis buffer (15 mM NaCl, 1 mM
172
MgCl2, 1 mM MnCl2, 2 mM CaCl2, 2 mM phenylmethylsulfonyl fluoride, and protease
173
inhibitor mixture (Sigma). The lysates were centrifuged at 12,000 rpm for 15 min at 4°C and the
174
protein concentration was measured with BCA reagent. Equal amounts of proteins from each
175
sample were separated on SDS-PAGE and transferred to nitrocellulose membranes. Membranes
176
were probed with the indicated primary antibodies and detected by incubation with species-
177
specific HRP-conjugated secondary antibodies. Immunoreactive protein bands were visualized
178
and imaged by enhanced chemiluminescent HRP substrate kit (Pierce, Rockford, IL). The bands
179
were scanned and densitometric quantification was performed using the FluorChem FC2 and
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
8
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
189
primary antibodies against the specific proteins and subsequently stained with secondary
190
antibodies conjugated to Alexa 488 or 594. The stained cells were mounted in mounting medium
191
with DAPI (4′,6′-diamidino-2-phenylindole) (Invitrogen) and visualized with a Nikon 80i
192
fluorescent microscope equipped with Metamorph digital imaging software.
193
Proximity Ligation Assay: Proximity Ligation Assay (PLA) was performed using the Duolink
194
in situ detection kit (Sigma) as per the manufacturer’s instructions (33, 34). Briefly, uninfected
195
and infected cells fixed with 4% paraformaldehyde solution for 15 min, permeabilized with
196
Triton X- 100 for 5 min and washed with PBS, and incubated for 1h with the Duolink blocking
197
buffer. The cells were then incubated with pairs of primary antibodies in Duolink antibody
198
diluent solution and then labeled with Duolink PLA PLUS and MINUS probes for 1h at 37°C.
199
This was followed by hybridization, ligation, amplification, and detection using a red fluorescent
200
probe. The red fluorescent fluorophore-tagged oligonucleotides were visualized using a Nikon
201
Eclipse 80i microscope equipped with Metamorph digital imaging software.
202
Determination of KSHV entry by real-time DNA PCR: Target cells were infected with 20
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
9
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
212
previously. Briefly, total RNA was isolated from the lysate using RNeasy kit (QIAGEN),
213
quantified, and the ORF73 expression was detected by real-time reverse transcription PCR using
214
specific
215
dehydrogenase) gene expression was used to normalize the expression levels of ORF73.
216
Isolation of membrane fraction: Subcellular fractionation and cell membrane preparation were
217
performed as described previously (18, 35). Briefly, cells were homogenized with a Dounce-
218
homogenizer in homogenization buffer (250 mM sucrose, 20 mM HEPES, 10 mM KCl, 1 mM
219
EDTA, 1 mM EGTA and protease inhibitors). The homogenate was centrifuged at 3,000 rpm for
220
5 min, and the post-nuclear supernatant was centrifuged at 8,000 rpm for 5 min at 4ºC. The
221
supernatant was again centrifuged at 40,000 rpm for 1h at 4ºC, and the cytosolic (supernatant)
222
and membrane (pellet) fractions were separated. The membrane fractions were lysed in RIPA
223
buffer and used for Western blot.
224
Analysis of dextran uptake: For the dextran uptake study, HMVEC-d cells were incubated with
225
Texas Red labeled dextran (40 kDa, 0.5 mg/ml; Invitrogen) and KSHV for 30 min. The cells
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.
primers
and
TaqMan
probes
(29).
GAPDH
(glyceraldehyde-3-phosphate
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
10
230
each were counted for each experiment and the results displayed as percentage of dextran
231
positive cells.
232
Intracellular pH measurement: An equal number of cells in 96 well plates were loaded with
233
BCECF-AM (bis-(2-carboxy-ethyl)-5-(and-6)-carboxyfluorescein, acetoxy-methyl, 10 μM) in
234
HBSS for 30 min. The cells were washed with HBSS to remove the free dye, and then infected
235
with KSHV for 2, 5, and 10 min at 37°C. Immediately after infection, BCECF fluorescence
236
excited with a 485/20 filter and the emission was measured with a 528/20 filter on a BioTek
237
Synergy2 HT microplate reader. The average readings for each time point were calculated and
238
the changes in pH expressed as relative fluorescent units.
239
Statistical analyses.
240
Statistical significance was calculated using a two tailed Student’s t-test. P < 0.05 was
241
considered significant.
242 243 244 245 246 247 248 249 250 251 252
11
253
RESULTS
254
Hrs knockdown inhibits KSHV entry and gene expression in HMVEC-d cells.
255
To explore the role of Hrs in KSHV infection, we performed Hrs knockdown in
256
HMVEC-d cells using two different lentiviral Hrs shRNAs (Hrs shRNA1 and 2). HMVEC-d
257
cells were transduced with control and Hrs shRNAs and the efficiency of knockdown was
258
determined 48h after transduction by Western blotting with anti-Hrs antibody. As shown in
259
figure 1A, both the Hrs shRNA1 and Hrs shRNA2 completely knockdown endogenous Hrs
260
expression. To address whether Hrs inhibition affects the entry of KSHV, control and Hrs
261
shRNA transduced HMVEC-d cells were infected with KSHV for 2h, DNA was isolated and the
262
internalized viral DNA copy numbers were determined by measuring viral ORF73 DNA copy
263
numbers by real-time DNA PCR. Compared to the control shRNA transduced cells (copy
264
number 18009 ± 938), the entry of KSHV was significantly reduced (~65-70 %) in both Hrs
265
shRNA1 (4427 ± 49) and Hrs shRNA2 (5330± 214) knockdown cells. Heparin-treated virus,
266
which cannot bind and enter the cells, was used as a control (Fig. 1B). We next investigated
267
whether Hrs knockdown also results in decreased viral gene expression. Control and Hrs shRNA
268
transduced HMVEC-d cells were infected with KSHV for 24h, and viral gene expression of the
269
latency associated ORF73 gene was determined by real-time RT-PCR analysis. Similar to the
270
entry results, the expression of ORF73 gene in both Hrs shRNA1 (copy number 7742 ± 2180)
271
and shRNA2 (8364 ± 963) was reduced compared to control cells (41135 ± 680). Hrs shRNA
272
cells showed 70–80% inhibition of ORF73 gene expression (Fig. 1C), suggesting that the
273
decreased viral entry results in decreased gene expression in Hrs knockdown cells.
274
Since macropinocytosis is the major pathway of KSHV entry leading to a productive
275
infection in HMVEC-d cells (16, 18), we asked whether the decreased entry in Hrs shRNA cells
12
276
is correlated with macropinocytosis. The macropinocytosis of KSHV in the control and Hrs
277
shRNA cells was assessed by incubating the cells with the macropinocytosis marker dextran
278
(Texas Red labeled) in the presence or absence of virus for 30 min. Compared to the uninfected
279
cells, KSHV infection resulted in a considerable increase in dextran uptake in the control shRNA
280
transduced cells (Fig. 1D, upper and middle panel). However, Hrs shRNA cells incubated with
281
dextran and KSHV showed a substantial decrease in dextran uptake compared to the control
282
shRNA cells (Fig. 1D, middle and lower panel). Quantitative analysis performed by counting the
283
number of dextran positive cells in the control and Hrs shRNA cells confirmed the decreased
284
dextran uptake in Hrs shRNA cells (Fig. 1E). This result suggested that Hrs plays a role in
285
macropinocytosis mediated entry of KSHV into endothelial cells.
286
In addition to macropinocytic mode of entry in endothelial cells, KSHV is known to enter
287
by clathrin mediated endocytosis in other target cells such as HFF, HEK293 and B-cells. To
288
assess whether Hrs is involved in clathrin mediated endocytosis of KSHV, we examined the
289
effect of Hrs knockdown on the entry of KSHV in HFF cells (Fig.1.F). HFF cells transduced
290
with control or lentiviral Hrs shRNA were infected with KSHV for 2h, and the entry of KSHV
291
was determined by measuring viral ORF73 DNA copy numbers by real-time DNA PCR.
292
Interestingly, real-time PCR analysis demonstrated that Hrs knockdown had no significant effect
293
on the entry of virus in HFF cells (Fig.1.F). These results suggest that Hrs plays a specific role in
294
regulating macropinocytosis-mediated entry of KSHV, but not in clathrin mediated entry.
295
Hrs localizes to the plasma membrane of KSHV infected cells.
296
To ascertain the mechanism of Hrs function in the entry process of KSHV, we next
297
analyzed the cellular localization of endogenous Hrs protein in the infected cells by
298
immunofluorescent labeling. HMVEC-d cells were infected with KSHV for 5, 10 and 30 min
13
299
and then stained for the KSHV envelope glycoprotein gB and host Hrs protein. As shown in
300
figure 2A, Hrs was distributed throughout the cytoplasm in the uninfected cells. In contrast, at 5
301
and 10 min post-KSHV infection, Hrs localized predominantly in the plasma membranes (Fig.
302
2A, arrow heads), and the KSHV gB staining (Fig. 2A, block arrows) was also observed in
303
similar regions of the infected cell membrane. After 10 and 30 min of infection, in addition to
304
membrane staining, we observed cytoplasmic staining as well as colocalization of Hrs with
305
KSHV-gB in the infected cells (Fig. 2A, yellow spots).
306
To confirm the membrane localization of Hrs in the infected cells, plasma membranes
307
were isolated from uninfected and infected cells and tested for the presence of Hrs using a
308
specific anti-Hrs antibody. As shown in figure 2B, first panel, lanes 1-5, detection of Hrs in the
309
plasma membrane fractions confirmed the membrane localization with a pattern similar to that of
310
the IFA results in figure 2A. However, in the cells infected with heparin treated virus in which
311
KSHV cannot bind and enter the cells (36), we did not observe the induction of membrane
312
translocation (Fig. 2B, lane 6). This indicates that KSHV binding and interaction with receptors
313
are essential for the translocation of Hrs to the membrane. The purity of the membrane fraction
314
was assessed by the detection of membrane positive marker Na, K-ATPase in the purified
315
membrane fractions (Fig. 2B, second panel). Additionally, the absence of ER membrane marker
316
calnexin by Western blot indicated that the fraction was not contaminated with the internal
317
membrane proteins (Fig. 2B, third panel). Western blot of whole cell lysate using Hrs antibody
318
showed no variation in the total Hrs protein level (Fig. 2B, fourth panel). The recruitment of Hrs
319
to the plasma membrane of the infected cells indicated that Hrs may be involved in the initial
320
stages of macropinosome formation and cytoplasmic entry of the virus.
14
321
To further evaluate whether Hrs affects the cytoplasmic entry of KSHV, we examined the
322
localization of viral particles in control and Hrs shRNA cells. Cells infected with KSHV for 30
323
min were stained with rhodamine-phalloidin for filamentous actin and KSHV-gB to visualize the
324
localization of KSHV. In control cells infected with KSHV, virus particles were detected at the
325
nuclear periphery by 30 min post-infection, whereas in Hrs shRNA cells the virus particles failed
326
to enter the cells and were found stuck at the cell surface (Fig. 2C). This result indicated that
327
there is a significant correlation between the membrane recruitment of Hrs and the early stages
328
of KSHV entry by macropinocytosis.
329
Hrs localizes to the membrane by interacting with α-actinin-4 in the infected cells.
330
Macropinocytosis is an actin driven process that depends on actin polymerization and
331
membrane ruffling (37). As membrane associated Hrs affects the macropinocytosis of KSHV, we
332
rationalized that membrane localization may be achieved through the interaction of Hrs with an
333
actin binding protein that is localized to the plasma membrane. It has been shown that Hrs is a
334
binding partner of α-actinin-4 (38), a cytoskeleton scaffolding protein that transiently associates
335
with macropinosomes (39). Moreover, we have reported before that α-actinin-4 formed a
336
complex with the membrane associated proteins EphA2, myosin IIA, and CIB1 in KSHV
337
infected cells (15). Since α-actinin-4 primarily cross-links F-actin and forms α-actinin-4-F-actin
338
network surrounding macropinosomes, we hypothesized that the interaction of α-actinin-4 with
339
Hrs may localize Hrs to the membrane and influence macropinosome formation. To test this
340
hypothesis, we first determined whether KSHV infection results in the recruitment of α-actinin-4
341
to the membrane of infected cells. Immunofluorescence analysis of uninfected and KSHV
342
infected cells with α-actinin-4 and KSHV envelope glycoprotein gpK8.1A antibody
343
demonstrated that the virus particles colocalize with α-actinin-4 at 10 min post infection at the
15
344
membrane of infected cells (Fig. 3A). To evaluate whether α-actinin-4 and Hrs could associate
345
with each other at the membrane, we examined the colocalization of Hrs and α-actinin-4 in
346
KSHV infected cells. Immunofluorescent analysis of uninfected and cells infected with KSHV
347
for 10 min demonstrated that Hrs colocalizes with α-actinin-4 at the membrane of infected cells
348
(Fig. 3B).
349
To visualize that the interaction of α-actinin-4 with Hrs leads to the recruitment of Hrs to
350
the cell membrane of infected cells, we used a proximity ligation assay (PLA) with antibodies
351
against Hrs and α-actinin-4. PLA enables the identification of the association between two
352
proteins which are located in close proximity (
