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. 2009 May;83(10):4895-911.
doi: 10.1128/JVI.02498-08. Epub 2009 Mar 11.

Kaposi's sarcoma-associated herpesvirus utilizes an actin polymerization-dependent macropinocytic pathway to enter human dermal microvascular endothelial and human umbilical vein endothelial cells

Hari Raghu  1 , Neelam Sharma-Walia, Mohanan Valiya Veettil, Sathish Sadagopan, Bala Chandran
Affiliations

Kaposi's sarcoma-associated herpesvirus utilizes an actin polymerization-dependent macropinocytic pathway to enter human dermal microvascular endothelial and human umbilical vein endothelial cells

Hari Raghu et al. J Virol. 2009 May.

Abstract

Kaposi's sarcoma-associated herpesvirus (KSHV) utilizes clathrin-mediated endocytosis for its infectious entry into human foreskin fibroblast (HFF) cells (S. M. Akula, P. P. Naranatt, N.-S. Walia, F.-Z. Wang, B. Fegley, and B. Chandran, J. Virol. 77:7978-7990, 2003). Here, we characterized KSHV entry into primary human microvascular dermal endothelial (HMVEC-d) and human umbilical vein endothelial (HUVEC) cells. Similar to the results for HMVEC-d cells, KSHV infection of HUVEC cells also resulted in an initial high level and subsequent decline in the expression of the lytic switch gene, ORF50, while latent gene expression persisted. Internalized virus particles enclosed in irregular vesicles were observed by electron microscopy of infected HMVEC-d cells. At an early time of infection, colocalization of KSHV capsid with envelope was observed by immunofluorescence analysis, thus demonstrating endocytosis of intact enveloped virus particles. Chlorpromazine, an inhibitor of clathrin-mediated endocytosis, and filipin (C(35)H(58)O(11)), a caveolar endocytosis inhibitor, did not have any effect on KSHV binding, entry (DNA internalization), or gene expression in HMVEC-d and HUVEC cells. In contrast to the results for HFF cells, virus entry and gene expression in both types of endothelial cells were significantly blocked by macropinocytosis inhibitors (EIPA [5-N-ethyl-N-isoproamiloride] and rottlerin [C(30)H(28)O(8)]) and by cytochalasin D, which affects actin polymerization. Inhibition of lipid raft blocked viral gene expression in HMVEC-d cells but not in HUVEC or HFF cells. In HMVEC-d and HUVEC cells, KSHV induced the actin polymerization and formation of lamellipodial extensions that are essential for macropinocytosis. Inhibition of macropinocytosis resulted in the distribution of viral capsids at the HMVEC-d cell periphery, and capsids did not associate with microtubules involved in the nuclear delivery of viral DNA. Internalized KSHV in HMVEC-d and HUVEC cells colocalized with the macropinocytosis marker dextran and not with the clathrin pathway marker transferrin or with caveolin. Dynasore, an inhibitor of dynamin, did not block viral entry into endothelial cells but did inhibit entry into HFF cells. KSHV was not associated with the early endosome marker EEA-1 in HMVEC-d cells, but rather with the late endosome marker LAMP1, as well as with Rab34 GTPase that is known to regulate macropinocytosis. Silencing Rab34 with small interfering RNA dramatically inhibited KSHV gene expression. Bafilomycin-mediated disruption of endosomal acidification inhibited viral gene expression. Taken together, these findings suggest that KSHV utilizes the actin polymerization-dependent, dynamin-independent macropinocytic pathway that involves a Rab34 GTPase-dependent late endosome and low-pH environment for its infectious entry into HMVEC-d and HUVEC cells. These studies also demonstrate that KSHV utilizes different modes of endocytic entry in fibroblast and endothelial cells.

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Figures

FIG. 1.
FIG. 1.
Morphological observations of KSHV entry into HMVEC-d cells. (A) Electron microscopy of KSHV entry. HMVEC-d cells were incubated with purified KSHV at 37°C for different times, washed with PBS, and fixed in 2% glutaraldehyde. Thin sections were made for ultrastructural analysis by transmission electron micrography. Electron micrographs obtained at 5 min p.i. are shown here. Black arrow indicates a virus at the cell membrane. Red arrows indicate virion particles in endocytic vesicles. White arrow indicates a virion envelope in contact with endocytic vesicle membrane and in the process of fusion and release of the dark core. Magnifications: a, b, and c, ×64,000; d, ×82,000. Cy, cytoplasm; N, nucleus. (B) Immunofluorescence examination of KSHV entry. HMVEC-d cells grown in eight-chamber slides were infected with KSHV at an MOI of 10 (10 DNA copies/cell) for 1 h at 4°C and shifted to 37°C for the indicated times. Cells were fixed with 4% paraformaldehyde, permeabilized with 0.5% Triton X-100, blocked with 5% BSA, washed, and incubated with mouse anti-KSHV envelope glycoprotein gpK8.1A mAb and rabbit anti-KSHV capsid ORF65 protein immunoglobulin G antibody for 30 min at room temperature. After being washed, cells were incubated with anti-mouse Alexa Fluor 488 (green) and anti-rabbit Alexa Fluor 594 (red), respectively, for 30 min at room temperature, washed, mounted in DAPI, and visualized under a Nikon fluorescent microscope. The cell whose enlarged merged image is shown on the right is boxed in panels a to c and e to g (red arrow). White arrows indicate colocalization of ORF65 and gpK8.1A, and white arrowheads indicate free capsids. Magnification, ×80.
FIG. 2.
FIG. 2.
Effect of endocytic inhibitors on KSHV gene expression, binding, and entry in HMVEC-d and HFF cells. (A and B) Cells were left untreated or pretreated with endocytic inhibitors for 1 h at 37°C, washed, and infected with KSHV at an MOI of 10. Total RNA was isolated at 2 h and 24 h p.i., and 50 ng of DNase-treated RNA/μl was subjected to real-time RT-PCR with ORF73 and ORF50 gene-specific primers and TaqMan probes. Known concentrations of DNase-treated, in vitro-transcribed ORF50 and ORF73 transcripts were used in real-time RT-PCR to construct a standard graph from which the relative copy numbers of viral transcripts were calculated and normalized to the amount of GAPDH. Histograms depict KSHV ORF73 and ORF50 gene RNA copy numbers in untreated cells (KSHV) or cells in the presence of indicated nontoxic concentrations of chlorpromazine (Chlor), EIPA, rottlerin (Rot), cytochalasin D (CytoD), filipin, cholera toxin B (CTB), MβCD, or nystatin (nys) in HMVEC-d (A) and HFF (B) cells. Each reaction was done in duplicate, and each bar represents the mean ± standard deviation of the results of three independent experiments. (C, D, and E) Effect of endocytic inhibitors on KSHV binding (C) and internalization (D) in HMVEC-d cells and on internalization (E) in HFF cells. (C) HMVEC-d cells grown in 24-well plates were either left untreated or pretreated with various nontoxic concentrations of agents for 1 h at 37°C and incubated with a fixed concentration of [3H]thymidine-labeled virus for 1 h at 4°C. As a control, labeled KSHV was preincubated with 100 μg of heparin/ml for 1 h at 37°C before being added to the cells. After incubation, cells were washed, lysed, and precipitated with trichloroacetic acid and the cell-associated-virus radioactivity (in cpm) was counted. The cell-associated cpm in the presence of drugs compared to virus binding to untreated cells was calculated as the percent inhibition of virus binding. Each reaction was done in triplicate, and each bar represents the average ± standard deviation of the results of three independent experiments. (D and E) Cells grown in six-well plates were either left untreated or preincubated with drugs at 37°C for 1 h. Cells were incubated with KSHV for 2 h, washed to remove unbound virus, treated with trypsin-EDTA for 5 min at 37°C to remove unbound, noninternalized virus, and washed, and the total DNA was isolated. KSHV preincubated with 100 μg of heparin/ml for 1 h at 37°C before being added to the cells was used as a control. KSHV ORF73 DNA copy numbers were estimated by real-time DNA PCR (21). Each reaction was done in duplicate, and each bar represents the mean ± standard deviation of the results of three experiments.
FIG. 3.
FIG. 3.
KSHV trafficking after treatment with macropinocytic inhibitor EIPA. HMVEC-d cells grown in eight-chamber slides were left untreated or treated with 0.25 μg EIPA, washed, infected with KSHV at an MOI of 100 for 30 min (30′) at room temperature, washed, fixed, permeabilized, blocked in 5% BSA, incubated in BSA with primary antibodies to tubulin and KSHV capsid ORF65 protein for 1 h at room temperature, washed, incubated with secondary antibodies to tubulin (green) and ORF65 (red), washed, mounted, and viewed under an immunofluorescence microscope. Pink arrowheads indicate KSHV capsids, red arrows indicate tubulin, and white arrowheads indicate colocalization of KSHV capsids with microtubules. Magnification, ×80. Boxed areas in c and g are shown enlarged in d and h.
FIG. 4.
FIG. 4.
KSHV induces actin polymerization in HMVEC-d (A) and HUVEC (B) cells. HMVEC-d and HUVEC cells were left uninfected or infected with KSHV (MOI of 10) at 37°C for different times (', min), fixed, permeabilized, and stained for polymerized actin by using Alexa Fluor 488-labeled phalloidin for 30 min at room temperature. Red arrowheads indicate the accumulated actin stress fibers, white arrows indicate hair-like membrane filopodial extensions, and white arrowheads indicate sites of membrane ruffling. Magnification, ×40.
FIG. 5.
FIG. 5.
Effects of endocytic inhibitors on KSHV entry into HUVEC cells. (A) Kinetics of KSHV entry into HUVEC cells. HUVEC cells were infected with KSHV (100 DNA copies per cell), and amounts of internalized viral DNA at different time points were estimated as described in the Fig. 2D and E legend. Each reaction was done in duplicate, and each bar represents the mean ± standard deviation of the results of three experiments. (B) Kinetics of KSHV DNA delivery into infected-cell nuclei. Nuclear fractions from HUVEC cells infected with KSHV at 100 DNA copies per cell for the indicated times (', min) were isolated, and total DNA extracted, normalized to 100 ng/5 μl, and analyzed by real-time DNA PCR with KSHV ORF73 primers. Each reaction was done in duplicate, and each bar represents the mean ± standard deviation of the results of three experiments. (C) Kinetics of KSHV latent gene (ORF73) and lytic gene (ORF50, K8, and gpK8.1) expression in HUVEC cells. For RNA isolation, PCR primers, and methodology, refer to Materials and Methods. Each sample was measured in triplicate, and data were analyzed by the threshold cycle method for comparing relative expression results. For each experiment, PCR amplifications without cDNA were performed as negative controls. (D) Effect of endocytic inhibitors on KSHV gene expression in HUVEC cells. HUVEC cells grown in six-well plates were either left untreated or preincubated with various nontoxic concentrations of agents for 1 h at 37°C, washed, and incubated with KSHV for 2 h, and total RNA was isolated at 8 h and 48 h p.i. and subjected to real-time RNA PCR. Histogram depicts the percent inhibition in KSHV ORF73 and ORF50 gene RNA copy numbers in the presence of indicated drugs, obtained by comparison with copy numbers in cells incubated with virus alone. Each reaction was done in duplicate, and each bar represents the mean ± standard deviation of the results of three experiments. (E) Effect of endocytic inhibitors on KSHV internalization in HUVEC cells. HUVEC cells grown in six-well plates were either left untreated or preincubated with various nontoxic concentrations of agents at 37°C for 1 h and infected with KSHV for 2 h, and the amount of internalized viral DNA at different time points was estimated as described in the Fig. 2D and E legend. The data are represented as the percent inhibition of KSHV DNA internalization in comparison with that in cells incubated with virus alone. Each reaction was done in duplicate, and each bar represents the mean ± standard deviation of the results of three experiments. Chlor, chlorpromazine; Rot, rottlerin; CytoD, cytochalasin D; CTB, cholera toxin B; nys, nystatin.
FIG. 6.
FIG. 6.
KSHV colocalizes with macropinocytic marker dextran and not with transferrin and caveolin. (A and D) Colocalization of KSHV with dextran. Uninfected and infected HMVEC-d (A) and HUVEC (D) cells were incubated at 37°C with Texas Red-labeled dextran and KSHV for 5, 10, and 15 min. Cells were washed in HBSS, fixed with 2% paraformaldehyde, permeabilized with 0.2% Triton X-100, blocked in 5% BSA, and then incubated with anti-gpK8.1A mAb followed by Alexa Fluor 488-labeled goat anti-mouse secondary antibody. Magnification, ×40. White arrows indicate colocalization of KSHV with dextran. (B and E) KSHV does not colocalize with transferrin. Uninfected cells and cells infected with KSHV and Alexa Fluor 594-conjugated transferrin were examined with anti-gpK8.1A antibody. Magnification, ×40. Yellow arrows indicate virus particles not colocalized with transferrin. (C) KSHV does not colocalize with caveolin. Uninfected and infected cells were incubated with anti-gpK8.1A and anticaveolin antibodies for 1 h at room temperature. The staining was visualized by incubation with Alexa Fluor 488-labeled antibody for gpK8.1A and Alexa Fluor 594-labeled secondary antibody for caveolin. Magnification, ×40. Yellow arrows indicate virus particles not colocalized with caveolin. Boxed areas are shown enlarged in the right-hand panels. ', min; UN, uninfected.
FIG. 6.
FIG. 6.
KSHV colocalizes with macropinocytic marker dextran and not with transferrin and caveolin. (A and D) Colocalization of KSHV with dextran. Uninfected and infected HMVEC-d (A) and HUVEC (D) cells were incubated at 37°C with Texas Red-labeled dextran and KSHV for 5, 10, and 15 min. Cells were washed in HBSS, fixed with 2% paraformaldehyde, permeabilized with 0.2% Triton X-100, blocked in 5% BSA, and then incubated with anti-gpK8.1A mAb followed by Alexa Fluor 488-labeled goat anti-mouse secondary antibody. Magnification, ×40. White arrows indicate colocalization of KSHV with dextran. (B and E) KSHV does not colocalize with transferrin. Uninfected cells and cells infected with KSHV and Alexa Fluor 594-conjugated transferrin were examined with anti-gpK8.1A antibody. Magnification, ×40. Yellow arrows indicate virus particles not colocalized with transferrin. (C) KSHV does not colocalize with caveolin. Uninfected and infected cells were incubated with anti-gpK8.1A and anticaveolin antibodies for 1 h at room temperature. The staining was visualized by incubation with Alexa Fluor 488-labeled antibody for gpK8.1A and Alexa Fluor 594-labeled secondary antibody for caveolin. Magnification, ×40. Yellow arrows indicate virus particles not colocalized with caveolin. Boxed areas are shown enlarged in the right-hand panels. ', min; UN, uninfected.
FIG. 7.
FIG. 7.
KSHV entry in HMVEC-d and HUVEC cells is dynamin independent. (A) HMVEC-d cells showing transfection with WT dynamin. Cells were transfected with 1 μg of GFP-WT dynamin plasmid or 1 μg of GFP-K44A dynamin plasmid and observed under a microscope after 24 h. a and c, GFP; b and d, phase-contrast microscopy. Magnification, ×10. (B) HMVEC-d cells were transfected with 1 μg of GFP-WT or GFP-K44A dynamin plasmid. After 24 h, cells were infected with KSHV at an MOI of 10 for different times and KSHV internalization was measured by real-time DNA PCR. Histogram shows internalized copy numbers of KSHV in HMVEC-d cells transfected with WT and K44A dynamin plasmids. (C and D) Dynasore did not affect internalization in HUVEC (C) and HMVEC-d (D) cells. HUVEC cells and HMVEC-d cells grown in six-well plates were treated with 80 μM dynasore for 1 h at 37°C and infected with KSHV at an MOI of 10 for different times, and KSHV internalization measured by real-time DNA PCR for ORF73 gene. (E) Dynasore treatment affects KSHV internalization in HFF cells. Cells grown in six-well plates were treated with 100 μM dynasore and infected with KSHV at an MOI of 10 for different times, and KSHV internalization measured by real-time DNA PCR. Histogram shows the internalized copy numbers of KSHV DNA with and without dynasore treatment. Each reaction was done in duplicate, and each bar represents the mean ± standard deviation of the results of three experiments.
FIG. 8.
FIG. 8.
KSHV trafficking in HMVEC-d cells and association with early and late endosomes. (A) Uptake of transferrin. Uninfected cells were pulsed with Alexa Fluor 594-tagged transferrin (25 μg/ml) for 5 min or 30 min at room temperature, fixed, permeabilized, washed in PBS, blocked with 5% BSA, and incubated with anti-mouse EEA-1 antibodies for 1 h at room temperature. The cells were washed, reacted with secondary antibodies, washed, mounted in antifade reagent with DAPI, and visualized under a fluorescence microscope. Red arrows indicate colocalization of transferrin with EEA-1. Magnification, ×40. (B and C) HMVEC-d cells grown in eight-chamber slides were infected with KSHV at an MOI of 10 for the indicated times, fixed, permeabilized, and stained with mouse anti-EEA-1 or anti-LAMP-1 and rabbit anti-ORF65 antibodies for 1 h at room temperature. Cells were washed, incubated with anti-mouse Alexa Fluor 488 (green) and anti-rabbit Alexa Fluor 594 (red) antibodies for 30 min at room temperature, washed, mounted in DAPI, and visualized. Red arrows indicate colocalization of KSHV capsid and LAMP-1. Magnification, ×40. Boxed areas are shown enlarged in the right-hand panels. ', min; UN, uninfected.
FIG. 9.
FIG. 9.
KSHV trafficking in HMVEC-d cells and association with Rab34. (A) HMVEC-d cells grown in eight-chamber slides were infected with KSHV at an MOI of 10 for different times, washed, fixed in 4% paraformaldehyde, permeabilized, and stained with goat anti-Rab34 antibodies and with anti-gpK8.1A mAb for 1 h at room temperature. The cells were washed and incubated with anti-mouse Alexa Fluor 488 (green) and anti-rabbit Alexa 594 Fluor (red) antibodies, washed, mounted, and visualized with a confocal laser scanning microscope, and the data analyzed by using Olympus Fluoview software. Arrows indicate areas of colocalization of virion particles with Rab34. Magnification, ×40. ', min. (B) Histogram representing percent inhibition of Rab34 gene expression in HMVEC-d cells after 24 h and 48 h of transfection with si-Rab34 and si-C as described in Materials and Methods. Percent inhibition was calculated using Rab34 expression in si-C-transfected HMVEC-d cells as 100%. Each bar represents the average ± standard deviation of the results of three independent experiments. (C) Histogram representing the percent inhibition of ORF50 and ORF73 gene expression upon Rab34 silencing in HMVEC-d cells. The percent inhibition was calculated by considering levels of ORF50 or ORF73 in si-C-transfected cells infected with KSHV to be 100%. Each bar represents the average ± standard deviation of the results of three independent experiments.
FIG. 10.
FIG. 10.
KSHV requires a low-pH environment for infection. HMVEC-d (A) and HUVEC (B) cells were left untreated or pretreated with nontoxic concentrations of bafilomycin (Baf) or NH4Cl for 1 h at 37°C, washed, and infected with KSHV at an MOI of 10 (10 DNA copies/cell), and total RNA isolated at the indicated time points. Fifty nanograms of DNase-treated RNA/μl was subjected to real-time RT-PCR as described in the Fig. 2A and 5D legends. Histograms depict the percent inhibition of KSHV ORF73 and ORF50 gene RNA copy numbers in the presence of indicated nontoxic concentrations of drugs, calculated in comparison to copy numbers in cells incubated with virus alone. Each reaction was done in duplicate, and each bar represents the mean ± standard deviation of the results of three independent experiments.
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