Kaposi's sarcoma-associated herpesvirus (human herpesvirus 8) infection of human fibroblast cells occurs through endocytosis

Abstract

Kaposi's sarcoma (KS)-associated herpesvirus or human herpesvirus 8 (HHV-8) DNA and transcripts have been detected in the B cells, macrophages, keratinocytes, and endothelial and epithelial cells of KS patients. In vitro, HHV-8 infects human B, endothelial, epithelial, and fibroblast cells, as well as animal cells, and the infection is characterized by (i) absence of lytic replication by the input virus and (ii) latent infection. For its initial binding to target cells, HHV-8 uses ubiquitous heparan sulfate molecules via its envelope-associated glycoproteins gB and gpK8.1A. HHV-8 also interacts with the alpha3beta1 integrin via its glycoprotein gB, and virus binding studies suggest that alpha3beta1 is one of the HHV-8 entry receptors (S. M. Akula, N. P. Pramod, F. Z. Wang, and B. Chandran, Cell 108:407-419, 2002). In this study, morphological and biochemical techniques were used to examine the entry of HHV-8 into human foreskin fibroblasts (HFF). HHV-8 was detected in coated vesicles and in large, smooth-surfaced endocytic vesicles. Fusion of viral envelope with the vesicle wall was also observed. In immune electron microscopy, anti-HHV-8 gB antibodies colocalized with virus-containing endocytic vesicles. In fluorescence microscopic analyses, transferrin was colocalized with HHV-8. HHV-8 infection was significantly inhibited by preincubation of cells with chlorpromazine HCl, which blocks endocytosis via clathrin-coated pits, but not by nystatin and cholera toxin B, which blocks endocytosis via caveolae and induces the dissociation of lipid rafts, respectively. Infection was also inhibited by blocking the acidification of endosomes by NH(4)Cl and bafilomycin A. Inhibition of HHV-8 open reading frame 73 gene expression by chlorpromazine HCl, bafilomycin A, and NH(4)Cl demonstrated that the virions in the vesicles could proceed to cause an infection. Taken together, these findings suggest that for its infectious entry into HFF, HHV-8 uses clathrin-mediated endocytosis and a low-pH intracellular environment.

FIG. 1.

EM observation of HHV-8 entry into HFF via endocytic vesicles. HFF (10⁶) were incubated with GFP-HHV-8 for 60 min at 4°C. Infection was initiated by shifting the temperature to 37°C. After 0 (A), 5 (B), and 15 (C, D, E, and F) min at 37°C, cells were washed in PBS and fixed in 2% glutaraldehyde. Thin sections were made for ultrastructural analysis by transmission EM. Virion particles at various stages of binding and entry and in endocytic vesicles are indicated by arrows. The arrowheads in panels E and F indicate the envelope of a virus particle in contact with the endocytic vesicle membrane and in the process of fusion, respectively.

FIG. 2.

IEM observation of HHV-8 entry into HFF via endocytic vesicles. HFF were incubated with GFP-HHV-8 for 60 min at 4°C. Infection was initiated by shifting the temperature to 37°C. After 0 (A) and 5 (B, C, and D) min at 37°C, cells were washed in PBS and fixed in 4% paraformaldehyde and thin sections were made. Grids containing the sections were incubated with rabbit anti-HHV-8 envelope glycoprotein gB IgG antibodies for 1 h at room temperature and then incubated at room temperature for 30 min with anti-rabbit antibodies conjugated with 15-nm gold particles. The grids were washed in PBS and distilled H₂O, counterstained, and viewed under an EM. Arrows indicate the gold particles identifying the location of gB near the enveloped virions.

FIG. 3.

Colocalization of FITC-HHV-8 and TRITC-transferrin in endosomes. HFF were incubated at 37°C with TRITC-labeled transferrin and FITC-labeled HHV-8 for 5 (A and B) and 15 (C) min, respectively. Cells were washed in PBS, fixed with 2% paraformaldehyde at 4°C for 10 min, washed, mounted in antifade reagent, and analyzed under a confocal microscope with appropriate filters. Magnification, ×62. The arrowheads and arrows indicate internalized HHV-8 and transferrin, respectively.

FIG. 4.

Inhibition of GFP-HHV-8 infection by chlorpromazine HCl. (A) HFF monolayers in eight-well chamber slides were incubated with DMEM containing no drug or chlorpromazine HCl (10 μg/ml), nystatin (100 μg/ml), or CTB (100 μg/ml) for 1 h at 37°C before infection with GFP-HHV-8. After incubation for 2 h at 37°C with the virus in the presence of inhibitors, cells were washed and further incubated with growth medium for 3 days at 37°C. Green fluorescent cells, indicative of GFP-HHV-8 entry and infection, were counted. In the absence of inhibitors, approximately 300 green fluorescence-expressing HFF per well were detected. A similar procedure was used for infection of HFF in 24-well plates with HSV-2. Cells and supernatant from HSV-2 infection were collected after 2 days, and the HSV-2 TCID₅₀ was quantitated by titration in HFF. Approximately 10^5.8 TCID₅₀ of HSV-2 was produced in HFF in the absence of inhibitors. Data are presented as the percentage of inhibition of virus infectivity obtained when cells were incubated with the virus without inhibitors. Each reaction was done in triplicate, and each point represents the average ± the standard deviation of three experiments. (B) HFF were incubated with DMEM containing chlorpromazine HCl (10 μg/ml) at 37°C for 1 h and then incubated at 37°C with TRITC-labeled transferrin and FITC-labeled HHV-8 in the presence of chlorpromazine HCl (10 μg/ml) for 15 min. Cells were washed in PBS, fixed with 2% paraformaldehyde at 4°C for 10 min, washed, mounted in antifade reagent, and analyzed under a confocal microscope with appropriate filters. Magnification, ×62.

FIG. 5.

Effects of NH₄Cl and BFLA1 on HHV-8 binding and infection. HFF monolayers were incubated with DMEM or DMEM containing various concentrations of NH₄Cl (A) and BFLA1 (B) for 1 h at 37°C. These cells were infected with GFP-HHV-8 in the presence or absence of respective inhibitors at 37°C for 2 h, washed twice with DMEM, and further incubated with growth medium at 37°C. After 3 days, infection was monitored by counting of green fluorescent cells. In the absence of inhibitors, approximately 300 green fluorescence-expressing HFF per well were detected. Data are presented as the percentage of inhibition of virus infectivity obtained when the cells were incubated with the virus without inhibitors. Each reaction was done in triplicate, and each point represents the average ± the standard deviation of three experiments. (C) HFF were treated with DMEM or DMEM containing different inhibitors (chlorpromazine HCl at 10 μg/ml, nystatin at 100 μg/ml, CTB at 100 μg/ml, NH₄Cl at 50 mM, or BFLA1 at 50 nM) at 37°C for 1 h. Cells were washed three times with DMEM containing 5% fetal bovine serum. Purified [³H]thymidine-labeled GFP-HHV-8 (2,500 cpm), along with the inhibitors, was added to the cells and incubated at 4°C for 1 h. Inhibition of radiolabeled HHV-8 binding by heparin and chondroitin sulfate C was performed by incubation of a constant quantity of purified [³H]thymidine-labeled virus (2,500 cpm) with 10 μg of soluble heparin or chondroitin sulfate C per ml at 37°C for 1 h. These mixtures were then added to HFF and incubated at 4°C for 1 h. After incubation, cells were washed five times with DMEM and lysed with 1% sodium dodecyl sulfate and 1% Triton X-100, and the radioactivity was precipitated with trichloroacetic acid and counted. In the absence of inhibitors, approximately 21% of the input HHV-8 radioactivity became associated with the cells. Each reaction was done in triplicate, and each point represents the average ± the standard deviation of three experiments.

FIG. 6.

Chlorpromazine HCl, NH₄Cl, and BFLA1 inhibit infectious entry of HHV-8. (A) HFF were left untreated or treated with various inhibitors at 37°C for 1 h and then infected with GFP-HHV-8 in the presence or absence of inhibitors at 37°C for 2 h. These cells were washed twice with DMEM and incubated with growth medium at 37°C. At 48 h postinfection, total RNA was isolated and reverse transcribed by RT-PCR with random hexamers and the cDNA was subjected to PCR analysis with different primers specific to HHV-8 ORF73 or the GFP and human β-actin genes (Table 1). The PCR-amplified products were resolved in a 1.2% agarose gel and transferred onto nylon membranes, and Southern blot hybridization was performed with different digoxigenin-labeled internal oligonucleotide probes to detect expression of the ORF73 (307 bp), GFP (328 bp), and human β-actin (838 bp) mRNAs. Lanes: 1, uninfected HFF; 2, HFF infected with GFP-HHV-8 in the absence inhibitors; 3 to 7, GFP-HHV-8 infection of HFF incubated with chlorpromazine HCl (10 μg/ml), nystatin (100 μg/ml), CTB (100 μg/ml), NH₄Cl (50 mM), and BFLA1 (50 nM), respectively. (B) The intensities of bands in the absence or presence of the inhibitors in panel A were scanned, the intensities were assessed with the ImageQuant software program (Molecular Dynamics), and HHV-8 ORF73 and GFP gene expression was quantitated. Each point represents the average ± the standard deviation of three experiments.