Broad Target Cell Selectivity of Kaposi's Sarcoma-Associated Herpesvirus Glycoprotein-Mediated Cell Fusion and Virion Entry ⁎ Johnan A.R

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Broad target cell selectivity of Kaposi's sarcoma-associated herpesvirus glycoprotein-mediated cell fusion and virion entry ⁎ Johnan A.R. Kaleeba, Edward A. Berger

Laboratory of Viral Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Building 4, Room 237, Bethesda, MD 20892, USA Received 23 November 2005; returned to author for revision 21 April 2006; accepted 15 June 2006 Available online 2 August 2006

Abstract

The molecular mechanism of Kaposi's sarcoma-associated herpesvirus (KSHV, human herpesvirus 8) entry is poorly understood. We tested a broad variety of cell types of diverse species and tissue origin for their ability to function as targets in a quantitative reporter gene assay for KSHV- glycoprotein-mediated cell fusion. Several human, non-human primate, and rabbit cell lines were efficient targets, whereas rodent and all human lymphoblastoid cell lines were weak targets. Parallel findings were obtained with a virion entry assay using a recombinant KSHV encoding a reporter gene. No correlation was observed between target cell activity and surface expression of α3β1 integrin, a proposed KSHV receptor. We hypothesize that target cell permissiveness in both the cell fusion and virion entry assays reflects the presence of a putative KSHV fusion–entry receptor. Published by Elsevier Inc.

Keywords: Human herpesvirus 8; Host range; Virus glycoprotein; Cell fusion assay; Virus entry assay; Vaccinia virus expression system; β-galactosidase; Enhanced green fluorescent protein; Virus receptor; Integrin

Introduction more cellular entry receptors (Spear and Longnecker, 2003). In most cases, these glycoprotein–receptor interactions lead to Kaposi's sarcoma-associated herpesvirus (KSHV, a gamma- direct fusion between the virion and plasma membranes; herpesvirus formally designated human herpesvirus 8) is alternatively, in some instances, they may induce virion etiologically linked to the pathogenesis of Kaposi's sarcoma, internalization into endosomes where the mildly acidic pH peripheral effusion lymphoma, and multicentric Castleman's triggers viral glycoprotein-induced fusion with the endosomal disease (Bubman and Cesarman, 2003). These pathologies membrane. By either route, the viral nucleocapsid is introduced occur more frequently in people with acquired immunodefi- into the cytotosol, thereby setting the stage for the subsequent ciency syndrome (AIDS) and in other settings of severe steps of the virus life cycle. immunodeficiency. Whereas the clinical burden of KSHV is KSHV infection in culture has been studied using both cell- underscored by the plethora of virus-associated syndromes, free virus and cell–cell transmission systems (Bubman and little is known about how the virus enters cells. Entry Cesarman, 2003). Analyses of KSHV entry have yielded mechanisms of enveloped viruses invariably involve fusion evidence for both direct fusion and endocytic mechanisms between viral and cellular membranes, mediated by specific (Akula et al., 2003; Dezube et al., 2002; Inoue et al., 2003; virus-encoded glycoproteins (Earp et al., 2004). For herpes- Pertel, 2002). In the present report, we employed two distinct viruses, initiation of infection generally involves virion reporter gene assay systems to analyze the activity and attachment to the target cell surface followed by specific selectivity of the processes involved in the first step in the interactions between viral envelope glycoproteins and one or infection cycle: a cell fusion assay to quantify KSHV glycoprotein function and a virion entry assay to measure the ⁎ Corresponding author. Fax: +1 301 480 1147. entry-dependent delivery of a constitutively activatable reporter E-mail address: [email protected] (E.A. Berger). gene. Both systems revealed that cells from diverse species and

0042-6822/$ - see front matter. Published by Elsevier Inc. doi:10.1016/j.virol.2006.06.009 8 J.A.R. Kaleeba, E.A. Berger / Virology 354 (2006) 7–14 tissue types could serve as KSHV targets. The results provide support for a putative KSHV fusion–entry receptor(s), distinct from the cellular molecules previously implicated in KSHV attachment and entry.

Results

Cell fusion mediated by effector cells expressing KSHV glycoproteins

To directly study the functional activity of KSHV glycoproteins, a vaccinia-based cell fusion assay system was devised in which fusion of “effector cells” expressing the required KSHV glycoproteins with “target cells” presumably expressing the appropriate receptor(s) activated the LacZ reporter gene. Cell fusion assays require only the minimal molecular components of both virus and cell involved in the fusion mechanism, independent of downstream post-entry events in the virus replication cycle. Such analyses have proven particularly valuable for studying viruses that enter by direct fusion with the plasma membrane, the major entry mechanism for members of the Herpesviridae family (Spear and Longnecker, 2003). Indeed, cell fusion assays, in several cases using the minimal number of essential recombinant viral glycoproteins, have been developed for all three herpesvirus subfamilies: alpha (Klupp et Fig. 1. Surface expression and functional activity of KSHV glycoproteins on al., 2000; Maresova et al., 2001; Muggeridge, 2000; Pertel et al., TPA-activated BCBL-1 cells. (A) Surface expression analyzed by flow 2001; Tiwari et al., 2004; Turner et al., 1998), beta (Kinzler and cytometry. BCBL-1 cells were cultured in the absence (blue) or presence of TPA (5 ng/ml) for 24 h (green), or 48 h (red), then stained with rabbit peptide Compton, 2005; Santoro et al., 1999), and gamma (Haan et al., antisera (gBN3,gH2,gL2, K8.1N3), followed by FITC-conjugated goat anti- 2001; Pertel, 2002; Wu et al., 2005). rabbit IgG. As background controls, cells were stained with the corresponding Two types of effector cells were employed in the present pre-immune sera (black). (B) Functional activity analyzed by cell fusion assay. studies: BCBL-1 cells chronically infected with KSHVand cells BCBL-1 effector cells (+BCBL-1) pretreated with TPA for the indicated times expressing recombinant KSHV glycoproteins. BCBL-1 cells were mixed with HeLa target cells; as a background control, the BCBL-1 cells were omitted (−BCBL-1). Cell fusion was assayed by measuring β-gal by both harbor the KSHV genome in a latent state; treatment with in situ staining (photomicrographs) and calorimetric assay of NP-40 lysates of phorbol esters induces expression of lytic phase genes, parallel co-cultures (values below, duplicate samples±SD). including those encoding the glycoproteins of the viral envelope. The expression patterns of gB, gH, gL, and K8.1A on the surface of BCBL-1 cells were analyzed by flow assay of nonionic detergent cell lysates (Fig. 1B). Low-level cytometry, using polyclonal antisera against peptides represen- fusion was observed with uninduced BCBL-1 effectors, and the ting sequences from each glycoprotein. These glycoproteins signal was greatly increased with BCBL-1 effectors that had were chosen because they are detected in purified KSHV virion been pre-activated with TPA for 24–48 h; no β-gal was detected preparations (Bechtel et al., 2005; Naranatt et al., 2002; Zhu et in the absence of BCBL-1 effectors. This fusion pattern is al., 2005) and have been directly implicated in virus attachment consistent with the KSHV surface glycoprotein expression data and/or entry (Akula et al., 2001a; Birkmann et al., 2001; described above. Naranatt et al., 2002; Wang et al., 2001, 2003) as well as Several criteria were employed to verify that the fusion membrane fusion (Pertel, 2002). As shown in Fig. 1A, surface observed with BCBL-1 effector cells reflected the activity of the expression of the four glycoproteins was low but detectable in surface KSHV glycoproteins. The cell fusion effector activities untreated BCBL-1 cells; the levels were greatly enhanced by were compared for KSHV-positive (chronically infected 12-O-tetradecanoyl-phorbol-13-acetate (TPA) treatment in a lymphoma-derived BC-3, BC-1, and BCBL-1) versus KSHV- time-dependent fashion over 24–48 h, as assessed by both negative (Burkitt's lymphoma-derived CA-46 and Ramos RA-1, fluorescence intensity and the percentage of positive cells and NIH-3T3 murine fibroblast) cell lines. These effector cells (approaching 100% for all). Immunoblot analyses of cell lysates were either untreated, pretreated with TPA for 24 h, or untreated confirmed low-level expression of all four glycoproteins in and transfected with plasmids encoding the KSHV glycopro- untreated cells and progressive increases over the 24–48 h teins gB, gH, gL, and K8.1A. Flow cytometry analyses (not period of TPA treatment (not shown). The ability of BCBL-1 shown) confirmed low surface expression of gB, gH, gL, and cells to function as effectors in the cell fusion assay was K8.1A on untreated KSHV-positive cell lines and increased demonstrated by measuring β-galactosidase (β-gal) activity expression upon TPA treatment or plasmid transfection; by both by in situ staining of fixed cells and by spectrophotometric contrast, the viral glycoproteins were undetectable on the J.A.R. Kaleeba, E.A. Berger / Virology 354 (2006) 7–14 9

KSHV-negative cell lines (untreated or TPA-treated) but were efficiently expressed upon plasmid transfection. For each of the KSHV-positive effectors (Fig. 2A), significant fusion was observed with untreated cells and was enhanced several fold by either TPA pretreatment or KSHV glycoprotein transfection. By contrast, the KSHV-negative effectors (Fig. 2B) failed to fuse, without or with TPA pretreatment, but were rendered highly fusogenic by KSHV glycoprotein transfection. Thus, the non- fusogenic phenotypes of the KSHV-negative effectors were due not simply to their inherent inability to undergo membrane fusion or to support the reporter gene readout, but instead to their lack of surface molecular components capable of inducing cell fusion. As a direct test of whether fusion mediated by TPA-activated BCBL-1 effectors reflected KSHV glycoprotein function, anti- peptide antibodies (IgG fractions from rabbit antisera) against the glycoproteins of interest were analyzed for their effects on fusion (Fig. 3A). Anti-gBN1 against a peptide based on the N-terminal extracellular segment of gB strongly inhibited KSHV fusion in a dose-dependent manner, as did anti-gH2 against a peptide derived from gH. Dose-dependent inhibition Fig. 3. Specificity of cell fusion mediated by BCBL-1 effectors. (A) Specific was also observed with antibodies against peptides derived from inhibition of fusion by antibodies to KSHV glycoproteins. Effectors (TPA- gL and K8.1A, although the effects were less pronounced. As treated BCBL-1 cells) were pre-incubated with the indicated concentrations of ○ expected, no fusion inhibition was observed with anti-gBC purified IgG from rabbit antisera against the gB N-terminus (gBN1, ), gH (gH2, ⋄ □ ▵ X against a peptide based on the cytoplasmic C-terminal segment ), K8.1A (K8.1N3, ), gL (gL2, ), and gB C-terminus (gBC, ). After of gB. The corresponding pre-immune IgG fractions had no washing, the effectors were assayed for cell fusion with PCI-13 targets. (B) Specificity of fusion mediated by KSHV-glycoprotein-transfected effectors effect (not shown). As specificity controls (not shown), the versus TPA-activated BCBL-1 effectors. Murine F-515 cells transfected with same antibodies had no effect on HIV-1 fusion, i.e. between control vector (open bars) or a combination of plasmids encoding gBΔ, gH, gL, effectors expressing vaccinia-encoded HIV-1 Env glycoprotein and K8.1A (hatched bars), or BCBL-1 cells treated with TPA for 24 h (black and targets expressing vaccinia-encoded CD4 and coreceptor; bars) were used as effectors; fusion assays were performed with the indicated target cells. In both panels, β-gal activity in NP-40 cell lysates was measured (duplicate samples±SD).

conversely, an anti-Env MAb potently inhibited HIV-1 fusion but had no effect on KSHV fusion. As a final validation for using TPA-activated BCBL-1 cells as effectors in cell fusion assays, their ability to fuse with different target cells was compared to that of effectors expressing recombinant KSHV glycoproteins (Fig. 3B). With both types of effector cells, minimal fusion occurred with NIH- 3T3 (murine) targets, whereas potent fusion was obtained with BSC-1 (African green monkey, AGM) as well as HeLa (human) and PCI-13 (human) targets. Taken together, these results validate the use of either TPA-activated BCBL-1 cells or recombinant glycoprotein-expressing cells as effectors in KSHV cell fusion assays. The former were chosen for the studies described below because they consistently expressed the required glycoproteins without the potential variability associated with cotransfection of multiple plasmids.

Broad target cell selectivity of KSHV-glycoprotein-mediated Fig. 2. Comparison of cell fusion effector activities of KSHV-positive (A) or fusion KSHV-negative (B) cell lines. Effectors were prepared from the indicated cell lines that were untreated (open bars), TPA-treated for 24 h (black bars), or An extensive panel of primary cells and established cell lines untreated and transfected with a combination of plasmids encoding gBΔ, gH, gL, and K8.1A (hatched bars). Effector cells were mixed with human PCI-13 targets, of different tissue and species origin were tested for their ability and cell fusion was assessed by measuring β-gal in NP-40 lysates (duplicate to serve as cell fusion targets with TPA-treated BCBL-1 samples±SD). effectors (Table 1). Based on the β-gal activities, a fusion index 10 J.A.R. Kaleeba, E.A. Berger / Virology 354 (2006) 7–14

Table 1 were highly permissive fusion targets for BCBL-1 effectors Fusion permissiveness of various target cell lines with TPA-activated BCBL-1 expressing recombinant HIV-1 Env glycoprotein, but remained effectors weak KSHV fusion targets (i.e. BCBL-1 effectors lacking HIV-1 Species Target cell lines β-gal Fusion Env). We also tested lymphoblastoid cells that showed Weak type (OD/ index a Derivative tissue Name 3 KSHV fusion target activity. Fig. 4 (right panel) shows that min×10 ) expression of recombinant CD4 and coreceptor on CA46 Rodent Mouse embryo fibroblast NIH-3T3 4 Weak Burkitt's lymphoma cells rendered them highly permissive for Mouse skin tumor F-515 6 Syrian baby hamster kidney BHK-21 9 HIV-1 fusion, although they remained Weak targets for KSHV fibroblast fusion. As expected, PCI-13 cells expressing recombinant CD4 and coreceptor were highly permissive with BCBL-1 effectors Rabbit Kidney epithelium RK-13 576 Strong lacking (i.e. KSHV fusion) or expressing HIV-1 Env. Additional experiments (not shown) demonstrated similar phenomena with Monkey Kidney fibroblast CV-1 158 Strong Kidney epithelium BSC-1 302 K-562 leukemia cells and BJAB B cell lymphoma cells, which Kidney epithelium Vero 1241 were rendered highly permissive as HIV-1 fusion targets upon expression of recombinant CD4 and coreceptor. Thus, the Weak Human Acute monocytic leukemia THP-1 1 Weak target cell phenotype of these cells was specific for KSHV fusion Chronic myelogenous leukemia K-562 7 and did not simply reflect their general refractoriness to Acute lymphoblastic leukemia HSB-2 6 Acute lymphoblastic leukemia KG-1 2 membrane fusion or their inability to support the vaccinia- Lymphoblastic Burkitt's Ramos- 3 based reporter gene readout of this assay system. lymphoma RA 1 Lymphoblastic Burkitt's CA46 6 Target cell selectivity of KSHV virion entry parallels KSHV cell lymphoma fusion Colon carcinoma CoLau-467 4 Hepatocellular carcinoma Hu-H7 4 Umbilical vein endothelium HUVEC 81 Moderate As a complementary method to assess the target cell Dermal microvascular HMVEC-d 57 selectivity of KSHV glycoprotein activity, a virus entry assay endothelium was employed, using a recombinant KSHV containing the Muscle spindle RD 136 enhanced green fluorescent protein (EGFP) gene linked to a rhabdomyosarcoma Renal epithelium (Ad-E1A, 293T 71 constitutive cellular promoter (Vieira and O'Hearn, 2004). SV40T) Since EGFP expression was strictly dependent on virion entry Foreskin fibroblast HFF 43 but did not require expression of any KSHV genes, this assay Epithelial cervical HeLa 92 accurately reflected the target cell selectivity of virus entry as adenocarcinoma long as the cells examined were permissive for the post-entry Ovarian adenocarcinoma SK-OV-3 158 Strong Head/Neck squamous cell PCI-13 988 steps essential for the reporter gene readout. Fig. 5 shows carcinoma representative KSHV virion entry data for several target cell Epithelial astrocytic U-87 MG 759 glioblastoma Oligodendroglioma MO3.13 458 Melanoma Mel-1700 408 a Arbitrary cut-offs (β-gal, OD/min×103) for Weak: 0–10; Moderate: 11–150; and Strong: >150. was assigned to each target cell type (Weak, Moderate, Strong). All three rodent cell lines tested (hamster, mouse) scored Weak, whereas the single rabbit and three AGM cell lines scored Strong or Moderate. Among the panel of human cell types tested, the fusion index ranged from Weak to Strong, with all the lymphoblastoid cell lines falling in the Weak category. It was important to test whether the Weak target cell per- Fig. 4. Specificity of target cell non-permissiveness for KSHV fusion. (Left missiveness of certain cell types was selective for KSHV- panel) NIH-3T3 and F-515 cells (both weakly permissive for KSHV fusion) were infected with recombinant vaccinia viruses encoding CD4 and CXCR4 and glycoprotein-mediated cell fusion and did not simply reflect tested for their ability to function as targets for HIV-1 fusion with TPA-treated artifactual aspects of the cell fusion assay. To this end, we BCBL-1 effector cells expressing vaccinia-encoded HIV-1 Env, IIIB isolate (+). examined the ability of several Weak KSHV fusion targets to (Right panel) CA46 and PCI-13 cells (Weak and Strong targets for KSHV function in an independent cell fusion system, i.e. as targets for fusion, respectively) were infected with recombinant vaccinia viruses encoding fusion mediated by HIV-1 Env glycoprotein when expressing CD4 and CCR5 and tested for their ability to function as targets for HIV-1 fusion with TPA-treated BCBL-1 effector cells expressing vaccinia-encoded HIV-1 the corresponding recombinant receptors (CD4 and coreceptor Env, Ba-L isolate (+). In both panels, controls were performed with TPA-treated CXCR4 or CCR5). Fig. 4 (left panel) shows that murine NIH- BCBL-1 effectors not expressing HIV-1 Env (−). β-gal was measured in NP-40 3T3 and F515 cells expressing recombinant CD4 and coreceptor cell lysates (duplicate samples±SD). J.A.R. Kaleeba, E.A. Berger / Virology 354 (2006) 7–14 11

Fig. 5. Susceptibility to KSHV entry of various target cells. The indicated target cells were pre-incubated with a 1:4 dilution of concentrated supernatant containing recombinant rKSHV.219 virions then washed and cultured. Virus entry was monitored between 2 and 5 days by flow cytometry to detect EGFP-positive cells (open histograms). Uninfected cells cultured for comparable times served as negative controls (filled histograms). Fusion index is based on the assignments in Table 1. types, based on flow cytometry analysis of EGFP expression both KSHV-glycoprotein-mediated cell fusion and KSHV following exposure to KSHV virions. A consistent correlation virion entry, consistent with the interpretation that both was observed between target cell susceptibility in the KSHV processes reflect the same underlying mechanism of KSHV virion entry assay and permissiveness for KSHV fusion. Thus, glycoprotein function. Our findings are in close agreement of the nine cell lines examined, six supported KSHV entry as with a recent analysis of the host range for KSHV virion illustrated by the prominent peaks of EGFP-positive cells (Mel- infectivity in vitro, which measured expression of the viral 1700, Vero, BSC-1, HeLa, RD, HFF); these findings parallel the ORF73 gene as a marker of entry and latent phase gene Strong or Moderate activities of these cell lines as KSHV fusion expression (Bechtel et al., 2003). In that study, all adherent targets (Table 1). By contrast, three cell lines (BHK-21, K-562, cell lines tested were shown to be susceptible to infection THP-1) did not support KSHVentry, consistent with their Weak (including human HeLa and 293 cells as well as monkey CV-1 target activities for KSHV cell fusion (Table 1). cells, all of which scored Moderate or Strong in our fusion assay); by contrast, the murine and hamster cell lines Lack of correlation between KSHV target cell permissiveness examined (including NIH-3T3 cells studied herein) were and α3β1 integrin expression considerably less susceptible, consistent with our observation of Weak fusion activities of rodent target cells. Interestingly, The α3β1 integrin has been implicated as a receptor for all the human lymphoblastoid cells tested (including B-cell KSHV entry (Akula et al., 2002). Therefore, it was important to lymphomas such as RAMOS cells analyzed in this report) compare surface expression of this integrin complex on human were reportedly resistant to infection, in agreement with our target cell types with varying permissiveness as KSHV targets. demonstration of Weak target cell activities of such cell types The data in Fig. 6 do not indicate a correlation between these in both the cell fusion and entry assays. These latter results parameters. Thus, RD cells, which were Moderate targets for remain puzzling since B cells are believed to be major sites for KSHV fusion (Table 1, Fig. 6) and virion entry (Fig. 5), KSHV infection in infected people (Bubman and Cesarman, displayed negligible α3β1 integrin surface expression (Fig. 6); 2003). Additional experiments are required to identify the by contrast, the Weak fusion targets HSB-2 and CoLau-467 various factors contributing to differences between in vitro and (Table 1, Fig. 6) expressed very high surface levels of α3β1 in vivo findings, e.g. altered characteristics of transformed cell integrin (Fig. 6). lines, possible influences of cell activation and changes associated with adaptation to cell culture. Discussion We favor the hypothesis that a major route for KSHV entry involves a sequence of distinct molecular events that culminate In the present study, we demonstrate that a broad range of in direct fusion of the virion envelope with the target cell plasma cell types from different tissues and species are permissive for membrane. In this model, the mechanistic cascade is initiated 12 J.A.R. Kaleeba, E.A. Berger / Virology 354 (2006) 7–14

followed by low-pH activation of viral glycoprotein function (Akula et al., 2002, 2003; Inoue et al., 2003; Wang et al., 2003). Such a context-dependent mechanism, which has been demonstrated for herpes simplex virus type I (Nicola et al., 2003), would likely involve molecular events distinct from those involved in the direct fusion pathway. Several types of cell surface molecules have been proposed to function as KSHV receptors, most notably α3β1 integrin in human dermal microvascular endothelial cells and human foreskin fibroblasts (Akula et al., 2002) and heparan sulfate (Akula et al., 2001b; Birkmann et al., 2001). Each has been shown to interact with specific KSHV glycoproteins, the former with gB (Wang et al., 2003) and the latter with both gB (Akula et al., 2001a) and K8.1A (Birkmann et al., 2001; Wang et al., 2001). However, our results do not indicate a direct role for α3β1 integrin in KSHV-glycoprotein-mediated cell fusion or in KSHV virion entry, at least for the cell types examined in this report. Nonetheless, the possible involvement of these surface molecules can be considered in a broader context since the internalization of enveloped viruses from many families (including other herpesviruses) can be strongly influenced by various integrins (Triantafilou et al., 2001) and by sulfated proteoglycans (Liu and Thorp, 2002; Shukla and Spear, 2001). The recent report that DC-SIGN facilities KSHV entry into myeloid dendritic cells and macrophages (Rappocciolo et al., 2006) is also noteworthy in view of emerging evidence implacating C-type lectins in interactions of many viruses with the cell surface of target cells (Altmeyer, 2004). These surface entities play important roles in facilitating entry of many viruses, in some cases by serving as attachment factors to enhance virus adsorption to the cell; in addition, they may participate in signaling pathways that influence infectivity in ways that do not directly involve the specific entry mechanism. These considerations highlight the difficulties in defining Fig. 6. Surface expression of α3β1 integrin on various human cells lines. The the molecular elements that comprise the essential entry indicated cells were analyzed by flow cytometry using murine MAb P1B5 machinery and in distinguishing them from factors that play against human α3β1 integrin (open histograms). Mouse IgG1 was used as an isotype control (black histograms). Cell fusion activities (β-gal in NP-40 cell important accessory roles. We believe that the prevailing data lysates, mean of duplicate samples) were obtained with the same target cell point to the existence of a specific receptor(s) underlying the preparations and are consistent with the fusion index as assigned in Table 1. primary KSHV entry route that is distinct from the molecules implicated thus far. Indeed, by exploiting the ability of the cell upon binding of a particular viral glycoprotein(s) to a specific cell fusion and virion entry assays to isolate the functional surface “fusion–entry” receptor(s). Indeed, for glycoproteins activities of the participating viral glycoproteins, we have from several herpesviruses, cell fusion assays have documented recently identified a KSHV fusion–entry receptor, a protein the absolute requirement for such a receptor (Browne et al., whose known properties suggest possible interactions with the 2001; Pertel et al., 2001; Santoro et al., 1999; Terry-Allison et integrin system (Kaleeba and Berger, 2006). al., 2001; Tiwari et al., 2004). The simplest interpretation of the present results is that KSHV target cell permissiveness is Materials and methods dependent on surface expression of a specific KSHV fusion– entry receptor(s) and that the non-permissive targets display Cell culture insufficient levels of such a receptor. However, we cannot rule out an alternative explanation that non-permissiveness results Mouse tumor F-515 and human Mel-1700, SK-OV-3, PCI-13, from a dominant negative inhibitory factor(s) selective for CoLau-467, and THP-1 cells were provided by Maurice KSHV or that factors besides receptor expression contribute to Zauderer and Ernest Smith (Vaccinex, Inc., Rochester, NY). the differences in fusion and entry observed in our assay Primary HMVEC-d and HUVEC cells were obtained from systems. Our results also do not exclude the possibility that, in Clonetics Inc. (Walkersville, MD). All other cell lines were some instances, e.g. with certain target cell types, KSHV might obtained from either the American Type Culture Collection initiate infection via the alternative entry route of endocytosis (Manassas, VA) or the AIDS Research and Reference Reagent J.A.R. Kaleeba, E.A. Berger / Virology 354 (2006) 7–14 13

Repository (Bethesda, MD). Cells were maintained as recom- originally developed to study HIV Env (Nussbaum et al., mended by the suppliers. For TPA treatment, cells were 1994) and subsequently applied to glycoproteins from many cultured in the presence of 5 ng/ml TPA, which for BCBL-1 families of enveloped viruses (Dey and Berger, 2003). Briefly, cells was sufficient to induce surface expression of KSHV two populations of single cell suspensions were prepared: glycoproteins but suboptimal for production of progeny KSHV “effector cells” expressing KSHV glycoproteins (endogenous particles. or transfected) and vaccinia-encoded T7 RNA polymerase, and “target cells” containing the vaccinia-encoded E. coli LacZ Antibodies against KSHV glycoproteins gene linked to the T7 promoter. Effector and target cells were mixed (typically 1×105 each, in 96-well flat-bottom plates, Rabbit polyclonal antibodies to KSHV glycoproteins were 0.2 ml total volume per well) and incubated at 37 °C for 1.5– generated using synthetic peptides predicted to be both immuno- 4h.β-gal activity was measured either by colorimetric assay genic and/or surface-exposed on the respective mature glycopro- of enzyme activity in 0.5% nonidet P-40 (NP-40) cell lysates teins, according to weighted scores from hydrophilicity, surface using as substrate chlorophenol red-β-D-galactopyranoside or probability, and backbone flexibility algorithms integrated in the by in situ staining with 4-chloro-3-indolyl-β-D-galactopyranoi- MacVector 7.0 software (Oxford Molecular-Accelrys, Burling- side substrate. For antibody inhibition studies, effector cells ton, MA). Each peptide was designed with an additional C- were pre-incubated with the indicated antibodies for 60 min at terminal Cys residue for conjugation to keyhole limpet 4 °C prior to mixing with target cells. All β-gal values shown hemocyanin. New Zealand White rabbits were immunized for cell lysate assays represent the mean of duplicate assay with 0.5 mg of conjugated peptide (Covance BP, Inc., PA). IgG wells, ±SD. was purified from antisera on protein-A Sepharose 4B columns (Amersham-Pharmacia Biotech, Piscataway, NJ) according to KSHV virion entry assay standard procedures. Designations and sequences of the peptides were as follows: gBN1 (gB 282–298, SDRGTSPTPQN- KSHV virion entry was measured with the recombinant RIFVET), gBN3 (gB 22–41, GAAHSRGDTFQTSSSPTPPG), virus rKSHV.219 (provided by Jeffrey Vieira), which and gBC (gB 797–812, LQQEERQKADDLKKST); gH2 (gH contains the EGFP reporter gene under control of the 229–247, LPSLKGHATYDELTFARNA); gL2 (gL 139–158, constitutive cellular promoter for human elongation factor 1- AMNRTGSVSGSQTRAKSSSR); K8.1N3 (K8.1 44–59, GQ- alpha. Virion stocks were prepared by concentrating the VYQDWLGRMNCSYE). supernatants of infected Vero cells as previously described (Vieira and O'Hearn, 2004). The indicated target cells were Recombinant protein expression pre-incubated with rKSHV.219 virions for 90 min at 37 °C then washed and cultured in DMEM supplemented with Vaccinia-virus-based technology was employed for recom- 10% Fetal Bovine Serum. Virion entry was monitored at 2– binant protein expression (Moss et al., 1998) and for cell fusion 5 days by flow cytometry to detect EGFP-positive cells. assays (Dey and Berger, 2003) (see those review articles for Each infection was performed in duplicate, with equivalent original citations regarding details of specific recombinant results. vaccinia viruses). Infections of the indicated cells were performed at an MOI of 5 for each virus; the cells were Analysis of surface KSHV glycoproteins and integrins incubated overnight in suspension at 31 °C to allow protein expression then washed for subsequent analyses. The following BCBL-1 cells were pre-blocked with rabbit IgG and anti- vaccinia recombinants containing the indicated cDNA linked to human CD32 (Fcγ-RII) blocker (Stem cell Technologies, Inc., a vaccinia early/late promoter were employed: vTF7-3, Vancouver, Canada) then stained with primary antibodies (1:50 bacteriophage T7 RNA polymerase, vCB3 encoding human dilution of rabbit anti-peptide antisera against the indicated CD4, vCBYF1-fusin and vHC-1 encoding HIV-1 coreceptors KSHV glycoprotein) followed by fluorescein-isothiocyanate- (CXCR4 and CCR5, respectively), and vCB41 and vCB43 conjugated anti-rabbit secondary antibody (Dako, Dakopatts, encoding HIV-1 Env (strain IIIB, CXCR4-restricted and strain High Wycombe, UK). Cells were washed, resuspended in Ba-L, CCR5-restricted, respectively). For expression of KSHV staining media supplemented with 2% paraformaldehyde, and glycoproteins, the pVOTE 2.1 expression vector (Ward et al., analyzed on a FACScan flow cytometer (Becton Dickinson, 1995) containing the T7 promoter and regulatory elements from San Jose, CA). Layout and statistical parameters were analyzed the E. coli Lac operon was used to generate the following by FlowJo v4.5.9 software (Tree Star, Ashland, OR). To plasmids encoding cDNAs for KSHV gH (pJK2-gH), gL analyze surface expression of α3β1 integrin, cells were (pJK3-gL), K8.1A (pJK4-K8.1A), and truncated gB lacking the harvested in non-enzymatic cell dissociation reagent (Sigma) C-terminal 58 amino acids (Pertel et al., 2001) (pJK1-gBΔ). and stained with murine MAb P1B5 against human α3β1 integrin (IgG1, Chemicon International, Temecula, CA) at 4 °C Cell fusion assay for 30 min; murine IgG1 was used as a control primary antibody. After two washes, cells were incubated in a 1:100 KSHV-glycoprotein-mediated cell fusion was measured dilution of FITC-conjugated anti-mouse IgG and analyzed by using a vaccinia-virus-based reporter gene activation assay flow cytometry. 14 J.A.R. Kaleeba, E.A. Berger / Virology 354 (2006) 7–14

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