Journal: Journal of Virology
Article Title: Kaposi Sarcoma-Associated Herpesvirus Glycoprotein H Is Indispensable for Infection of Epithelial, Endothelial, and Fibroblast Cell Types
doi: 10.1128/JVI.00630-19
Figure Lengend Snippet: rKSHVΔgH-eGFP does not infect epithelial, endothelial, or fibroblast cells, but infection of B cells remains equivocal. (a) Indicated cell types were seeded (5 × 104/well) in 48-well plates in triplicate and incubated with purified viruses (102 to 104 viral genome copies per cell) obtained from induced iSLK rKSHV WT-eGFP, iSLK rKSHVΔgH-eGFP, or iSLK rKSHVgH-eGFP Rev cells. After 48 h, eGFP+ cells were analyzed using flow cytometry (left panels) and imaged using EVOS cell imaging fluorescence microscopy (right panels) to determine viral infectivity. Representative flow cytometry plots and micrographs are shown (n ≥ 3 independent experiments). (b to d) Tonsil-derived primary fibroblasts from four donors or MC116 cells were infected by spinoculation at 1,500 × g for 1 h at room temperature with rKSHV WT-eGFP, rKSHVΔgH-eGFP, or rKSHVgH-eGFP Rev. Mock viral infection was performed using purified iSLK rKSHV WT-eGFP inactivated in 2% buffered formaldehyde in PBS for 60 min at 37°C. At day 2 or 6 postinfection, tonsil-derived fibroblast cells or MC116 cells were analyzed; viable cells were gated from single-cell populations, and eGFP+ cells were gated from viable cell populations. Tonsil-derived primary fibroblast cells (b) were not susceptible to infection by rKSHVΔgH-eGFP, whereas limited infection of MC116 B cells by rKSHVΔgH-eGFP (c and d) was observed. MC116 cells infected with viruses were imaged using EVOS cell imaging fluorescence microscopy. (e) (Top panel) Percent infection of MC116 cells with formaldehyde-inactivated rKSHV WT-eGFP (mock), rKSHV WT-eGFP, rKSHVΔgH-eGFP, or rKSHVgH-eGFP Rev from three replicates. (Bottom panel) MC116 cells infected with formaldehyde-inactivated rKSHV WT-eGFP (mock), rKSHV WT-eGFP, or rKSHVΔgH-eGFP were FACS sorted to enrich for eGFP expression. Sorted cells were grown for 4 days, lysed, separated on 4 to 12% SDS-PAGE gels, and then analyzed by immunoblotting for the expression of KSHV latent (LANA1) and lytic (K8.1) genes along with cellular housekeeping gene β-actin as control. (f) RT-qPCR confirmation of LANA1 and K8.1 gene expression. cDNA was synthesized from 100 ng total RNA extracted from MC116 cells infected with formaldehyde-inactivated rKSHV WT-eGFP (mock), rKSHV WT-eGFP, or rKSHVΔgH-eGFP (FACS sorted and enriched for eGFP expression). Three microliters of the resulting cDNA was used for RT-qPCR with KSHV LANA1 and K8.1 gene-specific primers, and rKSHV WT-eGFP DNA was used as the standard for quantification. RNA extracted from induced iSLK rKSHV WT-eGFP served as a positive control. Average CT values obtained using GAPDH primers in individual samples are indicated below the graph. Samples were analyzed in triplicate, and the experiment was repeated three times. (g) Immunoblot analysis of known gH/gL cellular receptors mediating KSHV infection in permissive human cells tested for infection. From each cell type, 1 × 106 cells were lysed, separated on 4 to 12% SDS-PAGE gels, and then analyzed by immunoblotting using specific monoclonal antibodies against EphA2 (top), EphA4 (middle), or actin (bottom; loading control). (h) Flow cytometry analysis of EphA7 receptor expression in MC116, HEK-293, and iSLK cells. P values were calculated using a Kruskal-Wallis nonparametric test and showed there was no difference among virus-infected MC116 cells in panels e and f.
Article Snippet: Human embryonic kidney epithelial cells (HEK-293), human cervical epithelial cells (HeLa), human umbilical vein endothelial cells (HUVEC), human foreskin fibroblasts (HFF-1), a human EBV- and KSHV-negative lymphoblastoid B cell line (MC116), Chinese hamster ovary epithelial-like cells (CHO-K1), African green monkey endothelial cells (Vero), mouse fibroblast cells (NIH 3T3), and Syrian golden baby hamster kidney fibroblasts (BHK-21) were obtained from the American Type Culture Collection (ATCC; Manassas, VA).
Techniques: Infection, Incubation, Purification, Flow Cytometry, Imaging, Fluorescence, Microscopy, Derivative Assay, Expressing, SDS Page, Western Blot, Control, Quantitative RT-PCR, Gene Expression, Synthesized, Positive Control, Bioprocessing, Virus
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