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Pacific Biosciences exonuclease iii
rKSHVΔgH-eGFP does not infect epithelial, endothelial, or fibroblast cells, but infection of B cells remains equivocal. (a) Indicated cell types were seeded (5 × 10 4 /well) in 48-well plates in triplicate and incubated with purified viruses (10 2 to 10 4 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 <t>37°C.</t> 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 <t>three</t> 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 C T 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 × 10 6 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.
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1) Product Images from "Kaposi Sarcoma-Associated Herpesvirus Glycoprotein H Is Indispensable for Infection of Epithelial, Endothelial, and Fibroblast Cell Types"

Article Title: Kaposi Sarcoma-Associated Herpesvirus Glycoprotein H Is Indispensable for Infection of Epithelial, Endothelial, and Fibroblast Cell Types

Journal: Journal of Virology

doi: 10.1128/JVI.00630-19

rKSHVΔgH-eGFP does not infect epithelial, endothelial, or fibroblast cells, but infection of B cells remains equivocal. (a) Indicated cell types were seeded (5 × 10 4 /well) in 48-well plates in triplicate and incubated with purified viruses (10 2 to 10 4 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 C T 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 × 10 6 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.
Figure Legend 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 × 10 4 /well) in 48-well plates in triplicate and incubated with purified viruses (10 2 to 10 4 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 C T 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 × 10 6 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.

Techniques Used: Infection, Incubation, Purification, Flow Cytometry, Cytometry, Imaging, Fluorescence, Microscopy, Derivative Assay, FACS, Expressing, SDS Page, Quantitative RT-PCR, Synthesized, Positive Control

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Incubation:

Article Title: Base modifications affecting RNA polymerase and reverse transcriptase fidelity
Article Snippet: .. Then, 50 U Exonuclease III (Escherichia coli ) and 5 U Exonuclease VII were added to the reaction and incubated at 37°C for 1 h. SMRTbell libraries were cleaned using a Zymo column, then additionally size selected with AMPure PB beads (Pacific Biosciences) using 1× volume (500 bp libraries) or 0.6× volume (1 kb libraries). .. Pacific Biosciences Binding Calculator was used to generate a protocol for annealing sequencing primers and polymerase binding using the DNA/Polymerase Binding Kit P6 v2 (Pacific Biosciences) and default settings.

Article Title: Kaposi Sarcoma-Associated Herpesvirus Glycoprotein H Is Indispensable for Infection of Epithelial, Endothelial, and Fibroblast Cell Types
Article Snippet: .. Briefly, the DNA was incubated with exonuclease VII at 37°C for 15 min to remove single-stranded DNA, and any possible DNA damage was repaired using a DNA damage repair mix at 37°C for 20 min. Blunt-ended DNAs were treated with end-repair mix at 25°C for 5 min and ligated with 1 μM annealed blunt adaptors using 0.75 U/μl ligase at 25°C overnight, and then the ligase was inactivated by incubation at 65°C for 10 min. To remove failed ligation products, samples were treated with exonuclease III and VII at 37°C for 1 h. To purify the DNAs and ligated products, 0.45× AMPure PB beads from Pacific Biosciences were applied. .. The final MagBead complexes were loaded into a PacBio RSII machine for SMRT sequencing, with one SMRT cell allocated to the complex for a 6-h running time.

Ligation:

Article Title: Kaposi Sarcoma-Associated Herpesvirus Glycoprotein H Is Indispensable for Infection of Epithelial, Endothelial, and Fibroblast Cell Types
Article Snippet: .. Briefly, the DNA was incubated with exonuclease VII at 37°C for 15 min to remove single-stranded DNA, and any possible DNA damage was repaired using a DNA damage repair mix at 37°C for 20 min. Blunt-ended DNAs were treated with end-repair mix at 25°C for 5 min and ligated with 1 μM annealed blunt adaptors using 0.75 U/μl ligase at 25°C overnight, and then the ligase was inactivated by incubation at 65°C for 10 min. To remove failed ligation products, samples were treated with exonuclease III and VII at 37°C for 1 h. To purify the DNAs and ligated products, 0.45× AMPure PB beads from Pacific Biosciences were applied. .. The final MagBead complexes were loaded into a PacBio RSII machine for SMRT sequencing, with one SMRT cell allocated to the complex for a 6-h running time.

Article Title: Long-read sequencing across the C9orf72 ‘GGGGCC’ repeat expansion: implications for clinical use and genetic discovery efforts in human disease
Article Snippet: .. Failed ligation products were removed by treatment with Exonuclease III and VII (Pacific Biosciences). .. Asymmetric SMRTbell templates were then enriched with MagBeads and buffers from MagBead Kit v2 (Pacific Biosciences, PN 100–676-500).

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    Pacific Biosciences exonuclease iii
    rKSHVΔgH-eGFP does not infect epithelial, endothelial, or fibroblast cells, but infection of B cells remains equivocal. (a) Indicated cell types were seeded (5 × 10 4 /well) in 48-well plates in triplicate and incubated with purified viruses (10 2 to 10 4 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 <t>37°C.</t> 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 <t>three</t> 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 C T 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 × 10 6 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.
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    rKSHVΔgH-eGFP does not infect epithelial, endothelial, or fibroblast cells, but infection of B cells remains equivocal. (a) Indicated cell types were seeded (5 × 10 4 /well) in 48-well plates in triplicate and incubated with purified viruses (10 2 to 10 4 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 <t>37°C.</t> 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 <t>three</t> 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 C T 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 × 10 6 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.
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    rKSHVΔgH-eGFP does not infect epithelial, endothelial, or fibroblast cells, but infection of B cells remains equivocal. (a) Indicated cell types were seeded (5 × 10 4 /well) in 48-well plates in triplicate and incubated with purified viruses (10 2 to 10 4 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 <t>37°C.</t> 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 <t>three</t> 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 C T 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 × 10 6 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.
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    rKSHVΔgH-eGFP does not infect epithelial, endothelial, or fibroblast cells, but infection of B cells remains equivocal. (a) Indicated cell types were seeded (5 × 10 4 /well) in 48-well plates in triplicate and incubated with purified viruses (10 2 to 10 4 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 C T 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 × 10 6 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.

    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 × 10 4 /well) in 48-well plates in triplicate and incubated with purified viruses (10 2 to 10 4 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 C T 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 × 10 6 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: Briefly, the DNA was incubated with exonuclease VII at 37°C for 15 min to remove single-stranded DNA, and any possible DNA damage was repaired using a DNA damage repair mix at 37°C for 20 min. Blunt-ended DNAs were treated with end-repair mix at 25°C for 5 min and ligated with 1 μM annealed blunt adaptors using 0.75 U/μl ligase at 25°C overnight, and then the ligase was inactivated by incubation at 65°C for 10 min. To remove failed ligation products, samples were treated with exonuclease III and VII at 37°C for 1 h. To purify the DNAs and ligated products, 0.45× AMPure PB beads from Pacific Biosciences were applied.

    Techniques: Infection, Incubation, Purification, Flow Cytometry, Cytometry, Imaging, Fluorescence, Microscopy, Derivative Assay, FACS, Expressing, SDS Page, Quantitative RT-PCR, Synthesized, Positive Control