Fig 1.
Cell viability time course of neuroblastoma cells infected by Zika virus.
Neuroblastoma cell lines IMR-32, SMS-KAN, SK-N-Be(1), SK-N-AS, LA-N-6, and CHLA-42, as well as Vero cells (infection control), were infected with Zika virus strain PRVABC59 and monitored for 10 days. Cell viability was assessed by MTS assay every two days for 10 days total. All infections were performed with MOI = 10 and the results shown are compared to control cells treated with non-infected conditioned media for each cell line. Data shown are the composite of experiments performed in triplicate, with error bars representing standard deviation.
Fig 2.
Permissiveness of neuroblastoma cells to Zika viral infection.
A) Western blot analysis of Zika virus infections in neuroblastoma cells. Total protein analysis was performed for Zika Envelope protein and NS1 (Non-Structural 1) protein compared to GAPDH control. The neuroblastoma cell lines tested included IMR-32, SMS-KAN, SK-N-AS, LA-N-6, SK-N-Be(1), and CHLA-42, using Vero cells as an infection control. All cells were compared to control cells treated with non-infected conditioned media. Samples were assessed 3 days after infection (MOI = 10). These results were representative of the combined data of experiments performed in triplicate. B) Viral Titer (TCID50) assays of IMR-32 and SK-N-AS cells at Day 2 and 3 post-infection. Data is composed of three biological replicates examined in sextuplicate, with error bars representing standard deviation. ** p > 0.05, Student’s t-test. C) Immunofluorescence labeling of Zika viral Envelope protein in IMR-32 and SK-N-AS cells at Day 3 post-infection. Envelope staining is in red (Alexa Fluor 647) and nuclei are stained in blue (DAPI). Samples are also shown together (merged). Cells were scanned using a Nikon A1R VAAS laser point- and resonant-scanning confocal microscope. Images are at a magnification of 40x with a 4x zoom.
Fig 3.
Analysis of CD24 expression in human neuroblastoma cells.
A) Schematic of the alignment of CD24 splice variants 1 and 7. B & C) Absolute quantification of CD24 expression by quantitative real-time PCR of total RNA acquired from neuroblastoma cells, measuring CD24 splice variants 1 (B) and 7 (C). Copy number values were normalized to the corresponding GAPDH values to determine the relative copy number. ** p > 0.05, Student’s t-test. D) Western blot analysis of CD24 expression in the total cell lysates of neuroblastoma cells. GAPDH was used as a loading control. All results are representative of the combined data of experiments performed in triplicate, with error bars representing standard deviation.
Fig 4.
Analysis of the role of CD24 in Zika-virus infected neuroblastoma cells.
A) Western blot analysis of siRNA-mediated knock-down of CD24 expression in IMR-32 cells. Samples include Negative Control siRNA and CD24 siRNA. B) Western blot analysis of the expression of Envelope protein and NS1 (Non-Structural 1) protein in IMR-32 cells after siRNA-mediated knock-down of CD24 expression, 96 hours after Zika infection (MOI = 10). Samples include control cells treated with non-infected conditioned media and infected IMR-32 cells transfected with either Negative Control siRNA or CD24 siRNA. C) Western blot analysis of CD24 expression in the human neuroblastoma cell line SK-N-AS, comparing wild type (WT) to stably selected “Vector Only” (VO), CD24 variant 1 (V1), and CD24 variant 7 (V7). D) Western blot analysis of Zika NS1 protein expression 96 hours after Zika infection in CD24-stably expressing SK-N-AS cells, comparing wild type (WT) to stably selected Vector Only (VO), CD24 variant 1 (V1), and CD24 variant 7 (V7). GAPDH was used as a load control for all experiments. All results are representative of the combined data of experiments performed in triplicate.
Fig 5.
CD24 expression renders SK-N-AS cells susceptible to Zika virus-induced cytotoxicity.
A) Cell viability and B) Apoptosis assays comparing Zika virus-infected and uninfected cells. Wild-type (WT) SK-N-AS cells, “Vector Only” (VO) SK-N-AS cells, and SK-N-AS cells expressing CD24 variant 1 (CD24 V1) and CD24 variant 7 (CD24 V7) were infected (MOI = 10) for 96 hours, and then subjected to MTS (Fig 5A) and caspase 3/7 (Fig 5B) assays. Zika infected cells were compared to control cells treated with non-infected conditioned media. The results are representative of the combined data of experiments performed in sextuplicate (n = 6), with error bars representing standard deviation. ** p > 0.05, Student’s t-test. C) Viral Titer (TCID50) assays of SK-N-AS/VO, SK-N-AS/CD24 v1, and SK-N-AS/CD24 v7 cells at Day 2 and 3 post-infection. Data is composed of three biological replicates examined in sextuplicate, with error bars representing standard deviation. ** p > 0.05, Student’s t-test. C) Immunofluorescence labeling of Zika viral Envelope protein in SK-N-AS/VO, SK-N-AS/CD24 v1, and SK-N-AS/CD24 v7 cells at Day 3 post-infection. Envelope staining is in red (Alexa Fluor 647) and nuclei are stained in blue (DAPI). Samples are also shown together (merged). Cells were scanned using a Nikon A1R VAAS laser point- and resonant-scanning confocal microscope. Images are at a magnification of 40x with a 4x zoom.
Fig 6.
Zika virus strains PRVABC59, MR766 and IBH 30656 all induce severe cytopathic effects in CD24-expressing SK-N-AS cells.
Wild-type (WT) SK-N-AS cells, Vector Only (VO) SK-N-AS cells, and SK-N-AS cells expressing CD24 variant 1 (CD24 v1) and CD24 variant 7 (CD24 v7) were infected with Zika virus reference strains PRVABC59, MR 766 and IBH 30656 (MOI = 10). Zika infected cells were compared to control cells treated with non-infected conditioned media. After 96 hours, cellular ATP levels were measured and normalized to cell number. The results are representative of the combined data of experiments performed in triplicate, with error bars representing standard deviation. ** p > 0.05, Student’s t-test.