Persistence of Coxsackievirus B4 in Pancreatic β Cells Disturbs Insulin Maturation, Pattern of Cellular Proteins, and DNA Methylation

Figure 1. Infection of pancreatic β cells by CV-B4 E2. Rat pancreatic β cells (INS-1 cell line) (a) infected with CV-B4 E2 at an MOI of 0.01 (b) The cytopathic effect (CPE) observed 72 h p.i, followed by lysis of most cells. (c) Only a few cells survived the lytic phase of infection. (d) Culture was maintained, and the cells organized into clusters. The cells were observed under an inverted light microscope (magnification ×100). (e) The viability of the cells was evaluated using the Uptiblue™ assay. The results are expressed as % relative to mock-infected cells (for which the viability index is 100%). The values correspond to the means ± SD of 10 independent experiments.

Figure 2. Persistence of CV-B4 E2 in cultures of pancreatic β cells. Cultures of pancreatic β cells (INS-1 cell line) infected with CV-B4 E2 were monitored over time. Culture supernatant and cells were recovered. (a) Infectious viral particles detected via titration in the culture supernatant. The limit of detection of the infectious titer (1.5) is indicated by the dotted line. The values are the mean ± SD of three independent experiments. (b) Intracellular enteroviral RNA detected using RT-PCR, followed by agarose gel electrophoretic migration of the amplification products. MW: Size marker (DNA Ladder 100 bp ®). A 435-bp (EV) band corresponds to the amplification product of enteroviral (EV) RNA obtained through RT-PCR. β-actin was used as a control for the presence of RNA and corresponds to a band of 182 bp. (c) CV-B4-infected INS-1 cells were stained for intracellular VP1 with mouse anti-enterovirus VP1 and Alexa Fluor™ 488-conjugated rabbit anti-mouse antibody using an indirect immunofluorescence assay. The nuclei were stained with Hoechst Dye 33342® (original magnification ×20). Pictures from one representative experiment out of three are shown.

Figure 3. Pancreatic β cells persistently infected with CV-B4 E2 secrete insulin. Cultures of pancreatic β cells (INS-1 cell line) infected with CV-B4 E2 or uninfected were maintained for 90 days. (a) The cells were cultured for 2 h in RPMI-1640 medium, followed by the removal of the supernatant and cells. The presence of insulin in the culture supernatants of infected or mock-infected cells was evaluated using ELISA. The amount of insulin present in the culture supernatants was normalized by the total amount of DNA extracted from the cells. Values are the mean ± SD of 10 independent experiments. (b) CV-B4 infected INS-1 cells were fixed, permeabilized, and double-stained for intracellular VP1 (in green) and insulin (in red) with mouse anti-enterovirus VP1 and guinea pig anti-insulin antibodies using an indirect immunofluorescence assay. The nuclei (in blue) are stained with Hoechst Dye 33342® (original magnification ×40). Pictures from one representative experiment out of three are shown.

Figure 4. Profile of proinsulin, insulin, and pro-hormone convertase 2 in pancreatic β cells persistently infected with CV-B4 E2. Mock and pancreatic β cells (INS-1 cell line) persistently infected with CV-B4 E2 were cultured for 2 h in RPMI-1640 medium, and the supernatant was recovered. The cells were then lysed to extract total DNA and RNA (a) The concentrations of proinsulin and insulin in the supernatants of cultures were evaluated using ELISA. The ratios of proinsulin (PI) and insulin (I) concentrations were calculated. The results are expressed as % of the PI:I ratio compared with the PI:I ratio obtained with mock-infected cultures (control cells). The PI:I ratio of mock-infected cells is represented by a dotted line. Values are the mean ± SD of 10 independent experiments, * p < 0.05. (b) Pro-hormone convertase 2 (PCSK2) mRNA was quantified using RT-qPCR. Normalization was performed with β-actin mRNA. The relative expression in mock versus CV-B4 E2-infected INS-1 cells was determined with the 2−ΔΔCt formula. The value of PCSK2 expression in uninfected cells is represented by a dotted line. The values are the mean ± SD of three independent experiments encompassing 4 to 6 replicates…

Figure 5. The DNA of pancreatic β cells persistently infected with CV-B4E2 was hypermethylated. Pancreatic β cells (INS-1 cell line) persistently infected with CV-B4 E2 or mock-infected were lysed to extract total DNA. The level of methylated cytosines (5′mC) in DNA of infected and mock-infected cells was measured using ELISA. The results are expressed as % of the 5′ mC level in DNA of mock-infected cells. The methylation value of mock-infected cells is represented by a dotted line. The values are the mean ± SD of four independent experiments, * p < 0.05.