Persistence of Coxsackievirus B4 in pancreatic ductal-like cells results in cellular and viral changes

Abstract

Introduction: Although known as cytolytic viruses, group B coxackieviruses (CVB) are able to establish a persistent infection in vitro and in vivo. Viral persistence has been reported as a key mechanism in the pathogenesis of CVB-associated chronic diseases such as type 1 diabetes (T1D). The impact of CVB4 persistence on human pancreas ductal-like cells was investigated.

Methods: A persistent CVB4 infection was established in ductal-like cells. PDX-1 expression, resistance to CVB4-induced lysis and CAR expression were evaluated. The profile of cellular microRNAs (miRNAs) was investigated through miRNA-sequencing. Viral phenotypic changes were examined, and genomic modifications were assessed by sequencing of the viral genome.

Results: The CVB4 persistence in ductal-like cells was productive, with continuous release of infectious particles. Persistently infected cells displayed a resistance to CVB4-induced lysis upon superinfection and expression of PDX-1 and CAR was decreased. These changes were maintained even after virus clearance. The patterns of cellular miRNA expression in mock-infected and in CVB4-persistently infected ductal-like cells were clearly different. The persistent infection-derived virus (PIDV) was still able to induce cytopathic effect but its plaques were smaller than the parental virus. Several mutations appeared in various PIDV genome regions, but amino acid substitutions did not affect the predicted site of interaction with CAR.

Conclusion: Cellular and viral changes occur during persistent infection of human pancreas ductal-like cells with CVB4. The persistence of cellular changes even after virus clearance supports the hypothesis of a long-lasting impact of persistent CVB infection on the cells.

Keywords: CAR; Coxsackievirus B4; PDX-1; miRNA; pancreatic cells; persistence.

Figure 1.

Characterization of persistent CVB4 infection in Panc-1 cells. A persistent CVB4 infection was established in Panc-1 cells. Cells were observed under an inverted microscope (initial magnification X 200) (a). Viral titer in supernatants was determined using end point dilution assay (b). Intracellular viral RNA was quantified using a real-time RT-qPCR (c). PDX-1 mRNA was assessed during persistent infection by using real-time RT-qPCR and expressed as fold-change as compared with uninfected cells (d). Results presented (b-e) are mean +/− SD of 3 independent persistent infections.

Figure 2.

Acute CVB4 superinfection of persistently infected cells did not induce cell lysis. CVB4 and uninfected cells were infected with CVB4 at a MOI of 10. Cell viability was assessed at 48h post infection by using the cristal violet assay (a). Supernatants were collected and viral progeny was determined (b), Cells were harvested, washed, and intracellular viral RNA was quantified by using RT-qPCR (c). Results are mean+/−SD of 3 independent experiments.

Figure 3.

CAR expression is significantly decreased during persistent infection. CAR mRNA was quantified by real-time RT-qPCR during persistent infection. Results are mean+/−SD of 3 independent experiments.

Figure 4.

Persistence of changes induced in CVB4 persistently infected cells after virus clearance. CVB4 persistent infection was cured using fluoxetine, and then PDX-1 mRNA expression was evaluated in CVB4 peristently infected cells that were cured (a). Cured cells and untreated cells were infected with CVB4 at a MOI of 10. Cell viability (b), viral progeny (c), and intracellular viral RNA (d) were investigated. CAR mRNA was quantified by real-time RT-qPCR (e). The membrane expression of CAR was evaluated by flow cytometry (f). Results are mean+/−SD of 3 independent experiments, and one representative experiment is shown for flow cytometry.

Figure 5.

Cellular microRNA profile during CVB4 persistent infection. MiRNA sequencing was performed on CVB4 and uninfected Panc-1 cells. The profile is compared between CVB4 and uninfected cells (a). MiRNAs with a fold change ≥ 3 and p<0.05 were considered as differentially expressed (b). The fold-change of miR-146a and miR-23b expression in persistently infected cells determined by miR-sequencing is shown (c). Taqman RT-qPCR was used to to quantify miR-146a and miR-23b in CVB4 acutely and persistently infected cells. RT-qPCR results are mean +/− SD of 3 independent experiments (d).

Figure 6.

Impact of miRNA mimic transfection on virus replication and CAR mRNA expression in Panc-1 cells. Panc-1 cells were transfected with miR-146a, miR-138 and miR-23b mimics at 20nM, and then infected with with CVB4 at MOI of 1, 24h after transfection. Levels of MiRNAs were quantified in cells (a-c). Viral titers in supernatants (d) and intracellular viral RNA levels (e) were determined at 48h post infection. MiR-146a mimic was transfected in Panc-1 cells at 50 nM, which were subsequently inoculated with CVB4 at MOI of 0.01. The expression of miR-146a (f) and CAR mRNA (g) was assessed in cells. Results are mean+/−SD of 3 independent experiments.

Figure 7.

Characterization of persistent infection derived virus. The aspect of the plaque induced by the persistent infection derived virus and the parental virus were compared (a). The ratio between the viral titer and the viral RNA load was evaluated for both viruses (b).

Figure 8.

CVB4 footprints on CAR during persistent infection in Panc-1 cells. Mutations with a frequency ≥ 20% obtained by deep sequencing of 2 CVB4 independent acute infections and 2 independent persistent infections in Panc-1 cells, were integrated in the sequence of the CVB4E2 published strain (Accession number: AF311939.1). Translated amino acid sequences were obtained using blastx (NCBI). VP1(a) and VP2(b) sequences were aligned along with sequences corresponding to CVB3/28 and CVB3/Nancy strains, using Uniprot software. Footprints of the virus on CAR are shown in blue boxes.

Figure 9.

Modeling of interaction between CAR and Coxsackievirus B VP1 north canyon rim. (a-b) Interaction between CAR and Coxsackievirus B3. VP1 model (PDB:1COV) was used. CAR residues that represent the virus footprint (as described by Organtini et al., 2014) have been modeled using CAR D1 domain (PDB:1F5W) as template and are shown in red. VP1 north canyon rim residues are shown in blue. Only Hydrogen bonds that link CAR to a VP1 amino-acid involved in CAR recognition (Organtini et al., 2014) are displayed. Bounds were observed with ASN 211, ASN 212, THR 215 and TYR 217. (c-d) Interaction between CAR and Coxsackievirus B4 E2. The CVB4 E2 strain used in our laboratory (stock virus) was sequenced on a Ion PGM™ deep sequencing platform, and mutations with a prevalence higher that 20% were introduced in the CVB4 E2 published full genome sequence (AF311939.1). The VP1 model was built using PDB:1COV as template. Bounds were observed with 3 VP1 amino-acids (ASN 214, ASN 215, GLY 217) including 2 residues observed in CVB3 model (ASN 214, ASN 215 that correspond to ASN 211 and ASN 212 in CVB3 reading frame). (e-f) Interaction between CAR and Coxsackievirus B4 derived from persistent infection in Panc-1 cells (PIDV). PIDV sequence was determined as described above. The VP1 model was built using PDB:1COV as template. Bounds were observed with the same residues (ASN 214, ASN 215, GLY 217) as compared with the initial stock virus. Nucleotides sequences were translated in proteins using BlastX (NCBI). Models were created using Swiss model (https://swissmodel.expasy.org/). Docking data were obtained from Zdock server (zdock.umassmed.edu/) and interactions investigated using Biova/Discovery Studio 2016 software (Accelry Inc.). Conventional hydrogen bounds are shown in green dotted and carbon hydrogen bound in black dotted.