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

Abstract

Coxsackievirus-B4 (CV-B4) can persist in pancreatic cell lines and impair the phenoytpe and/or gene expressions in these cells; however, the models used to study this phenomenon did not produce insulin. Therefore, we investigated CV-B4 persistence and its consequences in insulin-producing pancreatic β cells. The insulin-secreting rat β cell line, INS-1, was infected with CV-B4. After lysis of a large part of the cell layer, the culture was still maintained and no additional cytopathic effect was observed. The amount of insulin in supernatants of cell cultures persistently infected with CV-B4 was not affected by the infection; in fact, a larger quantity of proinsulin was found. The mRNA expression of pro-hormone convertase 2, an enzyme involved in the maturation of proinsulin into insulin and studied using real-time reverse transcription-polymerase chain reaction, was inhibited in infected cultures. Further, the pattern of 47 cell proteins analyzed using Shotgun mass spectrometry was significantly modified. The DNA of persistently infected cell cultures was hypermethylated unlike that of controls. The persistent infection of INS-1 cells with CV-B4 had a deep impact on these cells, especially on insulin metabolism. Cellular changes caused by persistent CV-B4 infection of β cells can play a role in type 1 diabetes pathogenesis.

Persistent coxsackievirus B infection and pathogenesis of type 1 diabetes mellitus

“…By contrast, in the carrier-state persistent infection, only a small proportion of the cells are considered to be infected but high titres of virus particles are produced 76 , 77 . Carrier-state CVB persistent infection has been reported in human pancreatic islets 23 and in pancreatic cell lines 26 – 28 , 93 , 94 . Enteroviruses and especially CVBs were thought to be released from infected cells by cell lysis; however, evidence indicates that these viruses can use autophagosome-like vesicles, cellular protrusions or extracellular vesicles for non-lytic viral egress 95 , 96 .…”

“…Inhibition of PCSK2 (an enzyme involved in the maturation of proinsulin to insulin) and induction of DNA hypermethylation have been reported in an insulin-producing rat β-cell line (INS-1), during persistent infection with CVB4 E2 (ref. 28 ).…”

“…CVBs are cytolytic viruses , but they are also able to establish a persistent infection in vitro in human primary pancreatic islets, ductal cells, thymic epithelial cells (TECs) and monocyte-derived macrophages or in human and mouse pancreatic cell lines and TEC lines 23 – 28 and in vivo in mice for several months 29 . Of note, persistent infection in vitro and in vivo in mice results in induction of structural or functional alterations in pancreatic and immune cells and development of autoimmunity towards islets 23 – 29 .…”

Early Emergence of 5′ Terminally Deleted Coxsackievirus-B3 RNA Forms Is Associated with Acute and Persistent Infections in Mouse Target Tissues

“…Dystrophin degradation has been shown to be the consequence of the viral protease 2A activity, even in 5 TD EV-B infection [45]. Insulin production decrease could be linked to viral protease activity on cellular translator factors [46,47]. Such impairment of specialized cell functions has been shown to be directly linked to the development of dilated cardiomyopathy or type 1 diabetes in mice and humans [48][49][50][51].…”

Persistent Enterovirus Infection: Little Deletions, Long Infections

“…Eventually, there is a fluctuating population of cells and of virions, with the persistence of both in the closed culture. Although studies using carrier cultures can demonstrate mutations that may adapt the virus to a particular cell type and/or changes in the cells in the culture in adaption to the virus [ 36 , 37 ], this does not seem characteristic of in vivo infections of the heart or pancreas, in which the tissue is well differentiated and not dividing. In particular, persistent infections within the heart demonstrate an enterovirus persisting in the well-differentiated, nondividing cardiomyocytes [ 18 , 38 ].…”