Enterovirus Persistence in Cardiac Cells of Patients With Idiopathic Dilated Cardiomyopathy Is Linked to 5’ Terminal Genomic RNA-Deleted Viral Populations With Viral-Encoded Proteinase Activities

Background

Coxsackieviruses of group B are common human pathogens which are considered the main viral cause of acute myocarditis. In many patients, the cardiac inflammation becomes chronic and progress to dilated cardiomyopathy (DCM). Experimentally, expression of a non-infectious but replication-competent CVB3 variant in transgenic mice resulted in dilated cardiomyopathy (Wessely et al.). Expression of CVB3 2A proteinase was found to cause significant impairment of cardiomyocyte function via proteolytic cleavage of dystrophin, leading to a decrease in cell contractility, an increase in membrane permeability, and a focal spread of the virus to adjacent cells (Badorff et al.). In acutely CVB3-infected mice significantly higher amounts of cardiac plus-strand RNA compared to minus-strand RNA were observed in acute myocarditis whereas in chronic myocarditis a ratio of plus- and minus-strand RNA of 1:1 was noted in the myocardium (Klingel et al.) Interestingly, similar findings with low ratios of plus- to minus RNA strands were observed by Chapman et al. in a letal cardiac CVB2 infection harboring a 5’ terminal deletion (TD).

Methods

In the present study, next generation sequencing (NGS) of the 5’ terminal genomic RNAs of persistent Enterovirus (EV) populations was performed in explanted heart tissue samples of patients with unexplained end-stage DCM. Subsequently, synthetic CVB3 RNAs harboring 5’ TDs like those observed in persistent viral populations were generated in vitro and transfected into cultured human primary cardiomyocytes (HCM) in order to investigate viral replication and translation activities.

Results

The presence of EV RNA was detected by quantitative RT-PCR in 8 (33%) of 24 patients with IDCM. The 5’ terminal regions of EV RNAs isolated from persistent infections were characterized with a validated NGS strategy. The persistent EV-B strains harbored 5’ TDs ranging in size from 17 to 50 nucleotides and were detected either alone or associated with low proportions of intact 5’ genomic termini. Transfection of viral RNA into primary human cardiomyocytes demonstrated that deleted forms of genomic RNAs displayed early replication activities in the absence of detectable viral plaque formation, whereas mixed deleted and complete forms generated particles capable of inducing cytopathic effects at levels distinct from those observed with full-length forms alone. Importantly, deleted or full-length and mixed forms of viral RNA were capable of directing translation and production of the proteolytically active viral proteinase 2A in human cardiomyocytes.

Discussion

The data presented in this study support the findings of Lévêque et al. who demonstrated that 5’ UTR deletions reduce the binding of replication factors to positive-strand CVB3 RNA, resulting in nondetectable RNA replication activities in cardiomyocytes or HeLa cells.

As 20% of DCM samples harbored the viral 5’ deleted forms of RNA in cardiac tissues it is not excluded that the replication of these deleted forms are sustained during long-term persistent cardiomyocyte infections in patients with DCM. It can be hypothesized that the full-length viral RNA form could initially provide helper functions to the deleted RNA forms by providing viral proteins involved in replication and by stimulating the synthesis of negative-strand RNA intermediates.

Furthermore, it was evaluated how the presence of very low levels of replication- impaired, 5’ terminally deleted forms of EV RNA could mediate cellular pathology. It is known that 2Apro activity is responsible for dystrophin disruption in CVB3 infection. The results of this study suggest that persistent forms of EV-B 5’ deleted genomic RNAs are associated with the pathogenesis of unexplained DCM cases, possibly via viral 2Apro proteolytic activity. Thus, the search for specific inhibitors of the CV-B 2Apro activity should be intensified, being capable to restrict viral replication and inhibit the shutoff of host cell translation and the disruption of dystrophin to prevent disease progression to chronic myocarditis and DCM. However, other mechanisms contributing to the pathogenesis of chronic enterovirus myocarditis are not excluded, given that the infection is associated with the activation of the immune system finally resulting in the apoptosis of cardiomyocytes.