Enterovirus-cardiomyocyte interactions: impact of terminally deleted genomic RNAs on viral and host functions

Abstract

Group B enteroviruses, including coxsackievirus B3 (CVB3), can persistently infect cardiac tissue and cause dilated cardiomyopathy. Persistence is linked to 5 terminal deletions of viral genomic RNAs that have been detected together with minor populations of full-length genomes in human infections. In this study, we explored the functions and interactions of the different viral RNA forms found in persistently-infected patients and their putative role(s) in pathogenesis. Since enterovirus cardiac pathogenesis is linked to the viral proteinase 2A, we investigated the effect of different terminal genomic RNA deletions on 2A activity. We discovered that 5 terminal deletions in CVB3 genomic RNAs decreased the proteinase activity of 2A but could not abrogate it. Using newly-generated viral reporters encoding nano-luciferase, we found that 5 terminal deletions resulted in decreased levels of viral protein and RNA synthesis in singly-transfected cardiomyocyte cultures. Unexpectedly, when full-length and terminally deleted forms were co-transfected into cardiomyocytes, a cooperative interaction was observed, leading to increased viral RNA and protein production. However, when viral infections were carried out in cells harboring 5 terminally deleted CVB3 RNAs, a decrease in infectious particle production was observed. Our results provide a possible explanation for the necessity of full-length viral genomes during persistent infection, as they would stimulate efficient viral replication compared to that of the deleted genomes alone. To avoid high levels of viral particle production that would trigger cellular immune activation and host cell death, the terminally deleted RNA forms act to limit the production of viral particles, possibly as trans-dominant inhibitors. ImportanceEnteroviruses like coxsackievirus B3 are able to initiate acute infections of cardiac tissue and, in some cases, to establish a long-term persistent infection that can lead to serious disease sequelae, including dilated cardiomyopathy. Previous studies have demonstrated the presence of 5 terminally-deleted forms of enterovirus RNAs in heart tissues derived from patients with dilated cardiomyopathy. These deleted RNAs are found in association with very low levels of full-length enterovirus genomic RNAs, an interaction that may facilitate continued persistence while limiting virus particle production. Even in the absence of detectable infectious virus particle production, these deleted viral RNA forms express viral proteinases at levels capable of causing viral pathology. Our studies provide mechanistic insights into how full length and deleted forms of enterovirus RNA cooperate to stimulate viral protein and RNA synthesis without stimulating infectious viral particle production. They also highlight the importance of targeting enteroviral proteinases to inhibit viral replication while at the same time limiting the long-term pathologies they trigger.