Major 5′terminally deleted enterovirus populations modulate type I IFN response in acute myocarditis patients and in human cultured cardiomyocytes

Abstract

the french enterovirus Myocarditis Study Group (feMSG) * Major 5′terminally deleted (5′TD) group-B enterovirus (EV-B) populations were identified in heart biopsies of patients with fulminant myocarditis or dilated cardiomyopathy suggesting that these 5′TD forms are key drivers of host-cell interaction in EV cardiac infections. To date, early emergence of EV-B 5′TD forms and its impact on type 1 IFN response during acute myocarditis remains unknown. Using quantitative RACE-PCR assay, we identified major EV-B 5′tD RnA populations in plasma or heart samples of acute myocarditis cases. Deletions identified within the 5′ non-coding region of EV-B populations only affected secondary-structural elements of genomic RNA domain I and were distinguished in two major groups based on the extent of RnA structural deletions. proportions of these two respective EV-B 5′tD population groups were positively or negatively correlated with IFN-β levels in plasma samples of myocarditis patients. Transfection of synthetic CVB3/28 RNAs harboring various 5′terminal full-length or deleted sequences into human cultured cardiomyocytes demonstrated that viral genomic RnA domain i possessed essential immunomodulatory secondarystructural elements responsible for IFN-β pathway induction. Overall, our results highlight the early emergence of major EVB-TD populations which deletions affecting secondary-structures of RNA domain i can modulate innate immune sensing mechanisms in cardiomyocytes of patients with acute myocarditis. Group-B Enteroviruses (EV-B) belong to the Picornaviridae family and are recognized as major causes of aseptic meningitis, upper or lower respiratory tract infections and acute myocarditis cases diagnosed in neonates, infants and young adults 1,2. Between 10-20% of acute myocarditis cases will evolve into chronic myocarditis as well as dilated cardiomyopathy (DCM, prevalence = 7 cases / 100,000, second leading cause of heart transplantation worldwide after ischemic heart disease) 1. EV-B RNA genome is approximately 7,400 nucleotides (nt) in length and is flanked at the 5′ end by a highly conserved non-coding region (5′NCR) that is crucial for the initiation of the viral replication and translation activities 3. A study in 2008 of heart tissue from a patient who died of fulminant myocarditis demonstrated the presence of a group B Coxsackie virus type 2 (CVB-2) population with partial deletions at the 5′ terminus and proceeding inward 4. Similar findings had been described following CVB-3 replication in cell cultures and in mice 5,6. Bouin et al.7 identified EV-B populations characterized by 5′NCR RNA deletions ranging from 15 to 48 nt

Structures and Functions of Viral 5′ Non-Coding Genomic RNA Domain-I in Group-B Enterovirus Infections

“…Natural 5 TD viral genomes resulting in the loss of different structures ranging from stem "a", to stem-loop "b" or "c" or part of stem-loop "d" in the 5 terminal Cloverleaf RNA structure (domain-I) were described in in vitro and in vivo experimental models of EV-B infections. Recently, EV-B populations with 5 terminal genomic RNA deletions (5 TD) similar to those described in experimental models were associated with the development of myocarditis or DCM human cases [16,17]. These 5 TD EV-B populations were characterized as low replicating RNA forms with viral-encoded proteinase activities related with dystrophin cleavage in human cardiac tissues [16].…”

“…Moreover, low expression levels of replication complex cellular factors such as hnRNP-C could decrease viral genome replication activity levels of full-length ends of negative-strand viral RNA in various human target tissues and cells [18,98]. Overall, hnRNP-C or other unknown host proteins of RNP complexes formed with the 3 ends of negative-strand could provide an early replicative advantage of full-length viral RNA forms in specific cells and drive the development of enteroviral human infections [17,18,96]. Identification of new cell host proteins of viral RNP complexes could help in designing new therapeutic strategies either targeting or inhibiting host protein activities involving EV-B RNA replication in human infections.…”

“…In cultured human cardiomyocytes, the deleted viral RNA forms can produce viral proteinases as 2A pro whose levels impact on cell host protein synthesis machinery by cleavage of major host cell proteins as eukaryotic translation initiation factor 4G (eIF4G) [16,18,125,126]. Overall, natural 5 TD viral genomes resulting in the loss of different structures in the RNA domain-I could be major key players of host-cell interactions in EV-B human infections [17].…”

“…Moreover, EV-71 3C pro activity was described as capable to cleave Interferon Regulatory Factor 7 (IRF-7) inhibiting its ability to transactivate IFN-β expression by its phosphorylated form during early phase of viral infection [133] (Figure 5). Natural truncated forms of up to 50 nt in the 5 NCR of the EV-B in experimental or clinical infections [16,116] would allow the maintenance of the viral genome in the infected host cell by a modulated activation of the innate immune system [17] (Figure 6). Recently, these 5 TD EV-B RNA forms showed their ability, alone, to produce in silico, in vitro, and in vivo viral proteins such as the viral 2A proteinase resulting in 2A pro -specific eIF4G cleavage [16].…”

“…To date, the specific contributions of RIG-I or MDA5 to the anti-EV-B innate immune response remain to be investigated in various infected cells. Through transfection of synthetic complete or various 5 TD EV-B RNA forms into cultured human cardiac cells, it was demonstrated that natural deletions into the 5 RNA domain-I of EV-B can modify the RNA secondary-structural elements recognized by cytoplasmic RLRs such as RIG-I or MDA5 and modulate type I interferon (IFN) activation pathway induction [17]. Interestingly, similar natural 5 terminally deletions capable of modulating activation type I IFN response were characterized in patients with acute myocarditis.…”