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

Abstract

Background: Group B enteroviruses are common causes of acute myocarditis, which can be a precursor of chronic myocarditis and dilated cardiomyopathy, leading causes of heart transplantation. To date, the specific viral functions involved in the development of dilated cardiomyopathy remain unclear.

Methods: Total RNA from cardiac tissue of patients with dilated cardiomyopathy was extracted, and sequences corresponding to the 5' termini of enterovirus RNAs were identified. After next-generation RNA sequencing, viral cDNA clones mimicking the enterovirus RNA sequences found in patient tissues were generated in vitro, and their replication and impact on host cell functions were assessed on primary human cardiac cells in culture.

Results: Major enterovirus B populations characterized by 5' terminal genomic RNA deletions ranging from 17 to 50 nucleotides were identified either alone or associated with low proportions of intact 5' genomic termini. In situ hybridization and immunohistological assays detected these persistent genomes in clusters of cardiomyocytes. 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. Moreover, deleted or full-length and mixed forms of viral RNA were capable of directing translation and production of proteolytically active viral proteinase 2A in human cardiomyocytes.

Conclusions: We demonstrate that persistent viral forms are composed of B-type enteroviruses harboring a 5' terminal deletion in their genomic RNAs and that these viruses alone or associated with full-length populations of helper RNAs could impair cardiomyocyte functions by the proteolytic activity of viral proteinase 2A in cases of unexplained dilated cardiomyopathy. These results provide a better understanding of the molecular mechanisms that underlie the persistence of EV forms in human cardiac tissues and should stimulate the development of new therapeutic strategies based on specific inhibitors of the coxsackievirus B proteinase 2A activity for acute and chronic cardiac infections.

Keywords: dilated cardiomyopathy; enterovirus; genomic RNA deletion; genomics; infection; persistent infection; viral 2A protease; viruses.

Figure 1:. Schematic representation of the Enterovirus genome.

A. Depiction of the genomic RNA of coxsackievirus. Structural proteins comprise the viral capsid and non-structural proteins are involved in replication. B. Two dimensional representation of the CV-B3 5’ terminal RNA sequences.

Figure 2.. Detection and characterization of EV persistent infection in cardiac tissues of idiopathic DCM patients.

A. Total EV RNA levels (copies/μg of total nucleic acid extract) in cardiac tissues of EV positive IDCM patients (8 of 24 tested IDCM patients) and healthy controls (n=14) were quantified using a generic EV RT-qPCR. Dots represent patients mean values (n=8). L: Left Ventricle, R: Right Ventricle, S: Septum. **: P<0.01 by Mann-Whitney U test. B. Quantification (RT-qPCR) of total and minus EV RNA strands in tissues of 8 EV positive IDCM patients (n=13 samples) using specific primers to each RNA strand polarity. Data represent mean values ± SEM (n=13); RNA+/RNA− ratio values are indicated as an index above each sample. When RNA minus strand levels were below the threshold of quantification value, RNA+/RNA− ratios were not calculated. C. Linear regression curve between the log₁₀ genome copies/μg and RNA+/RNA− ratio values (n=6) (P=0.038; R²=0.70). D. In situ Hybridization (ISH) targeting viral RNA (upper panels) displaying EV RNA (red) in serial cardiac tissue sections of infected DCM. Nuclei are stained in blue; Bar scale = 50 μm. White square (DCM+ EV+) corresponds to a high magnification (X400) of a single EV positive cardiomyocytes (bar scale = 50 μm). E. Fluorescence signal due to in situ hybridization of viral RNA observed in each slide was quantitatively estimated using Image J software (Software free NIH) three times at two magnifications (X20 and X40). Briefly, thresholding techniques were successively performed to remove background noise and empty spaces and to select relevant fluorescent cytoplasmic foci due to in situ hybridization. Results were expressed as fluorescent pixels per 100,000. ***: P<0.001 by Mann Whitney U test.

Figure 3.. Characterization of persistent 5’terminal deleted EV populations

A. Proportion of sequence reads detected in each patient (n=7) for each group of viral forms: Full-length (FL), deleted from 17 to 36 nucleotides (17–36) and deleted from 37 to 50 nucleotides (37–50). If multiple samples were obtained from a patient, they are displayed independently. B. Proportion of sequence reads found in the whole cohort for Full Length (FL) viruses and deleted forms groups of 17–36 and 37–50 nucleotides. Black bars represent the mean values [min., max. values]. *P<0.001 by Mann-Whitney U test. C. Schematic representation of the 5’ terminal deletions of EV-B cloverleaf structure with its binding sites for host PCBP and viral 3CD proteins. D. Linear regression curve between the log₁₀ EV genome copies/μg and reads proportion of deleted forms (both 17–36 and 37–50 groups) in each sample (n=10) indicating a decrease in the viral load when proportion of deleted forms increased (R²=0.54; P=0.016).

Figure 4.. Analysis of early and late replication activities of 5’ terminally deleted, full-length, and mixed CV-B3/28 RNA populations in primary human cardiac cells.

A–C. Synthetic RNAs (100 ng) of full-length (red squares) or deleted of 50 5’ terminal nucleotides (black circles) of CV-B3/28 forms were transfected alone or in association (95% of d50 associated with 5% of FL, blue triangles) in primary human cardiomyocytes (HCM): A. Viral RNA genomic replication activity (total viral RNA (intra and extracellular production)) was assessed using a RT-qPCR assay at 0, 8, 12, 24 and 48 hr post transfection. Data represent the mean ± SEM. *: P< 0.05 by Mann Whitney U test (n=3). B. Quantification by RT-qPCR of total, minus and EV RNA plus and minus strand ratios at each time post-transfection (in hours). RNA+/RNA− ratio values are indicated as an index above each sample. Data represent the mean ± SEM. Statistical analysis were assessed on the RNA+/RNA− ratios, *: P< 0.05; **: P< 0.01 by Mann Whitney U test (n=3). C. Viral RNA replication inhibition assay using guanidine hydrochloride (GuHCl; 2 mM) and fluoxetine (F; 8 μM) was performed at 0, 2 and 8 hr (h) post transfection for each viral form (d50: empty circle, FL: empty square, and mixed d50 + FL: empty triangle). Data represent the mean ± SEM. *: P< 0.05; **: P< 0.01; according to repeated measures two-way ANOVA test. D. Analysis of intra- and extra-cellular infectious particle production at 0, 8, 12, 24 and 48 hr (h) post transfection (n=3). Data represent the mean ± SEM. *: P< 0.05 by Mann Whitney U test.

Figure 5.. Viral protein production and analysis of 2A^pro activity in CV-B3 transfected human cardiomyocytes.

A. Western blot assay detecting eIF4G and VP1 in human cardiomyocytes at 24 hr post transfection of CV-B3/28 FL RNA, CV-B3/28 d50 RNA and both forms together. Proteins were subjected to electrophoresis on an SDS-containing polyacrylamide gel.1: Mock transfected, 2: 125 ng CV-B3/28 FL, 3: 2375 ng CV-B3/28 d50, 4: 125 ng CV-B3/28 FL + 2375 ng CV-B3/28 d50. B. Signals detected for eIF4G cleavage fragment in Western blot were standardized using GAPDH and quantified. 1: Mock transfected, 2: 125 ng CV-B3/28 FL, 3: 2375 ng CV-B3/28 d50, 4: 125 ng CV-B3/28 FL + 2375 ng CV-B3/28 d50. VP1 signal standardized with total eIF4G detection. C. Signals detected for VP1 in Western blot were standardized using GAPDH and quantified. 1: Mock transfected, 2: 125 ng CV-B3/28 FL, 3: 2375 ng CV-B3/28 d50, 4: 125 ng CV-B3/28 FL + 2375 ng CV-B3/28 d50. D. Western blot assay detecting eIF4G and VP1 in human cardiomyocytes at 12 hr post transfection of CV-B3/28 FL RNA. Z-VAD-FMK is reported to inhibit CV-B3 2A^pro. Synthetic full length (FL) CV-B3/28 RNA was transfected in HCM and proteins were extracted at 24 hr. Proteins were subjected to electrophoresis on an SDS-containing polyacrylamide gel. E. Detection of caspase-3 activation: Western blot assay detecting eIF4G and VP1 and caspase-3 in human cardiomyocytes at 24 hr post transfection. Proteins were subjected to electrophoresis on an SDS-containing polyacrylamide gel. 1: Mock; 2: CV-B3/28 FL; 3: CV-B3/28 d50; 4: Staurosporin treated. F. Western blot assay targeting eIF4G and caspase-3 in human cardiomyocytes at 24 hr post transfection. Proteins were subjected to electrophoresis on an SDS-containing polyacrylamide gel. 1: Mock; 2: CV-B3/28 FL; 3: CV-B3/28 d50; 4: CV-B2/28 FL C107S. G. Western blot assay detecting eIF4G and VP1 in human cardiomyocytes at 24 hr post transfection of CV-B3/28 FL RNA or CV-B3/28 d50 RNA. Amounts of transfected RNA are indicated above lanes. Proteins were subjected to electrophoresis on an SDS-containing polyacrylamide gel. *: P<0.05; **: P<0.01, ***: P.<0.001. Apparent molecular weights are indicated on the right side of Western blot images.