Figure 1. Schematic of picornavirus genome organization. The positive-strand RNA has the viral protein VPg covalently linked to the 5 end of the genome. Both the 5 and 3 noncoding regions are highly structured and contain RNA secondary structural elements required for enterovirus translation initiation (Internal Ribosome Entry Site (IRES)) and RNA replication. Picornaviruses have a genome-encoded poly(A) tract at the 3 terminus of their RNA and express a single polyprotein that is proteolytically processed into precursor and mature viral proteins, required for replication of the virus, by the two proteinases 2A and 3Cpro. The polyprotein is segregated into three major regions. The capsid proteins are encoded in the P1 region, and the nonstructural proteins (viral proteins required for modification of the host cell environment, protein processing, and RNA replication) are encoded in the P2 and P3 regions (adapted from the Swiss Institute of Bioinformatics website / TD21 TD7028 TD30 TD ng of in vitro transcribed RNA HeLa S10 Cytoplasmic extract ATP 6 hours of incubation at 30C Visualization of proteins synthesized on polyacrylamide gel [ 35 S]-methionine Wild-type strainsTerminaly deleted strains 3CD VP3 2A 3AB 3Dpol VP1 2C Figure 2. In vitro translation assay. After 6h of incubation at 30C of viral RNA in the presence of a cytoplasmic extract of HeLa S10 cells and 35 S-labeled methionine, synthesized viral proteins (most significant are indicated to the right of the gel) are observed after migration on a SDS-Page gel. rLuciferase activity B- HCM C- HL-1 cells Control Wild-type strains Deleted strains P1 region (capside proteins) Rluc Inserting deletions A Figure 3. Construction and transfection of luciferase replicons into two types of cardiac myocytes. (A) The replicon consists of the viral genome in which the region encoding the capsid proteins (P1) was replaced with the gene encoding Renilla luciferase (kindly provided by Dr. F.J. Van Kuppeveld, Virology Division, Department of Infectious Diseases and Immunology, Faculty of Veterinary Medicine, Utrecht University, the Netherlands). Deletions of 7 to 49 nucleotides have been inserted at the 5 'end of the viral genome. Replicons were then transfected (B) in primary human cardiac myocytes or (C) in a continuous cell line of murine cardiac myocytes from an atrial tumor. Luciferase activity was measured at 2 hours post-transfection. Full-length replicons are indicated in blue and deleted replicons in red. rLuciferase activity HCM Control 2 Logs HL-1 Control 2 Logs Translation -RNA +RNA 3D pol Transfected RNA Luciferase signal amplification AB C Wild-type viruses Deleted viruses Figure 4. Luciferase activity compared between full-length and deleted replicons. Non-deleted replicons in blue and deleted replicon in red were transfected (A) in primary human cardiac myocytes or (B) in murine cardiomyocytes. Luciferase activity was measured from 2 to 8 hours post-transfection. The results presented are the product of three independent experiments. (C) Schematic representation of luciferase signal amplification mechanism by replicating transfected viral RNA. PV1 TD21 TD7 028 TD30 TD49 Positive RNA Double-stranded RNA + - Deleted strains Wild-type strains Positive control Figure 5. In Vitro replication assay of the viral RNA. Six hours after in vitro translation in the absence of [ 35 S]-methionine, the reaction mixture consisting of viral RNA (400 ng for the CVB3 wild-type strains and poliovirus; 400 and 800 ng for the deleted CVB3 strains) and HeLa S10 cytoplasmic extract was incubated for further 2h at 34 C in the presence of -[ 32 P] CTP. The purified RNA was then loaded on an agarose gel at 1.1%. Poliovirus 1 (PV1) is used here as a positive control. rLuciferase activity A- HCM 2 Logs B- HL-1 2 Logs C- HCM Control D- HL-1 Control TD7TD30TD21TD49CV-B3 0 CV-B3 28 Without guanidine hydrochloride With guanidine hydrochloride Wild-type strainsDeleted strains Figure 6. Impact of guanidine hydrochloride treatment on the luciferase activity measured after transfection of human and murine cardiac myocytes with deleted and not deleted replicons. Full-length (A and B) and deleted (C and D) luciferase replicons were transfected into human (HCM) (A and C) and murine (HL-1) (B and D) cardiac myocytes. Guanidine hydrochloride at a final concentration of 3 mM was added or not to the cell culture medium 30 minutes after transfection of the replicons. Luciferase activity was then measured from T0 to T8H post- transfection in the GuHCl treated (in red) or untreated (blue) cells. The results presented here are the product of three independent experiments. 28 TD7 TD21 TD30TD49 28 TD7 TD21TD30TD49 28TD7 TD21TD30TD49 + strand - strand Viral load in genomic RNA copy/ml of cell medium h PI 48h PI 0h PI Figure 7. Quantification by one step RT-qPCR of positive- and negative-strands of viral RNA in a kinetics of infection of primary human cardiac myocytes. The full-length CVB3-28 and CVB3 viruses deleted of 7, 21, 31 and 49 nucleotides used to achieve this infection were produced on human hepatocarcinoma cells Huh7.5 defective for type I interferon response. Cardiomyocytes infected in triplicate were collected at 0, 24 and 48 hours post- infection in 2 ml of cell culture medium after three cycles of freezing/thawing. Load of positive- (blue) and negative-strand (orange) of viral RNA is shown on the y-axe as the number of RNA copies per ml of cell culture medium. 3 end 2C A B Figure 8. Schematic representation of cellular proteins (PCBP2, hnRNPC) and viral proteins (2C, 3AB, 3CDpro, 2BC) binding (A) the 5 end of the viral genome in order to prime the synthesis of the negative-strand antigenomic RNA and (B) the 3' end of the antigenomic negative-strand for the synthesis of positive-strand genomic RNA. 28 0 TD30PV1 Positive control TD7 TD ng of PCBP2 RNA alone PV Figure 9. RNA mobility shift assay of full-length (0 and 28) and deleted positive-strand viral RNA (TD7, TD31 and TD49) in the presence of the cellular protein PCBP2. A RNA fragment of 110 nucleotides located at the 5 end of the genomic positive-strand viral RNA (stem-loop I or clover-leaf) was transcribed with 32 P-CTP and incubated in the absence (free probe=FP; lines 1) or in the presence of 250 (lines 2), 500 (lines 3) and 1000 ng (lines 4) of the protein PCBP2. The formation of a ribonucleoprotein complex (indicated by the black arrows) results in a migration delay of the radioactive nucleic acid probe on the agarose gel. The stem-loop IV of poliovirus 1 IRES was used as positive control for PCBP2 experiments. Figure 10. RNA mobility shift assay of full-length (strain 28) and deleted positive-strand viral RNA (TD7, TD21, TD31 and TD49) in the presence of the viral protein 3CD. A RNA fragment of 110 nucleotides located at the 5 end of the genomic positive-strand viral RNA (stem-loop I or clover-leaf) was transcribed with 32 P-CTP and incubated in the absence (free probe=FP) or in the presence of 50, 100, 150, 250, 500 and 1000 ng of the viral protein 3CD. The formation of a ribonucleoprotein complex (indicated by the black arrows) results in a migration delay of the radioactive nucleic acid probe on the agarose gel FP FP FP FP FP 28 TD30 TD7 TD49TD21 Figure 11. RNA mobility shift assay of full-length (strain 28) and deleted negative-strand viral RNA (TD7, TD21, TD31 and TD49) in the presence of the cellular protein hnRNPC. A RNA fragment of 750 nucleotides corresponding to the 3 end of the antigenomic negative-strand viral RNA was transcribed with 32 P-CTP and incubated in the absence (free probe=FP) or in the presence of 100, 250 and 500 of the recombinant protein hnRNPC. The formation of a ribonucleoprotein complex (indicated by the black arrows) results in a migration delay of the radioactive nucleic acid probe on the agarose gel. 28 TD30 TD7 TD49TD21 Free Probe Free Probe Free Probe Free Probe Free Probe