Identification and analysis of a tropic element within the 5' nontranslated region of echovirus 12

The ∼740 nucleotide 5′ nontranslated region (NTR) of human enteroviruses (EVs) is highly structured and contains an internal ribosome entry site (IRES) that stimulates translation of the viral open reading frame. Furthermore, cis-acting signals for RNA synthesis are known to exist within the initial 88 nucleotides of the 5′NTR. For certain EVs, the 5′NTR has been shown to influence virulence phenotype. We have constructed a viral chimera in which the putative IRES element of coxsackievirus B3 (CVB3) was exchanged with that of echovirus 12 (E12), a wild-type EV rarely associated with disease in humans. The resulting chimera, known as CE12, replicated similarly to CVB3 in human (HeLa) and simian cell lines, yet was unable to replicate in primary murine heart (MFHF) and liver (BNLCL2) cells. Utilizing a reverse genetics approach, the cell type-specific replication phenotype was localized to a 90 nucleotide region that comprises the predicted stem loop (SL) II within the E12 IRES. In addition, a revertant of CE12 containing mutations within the 5′NTR was isolated (CE12R) that had regained viability in the utilized murine cell lines. Using a murine model for CVB3-induced acute myocarditis, the putative E12 IRES was to found mediate an attenuated cardiovirulence phenotype. Experiments to assess the efficiency of viral IRESs derived from growth-permissive and restricted viruses revealed that all chimeric IRES elements containing E12 sequences were similarly active in MFHF cells regardless of whether they conferred viability in this cell type. Studies were performed to examine viral RNA synthesis in HeLa and MFHF cells. In HeLa cells, both CVB3 and CE12 exhibited similar kinetics of RNA synthesis. In contrast, kinetics and yield of CE12 RNA synthesis in MFHF cells were significantly reduced compared to those of CVB3. In vitro assays to characterize RNA-protein interactions indicated that the E12 SLII differed from that of CVB3 in its ability to interact with cellular proteins. These results suggest that (i) the EV SLII functions as a cis-acting element in viral RNA synthesis, (ii) cellular factors are involved in EV RNA synthesis, and (iii) EV 5′NTR sequences can function as intracellular determinants of tropism.