Coxsackieviral proteins functionally recognize the polioviral cloverleaf structure of the 5′-NTR of a chimeric enterovirus RNA: influence of species-specific host cell factors on virus growth

Abstract

The 5′-non-translated region (NTR) of enteroviruses contains secondary structures which do not only serve in the initiation of translation but also in the initiation of plus-strand RNA synthesis by binding of viral and cellular proteins. To investigate a very early step of enteroviral replication by cis- and trans-complementation, 220 nucleotides of the 5′-region of coxsackievirus B3 (CVB3) were exchanged with the corresponding region of poliovirus type 1 (PV1) to yield the chimeric virus CVB3[PV5′]. The viability of this chimera demonstrates that the polioviral cloverleaf structure of the 5′-NTR is functional in the replication of a chimeric CVB3 RNA. The HeLa-generated chimera reveals a 4-nucleotide deletion (nt 232–235) within a short direct repeat. Besides clearly reduced growth characteristics in all permissive cell lines, the chimera exhibits a small-plaque phenotype. The host range is changed since the virus grows well in human HeLa cells, but does not replicate in murine YAC-1 and Ltk cells, although these cell lines are permissive for the replication of both parental viruses. Moreover, in simian Vero, COS-1, or FRhK-4 cells the HeLa-generated chimera CVB3[PV5′] exhibits a strict temperature sensitivity at 39°C. After infection of simian cells with high m.o.i., in situ hybridization data reveal that the chimera replicates in single cells at almost normal rates indicating that only a small fraction of HeLa-generated virus is able to multiplicate in simian cell lines. After passaging the virus chimera in Vero cells two further mutations occur at nucleotide positions 185 and 227. Since this genome region is known to interact with viral proteins and several host cell factors during the initiation of replication and translation, interactions of such factors with either viral RNA or viral proteins may be disturbed but still functional at permissive temperatures in HeLa cells and simian cell lines, whereas murine cell lines are not permissive. These experiments suggest that phenomena like host range, tissue tropism and cell-type specificity may be explained as a complex interplay of cellular surface receptors and intracellular host factors. Such intracellular factors could be part of the enteroviral initiation complex during the plus-strand RNA synthesis or during translation initiation and could be expressed in a tissue-, organ- or species-specific way or might be regulated developmentally.