TRANSLATION AND REPLICATION PROPERTIES OF THE HUMAN RHINOVIRUS GENOMEIN-VIVO AND IN-VITRO

The poor translation efficiency of genome-length human rhinovirus RNA in vitro using HeLa cell extract-supplemented rabbit reticulocyte lysa te has hampered the study of rhinovirus IRES-mediated translation and polyprotein synthesis in a cell-free system. In contrast, the efficien t in vitro translation characteristics of poliovirus RNAs have ultimat ely allowed the programming of cell-free coupled translation/replicati on extracts which are able to produce infectious poliovirus particles in vitro. A possible explanation for the decreased burst size observed during the course of a rhinovirus infection, compared to poliovirus i nfection, is reduced levels of polyprotein synthesis in vivo. In order to test this hypothesis and extend in vitro translation/replication t echnology to the study of human rhinoviruses, a chimeric cDNA construc t was engineered which allowed the in vitro synthesis of T7 transcript s containing the intact poliovirus type 1 (PV1) 5' noncoding region (5 ' NCR) and initiation codon upstream of the human rhinovirus 14 (HRV14 ) polyprotein-coding region and 3'-terminal sequences. These chimeric RNAs translated efficiently in vitro and were used successfully to pro gram a cell-free replication extract. Unexpectedly, parental HRV14 RNA s also translated efficiently in the HeLa cell-free translation/replic ation extract but replicated less efficiently than the chimera in vitr o. The chimeric HRV14/PV1 RNAs were infectious and gave rise to a viru s with a growth phenotype similar to that of parental HRV14. Prelimina ry characterization of this chimeric virus suggests that the biologica l properties characteristic of rhinovirus in vivo are determined prima rily by the rhinovirus gene products. Although the translation efficie ncy of the HRV14 5' NCR may be a limitation in rabbit reticulocyte lys ate-based in vitro translation extracts, it does not appear to be a ma jor limiting determinant for growth of rhinovirus in vivo or replicati on in the HeLa cell-free extract. (C) 1997 Academic Press.