Translation and Replication Properties of the Human Rhinovirus Genomein Vivoandin Vitro

Abstract

The poor translation efficiency of genome-length human rhinovirus RNAin vitrousing HeLa cell extract-supplemented rabbit reticulocyte lysate has hampered the study of rhinovirus IRES-mediated translation and polyprotein synthesis in a cell-free system. In contrast, the efficientin vitrotranslation characteristics of poliovirus RNAs have ultimately allowed the programming of cell-free coupled translation/replication extracts which are able to produce infectious poliovirus particlesin vitro.A possible explanation for the decreased burst size observed during the course of a rhinovirus infection, compared to poliovirus infection, is reduced levels of polyprotein synthesisin vivo.In order to test this hypothesis and extendin vitrotranslation/replication technology to the study of human rhinoviruses, a chimeric cDNA construct was engineered which allowed thein vitrosynthesis of T7 transcripts 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 efficientlyin vitroand were used successfully to program a cell-free replication extract. Unexpectedly, parental HRV14 RNAs also translated efficiently in the HeLa cell-free translation/replication extract but replicated less efficiently than the chimerain vitro.The chimeric HRV14/PV1 RNAs were infectious and gave rise to a virus with a growth phenotype similar to that of parental HRV14. Preliminary characterization of this chimeric virus suggests that the biological properties characteristic of rhinovirusin vivoare determined primarily by the rhinovirus gene products. Although the translation efficiency of the HRV14 5′ NCR may be a limitation in rabbit reticulocyte lysate-basedin vitrotranslation extracts, it does not appear to be a major limiting determinant for growth of rhinovirusin vivoor replication in the HeLa cell-free extract.