Positive-sense single-stranded RNA virus interactions with the human host

Picornaviruses are animal viruses that possess a genome consisting of a single positive strand of RNA. The RNA genome contains an internal ribosomal entry site (IRES) that enables cap-independent translation of the encoded viral proteins. Poliovirus is a well characterized member of the picornavirus family that has been widely used as a model to study the interactions of positive-strand RNA viruses with host factors. Host factors participate in all known steps of the positive-strand RNA virus life cycle, including viral entry, gene expression, replication, translation, assembly, and release. Although the expression of several host factors has been shown to be regulated as a result of poliovirus infection, the precise mechanism and timing of these changes and the interactions of the virus with many of these host factors have not been elucidated. To study how a positive-sense single-stranded RNA virus modulates the host response, we have combined bioinformatic and genetic approaches.;To examine the host responses to viral infection, a bioinformatic approach was used. Previously, classical microarray assays examined host gene expression that was affected by poliovirus infection, but these results reflected steady state mRNA levels. We used HeLa cells expressing the Toxoplasma enzyme uracilphosphoribosyltransferase, generated by Chris Meiering in collaboration with Mike Cleary, to label newly synthesized viral and host RNA in HeLa cells after addition of 4-thiouracil before and at different times during poliovirus infection. The labeled RNA was isolated from total RNA after biotinylation and purification on streptavidin beads, and was used in microarray assays. This method of identifying newly synthesized RNAs allowed us to monitor a specific and immediate host response during specific stages of infection. Using this assay, we discovered a group of NFkB-regulated genes that escape the classical poliovirus-induced transcriptional shutoff. Furthermore, increased levels of infectious poliovirus were produced in cells with diminished expression of one of these genes, tumor necrosis factor alpha-induced protein 3 (A20). Thus, A20 provides a cellular protective effect against poliovirus production.;A second approach was utilized to analyze the viral response to modulation of host factors. A genetic siRNA-based screen, employed by Sara Cherry at Harvard University, uncovered genes that when suppressed in Drosophila cells, allowed the cells to grow and proliferate, but inhibited the production of the IRES-containing Drosophila C virus. Most of these genes encode ribosomal proteins, suggesting that Drosophila C virus can not compete for the translation apparatus when ribosomal proteins become limiting. We asked the question whether IRES-containing picornaviruses in general display a similar growth defect in cells expressing reduced amounts of specific ribosomal proteins. Thus, we used siRNA to lower intracellular levels of ribosomal gene ribosomal protein S6 (RpS6) and noted decreased overall translation in HeLa cells by 2.5-fold, whereas poliovirus production was decreased by 10-fold. These results argue that the growth of IRES-containing viruses that infect eukaryotic cells is sensitive to intracellular levels of the translation apparatus, displaying a novel and unexpected Achilles' heel for this class of viruses. To examine the step in viral mRNA translation that is inhibited in cells treated with RpS6 siRNA, we employed dicistronic luciferase reporter assays and found that translation mediated by poliovirus, hepatitis C virus, and encephalomyocarditis virus IRESes is diminished relative to cap-mediated translation in HeLa and primary human foreskin fibroblast (HFF) cultures. Thus, cap-mediated translation is more efficient than IRES-mediated translation during low abundance of RpS6.;Overall, these approaches have revealed virus-induced alterations in host gene expression and pointed to new targets for antiviral intervention.