RESUMO
Inevitably, viruses depend on host factors for their multiplication. Here, we show that hepatitis C virus (HCV) RNA translation and replication depends on Rck/p54, LSm1, and PatL1, which regulate the fate of cellular mRNAs from translation to degradation in the 5'-3'-deadenylation-dependent mRNA decay pathway. The requirement of these proteins for efficient HCV RNA translation was linked to the 5' and 3' untranslated regions (UTRs) of the viral genome. Furthermore, LSm1-7 complexes specifically interacted with essential cis-acting HCV RNA elements located in the UTRs. These results bridge HCV life cycle requirements and highly conserved host proteins of cellular mRNA decay. The previously described role of these proteins in the replication of 2 other positive-strand RNA viruses, the plant brome mosaic virus and the bacteriophage Qss, pinpoint a weak spot that may be exploited to generate broad-spectrum antiviral drugs.
Assuntos
Genoma Viral/genética , Hepacivirus/genética , Hepacivirus/fisiologia , Biossíntese de Proteínas , Proteínas/metabolismo , RNA Viral/genética , Replicação Viral , Regiões 3' não Traduzidas/metabolismo , Regiões 5' não Traduzidas/genética , Linhagem Celular Tumoral , RNA Helicases DEAD-box/metabolismo , Proteínas de Ligação a DNA/metabolismo , Endorribonucleases/metabolismo , Exorribonucleases/metabolismo , Inativação Gênica , Hepacivirus/patogenicidade , Humanos , Proteínas Associadas aos Microtúbulos/metabolismo , Conformação de Ácido Nucleico , Ligação Proteica , Proteínas Proto-Oncogênicas/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Viral/química , Proteínas de Ligação a RNA/metabolismo , Replicon/genética , Transdução de SinaisRESUMO
BACKGROUND & AIMS: The Hepatitis C virus (HCV) exhibits large genetic diversity, both on a global scale and at the level of the infected individual. A major underlying mechanism of the observed sequence differences is error-prone virus replication by the viral RNA polymerase NS5B. In addition, based on phylogenetic comparisons of patient-derived HCV sequences, there is evidence of HCV recombination. However, to date little is known about the frequency by which recombination events occur in HCV and under what conditions recombination may become important in HCV evolution. We, therefore, aimed to set up an experimental model system that would allow us to analyze and to characterize recombination events during HCV replication. METHODS: A neomycin-selectable, HCV replicon-based recombination detection system was established. HCV replicons were mutated within either the neomycin-phosphotransferase gene or the NS5B polymerase. Upon co-transfection of hepatic cells lines, recombination between the mutated sites is necessary to restore the selectable phenotype. RESULTS: Recombinants were readily detected with frequencies correlating to the distance between the mutations. The recombinant frequency normalized to a crossover range of one nucleotide was around 4 × 10(-8). CONCLUSIONS: An experimental system to select for HCV recombinants in cell culture was successfully established. It allowed deriving first estimates of recombinant frequencies. Based on these, recombination in HCV seems rare. However, due to the rapid virus turnover and the large number of HCV-infected liver cells in vivo, it is expected that recombination will be of biological importance when strong selection pressures are operative.