RESUMEN
The specific packaging of the hepatitis C virus (HCV) genome is hypothesised to be driven by Core-RNA interactions. To identify the regions of the viral genome involved in this process, we used SELEX (systematic evolution of ligands by exponential enrichment) to identify RNA aptamers which bind specifically to Core in vitro. Comparison of these aptamers to multiple HCV genomes revealed the presence of a conserved terminal loop motif within short RNA stem-loop structures. We postulated that interactions of these motifs, as well as sub-motifs which were present in HCV genomes at statistically significant levels, with the Core protein may drive virion assembly. We mutated 8 of these predicted motifs within the HCV infectious molecular clone JFH-1, thereby producing a range of mutant viruses predicted to possess altered RNA secondary structures. RNA replication and viral titre were unaltered in viruses possessing only one mutated structure. However, infectivity titres were decreased in viruses possessing a higher number of mutated regions. This work thus identified multiple novel RNA motifs which appear to contribute to genome packaging. We suggest that these structures act as cooperative packaging signals to drive specific RNA encapsidation during HCV assembly.
Asunto(s)
Genoma Viral/genética , Hepacivirus/genética , ARN Viral/genética , Ensamble de Virus/genética , Aptámeros de Nucleótidos/química , Aptámeros de Nucleótidos/genética , Aptámeros de Nucleótidos/metabolismo , Secuencia de Bases , Western Blotting , Línea Celular Tumoral , Regulación Viral de la Expresión Génica , Hepacivirus/metabolismo , Humanos , Mutación , Conformación de Ácido Nucleico , Motivos de Nucleótidos/genética , Unión Proteica , ARN Viral/química , ARN Viral/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Técnica SELEX de Producción de Aptámeros , Proteínas del Núcleo Viral/genética , Proteínas del Núcleo Viral/metabolismo , Proteínas Virales/genética , Proteínas Virales/metabolismoRESUMEN
Patients with acute vitiligo have low epidermal catalase expression/activities and accumulate 10(-3) M H(2)O(2). One consequence of this severe oxidative stress is an altered calcium homeostasis in epidermal keratinocytes and melanocytes. Here, we show decreased epidermal calmodulin expression in acute vitiligo. Since 10(-3)M H(2)O(2) oxidises methionine and tryptophan residues in proteins, we examined calcium binding to calmodulin in the presence and absence of H(2)O(2) utilising (45)calcium. The results showed that all four calcium atoms exchanged per molecule of calmodulin. Since oxidised calmodulin looses its ability to activate calcium ATPase, enzyme activities were followed in full skin biopsies from lesional skin of patients with acute vitiligo (n=6) and healthy controls (n=6). The results yielded a 4-fold decrease of ATPase activities in the patients. Computer simulation of native and oxidised calmodulin confirmed the loss of all four calcium ions from their specific EF-hand domains. Taken together H(2)O(2)-mediated oxidation affects calcium binding in calmodulin leading to perturbed calcium homeostasis and perturbed l-phenylalanine-uptake in the epidermis of acute vitiligo.