RESUMEN
Viral proteins bind to numerous cellular and viral proteins throughout the infection cycle. However, the mechanisms by which viral proteins interact with such large numbers of factors remain unknown. Cellular proteins that interact with multiple, distinct partners often do so through short sequences known as molecular recognition features (MoRFs) embedded within intrinsically disordered regions (IDRs). In this study, we report the first evidence that MoRFs in viral proteins play a similar role in targeting the host cell. Using a combination of evolutionary modeling, protein-protein interaction analyses and forward genetic screening, we systematically investigated two computationally predicted MoRFs within the N-terminal IDR of the hepatitis C virus (HCV) Core protein. Sequence analysis of the MoRFs showed their conservation across all HCV genotypes and the canine and equine Hepaciviruses. Phylogenetic modeling indicated that the Core MoRFs are under stronger purifying selection than the surrounding sequence, suggesting that these modules have a biological function. Using the yeast two-hybrid assay, we identified three cellular binding partners for each HCV Core MoRF, including two previously characterized cellular targets of HCV Core (DDX3X and NPM1). Random and site-directed mutagenesis demonstrated that the predicted MoRF regions were required for binding to the cellular proteins, but that different residues within each MoRF were critical for binding to different partners. This study demonstrated that viruses may use intrinsic disorder to target multiple cellular proteins with the same amino acid sequence and provides a framework for characterizing the binding partners of other disordered regions in viral and cellular proteomes.
Asunto(s)
Hepacivirus/fisiología , Hepatitis C/metabolismo , Proteínas Intrínsecamente Desordenadas/metabolismo , Proteínas del Núcleo Viral/metabolismo , Secuencia de Aminoácidos , Animales , Biología Computacional , Hepacivirus/química , Hepacivirus/genética , Hepatitis C/veterinaria , Interacciones Huésped-Patógeno , Humanos , Proteínas Intrínsecamente Desordenadas/química , Proteínas Intrínsecamente Desordenadas/genética , Datos de Secuencia Molecular , Nucleofosmina , Filogenia , Unión Proteica , Mapas de Interacción de Proteínas , Proteínas del Núcleo Viral/química , Proteínas del Núcleo Viral/genéticaRESUMEN
BACKGROUND: In October, 2008, legislation was implemented in the city and county of San Francisco, California, prohibiting the sale of tobacco products in pharmacies. OBJECTIVE: To characterise public awareness and perceptions of the ban on tobacco sales in San Francisco community pharmacies. METHODS: A brief, anonymous survey was used to assess public awareness and perceptions of a ban on tobacco sales approximately 1 year after implementation. Individuals were approached by researchers outside of chain pharmacies in San Francisco. Smokers and non-smokers were included, and participants did not have to be patrons of the pharmacy. RESULTS: Of 198 participants, 56% were in favor of the ban, 27% opposed it and 17% were undecided. A greater proportion of current tobacco users (81%) than former/never users (48%) were aware of the ban (p<0.001), and a lesser proportion were supportive of the ban (21% of current users vs 66% of former/never users; p<0.001). Most current tobacco users (88% of n=43) had not considered quitting smoking as a result of the ban. The majority of consumers indicated that the ban on cigarette sales did not influence their shopping behaviour at retail pharmacies. CONCLUSIONS: In the city and county of San Francisco, public support exists for prohibiting the sale of tobacco products in pharmacies.