RESUMO
The hepatitis C virus (HCV) non-structural protein 5B (NS5B) is an RNA-dependent RNA polymerase that is essentially required for viral replication. Although previous studies revealed important properties of static NS5B-RNA complexes, the nature and relevance of dynamic interactions have yet to be elucidated. Here, we devised a single molecule Förster Resonance Energy Transfer (SM-FRET) assay to monitor temporal changes upon binding of NS5B to surface immobilized RNA templates. The data show enzyme association-dissociation events that occur within the time resolution of our setup as well as FRET-fluctuations in association with stable binary complexes that extend over prolonged periods of time. Fluctuations are shown to be dependent on the length of the RNA substrate, and enzyme concentration. Mutations in close proximity to the template entrance (K98E, K100E), and in the center of the RNA binding channel (R394E), reduce both the population of RNA-bound enzyme and the fluctuations associated to the binary complex. Similar observations are reported with an allosteric nonnucleoside NS5B inhibitor. Our assay enables for the first time the visualization of association-dissociation events of HCV-NS5B with RNA, and also the direct monitoring of the interaction between HCV NS5B, its RNA template, and finger loop inhibitors. We observe both a remarkably low dissociation rate for wild type HCV NS5B, and a highly dynamic enzyme-RNA binary complex. These results provide a plausible mechanism for formation of a productive binary NS5B-RNA complex, here NS5B slides along the RNA template facilitating positioning of its 3' terminus at the enzyme active site.
Assuntos
Hepacivirus/enzimologia , RNA Viral/metabolismo , Proteínas não Estruturais Virais/metabolismo , Sequência de Bases , Benzimidazóis/farmacologia , Transferência Ressonante de Energia de Fluorescência , Modelos Moleculares , Mutação , Conformação de Ácido Nucleico , Ligação Proteica , Conformação Proteica , RNA Viral/química , RNA Viral/genética , Proteínas não Estruturais Virais/antagonistas & inibidores , Proteínas não Estruturais Virais/química , Proteínas não Estruturais Virais/genéticaRESUMO
Over 40,000 HIV-infected individuals live in St Petersburg, Russia. Population characteristics and barriers to care are largely undefined. 152 consecutive patients receiving HIV care at two sites completed a questionnaire in Spring 2011. Rates of chronic hepatitis C virus (HCV) and hepatitis B virus (HBV) infection, alcohol use, and rates of antiretroviral uptake were similar by gender. Males reported a higher history of injection drug use (80.3% vs. 48.7%; p<0.01) and tuberculosis infection (18.8% vs. 1.6%; p<0.01). Females were more likely to have had a child (63.3% vs. 31.5%; p<0.01) and be currently raising that child within their residence (49.3% vs. 15.3%; p<0.01). Unprotected sex (60.5% vs. 17.8%; p<0.01) and a history of sexually transmitted infection (37.7% vs. 20.3%; p=0.03) were more common in females. Females utilized social services more frequently (34.2% vs. 11.9%; p<0.01). There is a heavy burden of concurrent infectious disease, substance use and abuse, mental health illness, and need for social service support in this population. Important differences exist between genders in service uptake and utilization. Further evaluation of these differences may help inform the allocation of limited resources in this high HIV prevalence region of Russia.