Suppression of Viral RNA Binding and the Assembly of Infectious Hepatitis C Virus Particles In Vitro by Cyclophilin Inhibitors

Nonstructural protein 5A (NS5A) of hepatitis C virus (HCV) is an indispensable component of the HCV replication and assembly machineries. Although its precise mechanism of action is not yet clear, current evidence indicates that its structure and function are regulated by the cellular peptidylprolyl isomerase cyclophilin A (CyPA). CyPA binds to proline residues in the C-terminal half of NS5A, in a distributed fashion, and modulates the structure of the disordered domains II and III. Cyclophilin inhibitors (CPIs), including cyclosporine (CsA) and its nonimmunosuppressive derivatives, inhibit HCV infection of diverse genotypes, both in vitro and in vivo. Here we report a mechanism by which CPIs inhibit HCV infection and demonstrate that CPIs can suppress HCV assembly in addition to their well-documented inhibitory effect on RNA replication. Although the interaction between NS5A and other viral proteins is not affected by CPIs, RNA binding by NS5A in cell culture-based HCV (HCVcc)-infected cells is significantly inhibited by CPI treatment, and sensitivity of RNA binding is correlated with previously characterized CyPA dependence or CsA sensitivity of HCV mutants. Furthermore, the difference in CyPA dependence between a subgenomic and a full-length replicon of JFH-1 was due, at least in part, to an additional role that CyPA plays in HCV assembly, a conclusion that is supported by experiments with the clinical CPI alisporivir. The host-directed nature and the ability to interfere with more than one step in the HCV life cycle may result in a higher genetic barrier to resistance for this class of HCV inhibitors.     

Rapid Antiretroviral Therapy Initiation for Women in an HIV-1 Prevention Clinical Trial Experiencing Primary HIV-1 Infection during Pregnancy or Breastfeeding

During an HIV-1 prevention clinical trial in East Africa, we observed 16 cases of primary HIV-1 infection in women coincident with pregnancy or breastfeeding. Nine of eleven pregnant women initiated rapid combination antiretroviral therapy (ART), despite having CD4 counts exceeding national criteria for ART initiation; breastfeeding women initiated ART or replacement feeding. Rapid ART initiation during primary HIV-1 infection during pregnancy and breastfeeding is feasible in this setting.     

Care of the diabetic child in the community.

Brief admission of the new diabetic child and of a parent to an enlightened hospital for stabilisation, preliminary education, and familiarisation with hospital and community staff is well worth while. The greater the demand for constant control of the highest quality, the greater the need for a close understanding of the psychosocial factors concerned and for clinical skill. The nature of the home and the family relationships should in theory be available from the child''s general practitioner at the time of the first referral since he has so much information about the whole family. With the virtual disappearance, however, of mutual consultation in the patient''s home in many places, the opportunity for oral communication has declined, and availability on the telephone is not always easy. The busy general practitioner (far less an unknown physician from a deputising service without access to the records) has little time to write a comprehensive letter. In practice a relatively small hospital-based mobile team of specially experienced sisters who are keen to communicate in the home, the GP''s surgery, and the school makes a major contribution to the diabetic care of a young population vulnerable to major handicap in what should be the prime of life. Their cost effectiveness may be difficult to prove but it is not at all in doubt--especially when the sisters as in this area deal in the community with a wider range of chronic illnesses and handicaps in children.     

Methimazole and generation of oxygen radicals by monocytes: potential role in immunosuppression.

A study was conducted investigating the possibility that the immunosuppressive action of methimazole (the active metabolite of the antithyroid drug carbimazole) might be due to an effect on the production of oxygen radicals by monocytes. Techniques comprised measurement of luminol dependent chemoluminescence in monocytes and a spectrophotometric assay for production of hydrogen peroxide. The results showed definite inhibition of formation of oxygen radicals by resting and stimulated monocytes, which may explain the immunosuppressive action of the drug in Graves'' disease. The findings also suggest that the formation of oxygen radicals and the initiation of the immune response may be closely related.     

Stereospecificity and requirements for activity of the respiratory NADH dehydrogenase of Escherichia coli

The respiratory NADH dehydrogenase of Escherichia coli has been further amplified in vivo by genetic methods. The enzyme, a single polypeptide of Mr 47 200 of known amino acid sequence [Young, I. G., Rogers, B. L., Campbell, H. D., Jaworowski, A., & Shaw, D. C. (1981) Eur. J. Biochem. 116, 165-170], constitutes 10-15% of the total protein in the amplified membranes. In situ in the membrane, the enzyme contains 1 mol of FAD/mol of subunit and has a specific NADH:ubiquinone-1 oxidoreductase activity of approximately 1100-1200 units mg-1 at 30 degrees C, pH 7.5. The purified enzyme contains phospholipid, which remains closely associated with it during gel filtration on Sephacryl S-300 in the presence of 0.1% (w/v) cholate at low ionic strength. Under these conditions the enzyme is extensively aggregated (apparent Mr greater than 10(6]. This procedure yielded enzyme with a specific activity of 980 units mg-1, similar to the value observed in the membrane. This preparation contained less than 0.1 mol of Fe/mol of enzyme, confirming that Fe is not involved in reduction of ubiquinone 1 catalyzed by the enzyme. Neutron activation analysis of purified enzyme has demonstrated the absence of 35 trace elements including Se, Zn, Mn, Co, W, Cu, and Fe. The enzyme polypeptide, prepared completely free of phospholipid, FAD, and ubiquinone by gel filtration in the presence of sodium dodecyl sulfate, has been reactivated. The results show that the only components necessary for catalysis of ubiquinone-1 reduction by NADH in this system are the enzyme polypeptide, FAD, and phospholipid.(ABSTRACT TRUNCATED AT 250 WORDS)