The expression and processing of hepatitis C virus core protein was analyzed. Two protein bands, 21 kDa (P21), corresponding to the full-length core, and 19 kDa (P19), were detected as major products when core protein was expressed in the standard rabbit reticulocyte lysate system or in Sf9 insect cells. Core proteins with amino-terminal hexa-histidine tags were expressed which allowed the purification of the hexa-histidine P19 core with NI(2+)-NTA columns. With the help of mass spectrometry, the molecular weight of hexa-histidine-P19 was analyzed and its carboxy-terminus could be calculated. Fusion proteins of truncated core/core-E1 species fused to mouse dihydrofolate reductase (mDHFR) showed cleavage in the expected region. Cleavage sites could be determined by amino-terminal protein sequencing of the DHFR-fusion partner. Our data show that there are not one but two core products with an apparent molecular weight of about 19 kDa, ending either at amino acid leucine 179 or leucine 182, respectively. These cleavages in the hydrophobic, carboxy-terminal region of HCV core suggest processing by (a) recently proposed eucaryotic signal peptide peptidase(s) (F. Lyko et al. (1995) J. Biol. Chem. 270, 19873-19878). Furthermore, our results demonstrate that cleavage at these sites and the formation of the P19 species does not require previous processing at the signalase site (position 191/192) of the HCV-polyprotein.

The influence of ciprofloxacin, nalidixic acid, norfloxacin, novobiocin, and ofloxacin on elements of eucaryotic DNA replication was investigated in vitro. Each of the 4-quinolones, when present in amounts of more than 100 ,Ig/mi, reversibly inhibited the DNA synthesis performed by the 9S DNA polymerase a primase complex from calf thymus. Novobiocin at 500 gLg/ml or at higher concentrations irreversibly inactivated DNA polymerase a primase complex. The accuracy of in vitro DNA synthesis in the absence of repair mechanisms was determined from amber-revertant assays with 4X174aml6(+) DNA as template. The antimicrobial agents did not significantly increase the frequencies of base pairing mismatches during the course of replication, indicating that the basal mutation rate is not affected by novobiocin and the 4-quinolones. The Ki values of 50% inhibition of DNA topoisomerases from calf thymus by ciprofloxacin, norfloxacin, novobiocin, nalidixic acid, and ofloxacin were 300, 400, 1,000 or more, 1,000 or more, and 1,500 or more ,ug/ml, respectively, in the case of topoisomerase I, and the Ki values were 150, 300, 500, 1,000, and 1,300 ,ug/ml, respectively, in the case of topoisomerase II. The procaryotic topoisomerase II is approximately 100-fold more sensitive to inhibition by ciprofloxacin, norfloxacin, and ofloxacin than is its eucaryotic counterpart. Growth curves of lymphoblasts were recorded in the presence of ofloxacin and ciprofloxacin. Neither 1 nor 10 ,ug of ciprofloxacin or of ofloxacin per ml affected cell proliferation. Ofloxacin and ciprofloxacin at 100 ,ug/mI inhibited cell growth; 1,000 gig/mI led to cell death. No correlation exists between the antimicrobial and cytotoxic activities of the 4-quinolones.The nalidixic acid analogs ciprofloxacin, norfloxacin, and ofloxacin are highly active bactericidal agents, effective against a broad spectrum of gram-positive and gram-negative bacteria (3,22,31,37). The MICs generally range between 0.01 and 10 ,ug/ml. According to the outcome of the first clinical trials, the new 4-quinolones show promise as safe and effective antimicrobial agents (see Proc. 14th Int. Congr. Chemother., 1985).The antibacterial activity of the 4-quinolones is probably caused by the inhibition of DNA synthesis (15,35,38,40 Chemother., p. 49, 1985), ciprofloxacin, norfloxacin, and ofloxacin inhibit the ATP-dependent supercoiling activity and the ATP-independent relaxing activity of the DNA gyrase from Escherichia coli and Pseudomonas aeruginosa at concentrations ranging from 0.1 to 10 ,ugIml. The subunit A of DNA gyrase is assumed to be the direct target of the 4-quinolones (15, 35); however, recently published data provide evidence that binding to single-stranded regions of DNA may be the primary mechanism of drug action (38). The subunit B of DNA gyrase is inhibited by novobiocin (15, 35).The DNA gyrases belong to the type II topoisomerases which alter the topology of DNA by passing a DNA helix through a transient double-stranded break in another helical structure, changing the linkin...

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