More effective therapies are urgently needed against hepatitis C virus (HCV), a major cause of viral hepatitis. We used in vitro protein expression and microfluidic affinity analysis to study RNA binding by the HCV transmembrane protein NS4B, which plays an essential role in HCV RNA replication. We show that HCV NS4B binds RNA and that this binding is specific for the 3′ terminus of the negative strand of the viral genome with a dissociation constant (Kd) of ~3.4 nM. A high-throughput microfluidic screen of a compound library identified 18 compounds that substantially inhibited binding of RNA by NS4B. One of these compounds, clemizole hydrochloride, was found to inhibit HCV RNA replication in cell culture that was mediated by its suppression of NS4B’s RNA binding, with little toxicity for the host cell. These results yield new insight into the HCV life cycle and provide a candidate compound for pharmaceutical development.

Some species of ciliates undergo massive DNA elimination and genome rearrangement to construct gene-sized ''chromosomes'' in their somatic nucleus. An example is the extensively scrambled DNA polymerase ␣ gene that is broken into 48 pieces and distributed over two unlinked loci in Stylonychia. To understand the emergence of this complex phenomenon during evolution, we examined DNA polymerase ␣ genes in several earlier diverging species, representing evolutionary intermediates. Mapping these data onto an evolutionary tree suggests that this gene became extensively fragmented and scrambled over evolutionary time through a series of steps, each leading to greater complexity. Our results also suggest a possible mechanism for intron loss by deletion of intron sequences as DNA during development of the somatic nucleus.ciliate ͉ DNA polymerase ͉ hypotrich ͉ intron loss ͉ spirotrich C iliates comprise a diverse group of microbial eukaryotes, or protists. These organisms contain two types of nuclei: a somatic macronucleus that is active during vegetative growth and a germ-line micronucleus that is quiescent except during sexual recombination (1). During sexual conjugation, the two mating cells exchange haploid micronuclei, after which the old macronucleus degrades, and the new diploid micronucleus develops into the new macronucleus. Extensive loss of DNA from the long micronuclear chromosomes, coupled to DNA rearrangement, removes transposons and most intergenic spacer DNA between genes, as well as intragenic spacer DNA, known as internal eliminated segments (IESs). In spirotrichous (formerly hypotrichous) ciliates, the remaining DNA sequences, known as macronuclear destined segments (MDSs), can occupy as little as 2-5% of the germ-line genome. These mostly coding regions assemble together to form Ϸ24,000 types of DNA molecules in the somatic nucleus. Because their average size is just 2.2 kbp and they usually contain only one gene, they are sometimes called ''nano-chromosomes.'' Short telomeres are added to both ends before these molecules are amplified several-thousand-fold (1-3).Furthermore, the germ-line order of the coding regions in 20-30% of the genes in stichotrichous ciliates (a subset of spirotrichous ciliates) are permuted, or scrambled, relative to their linear order in the macronucleus. Although the mechanism of reordering these segments is not well understood, short direct repeats, called pointers, may guide the unscrambling process (1, 4). Invariably, one copy of a repeated word pair is present at the 3Ј end of germ-line segment n and at the 5Ј end of segment n ϩ 1, providing the information to link them together, with only one copy of the repeated word retained in the final DNA molecule (1). Although fragmented genes do occur in other organisms [examples include V(D)J recombination in the vertebrate immune system and split ribosomal RNA genes in the mitochondria of fungi (5) and protists (6, 7)], rarely are they sewn back together at the DNA level.Three different scrambled genes, actin I (8-10), ␣ telomer...

New classes of drugs are needed to combat hepatitis C virus (HCV), an important worldwide cause of liver disease. We describe an activity of a key domain, an amphipathic helix we termed 4BAH2, within a specific HCV nonstructural protein, NS4B. In addition to its proposed role in viral replication, we validate 4BAH2 as essential for HCV genome replication, and identify first generation small molecule inhibitors of 4BAH2 that specifically prevent HCV replication within cells. Detailed mechanistic studies reveal that the inhibitors target 4BAH2 function by either preventing 4BAH2 oligomerization or 4BAH2 membrane association. 4BAH2 inhibitors represent an exciting, additional class of compounds that has potential to effectively treat HCV.

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