The antiviral factor CPSF6-358 interferes with the nuclear entry of human immunodeficiency virus type 1 (HIV-1). HIV-1 acquires resistance to CPSF6-358 through the N74D mutation of the capsid (CA), which alters its nuclear entry pathway. Here we show that compared to wild-type (WT) HIV-1, N74D HIV-1 is more sensitive to cyclosporine, has increased sensitivity to nevirapine, and is impaired in macrophage infection prior to reverse transcription. These phenotypes suggest a difference in the N74D reverse transcription complex that manifests early after infection and prior to interaction with the nuclear pore. Overall, our data indicate that N74D HIV-1 replication in transformed cells requires cyclophilin A but is dependent on other interactions in macrophages.

Hepatitis C virus (HCV) is a blood-borne pathogen that poses a significant threat to public health worldwide. The genetic diversity and distribution of HCV genotypes in non-Western countries, particularly subSaharan Africa, is poorly documented. This study reports a phylogenetic analysis of core and NS5B gene sequences of 37 HCV strains sampled in Cameroon. A high level of genetic diversity of both genotypes 1 and 4 was found, indicating a unique pattern of long-term HCV infection that has not been observed elsewhere. These results lead to the hypothesis that these HCV genotypes originated and diversified in west Central Africa before spreading to other regions. INTRODUCTIONHepatitis C virus (HCV) is a leading global cause of liver disease. It is estimated that more than 170 million people worldwide are chronically infected with HCV and are at risk of developing liver cirrhosis and liver cancer (WHO, 1997).HCV is a single-stranded, positive-sense RNA virus, with a genome of 9?7 kb long containing a single ORF. The ORF encodes three structural proteins (including the capsid protein, core) and six non-structural proteins (including the RNA polymerase, NS5B). The ORF is flanked by two NCRs located at the 59 and 39 ends (Choo et al., 1991).HCV is genetically diverse and is classified into six major genotypes, each of which is divided further into several subtypes (Robertson et al., 1998). HCV genotypes differ at more than 30 % of nucleotides across the entire genome, while HCV subtypes vary at more than 20 % of sites (Simmonds, 1995). HCV genotypes show different geographical distributions and levels of diversity, reflecting their different epidemic histories and routes of transmission. HCV isolates from Western countries typically have limited sequence diversity, resulting from the recent introduction of a few strains (such as subtypes 1a, 1b and 3a) from endemic areas. These strains have spread rapidly through infected blood products and intravenous drug use (Pybus et al., 2001). The distribution of HCV genotypes in non-Western countries is less well documented, although small-scale surveys have found considerable sequence diversity of genotypes 1, 2 and 4 in African countries (Xu et al., 1994; Fretz et al., 1995;Ruggieri et al., 1996;Wansbrough-Jones et al., 1998;Jeannel et al., 1998), suggesting that these strains have been endemic to the continent for several hundred years (Smith et al., 1997; Pybus et al., 2001). The investigation of HCV genetic diversity in subSaharan Africa is, therefore, necessary to provide insights into the global epidemiology, epidemic history and origin of HCV. Furthermore, availability of more sequence data from subSaharan Africa may help to refine the HCV classification system.In this work we present a phylogenetic analysis of core and NS5B gene sequences obtained from 37 HCV strains isolated in Cameroon. We show that HCV genotypes 1 and 4 are both highly divergent in Cameroon, indicating the longterm presence of both strains in west Central Africa. This pattern of HCV endemicity i...

Helicases are ubiquitous motor proteins that separate and/or rearrange nucleic acid duplexes in reactions fueled by adenosine triphosphate (ATP) hydrolysis. Helicases encoded by bacteria, viruses, and human cells are widely studied targets for new antiviral, antibiotic, and anticancer drugs. This review summarizes the biochemistry of frequently targeted helicases. These proteins include viral enzymes from herpes simplex virus, papillomaviruses, polyomaviruses, coronaviruses, the hepatitis C virus, and various flaviviruses. Bacterial targets examined include DnaB-like and RecBCD-like helicases. The human DEAD-box protein DDX3 is the cellular antiviral target discussed, and cellular anticancer drug targets discussed are the human RecQ-like helicases and eIF4A. We also review assays used for helicase inhibitor discovery and the most promising and common helicase inhibitor chemotypes, such as nucleotide analogues, polyphenyls, metal ion chelators, flavones, polycyclic aromatic polymers, coumarins, and various DNA binding pharmacophores. Also discussed are common complications encountered while searching for potent helicase inhibitors and possible solutions for these problems.

A screen for hepatitis C virus (HCV) NS3 helicase inhibitors revealed that the commercial dye thioflavine S was the most potent inhibitor of NS3-catalyzed DNA and RNA unwinding in the 827-compound National Cancer Institute Mechanistic Set. Thioflavine S and the related dye primuline were separated here into their pure components, all of which were oligomers of substituted benzothiazoles. The most potent compound (P4), a benzothiazole tetramer, inhibited unwinding >50% at 2±1 μM, inhibited the subgenomic HCV replicon at 10 μM, and was not toxic at 100 μM. Because P4 also interacted with DNA, more specific analogs were synthesized from the abundant dimeric component of primuline. Some of the 29 analogs prepared retained ability to inhibit HCV helicase but did not appear to interact with DNA. The most potent of these specific helicase inhibitors (compound 17) was active against the replicon and inhibited the helicase more than 50% at 2.6±1 μM.

Typical assays used to discover and analyze small molecules that inhibit the hepatitis C virus (HCV) NS3 helicase yield few hits and are often confounded by compound interference. Oligonucleotide binding assays are examined here as an alternative. After comparing fluorescence polarization (FP), homogeneous time-resolved fluorescence (HTRF®; Cisbio) and AlphaScreen® (Perkin Elmer) assays, an FP-based assay was chosen to screen Sigma’s Library of Pharmacologically Active Compounds (LOPAC) for compounds that inhibit NS3-DNA complex formation. Four LOPAC compounds inhibited the FP-based assay: aurintricarboxylic acid (ATA) (IC 50 = 1.4 μM), suramin sodium salt (IC 50 = 3.6 μM), NF 023 hydrate (IC 50 = 6.2 μM) and tyrphostin AG 538 (IC 50 = 3.6 μM). All but AG 538 inhibited helicase-catalyzed strand separation, and all but NF 023 inhibited replication of subgenomic HCV replicons. A counterscreen using Escherichia coli single-stranded DNA binding protein (SSB) revealed that none of the new HCV helicase inhibitors were specific for NS3h. However, when the SSB-based assay was used to analyze derivatives of another non-specific helicase inhibitor, the main component of the dye primuline, it revealed that some primuline derivatives (e.g. PubChem CID50930730) are up to 30-fold more specific for HCV NS3h than similarly potent HCV helicase inhibitors.