Selecting a path against Hepatitis C Virus

Hepatitis C virus (HCV) currently affects ~5% of the world’s population and has relatively limited treatment options for infected patients. Genetic suppressor elements (GSE) derived from a gene or genome of interest can act as transdominant inhibitors of a particular biology function presumably by binding to and blocking an essential interaction surface for protein activity. Taking advantage of hepatoma cell line n4mBid, that supports all stages of the HCV life cycle and strongly report HCV infection by a cell-death phenotype, we developed an iterative selection/enrichment strategy for the identification of GSE against HCV. Using this strategy, a library expressing random fragments of the HCV genome was screened for sequences able to suppress HCV infection. A 244 amino acid gene fragment, B1, was strongly enriched after 5 rounds of selection. B1 has a very high net positive charge of 43 at neutral pH and a high charge-to-mass (kDa) ratio of 1.5. We show that B1 expression specifically inhibits HCV replication, apparently due to its high positive charge. We also show that recombinant positively charged proteins can inhibit HCV infection, when supplied in vitro.In addition, eGFP-fused B1 potently penetrates both adherent and suspension cells with >80% of cells taking up the protein. Importantly, we show that B1 not only facilitates cellular uptake, but allows protein cargo to reach sites of biological relevance. B1 also delivers non-covalently conjugated RNA and DNA across the cell membrane to cytosolic and nuclear sites, with efficiency comparable to commercially available cationic lipid reagents. Our data suggest that B1 utilizes cell-surface glycans and multiple competing endocytic pathways to enter and traffic through cells.During a separate screening carried out in our lab, we identified a TACR3 inhibitor SB 222200 that had significant HCV activity. We go on to show that both TACR1 and TACR3 receptors are expressed in the HCV-permissive Huh 7.5 cell line. We also show that both TACR1 and TACR3 inhibitors significantly inhibit HCV infection. These results point to the potential for TACR1 antagonists in treating patients infected with both HCV and HIV

A screen for genetic suppressor elements of hepatitis C virus identifies a supercharged protein inhibitor of viral replication

Genetic suppressor elements (GSEs) are biomolecules derived from a gene or genome of interest that act as transdominant inhibitors of biological functions presumably by disruption of critical biological interfaces. We exploited a cell death reporter cell line for hepatitis C virus (HCV) infection, n4mBid, to develop an iterative selection/enrichment strategy for the identification of anti-HCV GSEs. Using this approach, a library of fragments of an HCV genome was screened for sequences that suppress HCV infection. A 244 amino acid gene fragment, B1, was strongly enriched after 5 rounds of selection. B1 derives from a single-base frameshift of the enhanced green fluorescent protein (eGFP) which was used as a filler during fragment cloning. B1 has a very high net positive charge of 43 at neutral pH and a high charge-to-mass (kDa) ratio of 1.5. We show that B1 expression specifically inhibits HCV replication. In addition, five highly positively charged B1 fragments produced from progressive truncation at the C-terminus all retain the ability to inhibit HCV, suggesting that a high positive charge, rather than a particular motif in B1, likely accounts for B1’s anti-HCV activity. Another supercharged protein, + 36GFP, was also found to strongly inhibit HCV replication when added to cells at the time of infection. This study reports a new methodology for HCV inhibitor screening and points to the anti-HCV potential of positively charged proteins/peptides.The open access fee for this work was funded through the Texas A&M University Open Access to Knowledge (OAK) Fund

PD 404,182 Is a Virocidal Small Molecule That Disrupts Hepatitis C Virus and Human Immunodeficiency Virus

We describe a virucidal small molecule, PD 404,182, that is effective against hepatitis C virus (HCV) and human immunodeficiency virus (HIV). The median 50% inhibitory concentrations (IC(50)s) for the antiviral effect of PD 404,182 against HCV and HIV in cell culture are 11 and 1 μM, respectively. The antiviral activity of PD 404,182 is due to the physical disruption of virions that is accompanied to various degrees (depending on the virus and exposure temperature/time) by the release of viral nucleic acids into the surrounding medium. PD 404,182 does not directly lyse liposomal membranes even after extended exposure, and it shows no attenuation in antiviral activity when preincubated with liposomes of various lipid compositions, suggesting that the compound inactivates viruses through interaction with a nonlipid structural component of the virus. The virucidal activity of PD 404,182 appears to be virus specific, as little to no viral inactivation was detected with the enveloped Dengue and Sindbis viruses. PD 404,182 effectively inactivates a broad range of primary isolates of HIV-1 as well as HIV-2 and simian immunodeficiency virus (SIV), and it does not exhibit significant cytotoxicity with multiple human cell lines in vitro (50% cytotoxic concentration, >300 μM). The compound is fully active in cervical fluids, although it exhibits decreased potency in the presence of human serum, retains its full antiviral potency for 8 h when in contact with cells, and is effective against both cell-free and cell-associated HIV. These qualities make PD 404,182 an attractive candidate anti-HIV microbicide for the prevention of HIV transmission through sexual intercourse

Phenothiazines Inhibit Hepatitis C Virus Entry, Likely by Increasing the Fluidity of Cholesterol-Rich Membranes

Despite recent progress in the development of direct-acting antiviral agents against hepatitis C virus (HCV), more effective therapies are still urgently needed. We and others previously identified three phenothiazine compounds as potent HCV entry inhibitors. In this study, we show that phenothiazines inhibit HCV entry at the step of virus-host cell fusion, by intercalating into cholesterol-rich domains of the target membrane and increasing membrane fluidity. Perturbation of the alignment/packing of cholesterol in lipid membranes likely increases the energy barrier needed for virus-host fusion. A screening assay based on the ability of molecules to selectively increase the fluidity of cholesterol-rich membranes was subsequently developed. One compound that emerged from the library screen, topotecan, is able to very potently inhibit the fusion of liposomes with cell culture-derived HCV (HCVcc). These results yield new insights into HCV infection and provide a platform for the identification of new HCV inhibitors

Expression of B1 in Huh-7.5 cells.

<p>(<b>A</b>) Western blot of Flag-NS4Am-B1 and Flag-B1. (<b>B</b>) Confocal microscopic images of Huh-7.5 cells stably expressing Flag-NS4Am-B1 or Flag-B1 after immunohistochemical staining with anti-flag antibody.</p

Overview of GSE selection.

<p>(<b>A</b>) GSE selection scheme. Randomly fragmented libraries were prepared by limited digestion of the HCV genome and fragments of 100–200 bp were cloned into the lentiviral vector pV1 and packaged into VSV-G-pseudotyped lentiviral particles. Library pseudoparticles were delivered into n4mBid cells such that each library fragment was expressed on average in at least 1000 cells. These cells were then infected with HCV and passaged for a period of 9 days or until the number of surviving cells was ≤10% of the initial cell population. The specific HCV inhibitor 2′C-MA was then added to rescue the surviving cells. Library fragments were repackaged by transfecting the surviving cells with plasmids encoding VSV-G and HIV gag-pol and formed the basis of subsequent rounds of selection. (<b>B</b>) Table depicting the number of cells that survived the HCV challenge as a percentage of the total number of seeded cells during consecutive rounds of selection. (<b>C</b>) Agarose gel image of cDNA inserts harvested from repackaged pseudoparticles after 5 rounds of selection.</p

Plasmid constructs.

<p>Plasmid constructs.</p

Discovery and Characterization of a New Cell-Penetrating Protein

We describe a new cell-penetrating protein, B1, capable of delivering conjugated proteins and nucleic acids into mammalian cells. B1 is a 244-amino-acid product of a single-base frameshift in the gene encoding enhanced green fluorescent protein (eGFP). The molecule has a net positive charge of 43 and a very high charge-to-mass ratio of 1.5. eGFP-fused B1 potently penetrates both adherent and suspension cells with >80% of cells taking up the protein when exposed to concentrations as low as 1 μM. The protein was found to cluster in the paranuclear region of TZM-bl cells. Most importantly, we show that B1 not only facilitates cellular uptake but allows biomolecular cargo to reach sites of biological relevance. For example, baby hamster kidney cells underwent DNA recombination when exposed to B1-tagged Cre recombinase at protein concentrations as low as 2.5 μM, indicating potent nuclear delivery of functional protein cargos. Additionally, B1 delivers noncovalently conjugated RNA and DNA across the cell membrane to cytosolic and nuclear sites accessible to the cellular translation and transcription machinery, as gauged by detection of encoded reporter functions, with efficiency comparable to commercially available cationic lipid reagents. B1 appears to utilize cell-surface glycans and multiple competing endocytic pathways to enter and traffic through cells. These studies provide both a new tool for intracellular delivery of biomolecules and insights that could aid in the design of more effective cell penetrating proteins