Abstract
The hepatitis C virus (HCV) has infected some 170 million people worldwide, and is expected to pose a significant medical problem for the foreseeable future. No vaccine is presently available, and the current antiviral therapies (pegylated interferon-α and ribavirin) are characterized by limited efficacy, high costs, and substantial side effects. Initiation of infection requires attachment of the HCV virus to the cell surface followed by viral entry and represents a critical determinant of tissue tropism and pathogenesis. Small molecules that inhibit the virus at the stage of viral entry, for example, by blocking the interactions between viral envelope glycoprotein and cellular receptor or coreceptor or by inhibiting the viral fusion process, would serve as attractive antiviral drugs. Recent development of HCV pseudoparticles (HCVpp), displaying unmodified and functional HCV glycoprotein on the surface of retroviral core particles, has greatly facilitated studies of HCV entry and provides an essential tool for the identification and characterization of molecules that block HCV entry. We have adapted the HCVpp infection assay with HCVpp harboring a luciferase reporter to a 96-well format and screened a small-molecule compound library to identify inhibitors of HCV entry. Such active viral entry inhibitors have the potential to be first-in-class antiviral drugs that can be incorporated into combinations of multiple drugs with different targets for the treatment of chronic HCV infection.
Key Words
1 Introduction
The development of antiviral drugs for HCV has been focused largely on inhibition of virus replication. NS3, a protease, and NS5B, an RNA-dependent RNA polymerase, have been the major targets for HCV drug development (1,2), but because of HCV’s genomic plasticity, viral resistance is a looming issue, as it has been in HIV therapy. Identification of small-molecule compounds that inhibit other steps in HCV replication, such as virus binding, entry, and postentry events, will greatly expand our antiviral repertoire. Combination chemotherapy with different classes of inhibitors including entry inhibitors may result in synergistic inhibition of viral infection (3).
The mechanism by which HCV enters target cells has not been fully elucidated. HCV envelope proteins E1 and E2 are type I integral transmembrane proteins with extensively glycosylated ectodomains and are required for virus particle formation and infection of host cells. Several surrogate systems such as recombinant HCV envelope glycoproteins (4), HCV-like particles (5), and HCV pseudoparticles (HCVpp) (6,7) have been developed and used to study viral attachment, entry, and infection. More recently, cell-cultured infectious HCV (HCVcc) has been developed (8,–), but its use for study of HCV entry is confounded by multiple steps and multiple cycles of infection and, further, pertains so far to only one genotype (genotype 2a).
HCVpp is considered the most biologically relevant reporter system for the study of HCV entry. It closely mimics the entry and serological properties of native HCV infection, such as the tropism for primary human hepatocytes and hepatocyte cell lines, pH dependence of the infection process, and neutralization by patient sera as well as monoclonal antibodies (MAbs) specific for E2 (6,7,11–13). The involvement of human CD81 in HCV entry was confirmed in this surrogate system (14).
HCVpp consists of unmodified HCV envelope glycoproteins assembled onto retroviral core particles carrying a reporter gene such as luciferase, green fluorescence protein, or antibiotic resistance genes. Use of HCVpp harboring a luciferase reporter permits easy detection of productive viral entry by the very sensitive luciferase assay. Similar pseudovirus particles bearing other viral envelope proteins (e.g., VSV-G protein) can be used as controls for events irrelevant to entry.
Here we describe our experience with HCVpp and VSV-Gpp in screening small-molecule compounds for inhibition of HCV entry. The specificity of the vector system was tested with antibodies to CD81 (Fig. 22.1). Several compounds were identified that inhibited HCVpp infection but not VSV-G infection (Fig. 22.2). We further evaluated the compounds’ dose dependence, specificity (e.g., activity on HCVpp harboring envelope proteins of other HCV genotypes), and cellular toxicity (Alamar Blue assay) (Fig. 22.3). Finally, the compounds were tested in the HCVcc system (data not shown). We believe the relatively high-throughput HCVpp system can be used to discover compounds that selectively inhibit HCV entry.
2 Materials
2.1 Viral Production
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1.
pNL4-3 HIV proviral DNA (AIDS Reagent Program, NIH, Bethesda, MD).
-
2.
pcDNA-E1E2 1a and 2b [cloned from total RNA prepared from HCV-infected plasma as described previously (15)].
-
3.
pcDNA-VSV-G (a plasmid encoding the vesicular stomatitis virus (VSV) glycoprotein G protein).
-
4.
pAdVantage (Promega, Madison, WI).
-
5.
293T cells (American Type Culture Collection, Manassas, VA).
-
6.
293T cell-culture medium: Dulbecco’s modified Eagle’s medium (DMEM) (Gibco/BRL, Bethesda, MD) supplemented with 10% heat-inactivated fetal bovine serum (FBS; HyClone, Ogden, UT), 2 mM l-glutamine, 0.1 mM MEM nonessential amino acids.
-
7.
Medium for viral production and infection: DMEM, supplemented with 3% FBS, 1 mM MEM sodium pyruvate, 2 mM l-glutamine, 0.1 mM MEM nonessential amino acids.
-
8.
TransIT-LT1 transfection reagent (Mirus, Madison, WI).
-
9.
Opti-MEM medium (Invitrogen).
-
10.
Solution of trypsin (0.25%) from Gibco/BRL.
-
11.
CD81 antibody (BD Biosciences).
2.2 Viral Infection Assay
-
1.
Huh-7 cell line.
-
2.
Polybrene: Prepare a 10-mg/mL stock solution in deionized, sterile water. Filter-sterilize it and dispense 1 ml aliquots into sterile microcentrifuge tubes. Store at \(-20^{\circ}{\rm C}\) for long-term storage (Sigma).
2.3 Luciferase Assay
-
1.
Costar TC-treated solid white 96-well tissue-culture plate with flat bottom (Corning Incorporated, NY, USA).
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2.
Bright-Glo‡ Luciferase Assay System (Promega).
2.4 Alamar Blue Assay
-
1.
96-well Microtest tissue-culture plate, flat bottom with low-evaporation lid (Becton Dickinson Labware, Franklin Lakes, NJ).
-
2.
Alamar Blue reagent (Biosource).
3 Methods
3.1 HCVpp Production
Here, we outline the optimal procedure for producing HCVpp and VSV-Gpp harboring a luciferase reporter, including plasmid transfection, viral supernatant harvesting, and HCVpp quality evaluation.
3.1.1 Transfection of 293T Cells with HIV Proviral DNA, pcDNA-E1E2, or pcDNA-VSV-G and pAdVAtage
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1.
Plate \(5 \times 10^{5}\,293{\rm T}\) cells/well in 2 mL of 10% DMEM culture medium in a 6-well plate and culture overnight at \(37^{\circ}{\rm C}\) under 5% \({\rm CO}_{2}\), humidified. They should be approximately 80% confluent by the next day.
-
2.
In two sterile plastic tubes, prepare the following mixtures for each transfection sample:
-
a.
Add \(5\,\mu{\rm L}\) of Mirus TransIT-LT1 directly into \(100\,\mu{\rm L}\) of Opi-MEM Medium per well; mix gently by pipetting up and down.
-
b.
Add \(1\,\mu{\rm g}\)/well HIV-Luc, \(0.7\,\mu{\rm g}\)/well pcDNA-E1E2, and \(0.3\,\mu{\rm g}\)/well pAdVantage to \(100\,\mu{\rm L}\) of Opti-MEM Medium per well and mix. To generating VSV-Gpp, add \(1\,\mu{\rm g}\)/well HIV-Luc and \(0.7 \mu{\rm g}\)/well pcDNA-VSV-G to \(100\,\mu{\rm L}\) of Opti-MEM Medium per well and mix. DNA is suspended in sterile water at a concentration of \(0.2-3.0\,\mu{\rm g}/\mu{\rm L}\) (see Note 1).
-
a.
-
3.
Slowly mix “a” and “b” together with gentle vortexing.
-
4.
Incubate the DNA/LT1 complex at room temperature for 15–30 min to precipitate the DNA.
-
5.
Gently resuspend any DNA-lipid complexes by pipetting the suspension up and down; add the DNA suspension to 293T cells in 2 ml of culture medium in a drop-wise fashion, swirling gently to prevent the cells from being lifted from the plate and to distribute the DNA suspension evenly (see Notes 2 and 3).
-
6.
Return the tissue-culture plates to the \(37^{\circ}{\rm C}\) incubator.
-
7.
After 4-6 h of incubation, remove the medium from the plates and replace it with 2 mL/well of 3% FBS DMEM supplemented with 1 mM sodium pyruvate that has been prewarmed to \(37^{\circ}{\rm C}\).
-
8.
Incubate the cells at \(37^{\circ}{\rm C}\) under 5% \({\rm CO}_{2}\) for 48 h.
3.1.2 Harvest of Viral Supernatants
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1.
Harvest virus-containing supernatants of the transfected 293T cells at the 48-h time point.
-
2.
Refeed cells with 2 mL/well of 3% FBS DMEM with 1 mM sodium pyruvate and incubate them at \(37^{\circ}{\rm C}\) under 5% \({\rm CO}_{2}\) overnight. Harvest the supernatant at the 72-h time point.
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3.
Filter the viral supernatants through membranes of \(0.45\,\mu{\rm m}\) pore size to remove the cell debris (see Note 4).
-
4.
Store aliquots of the viral supernatants of desired volumes at \(-80^{\circ}{\rm C}\) (see Note 5).
3.1.3 Evaluation of the Quality of HCVpp
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1.
Seed a 96-well plate with Huh-7 cells at \(1 \times 10^{5}\) cells/well and incubate it at \(37^{\circ}{\rm C}\) under 5% \({\rm CO}_{2}\) overnight (see Notes 6 and 7).
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2.
Dilute CD81 antibody and control IgG to \(10\,\mu{\rm g}/{\rm mL}\) in 3% FBS DMEM culture medium (see Note 8).
-
3.
Remove the culture medium from the Huh-7 cells.
-
4.
Add \(50\,\mu{\rm L}\) of CD81, IgG, or 3% FBS DMEM culture medium to the cells and incubate them at room temperature for 30 min.
-
5.
Add \(50\,\mu{\rm L}\) of HCVpp or VGV-Gpp (1:100 dilution) with \(8\,\mu{\rm g}/{\rm mL}\) polybrene per well to the cells.
-
6.
Incubate at \(37^{\circ}{\rm C}\) under 5% \({\rm CO}_{2}\) for 72 h.
-
7.
Perform the luciferase assay (see Section 3.3). A sample result is shown in Fig. 22.1 (see Notes 9, 10, and 11).
3.2 Screening Small-Molecule Compounds
3.2.1 Compound Preparation
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1.
Dilute chemical compounds in DMSO to make \(1500\,\mu{\rm M}\) and \(500\,\mu{\rm M}\) stock solutions in clear 96-well plates.
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2.
Further dilute stock compounds to working concentrations as follows:
-
a.
Add \(12\,\mu{\rm L}\) of \(1500\,\mu{\rm M}\) stock solution to \(588\,\mu{\rm L}\) of 3% FBS DMEM culture medium to make a \(30\,\mu{\rm M}\) compound solution containing 2% DMSO.
-
b.
Add \(12\,\mu{\rm L}\) of \(500\,\mu{\rm M}\) stock solution to \(588\,\mu{\rm L}\) of 3% FBS DMEM culture medium to make a \(10\,\mu{\rm M}\) compound solution containing 2% DMSO.
-
c.
Add \(3.6\,\mu{\rm L}\) of \(500\,\mu{\rm M}\) stock solution and \(8.4\,\mu{\rm L}\) of DMSO to \(588\,\mu{\rm L}\) of 3% FBS DMEM culture medium to make a \(3\,\mu{\rm M}\) compound solution containing 2% DMSO.
-
d.
Add \(2.4\,\mu{\rm L}\) of \(500\,\mu{\rm M}\) stock solution and \(9.6\,\mu{\rm L}\) of DMSO to \(588\,\mu{\rm L}\) of 3% FBS DMEM culture medium to make a \(2\,\mu{\rm M}\) compound solution containing 2% DMSO.
-
e.
Add \(1.2\,\mu{\rm L}\) of \(500\,\mu{\rm M}\) stock solution and \(10.8\,\mu{\rm L}\) of DMSO to \(588\,\mu{\rm L}\) of 3% FBS DMEM culture medium to make a \(1\,\mu{\rm M}\) compound solution containing 2% DMSO.
-
a.
-
3.
As a negative control, add \(12\,\mu{\rm L}\) of DMSO to \(588\,\mu{\rm L}\) of 3% FBS DMEM culture medium to make a control cell-culture medium containing 2% DMSO.
3.2.2 Seeding the Wells
-
1.
Count Huh-7 cells and make a suspension of \(1 \times 10^{5}\) cells/mL in 10% FBS DMEM culture medium.
-
2.
Divide the cell suspension into aliquots of \(100\,\mu{\rm L}\) (\(1 \times 10^{4}\) cells) per well in two white 96-well tissue-culture plates for the luciferase assay and one clear plate for the cell-toxicity assay.
-
3.
Incubate the cells at \(37^{\circ}{\rm C}\) under 5% \({\rm CO}_{2}\) overnight.
3.2.3 HCVpp and VSV-Gpp Infection Assays
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1.
Flip plates over to empty wells of culture medium and tap them on a bench over paper (Kimwipes) to empty wells completely.
-
2.
Add \(50\,\mu{\rm L}\) of diluted compound to each well.
-
3.
Incubate plates at room temperature for 30 min.
-
4.
During the incubation, thaw the viral stock and dilute the appropriate amount of virus in 3% FBS complete medium. We usually dilute the VSV-Gpp stock 1:100 because of its high infectivity.
-
5.
Add polybrene to viral supernatants to yield \(8\,\mu{\rm g}/{\rm mL}\).
-
6.
Add \(50\,\mu{\rm L}\) of HCVpp or VSV-Gpp per well to the cells.
-
7.
Incubate the cells at \(37^{\circ}{\rm C}\) under 5% \({\rm CO}_{2}\) for 4 h.
-
8.
Remove the medium containing virus and compound, and add \(100\,\mu{\rm L}\) of fresh culture medium, and incubate the cells at \(37^{\circ}{\rm C}\) under 5% \({\rm CO}_{2}\) for 72 h.
3.3 Luciferase Assay
-
1.
Make Bright-Glo reagent by reconsititution of Bright-Glo Substrate with room temperature Bright-Glo buffer; avoid exposing it to light.
-
2.
Add \(100\,\mu{\rm L}\) of Bright-Glo reagent per well to cells.
-
3.
Incubate the plate at room temperature for 5 min in dark.
-
4.
Read the plates on a Veritase Luminometer.
3.4 Cell-Toxicity Assay
3.4.1 Treatment the Huh-7 Cells with Compounds
-
1.
Add \(50\,\mu{\rm L}\) of diluted compound per well to Huh-7 cells in a clear 96-well plate.
-
2.
Incubate the plate at room temperature for 30 min.
-
3.
Add \(50\,\mu{\rm L}\) of 30% FBS DMEM/well over the \(50\,\mu{\rm L}\) of compound in each well.
-
4.
Incubate the plate at \(37^{\circ}{\rm C}\) under 5% \({\rm CO}_{2}\) for 72 h.
3.4.2 Alamar Blue Assay
-
1.
Dilute \(10\times\) stock Alamar Blue reagent 1:5 in PBS (see Note 12).
-
2.
Add \(100\,\mu{\rm L}\) of \(2\times\) Alamar Blue reagent per well over the cells.
-
3.
Incubate the plate at \(37^{\circ}{\rm C}\) under 5% \({\rm CO}_{2}\) for 3–4 h.
-
4.
Read plates in Cytofluor plate reader (emission 590 nm and excitation 530 nm).
4 Notes
-
1.
We observed that cotransfection of pAdVantage increased the viral titer at least 10-fold, but when the VSV-Gpp vector is packaged, including pAdVantage in the transfection is not recommended, because high expression of VSV-G is toxic to cells. We recommend making HCVpp and VSV-Gpp at different times to avoid cross contamination.
-
2.
Transfection efficiency is the key to production of high-titer virus. Early-passage 293T stocks should be used; cells should be passed at high density and thoroughly trypsinized to prevent clumping; the cell density should be about 80% confluent at the time of transfection.
-
3.
293T cells are weakly adherent, especially after transfection, so all medium changes should be performed with extreme care.
-
4.
The filter used for viral purification should be cellulose acetate polysulfonic (low protein binding), not nitrocellulose. Nitrocellulose binds proteins in the retrovirus membrane and destroys the virus.
-
5.
Viral supernatants can generally be collected 48 h and 72 h after transfection if many cells are still attached to the plate and look healthy at these times. Minimal differences in viral titer are observed for harvests at these two times.
-
6.
To avoid the edge effect, we generally exclude the outer wells of 96-well plates from screening. To prevent the plates from drying out, add \(200\,\mu{\rm L}\) of PBS to each outer well to humidify plate.
-
7.
We observed that Huh-7 cells seeded in 3% FBS DMEM medium are infected by psuedoviruses more efficiently than that in 10% FBS DMEM medium.
-
8.
We noticed that antibodies with sodium azide affected the infection of both HCVpp and VSV-Gpp because of cell toxicity. We recommend dialyzing the antibodies to remove sodium azide before use.
-
9.
This protocol for viral production is optimized in a six-well plate format. The cotransfection experiment can be scaled up if a large volume of virus is desired.
-
10.
Viral vector is stable for a week at \(4^{\circ}{\rm C}\). Frozen at \(-80^{\circ}{\rm C}\), the virus can be stable for at least 6 months, but the titer will be decreased twofold by each freeze-thaw cycle. Any viral supernatant that is not used immediately upon harvesting should be frozen in aliquots at \(-80^{\circ}{\rm C}\).
-
11.
Luciferase activity is temperature dependent. The most convenient and effective method for thawing or temperature equilibrating cold reagent is to place it in a water bath at room temperature. Do not use a water bath above \(25^{\circ}{\rm C}\).
-
12.
Upon thawing Alamar Blue reagent, warm it to \(37^{\circ}{\rm C}\) and mix it well to assure complete redissolution. Store the Alamar Blue reagent away from light to prevent changes in its absorbance properties.
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Acknowledgements
The authors would like to thank Jing Zhang and Maureen Ibanez for technical assistance.
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© 2009 Humana Press, a part of Springer Science+Business Media, LLC
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Yang, JP., Zhou, D., Wong-Staal, F. (2009). Screening of Small-Molecule Compounds as Inhibitors of HCV Entry. In: Tang, H. (eds) Hepatitis C. Methods in Molecular Biology™, vol 510. Humana Press. https://doi.org/10.1007/978-1-59745-394-3_22
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DOI: https://doi.org/10.1007/978-1-59745-394-3_22
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