www.elsevier.com/locate/yexmp

Experimental and Molecular Pathology 76 (2004) 242–252

Interferon alpha-2b inhibits negative-strand RNA and protein expression

from full-length HCV1a infectious clone

Ramesh Prabhu,a Virendra Joshi,b Robert F. Garry,c Frank Bastian,a Salima Haque,a

Fredric Regenstein,b Swan Thung,d and Srikanta Dasha,*

aDepartment of Pathology and Laboratory Medicine, Tulane University Health Science Center, New Orleans, LA 70112, USAbDepartment of Medicine, Tulane University Health Science Center, New Orleans, LA 70112, USA

cMicrobiology and Immunology, Tulane University Health Science Center, New Orleans, LA 70112, USAdDepartment of Pathology and Laboratory Medicine, Mount Sinai School of Medicine, New York, NY 10029, USA

Received 18 December 2003

Available online 27 March 2004

Abstract

We have established a T7-based model system for hepatitis C virus (HCV) 1a strain, which involves the use of a replication-defective

adenovirus that carries the gene for T7 RNA polymerase and a transcription plasmid containing full-length HCV cDNA clone. To facilitate

high-level expression of HCV, sub-confluent Huh7 cells were first infected with adenovirus containing the gene for the T7 RNA polymerase

and then transfected with the transcription plasmid. As a negative control, part of NS5B gene of this clone was deleted which abolishes the

HCV RNA-dependent RNA polymerase and prevents replication of viral RNA. This model produces high levels of structural (core, E1, E2)

and nonstructural proteins (NS5), which were detected by Western blot analysis and immunofluorescence assay. Negative-strand HCV RNA

was detected only in the wild-type clone in the presence of actinomycin D, and no RNAwas detected with the NS5B deleted mutant control.

As a practical validation of this model, we showed that IFN a-2b selectively inhibits negative-strand RNA synthesis by blocking at the level

of protein translation. The inhibitory effect of IFN a-2b is not due reduction of transcription by T7 polymerase or due to intracellular

degradation of HCV RNA. This in vitro model provides an efficient and reliable means of assaying negative-strand RNA, protein processing,

and testing the antiviral properties of interferon.

D 2004 Elsevier Inc. All rights reserved.

Keywords: RNA polymerase; Interferon; HCV RNA

Introduction et al., 1992). The resulting polyprotein is cleaved by cellular

Hepatitis C virus (HCV) is an enveloped positive-strand-

ed RNA virus belonging to the Flaviviridae family

(Houghton et al., 1991, Miller and Purcell, 1990). The viral

genome is organized into a 5V untranslated region (5V UTR),a single large open reading frame (ORF) of approximately

3010 amino acids, and a 3V untranslated region (3V UTR)(Choo et al., 1989). The 5V UTR contains an internal

ribosome entry site (IRES), which mediates the cap-inde-

pendent translation of the polyprotein encoded by the ORF

(Brown et al., 1992; Bukh et al., 1992; Tsukiyama-Kohara

0014-4800/$ - see front matter D 2004 Elsevier Inc. All rights reserved.

doi:10.1016/j.yexmp.2004.01.004

* Corresponding author. Department of Pathology and Laboratory

Medicine, Tulane University Health Science Center, 1430 Tulane Avenue,

New Orleans, LA 70112, USA. Fax: +1-504-587-7389.

E-mail address: sdash@tulane.edu (S. Dash).

and viral proteases into four structural proteins (core, E1, E2

and P7), and six nonstructural proteins (NS2, NS3, NS4A,

NS4B, NS5A and NS5B) (Grakoui et al., 1993; Hijikata et

al., 1991; Lin et al., 1994; Major and Feinstone, 1997). The

3V UTR of HCV is composed of three distinct regions

consisting of (i) a short variable sequence, (ii) an adjoining

polyuridine tract of variable length, followed by (iii) a

highly conserved region of 98 nucleotides (Kolykhalov et

al., 1996; Tanaka et al., 1995, 1996). The highly conserved

5V and 3V UTR sequences are essential for the replication of

the viral genome (Friebe and Bartenschlager, 2002; Friebe et

al., 2001; Kolykhalov et al., 2000; Oh et al., 1999; Yanagi et

al., 1999; Yi and Lemon, 2003). Replication of the HCV

occurs at both ends of the viral RNA in two separate steps.

The first step involves replication of positive-strand RNA to

negative-strand RNA beginning at the 3V end of positive-

R. Prabhu et al. / Experimental and Molecular Pathology 76 (2004) 242–252 243

strand RNA. In the second step, replication of the negative-

strand RNA to positive-strand RNA is initiated at the 3V endof the negative strand (complementary to the 5V UTR). Full-cycle replication of HCV involves one round of RNA

synthesis at the 3V end of positive and negative-strand

RNA. The nonstructural proteins (NS3 to NS5B) are

enzymes encoded by viral genome that are essential for

replication of HCV. However, the mechanisms by which

these proteins regulate positive-strand and negative-strand

RNA synthesis are unknown.

The establishment of a HCV replication model is impor-

tant to determine the mechanism of HCV replication. Prior

studies (Bertolini et al., 1993; Cribier et al., 1995; Ito et al.,

1996; Kato et al., 1995; Lanford et al., 1994; Mizutani et al.,

1996; Shimizu et al., 1992, 1993, 1996) have attempted to

develop aHCVreplication system by either infecting cultured

cells in vitro or transfecting cultured cells with the in vitro

transcribed viral genomic RNA (Dash et al., 1997, 2001; Yoo

et al., 1995). None of these models produce high-level HCV

replication and can only be measured using very highly

sensitive techniques such as RT-PCR. Availability of infec-

tious clones for HCV facilitated the development of innova-

tive approaches to the experimental studies on hepatitis C

virus (Kolykhalov et al., 1997; Yanagi et al., 1997, 1998).

High level of HCVreplication in cell culture was possible due

to the development of sub-genomic replicon (Blight et al.,

2000, 2003; Lohmann et al., 1999). The HCV sub-genomic

replicon is a chimeric RNA containing sequence arranged in

the order of HCV 5V UTR sequence, the neomycin gene, the

encephalomyocardities virus IRES (EMC IRES), and the

nonstructural proteins NS3-NS5B and 3V UTR. ReplicatingRNA in the transfected Huh-7 cells develop resistance to G-

418 and form cell colonies, which provides an assay to study

full cycle replication of HCV.

Alternatively, vaccinia virus encoding T7 polymerase

(Chung et al., 2001) or replication-defective adenovirus that

carries the gene for T7 polymerase (Kalkeri et al., 2001;

Myung et al., 2001) along with a plasmid DNA encoding the

HCV cDNA has been used to express full-length HCV

genome. The T7 polymerase transcribes higher levels of

HCV genome, which is processed fully due to the presence

of ribozyme and T7 terminator. Use of replication-defective

adenovirus expressing T7 polymerase provides advantages

over the vaccinia virus T7 because it is less toxic to the cell,

allowing study of viral gene expression in vitro for a long

period of time. Previously, we had established a T7-based

expression model for HCV1b strain using replication-defec-

tive adenovirus that carries the gene for T7 RNA polymerase

(Myung et al., 2001). High levels of viral proteins, negative-

strand RNA and virus particles were detected in a hepatic cell

line after transfection with an infectious clone HCV1b strain.

We have extended this approach and established a full-length

replication model for the HCV1a strain in Huh-7 cells. We

demonstrated that Huh-7 cells transfected with a chimpanzee

infectious clone expressed high levels of viral protein and

negative-strand RNA by ribonuclease protection assay. We

showed here that IFN a-2b inhibits negative-strand RNA

synthesis and inhibits protein expression from a full-length

infectious clone for HCV1a strain.

Materials and methods

Infectious clones and transcription plasmids

Chimpanzee infectious clones, pCV-J4L6S (HCV1b) and

pCV-H77C (HCV1a), were obtained from Jens Bukh, Na-

tional Institute of Health (Yanagi et al., 1997, 1998). A

transcription plasmid (pNIH-HCV1a-Rz) was prepared

which contained a T7 promoter, full-length cDNA of HCV

genome (HCV1a) at the 5V end, followed by a cDNA copy of

autolytic ribozyme from anti-genomic strand of hepatitis

delta virus and T7 transcriptional terminator sequences at

the 3V end of HCV cDNA. The cloning experiments were

performed in several steps. First, we excised the EcoRI and

XbaI fragment of plasmid (pCV-J4L6S) and then cloned into

pcDNA3 (Invitrogen Corp., San Diego, CA). The resulting

plasmid was named pcDNA3-EcoRI-XbaI-NIH1b. In the

second step, a PCR strategy was used to introduce anti-

genomic delta ribozyme and T7 terminator sequences to the

3V end of pcDNA3-EcoRI-XbaI-NIH-1b plasmid. For this

purpose, a 330-bp DNA fragment, which included part of the

3V UTR HCV sequence, followed by anti-genomic hepatitis

delta ribozyme sequences and T7 terminator, was amplified

from the transcription vector 2.0 (obtained as a gift from

Andrew Ball, University of Alabama at Birmingham, Ala-

bama) (Pattnaik et al., 1992). The amplicon was digested with

NheI and cloned into the pcDNA3-EcoRI-XbaI-NIH1b plas-

mid using a unique NheI site such that the ribozyme cleavage

occurred just after the last nucleotide of the 3V UTR of HCV.

At the third step, EcoRI and XbaI fragment of the resulting

plasmid was removed and inserted into the original pCV-

J4L6S plasmid. This construct was named pNIH-HCV1b-Rz.

The pCV-J4L6S and pCV-H77C has similar 3V UTR sequen-

ces. At the fourth step, we removed the AflII–XbaI fragment

from this pNIH-HCV1b clone and placed it back into the

pCV-H77C (NIH-1a) clone. At this point, we realized that

there was another T7 promoter in pCV-H77C plasmid down-

stream of 3VUTR. Therefore, XbaI and SfiI fragment from the

pNIH-HCV1a-Rz plasmid was removed, blunt ended and

religated. The final transcription plasmid was called pNIH-

HCV1a-Rz. Control plasmid was prepared by removing a

HindIII and NdeI fragment (7862–9160) from pNIH-

HCV1a-Rz clone (pNIH-HCV1a-Rz-NS5 mutant). This mu-

tant construct should not produce NS5B protein (HCV

replicase), and therefore should not replicate. A schematic

diagram of both transcription plasmids is shown in Fig. 1.

DNA transfection and virus infection

The HCV1a and mutant 1a transcription plasmids were

transfected to Huh-7 cells in a two-step procedure described

Fig. 1. Diagrammatic representation of the transcription plasmids containing chimpanzee infectious clone HCV1a (pNIH-HCV1a-Rz) and its NS5B mutant

control (top). Nucleotide sequences at the 5V and 3V end of full-length RNA transcript generated in vivo (bottom).

R. Prabhu et al. / Experimental and Molecular Pathology 76 (2004) 242–252244

below. In the first step, Huh-7 cells were infected with

AdexCAT7 virus (Aoki et al., 1998) at a multiplicity of

infection (MOI) of 10. Two hours later, cells were trans-

fected with 10 Ag of the transcription plasmid using the

FuGENE 6 Reagent (Roche Molecular Biochemicals, Indi-

anapolis, IN). The success of HCV transfection to Huh-7

cells was determined by examining the production of pro-

teins, positive- and negative-strand RNA at 24–72 h post-

transfection depending on the experiment being performed.

Detection of viral proteins in transfected Huh-7 cells

The expression of viral protein in transfected Huh-7 cells

was carried out after 72 h by Western blot and immunoflu-

orescence assay. Transfected Huh-7 cells were harvested at

72 h post-transfection by trypsinization and washed with 10

mM phosphate-buffered saline (PBS), pH 7.4 (Sigma Co,

St. Louis, MO). Approximately 105 cells were immobilized

onto glass slides using cytospins. Following this step, cells

were fixed with chilled acetone at 4jC for 5 min and were

permeabilized with 0.05% saponin. The cells were then

blocked with PBS containing 2% BSA and one drop of goat

serum per milliliter of blocking reagent for 30 min. The cells

were then incubated with primary antibodies diluted in PBS

(1:500) containing 2% bovine serum albumin (BSA). After

this step, each slide was washed three times with PBS and

then incubated with a respective secondary antibody at

1:1000 dilution (goat anti-mouse-FITC for monoclonal

anti-HCV for core, E1, E2; Sigma Co.) and goat anti-

rabbit-FITC, NS3 and NS5B, from Accurate Chemical and

Scientific Corp., Westbury, NY) for 1 h at room tempera-

ture. Finally, they were washed again in PBS and mounted

with coverslips after addition of Slowfade Light Antifade

Kit (Molecular Probes, Eugene, OR). Slides were examined

with an Olympus IX70 microscope equipped with an

epifluorescence attachment. Antibodies used in these experi-

ments were obtained as a gift from Michael Houghton,

Chiron Corporation, Emeryville, CA (E1, E2), anti-NS5B,

Dupont Merck Co., Delaware, and anti-core antibody was

purchased from Affinity Biochemicals, Denver, CO.

To examine whether Huh-7 cells after transfection with

full-length clone accurately processed structural and non-

structural proteins, lysates were prepared from transfected

cells after 72 h and subjected to Western blotting using a

protocol originally described by our laboratory (Myung et

al., 2001). Briefly, cells were lysed in a buffer containing

150 mM sodium chloride, 50 mM Tris–HCl, 1% NP-40,

0.5% deoxycholate, 0.1% SDS and protease inhibitors

(Protease Inhibitor Cocktail, Roche Molecular Biochemi-

cals). Fifty micrograms of the cell lysate was separated by

SDS-PAGE and transferred onto nitrocellulose membranes

(Amersham, Arlington Heights, IL). The membranes were

blocked with PBS containing 5% nonfat dried milk and

0.1% Tween-20 for 1 h at room temperature. Then, the

membrane was incubated with primary antibody at a 1:1000

dilution for 1 h and then washed three times with 0.1%

Tween-20 in PBS. Following this step, the membranes were

incubated with their respective peroxidase-labeled second-

Fig. 2. Inducible expression system utilized to replicate full-length HCV1a

clone in Huh-7 cells. The two-step procedure involves first infection of

Huh-7 cells with replication-defective adenovirus carrying gene for the T7

RNA polymerase followed by transfection with a transcription plasmid

containing the full-length infectious clone of HCV.

R. Prabhu et al. / Experimental and Molecular Pathology 76 (2004) 242–252 245

ary antibody (ECLWestern blotting analysis system, Amer-

sham Pharmacia Biotech UK, Amersham PLC, Bucking-

hamshire, England) at a dilution of 1:1000 for 1 h. After this

step, membranes were washed three times with PBS and

developed using ECL Chemiluminescence Detection Kit

(Amersham Pharmacia Biotech UK, Amersham PLC).

Detection of positive- and negative-strand HCV RNA in the

transfected Huh-7 cells

We examined levels of HCV genomic RNA (positive

strand) and replicative intermediate (negative strand) in

Huh-7 cells that were transfected with either wild-type

plasmid or mutant plasmid. Cells were harvested at 0 and

72 h after transfection by the treatment of trypsin-EDTA.

Total RNAwas isolated by the GITC method. RNA extracts

were treated with DNaseI (Roche Molecular Biochemicals)

5 U/Ag of RNA for 1 h at 37jC to remove any residual

plasmid DNA templates. Ribonuclease protection assay

(RPA) was performed to detect presence of HCV positive

and replicative negative-strand RNA in transfected Huh-7

cells (Ambion Inc., Austin, TX). A sense riboprobe targeted

to the highly conserved 5V UTR of HCV genome was

transcribed from a HindIII digested plasmid construct

(PCR II-296 containing nts 45–341 of HCV) by T7 RNA

polymerase in the presence of 32P-UTP. The probe length

was 422 nts, with 126 nts derived from the plasmid vector

PCR II (Invitrogen Corp.). The same plasmid was linearized

with XbaI and used to prepare an anti-sense RNA probe to

detect positive-strand RNA. For RPA assays, approximately

1 � 106 cpm of the labeled anti-sense probe was added to

25 Ag of RNA sample and vacuum dried. Hybridization was

performed in 10 Al of the hybridization buffer after dena-

turing for 3 min at 95jC and then overnight incubation at

45jC. RNase digestion was performed in 200 Al of RNasecocktail (1:100) (Ambion Inc.) in a buffer consisting of 10

mM Tris, pH 7.5, 5 mM EDTA and 0.3 M NaCl for 1 h at

37jC. Reactions were stopped by the addition of 2.5 Al of25% SDS and 1 Ag of proteinase K at 37jC for 15 min.

Samples were extracted with phenol/chloroform and precip-

itated with ethanol. The pellet was air dried and resuspended

in 15 Al of gel loading buffer. The samples were then boiled

for 3 min and separated on an 8% acrylamide/8 M urea gel.

The gel was dried and exposed to X-ray film (Kodak, X-

OMAT-AR). Since HCV is a positive-stranded RNA virus

which replicates by synthesis of genome length negative-

strand RNA, the detection of negative-strand RNA is an

indication of RNA synthesis from the transcribed HCV

genome inside transfected cells.

HCV1a replication is sensitive to IFN a-2b

Alpha interferon has been used as a potent antiviral agent

against hepatitis C virus. We tested potential antiviral effect

of IFN a-2b in this DNA transfection models in Huh-7 cells

by adding this drug immediately after transfection. At the

beginning, we examined whether IFN a-2b treatment could

have an effect on the expression of transgene from a

replication-defective adenovirus. For this experiment, we

used a replication-defective adenovirus that carries the gene

for green fluorescence protein (Adv-GFP) provided by Jay

Kohl, Louisiana State University. Huh-7 cells were infected

with Adv-GFP virus at a multiplicity of infection (MOI) of

10 and then treated with increasing concentration of IFN a-

2b (10–1000 IU/ml). Expression of GFP in Huh-7 cells was

recorded at different time intervals. At the second step, we

examined whether IFN a-2b has an effect on the transcrip-

tion of HCV genome by T7 RNA polymerase. Huh-7 cells

were transfected with pNIH-HCV1a-Rz plasmid using the

two-step procedures then treated with varying concentra-

tions of IFN a-2b (10–1000 IU/ml). After 72 h, total RNA

was isolated from the IFN-treated cells, digested with

DNase1 and the levels of HCV positive-strand RNA were

examined by RPA. At the third step, we determined whether

IFN a-2b could have an effect on negative-strand RNA

synthesis. Huh-7 cells were transfected with pNIH-HCV1a-

Rz plasmid using the two-step procedures and then treated

with different concentrations of IFN a-2b. After 72 h, total

RNA was isolated, digested with DNase1 and tested for the

presence of negative-strand HCV RNA by RPA. At the final

step of these investigations, we examined the effect of IFN

a-2b on the expression of viral protein from the transfected

HCV1a plasmid. Huh-7 cells were transfected with pNIH-

HCV1a-Rz plasmid then treated with IFN a-2b. Protein

lysates were prepared from HCV-transfected cells and

Fig. 3. Immunofluorescence assay demonstrating expression of core, E1, E2, NS3 and NS5B proteins in Huh-7 cells. Transfected cells after 72 h were stained

using a monoclonal antibody against core, E1, and E2 and stained with polyclonal antibody for NS3 and NS5B proteins. More than 60% cells showed strong

cytoplasmic staining. Mock-transfected cells and HCV-transfected cells stained with a nonspecific antibody were negative.

R. Prabhu et al. / Experimental and Molecular Pathology 76 (2004) 242–252246

Western blotting for HCV core protein was performed using

standard protocol described previously.

Results

Detection of HCV proteins in the transfected Huh-7 cells

Experimental details involving the virus infection and

DNA transfection is summarized in Fig. 2. The HCV

Fig. 7A

genome encodes a large polyprotein, which is processed

into 10 different mature proteins by the combined action of

viral and cellular proteases. We examined production of

selected viral proteins (core, E1, E2, NS3 and NS5B) in the

transfected Huh-7 cells by immunofluorescence and West-

ern blot analysis. We first examined whether Huh-7 cells

transfected with the infectious clone produces high levels of

HCV proteins. Immunofluorescence experiments were car-

ried out to determine what percentage of cells express HCV

after transfection. Results of these experiments using full-

.

Fig. 4. Western blot analysis showing the structural and nonstructural proteins are accurately processed in Huh-7 cells transfected with full-length and NS5B

mutant plasmid. Molecular weight standards in kilodaltons are shown on the left. (A) Mock-transfected control. (B) Detection of core protein. (C) Detection of

E1 protein. (D) Detection of E2 protein. (E) Detection of NS5B protein in the transfected Huh-7 cells (only full-length is positive since most of the NS5B is

deleted in the mutant clone).

R. Prabhu et al. / Experimental and Molecular Pathology 76 (2004) 242–252 247

length and mutant clones are presented in Fig. 3. Fairly high

expression of core, E1, E2, NS3 and NS5B proteins was

detected in more than 60% of Huh-7 cells transfected with

wild type (pNIH-HCV1a-Rz) as well as with mutant clone

(pNIH-HCV1a-Rz-NS5 mutant). Expression of HCV pro-

teins was localized in the cytoplasm of transfected Huh-7

cells. Expression of NS5B protein was seen only in wild

type but not in Huh-7 cells transfected with mutant clone

Fig. 5. Strand specificity of ribonuclease protection assay (RPA). Positive- and n

transcription reaction. Ten-fold serially diluted HCV RNAwere hybridized with eit

targeted to the 5V UTR (nts 45–341). RPA was performed using a commercially

hybridized to positive-strand HCV RNA. Bottom panel shows that positive-strand

can detect HCV as low as 1000 molecules.

because most of the NS5B coding sequences were deleted in

the mutant clone. No fluorescence was observed in control

mock cells, which were stained with the same HCV anti-

bodies. This staining is specific since no signal was seen

when these cells were stained with another monoclonal

antibody against the FLAG peptide (Eastman Kodak Co.,

Massachusetts). To confirm that the cellular and virus-

encoded enzymes accurately processed HCV polyproteins

egative-strand full-length HCV RNA transcripts were prepared by in vitro

her a gel-purified negative-strand RNA probe or positive-strand RNA probe

available RPA kit. Upper panel shows that minus-strand RNA probe only

RNA probe only hybridized to negative-strand HCV RNA. The RPA assay

R. Prabhu et al. / Experimental and Molecular Pathology 76 (2004) 242–252248

after translation, lysates were prepared from transfected

Huh-7 cells and tested by Western blot analysis. We were

able to demonstrate the presence of core, E1, E2 (structural)

and NS5B (nonstructural) proteins in the transfected Huh-7

cells by Western blot analysis. The result of Western blot

experiment is shown in Fig. 4. A single protein of 21 kDa

was identified using an anti-core antibody in Huh-7 cells

transfected with wild type as well as mutant clone (Fig. 4B).

Equal processing of E1 (31 kDa) and E2 (70 kDa) proteins

of HCV was observed in Huh-7 cells transfected with wild

type as well as mutant transcription plasmid (Figs. 4C and

D). A single band of approximately 68 kDa was detected

with an anti-NS5B specific rabbit polyclonal antibody only

in the wild-type clone which was absent in the NS5B mutant

plasmid transfected Huh-7 cells (Fig. 4E). Lysates of mock-

transfected cells did not show any reaction with antibodies

used for Western blot analysis. Taken together, the results of

these experiments suggested that Huh-7 cells transfected

with mutant as well as wild-type full-length clone express

high levels of HCV proteins in Huh-7 cells. To determine

the levels of HCV in the transfected Huh-7 cells, HCV RNA

levels (full-length as well as mutant) were quantitated by

competitive RT-PCR (Dash et al., 2000). Results of these

experiments suggested that HCV transcription in this model

was exponentially increased up to 48 h (1 � 107 copies per

microgram of total RNA) and slightly decreased at 72 h (data

not shown).

Detection of positive and negative-strand RNA by

ribonuclease protection assay (RPA)

We have previously described a RPA method to detect

negative-strand RNA in HCV-transfected cells. The RPAwas

Fig. 6. (Panel A) Detection of HCV positive-strand RNA in transfected Huh-7 cel

full-length HCV1a clone (pNIH-HCV1a-Rz) or NS5B deleted (pNIH-HCV1a-R

isolated at 0 and 72 h after transfection and digested with DNase1. RPA was perf

levels of HCV RNA transcripts were produced in Huh-7 cells transfected with fu

strand RNA in transfected Huh-7 cells. After 24 h after transfection, actinomycin D

the transfected cells, digested with DNaseI. RPA was performed using a sense RN

only in full-length transfected Huh-7 cells and no RNA was found in the NS5B

performed using a probe targeted to the highly conserved 5VUTR region (46–341). Since this part of HCV genome has

several stem-loop structures, it was important to examine

strand specificity of the RNA probes used in the RPA

experiment. To evaluate the strand specificity of anti-sense

RNA probe used in RPA assay, it was hybridized to in vitro

transcribed positive and negative-strand genome length

HCV RNA and RPA was performed. Results presented in

the top panel of Fig. 5 show that anti-sense RNA probe

hybridized only to positive-strand HCV RNA but not with

negative-strand HCV RNA. To evaluate strand specificity of

sense RNA probe, it was hybridized to in vitro transcribed

positive- and negative-strand genome length HCV RNA and

RPA was performed. Results presented in bottom panel of

Fig. 5 show that sense RNA probe hybridized only with in

vitro transcribed negative-strand HCV RNA but not with

positive-strand HCV RNA. The sensitivity of RPA assay was

determined to be at the range of 1000 copies per assay. RPA

assay for both positive- and negative-strand HCV RNA

detection appears to be equally sensitive. We concluded

from these experiments that the RPA method used here is

sensitive and specific. Initially, we determined whether high-

level transcription of HCV genome occurs in Huh-7 cells

transfected with wild type (pNIH-HCV1a-Rz) and mutant

clones. RNA extracts were prepared at 0 and 72 h after

transfection and RPA was performed using a gel-purified

negative-strand RNA probe. Results shown in Fig. 6 (left

panel) demonstrate positive-strand RNA was detected in

Huh-7 cells transfected with full-length and mutant plasmid

at 72 h, which was absent at 0 time after transfection.

Since HCV is a positive-strand RNA virus, detection of

negative-strand RNA in transfected cells demonstrates the

replication of the virus. We examined the presence of

ls. Huh-7 cells were transfected with transcription plasmid containing either

z-NS5BD) clone using a two-step transfection procedure. Total RNA was

ormed using an anti-sense RNA probe targeted to the 5V UTR region. High

ll-length and NS5B mutant plasmid. (Panel B) Detection of HCV negative-

treatment was performed for additional 48 h. Total RNAwas isolated from

A probe targeted to the 5V UTR region. Negative-strand RNA was present

deleted control.

R. Prabhu et al. / Experimental and Molecular Pathology 76 (2004) 242–252 249

negative-strand HCV RNA in Huh-7 cells transfected with

wild type and mutant clone by RPA using a gel-purified

sense RNA probe targeted to 5V UTR region of HCV. There

are reports suggesting T7 RNA polymerase could transcribe

complementary RNA strand to some extent and thus may

give false positive results in the detection of negative-strand

RNA in this model. It is also possible that RNA polymerase

from the host cell could transcribe HCV RNA from inte-

grated HCV plasmid DNA. Considering these possibilities,

we have treated HCV-transfected cells with actinomycin D

for 24 h to inhibit all DNA-dependent RNA polymerase

activities. After that, RNA extracts were prepared and

digested with DNaseI and were subjected to RPA for

negative-strand HCV RNA detection. Results of these

experiments with two different concentration of actinomy-

cin D are shown in Fig. 6 (right panel) and indicate that

negative-strand RNA was present only in Huh-7 cells trans-

fected with full-length HCV genome. These results suggest

that Huh-7 cells transfected with full-length plasmid repli-

cate and produce high levels of negative-strand HCV RNA.

IFN a-2b inhibits negative-strand RNA by directly blocking

protein synthesis

Studies in our laboratory as well as other laboratories

have shown that HCV replication is sensitive to IFN a-2b(Chung et al., 2001; Frese et al., 2001; Guo et al., 2001).

The replication model described here involves the expres-

sion of T7 RNA polymerase from a replication-defective

adenovirus, which drives the transcription of HCV genomic

RNA. Therefore, possibility of direct effect IFN a-2b on the

expression of transgene (GFP) from a replication-defective

adenovirus was examined. Results of these studies presented

Fig. 7. Antiviral effects of IFN a-2b in HCV-transfected cell culture model.

(Panel A) IFN a-2b does not inhibit expression of green fluorescence

protein from a replication-defective adenovirus. Huh-7 cells were infected

with a replication-defective adenovirus carrying the gene for green

fluorescence protein at MOI of 10 and then treated with varying

concentrations of IFN a-2b (10–1000 IU/ml). Expression of GFP was

recorded under a fluorescence microscope. Photographs were taken after 24

h. (Panel B) IFN a-2b does not inhibit HCV RNA transcription from full-

length transcription plasmid. Huh-7 cells were transfected with full-length

transcription plasmid (pNIH-HCV1a-Rz) immediately after transfection

cells were treated with increasing concentrations of IFN a-2b (10–1000 IU/

ml). After 72 h, total RNAwas isolated from IFN-treated cells and digested

with DNaseI. RPA for HCV positive-strand RNA was performed using an

anti-sense probe targeted to the 5V UTR region. (Panel C) IFN a-2b inhibits

negative-strand RNA synthesis in transfected Huh-7 cells. Huh-7 cells were

transfected with full-length transcription plasmid (pNIH-HCV1a-Rz) and

subsequently treated with increasing concentrations of IFN a-2b (10–1000

IU/ml). After 72 h, total RNA was isolated from IFN-treated cells and

digested with DNaseI. RPA for HCV negative-strand RNA was performed

using a sense probe targeted to the 5V UTR region. (Panel D) IFN a-2b

inhibits translation of HCV core protein from full-length infectious clone.

Huh-7 cells were transfected with pNIH-HCV1a-Rz clone and subsequently

treated with IFN a-2b. After 24 h, cell lysates were made from the IFN-

treated cells and an aliquot of protein lysates was used to detect HCV core

protein by Western blot analysis. Core protein expression was completely

inhibited at 10 IU/ml.

in Fig. 7A, indicating that expression of transgene (GFP)

from a replication-defective adenovirus is not inhibited by

IFN a-2b treatment. It is possible that IFN a-2b could have

inhibited at the level of transcription of HCV RNA in Huh-7

cells by T7 polymerase from adenovirus. To exclude this

possibility, level of intracellular HCV positive-strand RNA

in IFN a-2b-treated Huh-7 cells was examined by RPA

experiment. Results shown in Fig. 7B indicate transcription

of HCV RNA from the plasmid DNAwas not inhibited due

to IFN a-2b treatment. We examined whether IFN a-2btreatment could prevent negative-strand HCV RNA synthe-

sis in the transfected Huh-7 cells. Results of this experiment

presented in Fig. 7C suggest IFN a-2b at concentrations of

10–1000 IU/ml completely inhibit HCV negative-strand

RNA synthesis. This concentration range of interferon

treatment did not show any variation in the levels of

GAPDH mRNA in Huh-7 cells. Antiviral effect of IFN a-2b at the level of viral protein translation was examined by

measuring core protein expression by Western blot analysis.

R. Prabhu et al. / Experimental and Molecular Pathology 76 (2004) 242–252250

Results of these experiments shown in Fig. 7D indicate IFN

a-2b inhibits HCV core protein translation at a concentra-

tion of 10 IU/ml. To make sure that equal amounts of

protein samples were loaded in each lane, the same mem-

brane was reacted with beta-actin monoclonal antibody

(Sigma Co.) and it appears to remain unaltered.

Discussion

Development of HCV replication models is possible due

to the availability of chimpanzee infectious clones that have

been developed for 1a and 1b strains. In this study, we

demonstrated that Huh-7 cells transfected with pCV-H77C

clone (chimpanzee infectious clone for HCV1a) and NS5B

deleted clone expressed high levels of HCV structural and

nonstructural proteins. It is shown that high levels of HCV

genome were transcribed in Huh-7 cells, which was detected

by ribonuclease protection assay. The ribonuclease protec-

tion assay used here is strand specific, although it is targeted

to the 5V UTR region. To examine whether HCV1a strain

replicates in the transfected Huh-7 cell, first negative-strand

RNAwas detected by RPA method. To avoid the possibility

that negative-strand RNA could have been produced from

the transfected cells due to activity of T7 polymerase, cells

were treated with actinomycin D. Actinomycin D is known

to inhibit DNA-dependent RNA polymerase. Detection of

negative-strand RNA in the presence of actinomycin D,

thus, is an indication of HCV RNA synthesis. We could

detect negative-strand HCV RNA in Huh-7 cells transfected

with only the full-length plasmid and not with the NS5

mutant plasmid. This result was not due to hybridization of

our probe to plasmid HCV cDNA since the protected

fragment was not seen at zero time point. To exclude the

possibility that plasmid DNA used for transfection could

have been carried during RNA extraction-produced reaction

in the RPA assay, RNA extracts were digested with DNaseI

and tested by RPA. Detection of negative-strand RNA in

this model is not due to lack of strand-specific hybridization

because the RPA method used here is strand specific.

Previously, we have performed similar experiments and

showed that negative-strand RNA was detected in HepG2

cells transfected with HCV1b clone. Prior studies (Chung et

al., 2001) have used this clone and have used a vaccinia T7

virus. They have demonstrated interferon alpha inhibits

negative-strand RNA synthesis in a different cell line.

The applicability of this in vitro model system was tested

using IFN a-2b. IFN a-2b has been shown to inhibit sub-

genomic replicon and full-length replicon in stable cell lines

(Blight et al., 2000; Chung et al., 2001; Frese et al., 2001; Guo

et al., 2001). Therefore, we validated our replication model to

determine whether IFN a-2b inhibits replication of HCV1a

strain in this system. Results of this study along with other

reports suggest that replication of HCV1a and HCV1b strains

can be inhibited by interferon alpha. These results show the

applicability of this model for future molecular studies. We

have searched for an explanation how IFN a-2b inhibits

replication of HCV1a clone. The inhibition of HCV replica-

tion in this model could have occurred due to blockage at the

level of RNA synthesis or by direct blockage at the translation

level. Since this T7 RNA-based expression system generates

high levels of positive-strand RNA, it is unlikely that all the

T7 RNA transcripts go through the full replication cycle. It is

not sufficient enough to claim that inhibition of protein

translation could have occurred due to blockage at the level

of RNA replication or RNA synthesis. Therefore, we believe

that IFN a-2b directly inhibits translation of HCV, which

inhibits negative-strand RNA synthesis of HCV1a strain,

since the production of viral proteins are absolutely required

for negative-strand RNA synthesis. In separate experiments,

we showed that HCV RNA transcription in Huh-7 cells was

not affected by IFN a-2b. The stability of HCV RNA tran-

scripts also appears not be affected due to IFN treatment.

Furthermore, we showed that expression of GFP from a

replication-defective adenovirus was not altered in cells

treated with different concentrations of IFN a-2b. There werereports indicating that expression of adenovirus E1A protein

could modulate IFN antiviral activity. Expression of adeno-

virus early region E1B, E2A, E3 or E4 proteins in cultured

cells has no effect on the antiviral activity of interferon

(Ackrill et al., 1991; Anderson and Fennie, 1987; Goodbourn

et al., 2000; Gutch and Reich, 1991; Theresa and Look,

2001). The replication-defective adenovirus used in our

expression model specifically lacks the E1A gene. There

were reports suggesting VA RNA produced in adenovirus-

infected cells could antagonize the antiviral action of inter-

ferons (Mathew and Shenk, 1991). It is unknown whether VA

RNAs are produced in cells infected with a replication-

defective adenovirus. However, in our experiments, we found

IFN treatment clearly inhibits HCV negative-strand RNA and

core protein. Therefore, we have no reason to doubt that a

replication system including replication-defective adenovirus

blocked the antiviral activity of interferon alpha. In conclu-

sion, we established a model, which produces high levels of

HCV protein and negative-strand RNA of HCV1a genome.

Future studies can be designed to utilize similar approaches to

directly compare replication efficiencies among different

HCV strains currently available.

Acknowledgments

This work was supported by NIH Grant CA54576,

CA89121 and partial support from the Tulane Cancer Center.

The authors wish to acknowledge following investigators for

their contributions to this study: (i) Cesar Fermin, PhD, and

Centralized Tulane Imaging Center (CTIC) in the Pathology

Department for fluorescence images; (ii) Tatsuo Miyamura,

Department of Virology, National Institute of Infectious

Diseases, Tokyo, Japan, forAdexCAT7 virus; (iii) Jens Bukh

and Robert Purcell for providing the infectious clones used in

this project; (iv) Michael Houghton, Chiron Corporation,

R. Prabhu et al. / Experimental and Molecular Pathology 76 (2004) 242–252 251

Emeryville, CA, for providing HCV antibodies used in this

project; (v) Andrew Ball, University of Alabama at

Birmingham, for providing vector 2.0 plasmid; (vi) Michael

M C Lai, University of Southern California, for his

continuous support and advice in this study. The authors

gratefully acknowledge Professor Michael MC Lai, Univer-

sity of Southern California, for reviewing the manuscript

before submission.

References

Ackrill, A.M., Foster, G.R., Laxton, C.D., Flavell, D.M., Stark, G.R., Kerr,

I.M., 1991. Inhibition of the cellular response to interferons by prod-

ucts of the adenovirus type 5 EIA oncogene. Nucleic Acids Res. 19,

4387–4393.

Aoki, Y., Aizaki, H., Shimoike, T., Tani, H., Ishii, K., Saito, I., Matsuura,

Y., Miyamura, T., 1998. A human liver cell exhibits efficient translation

of HCV RNAs produced by a recombinant adenovirus expressing T7

RNA polymerase. Virology 250, 140–150.

Anderson, K.P., Fennie, E.H., 1987. Adenovirus early region 1A modula-

tion of interferon antiviral activity. J. Virol. 61, 787–795.

Bertolini, L., Iacovacci, S., Ponzetto, A., Gorini, G., Battaglia, M., Carloni,

G., 1993. The human bone-marrow-derived B cell line CE, susceptible

to hepatitis C virus infection. Res. Virol. 144, 281–285.

Blight, K.J., Kolykhalov, A.A., Rice, C.M., 2000. Efficient initiation of

HCV RNA replication in cell culture. Science 290, 1972–1974.

Blight, K.J., Mckeating, J.A., Marcotrigiano, J., Rice, C.M., 2003. Efficient

replication of hepatitis C virus genotype 1a RNAs in cell culture. J.

Virol. 77, 3181–3190.

Brown, E.A., Zhang, H., Ping, L.H., Lemon, S.M., 1992. Secondary struc-

ture of the 5V nontranslated regions of hepatitis C virus and pestivirus

genomic RNA. Nucleic Acids Res. 20, 5041–5045.

Bukh, J., Purcell, R.H., Miller, R.H., 1992. Sequence analysis of the 5Vnoncoding region of hepatitis C virus. Proc. Natl. Acad. Sci. U. S. A.

89, 4942–4946.

Choo, Q.-L., Kuo, G., Weiner, A.J., Overby, L.R., Bradley, D.W.,

Houghton, M., 1989. Isolation of a cDNA derived from a blood-borne

non-A, non-B viral hepatitis genome. Science 244, 359–362.

Chung, R.T., He, W., Saquib, A., Contreras, A.M., Xavier, R.J., Chawla,

A., Wang, T.C., Schmidt, E.V., 2001. Hepatitis C virus replication is

directly inhibited by IFN-alfa in a full-length binary expression system.

Proc. Natl. Acad. Sci. U. S. A. 98, 9847–9852.

Cribier, B., Schmitt, C., Bingen, A., Kirn, A., Keller, F., 1995. In vitro

infection of peripheral blood mononuclear cells by hepatitis C Virus. J.

Gen. Virol. 76, 2485–2491.

Dash, S., Hiramatsu, N., Gerber, M.A., 1997. Transfection of HepG2 cells

with infectious hepatitis C virus RNA genome. Am. J. Pathol 151,

363–373.

Dash, S., Saxena, R., Myung, J., Rege, T., Tsuji, H., Gaglio, P., Garry,

R.F., Thung, S.N., Gerber, M.A., 2000. HCV RNA levels in hepato-

cellular carcinoma and adjacent non-tumorous livers. J. Virol. Methods

90, 15–23.

Dash, S., Kalkeri, G., McClure, H.M., Garry, R.F., Clejan, S., Thung, S.N.,

Murthy, K.K., 2001. Transmission of HCV to a chimpanzee using virus

particles produced in an RNA transfected hepG2 cell culture. J. Med.

Virol. 65, 276–281.

Frese, M., Pietschmann, T., Moradpour, D., Haller, O., Bartenschalager,

R., 2001. Interferon alfa inhibits hepatitis C virus subgenomic RNA

replication by an MxA-independent pathways. J. Gen. Virol. 82,

723–733.

Friebe, P., Bartenschlager, R., 2002. Genetic analysis of sequences in the 3Vnontranslated region of hepatitis C virus that are important for RNA

replication. J. Virol. 76, 5326–5338.

Friebe, P., Lohmann, V., Krieger, N., Bartenschalager, R., 2001. Sequences

in the 5VUTR nontranslated region of hepatitis C virus required for RNA

replication. J. Virol. 75, 12047–12057.

Goodbourn, S., Didcock, L., Randall, R.E., 2000. Interferons: cell signal-

ling, immune modulation, antiviral response and virus countermeasures.

J. Gen. Virol. 81, 2341–2364.

Grakoui, A., Wychowski, C., Lin, C., Feinstone, S.M., Rice, C.M., 1993.

Expression and identification of hepatitis C virus polyprotein cleavage

products. J. Virol. 67, 1385–1395.

Guo, J., Bichko, V.V., Seeger, C., 2001. Effects of alfa interferon on the

hepatitis C virus replicon. J. Virol. 75, 8516–8523.

Gutch, M.J., Reich, N.C., 1991. Repression of the interferon signal trans-

duction pathways by the adenovirus E1A oncoprotein. Proc. Natl. Acad.

Sci. U. S. A. 88, 7913–7917.

Hijikata, M., Kato, N., Ootsuyama, Y., Nakagawa, M., Shimotohno, K.,

1991. Gene mapping of the putative structural region of the hepatitis

C virus genome by in vitro processing analysis. Proc. Natl. Acad. Sci.

U. S. A. 88, 5547–5551.

Houghton, M., Weiner, A., Han, J.H., Kuo, G., Choo, Q.-L., 1991.

Molecular biology of the hepatitis C viruses: implications for diag-

nosis, development and control of viral disease. Hepatology 14,

381–388.

Ito, T., Mukaigawa, J., Zuo, Y., Hirabayashi, Y., Mitamura, K., Yasui, K.,

1996. Cultivation of hepatitis C virus in primary hepatocyte culture

from patients with chronic hepatitis C results in release of high titre

infectious virus. J. Gen. Virol. 77, 1043–1054.

Kalkeri, G., Khalap, N., Garry, R.F., Fermin, C., Dash, S., 2001. Hepatitis

C viral proteins affect cell viability and membrane permeability. Exp.

Mol. Pathol 71, 194–208.

Kato, N., Nakazawa, T., Mizutani, T., Shimotohno, K., 1995. Suscepti-

bility of human T-lymphotropic virus type I infected cell line MT-2 to

hepatitis C virus infection. Biochem. Biophys. Res. Commun. 206,

863–869.

Kolykhalov, A.A., Feinstone, S., Rice, C.M., 1996. Identification of a

highly conserved sequence element at the 3V terminus of hepatitis C

virus genome, RNA. J. Virol. 70, 3363–3371.

Kolykhalov, A.A., Agapov, E.V., Blight, K.J., Mihalik, K., Feinstone, S.M.,

Rice, C.M., 1997. Transmission of hepatitis C by intrahepatic inocula-

tion with transcribed RNA. Science 277, 570–574.

Kolykhalov, A.A., Mihalik, K., Feinstone, S.M., Rice, C.M., 2000. Hepatitis

C virus encoded enzyme activities and conserved RNA elements in the 3Vnon-translated region are essential for virus replication in vivo. J. Virol.

74, 2046–2051.

Lanford, R.E., Sureau, C., Jacob, J.R., White, R., Fuerst, T.R., 1994.

Demonstration of in vitro infection of chimpanzee hepatocytes with

hepatitis C virus using strand-specific RT-PCR. Virology 202,

606–614.

Lin, C., Lindenbach, B.D., Pragai, B.M., McCourt, D.W., Rice, C.M., 1994.

Processing in the hepatitis C virus E2-NS2 region: identification of p7

and two distinct E2-specific products with different C termini. J. Virol.

68, 5063–5073.

Lohmann, V., Korner, F., Koch, J.O., Herian, U., Theilmann, L., Bar-

tenschlager, R., 1999. Replication of subgenomic hepatitis C virus

RNAs in a hepatoma cell line. Science 285, 110–113.

Major, M.E., Feinstone, S.M., 1997. The molecular virology of hepatitis C.

Hepatology 25, 1527–1538.

Mathew, M.B., Shenk, T., 1991. Adenovirus-associated RNA and transla-

tion control. J. Virol. 65, 5657–5662.

Miller, R.H., Purcell, R.H., 1990. Hepatitis C virus shares an amino acid

sequence similarity with pestiviruses and flaviviruses as well as mem-

bers of two plant virus supergroups. Proc. Natl. Acad. Sci. U. S. A. 87,

2057–2061.

Mizutani, T., Kato, N., Ikeda, M., Sugiyama, K., Shimotohno, K., 1996.

Long-term human T-cell culture system supporting hepatitis C virus

replication. Biochem. Biophys. Res. Commun. 227, 822–826.

Myung, J., Khalap, N., Kalkeri, G., Garry, R.F., Dash, S., 2001. Induc-

ible model to study negative strand RNA synthesis and assembly of

R. Prabhu et al. / Experimental and Molecular Pathology 76 (2004) 242–252252

hepatitis C virus from a full-length cDNA clone. J. Virol. Methods

94, 55–67.

Oh, J.W., Ito, T., Lai, M.C., 1999. A recombinant hepatitis C virus RNA

dependent RNA polymerase capable of copying the full-length viral

RNA. J. Virol. 73, 7694–7702.

Pattnaik, A.K., Ball, L.A., Legrone, A.W., Wertz, G.W., 1992. Infectious

defective interfering particles of VSV from transcripts of a cDNA clone.

Cell 69, 1011–1020.

Shimizu, Y.K., Iwamoto, A., Hijikata, M., Purcell, R.H., Yoshikura, H.,

1992. Evidence for in vitro replication of hepatitis C virus genome in a

human T-cell line. Proc. Natl. Acad. Sci. U. S. A. 89, 5477–5481.

Shimizu, Y.K., Purcell, R.H., Yoshikura, H., 1993. Correlation between the

infectivity of hepatitis C virus in vivo and its infectivity in vitro. Proc.

Natl. Acad. Sci. U. S. A. 90, 6037–6041.

Shimizu, Y.K., Feinstone, S.M., Kohara, M., Purcell, R.H., Yoshikura, H.,

1996. Hepatitis C virus: detection of intracellular virus particles by

electron microscopy. Hepatology 23, 205–209.

Tanaka, T., Kato, N., Cho, M.J., Shimotohno, K., 1995. A novel sequence

found at the 3V terminus of hepatitis C virus genome. Biochem. Bio-

phys. Res. Commun. 215, 744–749.

Tanaka, T., Kato, N., Cho, M.J., Sugiyama, K., Shimotohno, K., 1996.

Structure of the 3V terminus of the hepatitis C virus genome. J. Virol.

70, 3307–3312.

Theresa, D.J., Look, D.C., 2001. Specific inhibition of interferon signal

transduction pathways by adenoviral infection. J. Biol. Chem. 276,

47136–47142.

Tsukiyama-Kohara, K., Iizuka, N., Kohara, M., Nomoto, A., 1992. Inter-

nal ribosome entry site within hepatitis C virus RNA. J. Virol. 66,

1476–1483.

Yanagi, M., Rurcell, R.H., Emerson, S.U., Bukh, J., 1997. Transcripts from

a single full-length cDNA clone of hepatitis C virus are infectious when

directly transfected into the livers of a chimpanzee. Proc. Natl. Acad.

Sci. U. S. A. 94, 8738–8743.

Yanagi, M., Claire, M.S., Shapiro, M., Emerson, S.U., Purcell, R.H., Bukh,

J., 1998. Transcripts of a chimeric clone of hepatitis C virus genotype

1b are infectious in vivo. Virology 244, 161–172.

Yanagi, M., Claire, M.S., Emerson, S.U., Purcell, R.H., Bukh, J., 1999. In

vivo analysis of the 3V untranslated region of the hepatitis C virus after in

vitro mutagenesis of an infectious clone. Proc. Natl. Acad. Sci. U. S. A.

96, 2229–2291.

Yi, M., Lemon, S.M., 2003. 3V untranslated RNA signals required for

replication of hepatitis C virus RNA. J. Virol. 77, 3557–3568.

Yoo, B.J., Selby, M.J., Choe, J., Suh, B.S., Choi, S.H., Joh, J.S.,

Nuovo, G.J., Lee, H.S., Houghton, M., Han, J.H., 1995. Transfec-

tion of a differentiated human hepatoma cell line (Huh7) with in

vitro-transcribed hepatitis C virus (HCV) RNA and establishment of

a long-term culture persistently infected with HCV. J. Virol. 69,

32–38.