Introduction

The interferons (IFN) are a family of proteins formally divided into types I and II. The former (including IFN and IFN), of which there are several isoforms, are produced by most cell types in response to virus infection and other stimuli. The latter (also called IFN) is mainly made by T lymphocytes and is important in activating certain T-cell and NK responses. Although IFN is an effective antiviral and antineoplastic agent, its therapeutic effectiveness has been limited by the high systemic toxicity of systemically administered recombinant IFN protein.

Recombinant human IFN (rhIFN) is less toxic, and is approved in the US for treatment of chronic hepatitis C virus (HCV) infection. In combination with ribavirin, rhIFN is administered intensively to patients with progressive liver damage due to chronic hepatitis C.¹ This approach is effective in controlling the progression of chronic HCV infection in slightly less than half of patients.² Among the reasons for treatment failure are the short half-life of rhIFN in the blood, the necessity of maintaining high blood levels of rhIFN in order to achieve therapeutic levels in the liver, unpleasant or toxic side effects, viral resistance to rhIFN that is associated with certain HCV genotypes, and the impermanence of the therapeutic effect in many cases if therapy is interrupted prematurely.^3,4

An approach to treatment in which cDNAs for IFNs and IFN are delivered directly to the liver, that is the therapeutic target, could theoretically magnify the local therapeutic effect without requiring high systemic levels or repeated administration of the toxic and short-lived proteins. Thus, gene delivery to the liver might potentially be an adjunct to current therapies. For such liver-directed gene therapy to be effective, a large number of hepatocytes should be transduced, the DNA delivered must remain in these cells indefinitely, and, if possible, expression of IFN should respond to the presence of the offending virus but otherwise be quiescent.

For these studies, we used recombinant gene delivery vectors derived from Tag-deleted simian virus-40 (rSV40 s). For gene delivery to the liver, rSV40 vectors have been shown to deliver enduring transgene expression to very high percentages of unselected hepatocytes, whether resting or dividing, in vitro or in vivo.^5,6

The goal of rendering transgene expression responsive to the presence of the offending HCV virus was approached by exploiting the reported ability of the HCV gene product NS5a to activate the cellular transcription factor NFB,⁷ as well as the known responsiveness of the promoter activity of the HIV-1 long terminal repeat (LTR) to NFB.⁸ Accordingly, we engineered rSV40 vectors to carry the HIVLTR as a promoter, to drive expression of murine and human IFN and IFN as transgenes. The effectiveness of HCV in activating these constructs to produce and secrete the several IFNs was tested, as was the ability of the IFNs so generated to activate paracrine signaling through IFN-signal response elements (ISRE) and the effect of IFN on detectable HCV transcript.

Results

HCV-induced expression of IFN delivered by SV[HIVLTR]IFN

Our strategy for these studies entailed transducing cells with SV[HIVLTR](IFN) vectors, then transfecting them with HCV cDNA to examine first the effectiveness of the transfected HCV cDNA in activating HIVLTR promoter activity. We then evaluated the effectiveness of the SV[HIVLTR](IFN) vectors in inhibiting HCV. These vectors were constructed as described previously⁹ and in Materials and methods. A map of a generic vector such as an SV[HIVLTR](IFN) vector, is shown in Figure 1a. The HCV cDNA-carrying plasmid used in these studies is shown in Figure 1b.

Figure 1.

(a) Genome map of a generic SV[HIVLTR](IFN). In all of these constructs, the HIV-1 LTR from HIV-1NL4-3 was used as a promoter to drive expression of the IFN cDNA in question. The LTR is positioned just 3' (counter-clockwise) to the SV40 early promoter (SV40-EP). Since it overlaps the SV40 origin of replication (ori), the SV40-EP must be retained intact in order to produce these vectors. To block transcription from it, four tandem polyadenylation signals (mpA) were inserted. The SV40 capsid genes (VP1, VP2, and VP3) and regulatory sequences are included in these vectors (enh=enhancer; ses=SV40 encapsidation signal), but are not expressed. (b) Structure of plasmid pRC/CMV-HCV, containing full genomic cDNA form of HCV, strain 1b, under the control of cytomegalovirus immediate-early promoter (CMV-IEP).

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This strategy relies upon the ability of transfected HCV cDNA to be transcribed upon transfection into liver cells. Normally, the HCV genome is transcribed as a single mRNA encoding a polyprotein that is subsequently post-translationally cleaved into its several substituents.¹⁰ We therefore transfected HepG2 cells with the HCV cDNA construct and tested for its transcription using nested RT-PCR, with both sets of PCR primers chosen to amplify the NS5a region of this transcript, as described in Materials and methods. We found that transfection of HCV cDNA results in its transcription, and documented that the observed PCR product was derived from the transcribed HCV mRNA (Figure 2). Sequence analysis of this RT-PCR product was performed (not shown), and documented that the amplicand was in fact the appropriate part of the coding sequence for NS5a.

Figure 2.

Detection of HCV transcripts by nested RT-PCR. HepG2 cells were transfected with pRC/CMV-HCV. Direct nested RT-PCR was performed on total RNA extracted from these cells 48 h post-transfection as described in Materials and methods. To ascertain the accuracy of these results, all samples were first treated with DNase; some were also treated with RNase before the reverse transcription step, as described in Materials and methods. Results are representative of two independent RT-PCR studies.

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In order to evaluate the potential therapeutic utility of rSV40-delivered, HIVLTR-driven IFN expression, we tested the time course of responsiveness to HCV transfection of IFN production and secretion by cells transduced with these constructs. Thus, HepG2 cells were transduced with SV[HIVLTR](IFN) constructs, then transiently transfected with HCV cDNA. IFN production was measured by ELISA in cell lysates and in culture supernatants. Although the specific IFN levels and kinetics of responsiveness to HCV varied among the different IFNs, transfection of the HCV genome into transduced cells significantly increased IFN, both intracellularly and secreted into culture media, as a function of time (shown in Figure 3 for mouse IFN) (CmIFN). The peak production and secretion of IFN induced by transient transfection with HCV cDNA occurred 2–3 days post-transfection.

Figure 3.

Effect of HCV on mIFN production and secretion. HepG2 cells were transduced with 100 MOI of SV[wtHIVLTR]mIFN, or with control vectors SV[muHIVLTR]mIFN or SV(BUGT), or mock-transduced. They were then transfected with HCV cDNA 4 days later. IFN protein concentrations were measured by ELISA 1, 2, 3, and 4 days post-transfection. (a) mIFN in cell lysates, expressed as pg/mg total protein. (b) mIFN in culture supernatants, expressed as pg/ml culture supernatant. Data represent means of independent experiments repeated a minimum of three times. Standard error bars are shown. For days 2 and 3, the differences between SV[wtHIVLTR](IFN)+HCV groups and all other groups were significant at the level of P<0.05 or better.

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HCV activation of HIVLTR occurs via NFB binding sites in the HIVLTR

It has been reported that HCV NS5a activates NFB, which, in turn, is known to activate the HIVLTR as a promoter.^7,8 In order to determine if NFB was involved in the activation of IFN expression demonstrated above, HepG2 cells were transduced with SV[wtHIVLTR](IFN) viruses, or with SV[muHIVLTR](IFN) vectors. Comparative production of IFNs was assayed by ELISA in culture supernatants and in cell lysates (shown in Figure 4 for hIFN). HCV activated wtHIVLTR to cause the cells to produce and secrete hIFN. Production and secretion of hIFN was comparable in cultures transduced with SV[wtHIVLTR](hIFN) but without transfected HCV cDNA, in cultures transduced with SV[muHIVLTR](hIFN) with or without transfected HCV cDNA, and in untransduced cultures. Cultures that had been treated with SV[wtHIVLTR](hIFN)+HCV cDNA consistently produced greater amounts of IFN, and secreted it at much higher levels than did any of the control groups: at the minimum P<0.05 for all comparisons between SV[wtHIVLTR](hIFN)+HCV and all other groups. These data are further illustrated by the time course studies shown in Figure 3, which include cells transduced with SV[muHIVLTR](hIFN) in comparison to the other treatment groups. These data also show that HCV did not activate significant hIFN secretion in cells receiving SV[muHIVLTR](IFN), in contrast to significant secretion by cells receiving SV[wtHIVLTR](IFN) in response to HCV.

Figure 4.

Activation of wtHIVLTR by HCV induces production and secretion of hIFN. HepG2 cells were transduced with 100 MOI of SV[wtHIVLTR]hIFN, or with control vectors SV[muHIVLTR]hIFN or SV(BUGT), or mock-transduced. They were then transfected with HCV cDNA 4 days later. HIFN protein concentrations were measured by ELISA 48 h post-transfection: in cell lysates (a), shown as pg IFN/mg total protein; and in culture supernatants (b) shown as pg IFN/ml culture supernatant. Data represent means of independent experiments repeated a minimum of three times. Standard error bars are shown. IFN production and secretion levels for SV[HIVLTR](IFN)+HCV groups were significantly different from all other groups (at the minimum P<0.05 for all such comparisons).

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Detection of IFN transcripts and proteins elicited by HCV in SV[wtHIVLTR](IFN)-transduced cells

The ELISA data described above were further assessed by Northern and Western analyses, respectively, of cell lysates and culture supernatants. Thus, HepG2 cells were transduced with SV[wtHIVLTR](IFN), HCV, or with SV[muHIVLTR](IFN), +HCV. Control cultures were transduced with SV(BUGT) (BUGT=human bilirubin-UDP-glucuronysyl-transferase) as a control rSV40 vector, or were mock-transduced. RNA from these cultures was harvested after transfection with HCV cDNA, electrophoresed, blotted, and hybridized with radiolabeled cDNA probes for the IFNs used under very stringent conditions (final wash: 0.1 SSC, 0.1% SDS, 55°C). Figure 5 shows representative results, using mIFN and mIFN as the transgenes; comparable results were obtained with hIFN and hIFN. Blots were reprobed for -actin as a loading control. The only cultures that demonstrated mIFN production by this assay were those that had been both transduced with SV[wtHIVLTR](IFN) and transfected with HCV cDNA. Other cultures were negative, save for small amounts of IFN mRNA in the group receiving SV[wtHIVLTR](IFN) without HCV challenge.

Figure 5.

HCV activation of HIV-1 LTR to yield mIFN and mIFN expression. HepG2 cells were transduced with the several SV[HIVLTR]IFN constructs shown, then transfected with HCV 4 days later, or mock-transfected. Total RNA was harvested from these cells 2 days after transfection, electrophoresed, blotted to nylon membranes, and then hybridized with the respective IFN cDNA probes, labeled with -32P-dCTP. Results are representative of three Northern blot analyses performed on three sets of cells transduced independently. They are also representative of comparable data seen with human IFN and IFN.

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Culture supernatants and cell lysates were also analyzed by Western blotting, using antisera specific for the individual IFNs. Figure 6 shows the representative studies from cultures transduced with rSV40 vectors carrying mIFN as a transgene. In both cell lysates and culture supernatants, the 21 kDa mIFN protein was mainly detected in cultures that had been both transduced with SV[wtHIVLTR](mIFN) and transfected with HCV cDNA. Slight intracellular IFN was observed in lysates from cells transduced with SV[wtHIVLTR](mIFN) but not challenged with HCV. The absence of detectable IFN in cultures transduced with SV[muHIVLTR](IFN) vectors underscores the importance of HCV activation of HIVLTR via NFB. However, the presence of cross-reactive proteins in all test groups may explain the apparent 'background' detection of 'IFN' in control groups in Figures 3 and 4 (and Figure 9).

Figure 6.

Production and secretion of mIFN, induced by HCV in SV[HIVLTR](mIFN)-transduced HepG2 cells. HepG2 cells were transduced with the SV[HIVLTR]IFN constructs shown, then transfected with HCV 4 days later. Total proteins from cell lysate or from medium were electrophoresed, immunoblotted, then probed with monoclonal antibodies against mouse IFN as described in Materials and methods. Following visualization of mouse IFN, blots of cell lysates were stripped and reprobed with antibody to -actin, as a loading control. These results are representative of two independent immunoblotting experiments carried out with these reagents, and of similar studies carried out with rSV40 vectors carrying cDNAs for the other IFNs studied.

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Figure 9.

Effect of HCV on mIFN production and secretion in PRH transduced with SV[HIVLTR]mIFN. Normal PRH cultures were treated with SV[HIVLTR](mIFN), then transfected with HCV cDNA 4 days later. At 2 days after transfection, cell lysates (a) and culture supernatants (b) were assayed for mIFN protein, expressed as pg IFN/mg protein (a) or as pg IFN/ml culture supernatant (b). Data represent means of independent experiments repeated a minimum of three times. Standard error bars are shown. IFN production (a) and secretion (b) for cells receiving SV[HIVLTR](IFN)+HCV were significantly different form all other groups: at the minimum, P<0.02 (a) and P<0.05 (b) for all such comparisons.

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Frequency of IFN-producing cells

The transduction efficiency with rSV40 vectors in unselected cultures is generally >98%.⁹ In order to determine the efficiency of induction of IFN expression in these cultures, HepG2 cells were transduced with SV[HIVLTR](IFN) vectors, then transfected with HCV. Control cultures were transduced, but not transfected, or were treated with a control rSV40 vector. Cells were then immunostained for the IFN whose cDNA was delivered to the cells. Approximately 10% of cultured cells demonstrably expressed the IFN proteins delivered (Figure 7a, b), that is transfection with HCV cDNA activated transduced IFN expression in approximately 10% of HepG2 cells.

Figure 7.

Assessment of transduction efficiency for hIFN and mIFN expression induced by HCV. (a) and (b) Immunochemical analysis. HepG2 cells were transduced with SV[wtHIVLTR]IFN or SV(BUGT), then transfected with HCV and 48 h later stained with mouse anti-hIFN, followed by ALEXA-conjugated rabbit anti-mouse IgG (a), or with mouse anti-mouse IFN, followed by ALEXA-conjugated rabbit anti-mouse IgG (b). Results are representative of two independent studies performed on two different sets of independently transduced and transfected cells. (c) Analysis by in situ RT-PCR. HepG2 cells were transfected with HCV. Direct in situ RT-PCR was performed 48 h post-transfection as described in Materials and methods. Results are representative of two independent in situ RT-PCR studies.

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Owing to the high transduction efficiency of rSV40 s, we attempted to correlate this percentage with the percentage of cells effectively transfected with HCV cDNA. For this, we used in situ RT-PCR to detect HCV transcript in cultures transfected with HCV cDNA. By this technique, approximately 10% of HepG2 cells in cultures transfected with HCV cDNA expressed HCV mRNA (Figure 7c).

We also asked if prior transduction with an rSV40 vector altered the transfection efficiency of transduced cells. That is, we asked whether the differences in IFN expression among the various study and control groups might represent differences in the success of the secondary transfection step that followed transduction. Accordingly, HepG2 cells were transduced with SV[wtHIVLTR](IFN), SV[muHIVLTR](IFN) or SV(BUGT), or mock-transduced. They were then transfected with pCMVluc, respectively carrying enhanced green fluorescent protein and luciferase as reporter genes. There was no significant difference in luciferase activity (Figure 8) between any of the experimental groups (P>0.1), indicating that transduction did not produce significant differences in transfection efficiency. These data were confirmed by quantitation of fluorescent cells on cotransfection with pT7egfp (data not shown).

Figure 8.

Assessment of HCV transfection efficiency in transduced cells. HepG2 cells were transduced with one of the several vectors indicated, then 2 days later were transfected with pCMVluc. Luciferase activity in cell lysates was measured thereafter, as described in Materials and methods, and is expressed as a percentage of the luciferase activity seen in cells that were mock-transduced and pCMVluc-transfected, s.e.m. from two independent experiments. All groups were statistically indistinguishable (P>0.1).

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Effectiveness of SV[HIVLTR](IFN) activation by HCV in primary hepatocytes

In the light of these results achieved using a hepatoblastoma cell line, we asked if SV[HIVLTR}(IFN) vectors would show comparable responsiveness to HCV in primary hepatocytes. Accordingly, PRH were cultured from normal rat livers, and transduced with the IFN-carrying vectors. Cultures were then 'challenged' by transfecting HCV cDNA, and intracellular and secreted IFN concentrations were measured by ELISA (shown in Figure 9 for mIFN).

Although levels of IFN made by primary hepatocytes in response to HCV were lower than were seen using HepG2 cells, similar patterns of responsiveness to HCV were evident. That is, liver cells that had been transduced with SV[wtHIVLTR](mIFN) and transfected with HCV produced and secreted more mIFN than did mock-treated cultures, cultures transduced similarly but not transfected with HIV, or cultures transduced with SV[muHIVLTR](mIFN). The relative magnitudes of these differences are comparable to those seen in the experiments described above. Differences between groups receiving SV[muHIVLTR](mIFN) +HCV and all other groups were highly significant: at the least, P<0.05 for all such comparisons.

The efficiency of transfection of these primary hepatocytes was about 5%, as observed using pCMV-eGFP plasmid (Figure 10a). Furthermore, the morphology of these cells was not appreciably altered by transduction+transfection (Figure 10b).

Figure 10.

Transfection efficiency and morphology of PRH. (a) PRH cells were transfected with pCMV-eGFP. Direct visualization was performed 48 h post-transfection. PRH before (upper picture) and after (lower picture) transduction with SV[wtHIVLTR]IFN and subsequent transfection with HCV cDNA. (b) Morphology of PRH cells before (above) and after (below) transduction with SV[wtHIVLTR](hIFN).

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Secreted, HIVLTR-driven IFN activates signaling in a paracrine fashion in untransduced cells

An important part of the antiviral activity of IFNs is their ability to activate cellular signaling pathways, which alter transcription of a number of genes. At the same time, since all hepatocytes in vivo would probably not be transduced with the IFN-carrying vector, it was important to establish whether IFN produced by cells that were both transduced with the SV[HIVLTR](IFN) vector and infected with HCV would secrete enough IFN to exert paracrine – possibly protective – effects on neighboring cells that might not have been transduced with the vector. That is, is there a possibility that SV[HIVLTR](IFN)-transduced, HCV-infected cells could protect neighboring, untransduced cells from HCV? Two different types of studies were used to test this possibility. In one series of studies, primary hepatocytes or HepG2 cells were transduced with SV[HIVLTR](mIFN), then challenged with transfected HCV DNA. In parallel, similar cells were transfected with pISRE-Luc, which carries the IFN-signal response element, driving the reporter gene luciferase. On the fourth day after HCV challenge, the pISRE-Luc-transfected cells were added to the cultures and the luciferase activity that was stimulated by the IFN made by the transduced cells was measured. In these studies (Figure 11a), luciferase activity in response to IFN made by cells that were transduced and challenged was substantially higher than in mock-transduced cultures. This increase was consistent and statistically significant (minimum significance for all such comparisons P<0.05).

Figure 11.

Paracrine IFN-induced biological responses in nontransduced cells. PRH (a) and HepG2 cells (b) were transduced with 100 MOI of SV[wtHIVLTR]mIFN, SV(BUGT), SV[muHIVLTR](mIFN), or mock-transduced. After 4 days, the cultures were transfected with HCV. In parallel, the same cell types were transfected with pISRE-Luc. (left panels) Supernatants from the transduced, HCV-transfected cell cultures were added to separate cultures of pISRE-Luc-transfected cells. Luciferase activity induced by IFNRE was measured on day 6 in a standard luminometric assay. (right panels) In coculture studies, pISRE-Luc-transfected cells were added to cultures of transduced, HCV-transfected cells. Luciferase activities were measured as indicated. Data represent means of independent experiments repeated a minimum of three times. Standard error bars are shown. Differences between SV[wtHIVLTR](IFN)+HCV and all other groups were significant at least at P<0.05 (a) and P<0.02 (b).

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An additional series of studies tested culture supernatants from transduced, HCV-challenged primary hepatocytes or HepG2 cells. These supernatants were added to separate cultures of pISRE-Luc-transfected cells of the same type. Again, similar patterns of paracrine activity were seen: supernatants from cells that were SV[HIVLTR](mIFN)-transduced and HCV-challenged induced much more luciferase activity than did supernatants from mock-transduced cells and consistently more luciferase activity than supernatants from cells that were transduced but not challenged (Figure 11b) (P<0.05).

SV[HIVLTR[(IFN)-delivered IFN production decreases HCV transcript

HCV does not easily replicate in cultured cells, so analysis of potential effectiveness of SV[HIVLTR](IFN)-delivered IFN production was approached using in situ RT-PCR analysis to visualize cells in which the transfected HCV genomes had produced transcripts. Thus, HepG2 cells were transduced with SV[wtHIVLTR](hIFN), or mock-transduced. They were then challenged with HCV by transfection of HCV cDNA, or mock-transfected. In situ RT-PCR was performed to visualize cells producing HCV mRNA. We found by in situ RT-PCR that numbers of cells positive for HCV transcript in cultures transduced with SV[wtHIVLTR](hIFN) and then challenged with HCV were much less than in cultures that had been mock-transduced and then transfected with HCV DNA (Figure 12).

Figure 12.

SV[HIVLTR](hIFN) decreases detectable HCV transcripts. HepG2 cells were transduced with SV(BUGT), or with SV[wtHIVLTR]hIFN at MOI=100, and 4 days later were transfected with HCV cDNA. One group was transfected with HCV cDNA and 24 h later treated with exogenous hIFN at 50 pg/ml. Cells expressing HCV transcripts were visualized by in situ RT-PCR, incorporating fluorescent nucleotides into the HCV RT-PCR product, as described in Materials and methods. These data are representative of two independent experiments.

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Discussion

The development of new strategies to manage chronic hepatitis C remains a major goal for the treatment of HCV-infected individuals. In this report, we describe a potential approach to HCV therapy using gene delivery to provide expression of IFN and IFN, in order to protect hepatocytes from HCV infection and replication.

IFN and IFN exert antiviral activities via several different mechanisms. One of their principal effects is to inhibit protein synthesis. Thus, IFN is produced in response to virus entry into the cell, possibly as a result of the activation of protein kinase R (PKR) by double-stranded viral RNAs (dsRNAs).^11,12,13 The expression of one IFN, induced in this fashion, may upregulate expression of other IFNs via receptor-mediated signaling pathways.^14,15 Viral dsRNAs also activate 5'-oligoadenylate synthetases (OAS), which in turn activate an enzyme that degrades 18S and 28S ribosomes, RNase L.¹⁶ Activated PKR phosphorylates and so inactivates eucaryotic translation initiation factor 2 (eIF2).^17,18 Both activation of RNase L and inactivation of eIF2 inhibit virus replication by decreasing production of viral (and cellular) proteins.

It has been reported that one or more signal transduction pathways, beginning with viral dsRNA, activate transcription of IFN and IFN, which in turn signal their abilities to inhibit virus infection and replication via a cognate receptor.^19,20,21,22 This receptor, on binding these type I IFNs, activates the Jak-STAT signaling cascade, to upregulate transcription of an array of cellular genes.^23,24,25 These IFNs have important effects on the immune system: they alter expression, activation, and localization of many cellular proteins, including MHC-I and -II, and TNF, that activate and execute antiviral T and NK cell responses.²⁶ IFN also stimulates Th1 responses to such Th1-tropic cytokines such as IL-12, IL-15, and IL-18, and mediates T-cell responses to IL-12-induced secretion of IFN.^26,27

IFN is generally made and secreted by activated T and NK lymphocytes.^28,29,30 It mediates many immune functions, and is important in activating cytotoxic responses against virus-infected cells. IFN also possesses antiviral activities that are mechanistically similar to those described for IFN and IFN. IFN receptor, like the receptor for type I IFNs, signals via the Jak-STAT pathway to activate transcription of an array of genes.^31,32 Also like type I IFNs, IFN activation of transcription may occur via transcription factor binding to ISRE.¹⁹

Owing to their potent effects on the immune system and on virus infections, as well as inhibitory effects on cell proliferation, IFNs have been tested as therapy for viral, neoplastic, and other diseases. rhIFN, tried in this mode, was far too toxic to be useful when administered systemically.³³ rhIFN is used effectively not only as therapy for viral illnesses such as hepatitis C, but also for several malignancies, particularly certain types of leukemia, myeloma, and some solid tumors that are otherwise resistant to drug therapies, such as malignant melanoma and renal cell carcinoma.³⁴ Although systemically administered rhIFN is less toxic than systemic rhIFN, treatment regimens using either have significant drawbacks in addition to toxicity. These include the need to maintain high blood concentrations of the recombinant protein in order to provide adequate therapeutic levels at disease sites, and uneven distribution of the proteins outside of the circulatory system.²⁰

Gene delivery could conceivably address these limitations, including the toxic side effects (especially of IFN) by allowing localized gene delivery to the liver, and by making IFN expression responsive to the presence of the offending virus. Thus we sought to provide the potential for permanent, HCV-responsive IFN expression at the site where the IFN is needed, without having to maintain comparable levels throughout the body. Gene delivery would be accomplished using rSV40 vectors, which are very efficient in permanently transducing hepatocytes in vitro and the liver in vivo.

We also report the use of HIV-1 LTR as an HCV-responsive promoter, to drive expression of IFNs.^6,35 The potential utility of HIVLTR as a promoter that could be activated by HCV was first tested using plasmid cotransfection with HCV cDNA+reporter plasmids incorporating HIVLTR driving either luciferase or chloramphenicol acetyl transferase. These studies (not shown) suggested that transducing with SV[HIVLTR](IFN), followed by transient transfection with HCV cDNA, might demonstrate effective gene delivery and IFN induction by HCV. Studies reported here repeatedly demonstrated that rSV40-delivered, HIVLTR-driven IFNs (IFN and IFN) are produced and secreted in response to the presence of HCV.

The incremental production and secretion of IFNs, as shown by ELISA, was generally 2- to 5-fold, compared to mock-transduced cells, or to cells that were transduced with SV[HIVLTR](IFN) but not transfected with HCV. Relative levels of IFN were comparable in all ELISAs, whether the transgenes were human or murine, and whether they were IFN or IFN. The levels of IFN (eg IFN) secreted in these studies are comparable to therapeutic levels for chronic HCV infection. The value of the ELISAs for these studies was their adaptation to testing the large numbers of samples generated, and their reproducibility and comparability to externally supplied quantitation standards. However, they carried the potential for detecting immunologically cross-reactive proteins besides the IFNs being tested. This possibility is underscored by the Western blotting studies, which demonstrated some cross-reactivity with other proteins. Our Northern analyses, which were performed under conditions of very high stringency, showed that virtually the only detectable IFN expression occurred in cells that had been both transduced with SV[wtHIVLTR](IFN) and challenged by transfection with HCV cDNA.

HIVLTR-driven gene expression is controlled by numerous cell regulatory proteins that interact with cis-acting sequences located in the HIV-1LTR. Among the multiple regulatory elements of the HIV-1 LTR, the B enhancer, which contains two copies of B elements at nucleotides 104–81, is considered the main inducible cis-acting element,³⁶ and therefore our study was mainly focused on NFB-mediated activation. Deletion or site-directed mutations of this regulatory sequence may affect LTR transcriptional activation induced by T-cell activation stimuli and by HIV Tat protein.^36,37,38 It has been widely demonstrated that the B enhancer element binds and responds to NFB/Rel family of transcription factors, which are induced by a number of stimuli such as mitogens, cytokines, and specific T-cell activators.³⁹ The core promoter region of the HIV-1 LTR also contains three tandem Sp1-binding sites located upstream of the TATA box. These Sp1-binding sites are necessary for basal and Tat-induced transcriptional activity.⁴⁰ Other transcription factors such as AP-1, or ATF-1 can activate HIV replication deleted in these Sp1 sites.^41,42

Members of the NF-AT family of transcription factors also bind an overlapping but distinct sequence at the B enhancer: NF-AT2 (also called NF-ATc) cooperates with NFB and Tat in HIV-1 LTR transcriptional activation.⁴³ These data may also help to explain why wtHIVLTR and muHIVLTR could be active in the absence of the direct NFB activator (HCV) in some situations.

The responsiveness of IFN to HCV was expected to reflect the ability of HCV NS5a to activate NFB,⁷ which in turn would upregulate HIVLTR as a promoter.⁸ We did not directly address the mechanism of HCV activation of NFB, but we found that mutating NFB binding sites in the HIVLTR eliminated HCV responsiveness in IFN production and secretion. Detectable IFN made by cells treated with SV[muHIVLTR](IFN)+HCV was comparable to levels seen in negative control cells and supernatants. Thus, these studies support the responsiveness of HIVLTR promoter activity to HCV, via NFB.

Although the transcriptional activity of the HIVLTR was strongly, consistently and significantly activated by HCV in all studies, some leakiness of the wt HIVLTR was observed. This may reflect HCV activation of other transcription factors, as indicated above. Thus, additional optimization of the HIVLTR as a promoter is desirable, and is underway, to avoid such low level increases in its HCV-independent transcriptional activation.

Although NFB has been shown to be activated by a large number of stimuli, our data suggest relatively specific responses to HCV. Transcriptional activation of the HIV-1 LTR via NFB has also been described for other viruses, such as herpes simplex virus type-1, cytomegalovirus, human T-cell leukemia virus, and human herpesvirus 8.^44,45,46,47 Therefore, in HIV carriers, disease progression may be directly influenced by coinfection with multiple viruses. Results presented here thus suggest that there may be complex interactions between HCV and HIV-1 replicative cycles, and that HCV may be a cofactor for HIV disease progression. Thus, the identification of HIVLTR as an HCV-responsive promoter is a potentially important advance in considering gene therapy of hepatitis C, but may also offer insight into the effects of the frequently observed coinfection of AIDS patients with HCV.

It should be noted in this context that SV[HIVLTR](IFN) strongly inhibits HIV.⁹ At the same time, since not all hepatocytes would be transduced by the rSV40 vector, it was important to understand whether IFNs produced by cells that were both transduced with rSV40 and infected with HCV were likely to protect cells that were not transduced, should the latter come into contact with HCV. Thus, we tested IFNs made by transduced and transfected cells for their ability to elicit detectable signaling via IFN receptors in naive neighboring cells. Levels of ISRE activation by IFNs produced by SV[HIVLTR](IFN)-transduced, HCV-transfected cells were consistently 25–50% greater than levels seen in control cells.

Since ISRE are activated by a variety of cellular transcription factors, many of which are not specific for IFN-signaled responses, it is not surprising that background ISRE activities seen in these studies were high. Thus, although such studies do not establish that naive cells would be protected, they suggest that the rSV40-delivered IFN response to HCV has paracrine effects on neighboring cells, via ISRE.

It is difficult to study HCV challenge in tissue culture (or, even, in experimental animals). For this reason, we delivered HCV to HepG2 human hepatoblastoma cells and to PRH by transfecting HCV cDNA. The effect we sought was production of HCV proteins, in particular NS5a. As attempts to demonstrate NS5a or other HCV proteins by immunostaining or Western blotting using commercial antibodies gave equivocal results (data not shown), we tested HCV transcription by in situ RT-PCR. Cells adherent to coated slides were used to visualize those cells in which HCV cDNA transfection elicited an HCV RNA. We were able determine that between 5 and 10% of cells in transfected cultures expressed HCV transcript detectably. In cultures transduced with SV[HIVLTR](IFN), and then transfected with HCV cDNA, the percentage of cells expressing the IFN protein as detected by immunostaining was comparable (5–10%). Thus, the transduction efficiency approached 100%, and the limiting factor in determining how many cells produced IFN was the transfection efficiency of HCV cDNA.

The amounts of IFN produced and secreted should be judged in this context: HepG2 cells secreted about 50 pg IFN/day and primary hepatocytes about 20 pg IFN/day. Lower production of IFN in primary cells may reflect differences in levels, or accessibility to activation, of NFB in transformed versus primary cells in response to HCV. Lower transfection efficiency for PRH than for HepG2 cells may also reflect the fact that about 20% of hepatocytes are naturally binucleate, which might affect transduction by rSV40 vectors.⁴⁸ The levels of IFN measured reflected IFN secretion by only 5–10% of cells in the culture – those cells that were also transfected with HCV. Had delivery of HCV been more effective, much more IFN secretion would probably have been observed.

Inhibition of HCV transcription was demonstrated by the semiquantitative approach of comparing the numbers of cells making detectable HCV transcript as visualized by in situ RT-PCR. These studies showed that the IFN delivered by SV[HIVLTR](mIFN), and elicited by HCV, decreased detectable HCV transcript in PRH. Although this is not a precise simulation of HCV infection in people, it provides a reasonable assessment of the efficacy of the IFN expressed in those hepatocytes in inhibiting HCV, since HCV does not replicate well in cultured cells.

Recombinant gene transfer vectors derived from SV40 virus (rSV40) are not subject to many of the problems that have limited gene delivery using other vector systems. rSV40s are made at a very high titers and infect – and so transduce – almost all nucleated cell types very efficiently, regardless of lineage or whether they are resting or dividing; they integrate and are not susceptible to transgene silencing.³⁵ They also elicit no detectable immune response by normal animals and so can be used to deliver multiple transgenes over time and in sequence.^49,50,51

The ability of rSV40 vectors to deliver long-term transgene expression to mostly quiescent liver cells has been illustrated in murine models, in vivo: immunochemical staining carried out 7 weeks after administration of SV(BUGT) showed that 60% of hepatocytes continued to express the BUGT transgene.⁶ Expression continued undiminished for 18 months.⁵¹

IFN has proven to be therapeutically efficacious in many cases of HCV infection. Our data suggest that SV[HIVLTR](IFN), perhaps in combination with SV[HIVLTR](IFN), may deliver IFN expression that responds to HCV and so may be useful in the treatment of HCV infection.

Materials and methods

Cell lines

The human hepatoblastoma cell line, HepG2, was pathogen and PPLO-free, and was maintained in Dulbecco's modified Eagle's medium (DMEM)+2 mM L-glutamine, 2 mM nonessential amino acids, 1 mM sodium pyruvate, streptomycin (100 g/ml), penicillin (200 U/ml), and 10%(v/v) bovine calf serum (NCS, Hyclone).

Freshly isolated PRH were grown in William's medium E, supplemented with 10% (v/v) bovine calf serum, streptomycin (100 g/ml), penicillin (200 U/ml), gentamicin (10 g/ml), 0.015 U insulin/ml, and 2 M dexamethasone.

All these cells were cultivated on rat type-1 collagen-treated plates.

COS-7 cells, used to package rSV40 vectors, were maintained in DMEM as described above.

Plasmids

A cDNA form of HCV, strain 1b, complete virus genome as pRC/CMV-HCV, was the kind gift of Dr Mark Feitelson (Department of Pathology, TJU) (Figure 1).

rSV40 vectors were made by cloning the transgene of interest (specific promoter) into a plasmid-carried modified SV40 genome.^52,53 The production of SV[wtHIVLTR](IFN) has been reported in the context of inhibition of HIV replication in lymphocytes.⁹ The other SV[HIVLTR](IFN) constructs were made similarly. Thus, the IFN cDNAs and the wt or mutant (doubly mutated at its NFB binding sites, so as to destroy NFB binding (muHIVLTR)) HIVLTRs were cloned into pT7() mpa. This plasmid carries an SV40 genome in which the Tag gene was replaced by a polylinker upstream of the SV40 polyadenylation site. Transcription from the SV40 early promoter (which overlaps the ori, and so cannot be deleted) is blocked by multiple tandem polyadenylation signals. All structures were verified by automated DNA sequencing (PE Applied Biosystems, Inc., Kimmel Cancer Center, TJU).

rSV40 vectors

Construction of recombinant SV40 derivative viruses (rSV40) for gene transfer has been described previously.⁵⁴ Briefly, rSV40 genomes were excised from the modified pT7blue (Novagen) carrier plasmid, gel purified, recircularized, and transfected into COS-7 cells. These cells supply all packaging functions in trans. Replication-incompetent vectors are isolated from COS-7 cell lysates, purified by ultracentrifugation and titered by in situ PCR, as described. Typical infectious titers for vectors prepared in this manner are between 10¹² and 2 10¹³ infectious units (IU)/ml.⁴¹ The rSV40 vectors used for these studies were named according to the promoter (wt or mutant HIVLTR, the IFN type and its species of origin). Thus, SV[wtHIVLTR](hIFN) carries wt HIVLTR+human IFN, the vector carrying mutant HIVLTR+mouse IFN is SV[muHIVLTR](mIFN), etc. Generically, these vectors are referred to as SV[HIVLTR]IFN (Figure 1). Control viruses for these studies have been reported: SV(HBS) and SV(BUGT), respectively carrying cDNAs for hepatitis B virus surface antigen (HBsAg) and human bilirubin-UDP-glucuronysyl-transferase.

Detection of HCV RNA by nested RT-PCR

Primers from the NS5A coding region of the HCV genome were selected for RT-PCR⁵⁵: 5'-TGGATGGAGTGCGGTTGCACAGGTA as the outer sense strand primer; 5'-TCTTTCTCCGTGGAGGTGGTATTGC as the outer antisense strand primer; 5'-CAGGTACGCTCCGGCGTGCA as the inner sense strand primer; 5'-GGGGCCTTGGTAGGTGGCAA as the inner antisense primer. The expected PCR products are 617 bp for the outer set and 571 bp for the inner set.

The specificity of the transfection with HCV was confirmed by nested PCR. The PCR was performed on the extracted RNA using the Rneasy Mini Kit (Qiagen, MA, USA) utilizing outer set of primers for the first round. The reaction mixture contained RT buffer (Invitrogen single step RT PCR kit) (50 mM Tris-HCl, 75 mM KCl, 1.5 mM MgCl₂) containing 10 mM dithiothreitol, 200 U of Superscript II Reverse transcriptase (Invitrogen), 40 U of RNase inhibitor (RNAsin; Pharmacia), 300 M each dGTP, dATP, dCTP, 5 U of platinum Taq polymerase (Invitrogene), and 0.2 M of each primer. The extracted RNA (5 l) was added to the mix. As a control, RNA was treated with 40 U of DNase (Promega) or 10 U of RNase One (Promega). The PCR was performed over 25 cycles, the first one consisting of 30 min at 58°C, followed by 25 cycles of 1 min at 94°C, 1 min at 60°C, and 1 min at 72°C, and finally 7 min at 72°C. The first round of product (5 l) was added to 45 l of PCR mix containing 1 buffer, 300 M each dNTP, 2.5 U of platinum Taq polymerase, and 0.2 M of inner set of primers. Thermocycling was performed over 25 cycles, each consisting of 1 min at 94°C, 1 min at 60°C, and 1 min at 72°C.

Detection of HCV RNA by in situ RT-PCR

HepG2 cells were spread on chamber, fixed in sterile saline, resuspended in ice-cold 10% buffered formaldehyde solution and kept for 2 h at 37°C and treated with Proteinase K at 37°C for 10 min, washed again and treated with 40 U of DNase (Invitrogen) for 4 h, at 37°C. As a control, cells were treated with 40 U of RNase. Permeabilized HepG2 cells were then washed in PBS and treated by RT buffer (Invitrogen single step RT PCR kit) (50 mM Tris-HCl, 75 mM KCl, 3 mM MgCl₂) containing 10 mM dithiothreitol, 200 U of Superscript II Reverse transcriptase (Invitrogene), 40 U of RNase inhibitor (RNAsin; Pharmacia), 1 M each dGTP, dATP, dCTP and 1 M of dUTP-FITC, platinum Taq polymerase and 50 pmol of the outer oligonucleotide primers of HCV. The tubes were incubated at 58°C for 45 min and at 94°C for 5 min to inactivate residual RT activity. The RT step was followed by first round PCR: 35 cycles of 1 min each at 94, 58 and 72°C, with the final extension done at 72°C for 10 min. At the end of RT-PCR, slides were incubated in PBS protected from light until the coverslips were removed. Slides were washed in 0.1 SSC at 37°C for 15 min, then analyzed under epifluorescence microscope for FITC emission.

IFN pathway reporter plasmid

To elucidate the activation of IFN-inducible Elements (ISRE) reporter plasmid ISRE-Luc, containing luciferase reporter gene driven by a basic promoter element (TATA box) plus five repeats of ISRE cis-enhancer element (Stratagene, La Jolla, CA, USA), was purchased.

All cloned plasmids were purified using the Qiaquick plasmid kit (Qiagen, Hilden, Germany). Nucleotide sequencing of constructed plasmids was confirmed by using an autosequencer (PE Applied Biosystems) and the standard dye termination.

Transduction and transfection experiments

For transduction with SV40-derived virus, HepG2 or PRH were treated once at an MOI of 100, as described. After 5 days, cells were transfected with 7.5 g HCV-cDNA-containing plasmid (Lipofectamine 2000, Invitrogen), according to the manufacturer's instructions, and then were cultured for 2–3 days.

Culture medium and cells were collected 48 h post-transfection. Cells were lysed on ice in 300 l of lysis buffer (50 mM TRIS, pH 7.4/150 mM NaCl/1% Nonidet P-40/1% sodium deoxycholate/1 protease inhibitor mix (Sigma)/0.5 mM sodium orthovanadate/0.02% sodium azide) for 5 min. Cell extracts was analyzed for luciferase activity and IFN expression.

Luciferase activity

Luciferase activity was measured using the Steady-Glo Luciferase Assay System (Promega), according to the manufacturer's instructions.

Determination of IFN by ELISA

Expression of all the IFNs was measured using their respective IFN ELISA kits (Antigenix America). This kit detects IFN using a sandwich immunoassay. In this assay, plates are coated with monoclonal antibodies against the particular IFN. Binding to that antibody was detected using an anti-secondary antibody conjugated to horseradish peroxidase (HRP). Tetramethyl-benzidine was used as a substrate to quantitate the antigen–antibody complex by subsequent color development.

Western blot analysis

10⁶ HepG2 cells either SV[HIVLTR]IFN-transduced, mock-transduced or expressing HBsAg or BUGT and subsequently transfected with HCV (if mentioned) were washed in PBS. Cells were lysed on ice in 300 l of lysis buffer (50 mM TRIS, pH7.4/150 mM NaCl/1% nonidet P-40/1% sodium deoxycholate/1 protease inhibitor mix (Sigma)/0.5 mM sodium orthovanadate/0.02% sodium azide) for 5 min. Cell culture medium was concentrated two times using Centricon-100 concentrators (Amicon, Bedford, MA, USA).

Equal amounts of protein were separated by 4–20% gradient Tris-glycine SDS/PAGE (Bio-Rad) and transferred to a PVDF-Plus membrane (Osmonics, Minnetonka, MN, USA). After overnight blocking (PBS (pH 7.4)/0.1% Tween-20 containing 5% nondry milk), blots were incubated with monoclonal anti-IFN antibody (Antigenix America) (1:400) in PBS (pH 7.4)/0.1% Tween-20 containing 5% nondry milk) for 1 h at room temperature. Goat anti-mouse IgG conjugated to HRP (Pierce) (1:10 000) was added, and blots were incubated for 1 h at room temperature. Each antibody incubation was followed by three extensive washes in PBS (pH 7.4)/0.1% Tween-20. Detection was by chemiluminescence (ECL Plus, Amersham) according to the manufacturer's instruction.

Northern blot analysis

Total RNA from 10⁶ HepG2 cells either SV[HIVLTR]IFN-transduced, mock- or SV-BUGT-transduced and subsequently transfected with HCV (if mentioned) was extracted using the RNA easy Mini Kit (Qiagen, CA, USA). Samples of 15 g of total RNA were electrophoresed in a 1% agarose/formaldehyde gel, transferred on a nylon filter (Nytran Super Charge, Schleicher and Schuell), UV cross-linked with a Stratalinker oven (Stratagene) and baked for 2 h at 80°C in a vacuum oven. Filters were prehybridized in 50% formamide, 5 SSPE, 20 mg/ml denatured salmon sperm DNA, 5 Denhardt's solution, 0.1% SDS at 42°C for 8 h, then hybridized under the same conditions with an IFN cDNA probe that had been labeled with ³²P-dCTP using a random priming labeling kit (Gibco BRL). Hybridization was performed at 42°C in 2 SSC overnight. After hybridization, filters were washed under high stringency conditions in 0.1 SSC, 0.1% SDS at 37–55°C for 30 min and signals were visualized using a phosphoimager (Molecular Dynamics Storm 840). To assess loading of various lanes, Northern blots were stripped and reprobed with a radiolabeled cDNA for human -actin.

Immunostaining

HepG2 cells (2 10⁵) HepG2 cells were grown on 12-well plates coated with collagen. At 5 days after transduction with either SV[HIVLTR]IFN or SV(BUGT) and subsequently transfection with HCV (if mentioned), cells were fixed with 2% paraformaldehyde for 1 h at room temperature, permeabilized with PBS/0.1%NP-40 for 10 min, blocked overnight at 4°C with the normal serum, corresponding to the animal source of the secondary antibody, and immunostained with anti-IFN antibodies for 1 h at 37°C. After extensive washes in PBS, corresponding FITC-conjugated secondary antibodies (donkey-anti-rabbit IgG; Molecular Probes) were used for 1 h at 37°C. Slides were extensively washed, mounted, and photographed at 400 magnification.

Statistical analysis

IFN levels and luciferase activity in HepG2 and PRH cells from various treatment groups and controls at various time points were compared by the Student's t-test or analysis of variance. Levels of statistical significance between the test group, on the one hand, and the several control groups, on the other, varied considerably. In each figure, the minimum level of statistical significance is mentioned (usually, P<0.05), but often comparisons between test and control groups typically reached much higher levels of significance (eg P<0.001 or better).

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Acknowledgements

We greatly appreciate the input of Drs J Roy Chowdhury, Pierre Cordelier, Mark Feitelson, Geetha Jayan, and Aleem Siddiqui. The assistance of Dr Jan Hoek's lab was helpful in establishing primary hepatocyte cultures. Dr Mark Feitelson (TJU) kindly supplied us with pRC/CMV-HCV; Dr Henry Wu (Immune Response Corp) provided us with the cDNA for hIFN 2b; Dr Janet S Butel (Baylor College of Medicine) gave us the plasmids carrying the original wtSV40 genome, from which rSV40 vectors were made. The current work was supported by NIH Grants AI41399, AI48244, and RR13156.