The current strategies for treatment of HCV liver disease are not yet satisfactory to the majority of HCV patients. Sustained viral response to interferon α
2 plus ribavirin combined therapy has been successful for only 10% among Egyptian HCV patients who are predominantly infected with genotype 4 [
10]. Data on the use of pegylated interferon in Egyptian patients infected with this HCV genotype have not yet been completed. Moreover, combination therapy has significant side effects and is poorly tolerated by individuals who are affected by other diseases, and the overall chances for a cure are less than 50%. Thus the development of alternative antiviral therapies is of paramount interest to many investigators and clinicians who are dealing with this devastating disease in Egypt. Unlike nonspecific antiviral treatment with interferon-α and ribavirin, target specific antiviral therapy would directly block viral replication and prevent continuing infection of liver. These potential therapies include nucleoside analogues [
14], bridged nucleic acids (BNA) [
15], inhibitors of viral proteases, helicases and polymerases [
16‐
18], and antisense phosphorothioate oligodeoxynucleotides (S-ODN). The latest therapeutic option i.e. S-ODN has received much attention from several investigators around the world [
8,
11]. However, as alluded to earlier, the lack of a reliable cell culture system allowing persistent in vitro virus propagation is still hampering screening of antiviral activity of these molecules and the development of effective therapies. Much of the struggle against HCV is caused by its genetically heterogeneous nature and the existence of quasispecies. Quasispecies are distinct but closely related variants of the virus and circulate in the infected individuals. This viral heterogeneity results from high error rate of NS5B gene-coded RNA-dependent RNA polymerase Because the liver is the main target for replication of HCV in vivo, the majority of cell types used for HCV replication in vitro were of a human hepatocyte origin; including human hepatoma, HuH7 [
19]; hepatoblastoma, HepG2 [
19,
20], fetal hepatocytes [
21,
22] or fused primary human hepatocytes with hepatoblastoma cells [
22]. In the present study, the reasons why we utilized HepG2 cells for HCV replication in vitro experiments are attributed to their similarities to primary human hepatocytes in their biosynthetic pathways. An additional advantage of HepG2 cells is the presence of a 66 K Da receptor protein for S-ODN that was purified from HepG2 cell membrane [
23], thus allowing reasonable uptake and cytosolic transfer of S-ODN in these cells. There have been several approaches for testing the efficacy of antiviral agents on HCV replication. Transfection of subgenomic viral cDNA fragments that were linked to reporter genes such as the firefly luciferase gene and expressing non structural and structural proteins in various expression systems have been reported [
8,
11,
24]. These viral constructs were not permissible for HCV replication. Alternatively, full-length cDNA clones were constructed from positive stranded viral RNA genomes and were found infectious to cells [
25,
26]. The viral RNA, produced presumably via transcription of transfected cDNA, is expected to be inactivated due to splicing and polyadenylation processes similar to all nuclear transcripts. Furthermore, a major problem that makes this approach suboptimal for the present study is its structural limitations in terms of the correct length and sequences at 3' and 5' ends of RNA molecules. The later comprises the components of the IRES, which is the main target for S-ODN in this study. The IRES is a highly structured RNA element that directs cap-independent translation of the HCV polyprotein from the 5' end of the plus strand RNA. Although the minus strand 3'-terminal region has the antisequence of the 5'-end of the plus strand, it doesn't fold into its mirror image [
27]. Several laboratories have shown that the 3'-terminal sequences of either strand RNA contributes essential biological functions for viral replication [
27‐
29]. We, therefore, hypothesized that the use of viral constructs from cDNA or viral RNA in transfection experiments will deprive the viral replication machinery from the action of host cellular factors, like polypyrimidine-tract binding protein, PTB [
30,
31] that was found to bind a cis acting element at the 3'end of HCV for viral replication. In the present study, an alternative in vitro system to test S-ODN antiviral function was made by utilization of a well defined HCV inoculum from positive serum in infection experiments to HepG2 cells. Native viral RNA genomes containing the IRES components at the 5' end for efficient translation of viral polyprotein precursor and intact PTB binding elements at the 3' end of the HCV genome for efficient viral replication were expected to provide a fairly natural intracellular system for HCV proliferation. Several reports have shown that the infection experiments in a variety of cells in culture were associated with transient viral replication and minimal viral yield, the in vitro system we describe here has been associated with moderate viral load ~10
4 viral copies per 10
6 HepG2 cells and prolonged viral replication as well as core and E1 expression for up to 130 days. Furthermore, culture media of these infected cells were found to be highly infectious to naïve cells (results not shown), indicating successful shedding out of infectious viral particles in cell surroundings.
Wide variability in the inhibitory potency of antisense S-ODN targeted against several viral sequences has been reported in a variety of in-vitro systems. The reasons for the limited success with the use of S-ODN against specific stem loop structures, particularly those constituting the IRES elements, is that some of these stem loops form a very stable secondary structures, so that the target motifs for S-ODN contain up to 75% paired RNA nucleotides [
8,
32] which may interfere with the inhibitory effect of S-ODN. Furthermore, the biological significance of certain stem loops in HCV translation is still not well understood. Site directed mutagenesis of stem loop1 has been previously shown that sequence conservation within this region is not essential for IRES activity [
14]. In contrast, stem loop IIId (264–282) was shown to contain the conserved sequence, GGG triplet, which is essential for proper IRES folding [
33] and viral translation from the AUG start site located at a distance. In the present study S-ODN structures were designed against the two phylogenetically conserved regions; the region comprising the AUG start codon (S-ODN1) and stem loop IIId (S-ODN2). The sequence data from local isolates revealed conservation at specific motifs related to proper folding and efficient translation i.e. IIId GGG (nucleotide 266–268) and AUG start codon (nucleotide 340–342) respectively. These data offer an advantage for antisense drugs to be a therapeutic option for most known genotypes of HCV. Earlier studies showed that IRES motifs were efficient targets for S-ODNs on constructs containing the 5'-UTR alone or with subgenomic fragments of the virus linked to luciferase reporter in either cell-free system [
8] or HepG2 cells [
8,
11,
24]. Our results using cells infected with native viral genome proved to be very sensitive for testing S-ODN inhibitory activity on viral translation/replication. The antisense S-ODN1 and S-ODN2 completely inhibited viral replication at concentrations as low as 1 μM, whereas the use of subgenomic construct in reticulocyte lysate showed inhibition of translation at > 4 μM concentration of the same S-ODN structures [
8] The present in-vitro system is advantageous in the sense that the use of higher concentration of S-ODN tend to be nonspecific for translation inhibition. The reason why the use of cell culture provides more sensitivity for S-ODN concentrations than cell-free systems in this and in another study (8) is related to the triggering of the intracellular RNAse H activity by the readily formed RNA-DNA hybrid between viral 5'-UTR RNA and S-ODN DNA molecules, a mechanism that facilitates elimination of viral RNA by RNAse H degradation.
Recent studies focused on the use of RNA interference (RNAi) as a new strategy against HCV showed similar success to the antisense oligodeoxynucleotides treatment in inhibiting viral replication in cell culture [
34,
35]. However for use of RNAi strategy in human patients several major issues have to be addressed. These include poor stability of dsRNA in circulation, dsRNA-induced interferon response resulting in shutting down general protein synthesis, off-target effects of dsRNA, and because of the exquisite sensitivity of RNAi strategy, generation of resistant viruses (escape viruses) due to a single nucleotide change in the target region [
36]. Thus, we believe that antisense strategy is more promising in combating HCV.
In summary, the results described in the present in-vitro system indicated that S-ODN1 has relatively more inhibitory potency than S-ODN2, a finding that supports earlier reports [
30]. The results described herein also demonstrate the establishment of an in vitro model for the replication of HCV Type 4; a major accomplishment in studying HCV which may facilitates the development of anti HCV therapeutics. Finally, the results also provide evidence that antisense phosphorothioate oligonucleotides targeting stem loop IIId and AUG translation initiation site are effective inhibitors for viral replication and represent potential prototype for treatment of HCV type 4 in liver pathology. Future direction will make use of enhanced delivery strategy of the antisense oligodeoxynucleotides by conjugation to arginine-rich peptides [
37]. We have successfully used such an approach to show specific inhibition of growth factor (EGF) as well as phorbol ester-mediated activation of MAP kinase and its phosphorylation of the transcription factor ELK in the nucleus [
38]. Such approach may enhance the efficacy of the antisense strategy in mediating the inhibition of HCV replication and thus eliminating the HCV as a dreadful disease that is devastating the Egyptian population.