Male or female patients (n =19; 18-60 years) with chronic active hepatitis C virus infection were included in this study. Patients were negative for HBsAg and HBsAb but positive for anti-HCV and HCV-RNA by RT- PCR. All had elevated ALT and AST levels and received combined therapy of pegylated IFNα-2b (100 &mgr;g/wk) plus ribavirin (800-1000 mg/d). Patients had normal values for blood counts, other liver functions, auto immune markers, T3, T4 and TSH, renal functions, blood sugar and α-fetoprotein. None of the patients had other causes of liver disease (e.g. α1 antitrypsin deficiency, Wilson’s disease, alcoholic or decompensated liver disease, obesity-induced liver disease, drug-related liver disease), no CNS trauma, or active seizures, no ischemic cardiovascular disease within the last six months or hemochromatosis. None was co-infected with HBV or schistosomiasis.

RNA extraction and reverse transcription-PCR of HCV RNA, using Qiagen single step RT-PCR kit (Qiagen, Inc., Chatsworth, CA, USA), were performed as described previously[1314]. Amplification of 266 bp was performed in a single step using primer pair; forward (nt 47-68) 5'-GTGAGGAACTACTGTCTTCACG-3' and reverse (nt 292-312) 5'-ACTCGCAAGCACCCTATC AGG-3'. Cloning of amplified products was done with TA cloning kit (Invitrogen Co., Carlsbad, CA). Fifteen clones from each subject were sequenced using the TRUGENE HCV 5-NC genotyping kit, Visible Genetics, Inc. (Toronto, Ontario, Canada) in conjunction with the Open Gene DNA sequencing system. The insert DNA was sequenced by CLIP sequencing which allows both directions of the target amplicon to be sequenced simultaneously in the same tube using two different dye labeled primers (Cy5.0 and Cy5.5) for each reaction. This method provides sequence information for both positive and negative DNA strands from a single reaction. The obtained sequences were then aligned with genotype 4a prototype using the ClustalW program (http://www.ch.embnet.org/software/clustalw.html). Among the 15 domain III sequences obtained for each patient only one sequence was found in the majority of the clones, thus representing the major quasispecies of domain III in each patient. Sequence diversities in each major quasispecies were compared with genotype 4a and various mutation types were scored.

Prediction of stem-loop structure and thermodynamic stability of the major quasispecies in each patient was performed using the MFOLD 3.2 program with Turner energies and selected constraints on base pairing as indicated[15]. The program was run on EFN server: 1996-2008, Michael Zuker, Rensselaer Polytechnic Institute (http://mfold.bioinfo.rpi.edu/cgi-bin/rna-form1.cgi).

The mean number of domain III quasispecies in pre-treatment samples of SVR and BT states was about 2 quasispecies per patient with no significant difference between both groups. The quasispecies complexity of domain III tripled in post-treatment samples of BT patients. All patients included in the study were genotyped as 4a except for one SVR patient infected with a hybrid 4a/1b genotype.

Different types of mutations were compared in both study groups with the prototype sequence of HCV 4a (Table 1). Insertion mutations in domain III were statistically more prevalent in SVR than BT patients. This is expected since insertions are known to dramatically alter the stem-loop structure by changing the Watson-Crick base pairing of stems, thus reducing molecular stability of the RNA or binding affinity to ribosomal proteins. The percentage of transition mutations was statistically higher in pre-treatment samples of BT than SVR. There were no statistical differences in percentage of transversion mutations between the two patient groups.

Alignment of domain III sequences derived from study patients revealed the presence of 19 point mutations scattered in stem-loops IIIa, b, c and d (Table 1). Percentage distribution of the identified mutations in different loops of domain III in SVR and BT patients is presented in Table 2. The majority of mutations, 14/19 (73.7 %), were simple substitutions while 5 (26.3%) were insertions. Substitutions included 42.8% transitions and 57.2% transversions. Most of the mutations (84.2%) were localized in 2 stem-loops; IIIb (nt 175-205) and IIId (nt 254-275), with 42.1% of the mutations in each loop. The remaining 15.8% of mutations were located in junction IIIa, b, c (10.5%) and in loop IIIa (5.3%). The SVR patients contain specific mutations (Figure 1) that were not detected in either pre-treatment BT groups or post-treatment BT patients (Table 1). These SVR specific mutations comprise 42.1% (8/19) of the total number of mutations detected in the studied patient population (Table 1). Interestingly, approximately two thirds of SVR (5/8) specific mutations were located in loop IIIb and one third (3/8) in IIId with only one exceptional mutational event (nt 243 in junction joining loops IIIa, b, c, d) that was detected outside loops IIIb and IIId. These two stem-loop structures play critical roles in directing viral protein translation via recruiting and regulating ribosomal subunit proteins and cellular initiation factors for accurate positioning of HCV RNA in the translational machinery of the host cell. On the other hand, mutational events in pre-treatment samples of the BT patients were detected in almost all loop structures of domain III, mostly located in loop IIId. Most notably loop IIId mutations were associated with decreased ΔG value (Table 1) indicating increased thermodynamic stability of the RNA structure, thus explaining resistance to therapy in the BT patient group. It is noteworthy that most of loop IIIb mutations in SVR were associated with increased ΔG value, indicating decreased thermodynamic stability of the viral RNA, therefore contributing to the multifactorial eradication of viral RNA in the SVR patient group. When comparing the mutational events in pre-treatment BT patients with those observed post-treatment, the number of mutational events known to induce significant elevation in thermodynamic stability (i.e. decreased ΔG in loop IIId) did not increase after treatment in BT patients. These results suggest that domain III-associated factors of viral breakthrough are determined by genetic events in the HCV genome before start of treatment rather than being acquired as a result of stress induced by IFNα therapy.

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Figure 1 Predicted secondary and tertiary structure of HCV IRES[23] with mutations observed in SVR patients shown.

Changes in the minimum free energy (ΔG) were detected for stem-loops IIIa, b, c and d spanning nucleotides 141 to 282. Predictive stability values were compared for genotype 1b (strain H77), genotype 4a (I.D. Y11604.1) and study samples derived from SVR and BT patients. The ΔG value of genotype 1b was -53.6 kcal/mol; more stable than genotype 4a (ΔG -47.0 kcal/mol) as illustrated in the secondary structure/thermodynamic stability prediction (Figure 2 and Figure 3). As shown in Table 1, 31.5% of the mutations have no effect on the ΔG value, 31.5% of the mutations were associated with increased ΔG i.e. reduced stability of the secondary structure, while 37% of the mutations were associated with decreased ΔG value i.e. increased stability of the RNA secondary structure. An interesting observation (Table 1 and Figure 1) is that mutations with no effect on ΔG values were located in the apical part of domain III secondary structure (nt 186-243), spanning mostly the upper part of loop IIIb involved in eIF3 binding. On the other hand, those mutations affecting the stability parameters were localized in the basal part of domain III encompassing loops IIIa/b and IIId. Most notably, IIIa/b (nt 158-199) mutations were associated with reduced stability and IIId (nt 254-275) mutations were associated with increased stability of the RNA structure. Taken together, the data relating to minimum free energy in SVR and BT patients indicate that the levels of thermodynamic stability are not sufficient parameters to predict response to IFNα treatment and suggest that other parameters involving affinity of RNA binding to ribosomal subunits play important roles in determining response to treatment.

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Figure 2 RNA secondary structure and thermodynamic stability of loops IIIa, b, c, & d from genotype 1b. The minimum free energy value (ΔG) was -53.6 kcal/mol.

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Figure 3 RNA secondary structure and thermodynamic stability of loops IIIa, b, c, & d from genotype 4a (prototype). The minimum free energy value (ΔG) was -47.6 kcal/mol.

The hepatitis C virus (HCV) is a major public health problem with about 200 million individuals currently infected with the virus (about 3% of the world’s population). So far, 11 genotypes and more than 70 subtypes have been identified. The only approved FDA treatment for chronic HCV infection is the combination therapy of pegylated interferon and ribavarin. The variable response of patients to therapy ranges between 28% and 60% and has been proposed to be affected by various host and viral factors. Investigating the effect of mutations within the HCV 5’UTR, the most conserved region in the viral genome, on response to therapy is important because this region is vital for initiation of viral polyprotein translation and the ability of HCV to replicate.

Although interferon and ribavirin are the only FDA approved drugs to treat HCV, they suffer from several drawbacks including severe side effects (including hematological abnormalities and neuropsychiatric symptoms), very high cost, and most importantly low therapeutic response. Consequently, factors that affect the response of HCV patients to therapy have to be addressed and results could be used for predicting response to therapy before initiation of treatment. Moreover, identification of viral factors that correlate with therapeutic response would contribute to other studies on viral and host factors. This could result in a global view and comprehensive understanding of how host and viral factors affect a patient’s response to therapy.

Recent reports, using clinical specimens or HCV replicon systems from different genotypes, have highlighted the effect of mutations in different domains of the viral genome (in particular the HCV 5’UTR) on patients’ response to therapy. However, limited studies have been done on genotype 4a. In the present article, The authors focused mainly on genotype 4a which is the predominant genotype in Egypt; found in over 90% of all HCV-infected patients. They showed that the thermodynamic stability of the HCV 5’UTR region is different among responders (sustained viral clearance) and breakthrough patients (who suffer relapse at the end of treatment). Additionally, their results indicate that response to therapy is related mainly to the position of mutations but not their frequency. Finally, thermodynamic stability of IRES was shown to have a direct influence on the binding of the viral genome to the host proteins, which results in initiation of the translation of the viral polyprotein.

The results of this study suggest that the presence of single nucleotide polymorphisms (SNPs) in certain positions had direct effect on the response of HCV patients to interferon therapy. Taking into consideration the positions of these mutations, different real-time PCR or other assays can be developed for detection of the SNPs to allow the prediction of the response to interferon therapy as a step for identification of patients who are more likely to respond to therapy.

5’UTR: non-coding region of HCV RNA; contains the internal ribosomal entry site (IRES); site of initiation of translation. IRES: (internal ribosomal entry site): a structure within the HCV RNA 5’UTR that binds directly to the ribosome to initiate translation. Cap-dependent translation: the mechanism of translation (protein synthesis) predominantly used for cellular proteins. Cap-independent translation: translation via an internal ribosomal entry site (IRES); the mechanism utilized by HCV. Eukaryotic initiation factors: cellular proteins involved in translation. SVR: sustained viral response; patients who show negative HCV PCR results after termination of therapy.

The authors present a sequence analysis study of HCV genotype 4a patients undergoing combination therapy. This study is of a substantial potential interest. In this work the authors propose that insertion mutations in domain III of the IRES region are more prevalent in sustained viral response patients compared with breakthrough patients and that such mutations may affect the ability of HCV virus to replicate by decreasing the thermodynamic stability of its RNA.