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  Table of Contents  
Year : 2015  |  Volume : 33  |  Issue : 5  |  Page : 143-148

Spontaneous clearance of chronic hepatitis C infection is associated with an internal ribosomal entry site IIId stem loop structure variant

1 Department of Microbial Biotechnology , National Research Center, Tahrir, Dokki, Cairo, Egypt
2 Department of Informatics and Systems, National Research Center, Tahrir, Dokki, Cairo, Egypt

Date of Submission09-Nov-2013
Date of Acceptance07-Jul-2014
Date of Web Publication6-Feb-2015

Correspondence Address:
N G Bader El Din
Department of Microbial Biotechnology , National Research Center, Tahrir, Dokki, Cairo
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/0255-0857.148835

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 ~ Abstract 

Aim: To investigate if any mutations in hepatitis C virus (HCV) internal ribosome entry site (IRES) can inhibit the translation of viral polyprotein. Materials and Methods: A 26-year-old male patient infected with HCV 10 years ago was followed up. After 9 years of chronic infection. The patient had managed to resolve the infection for a period of 9 months, after which the patient experienced a viral recurrence characterized by high viral load and diverse HCV quasispecies. The IRES structures of the viral strains that disappeared were comparable with those that are currently active using structural mutational analysis. Results: A novo mutational position 254 combined with a rarely observed mutation at position 253 in the stem of the IIId subdomain were observed and the new conformation had an octa-apical loop (AGUGUUGG) and a shift in the 3 ` GU from the loop to the stem. Conclusions: These mutations were found to be highly deleterious, and they affected the direct binding of the IIId loop to the 40S ribosomal subunit with a subsequent inhibition of translation of viral polyprotein and clearance of the virus.

Keywords: Domian III, Hepatitis C virus, internal ribosome entry site, ribonucleic acid secondary structure, spontaneous viral clearance, Triple reverse transcription polymerase chain reaction

How to cite this article:
Bader El Din N G, El Hefnawy M M, Omran M H, Dawood R M, El Abd Y, Ibrahim M K, El Awady M K. Spontaneous clearance of chronic hepatitis C infection is associated with an internal ribosomal entry site IIId stem loop structure variant . Indian J Med Microbiol 2015;33, Suppl S1:143-8

How to cite this URL:
Bader El Din N G, El Hefnawy M M, Omran M H, Dawood R M, El Abd Y, Ibrahim M K, El Awady M K. Spontaneous clearance of chronic hepatitis C infection is associated with an internal ribosomal entry site IIId stem loop structure variant . Indian J Med Microbiol [serial online] 2015 [cited 2020 Aug 12];33, Suppl S1:143-8. Available from:

 ~ Introduction Top

Hepatitis C virus (HCV) internal ribosome entry site (IRES) is a highly structured region that gives the virus an advantage over the host cell mRNAs by selectively attracting a portion of eukaryotic initiation factors (eIFs) which recruits the ribosomal machinery to start translation. [1] Different levels of variability exist in the HCV IRES. Some are crucial and their role has been evolutionary while others are conserved in all HCV isolates. [2] Determining the IRES secondary structure is a significant step in identifying its mechanism of action. [3] Structured domains, known as stem-loops II, III, and IV, have been identified based on chemical and enzymatic probing as well as phylogenetic comparisons. [2],[4] It was shown that the HCV IRES folds into a unique three dimensional structure in which functionally important stem loops are exposed, and contained at least two independently folded regions corresponding to 40S and eukaryotic initiation factor-3 (eIF3) binding sites. [5] The correct secondary and tertiary IRES RNA folding is essential for correct binding to cellular proteins. RNA secondary structures have been shown to be very sensitive to mutations and any RNA structural re-conformations were shown to cause deleterious effects on function. [6]

Prevalence of HCV in Egypt exceeds 18% of its population and most infections are caused by genotype 4. Interest in the HCV IRES has thrived because of its essential role in translation, thus making it an attractive target for new antiviral agents. [7] This study attempts to investigate changes in the sequence of IRES IIId stem loop as one of the viral factors that may cause clearance of the HCV infection. The results showed a GC to AG double mutation at positions 253 and 254 within subdomain IIId which induced shuffling of the triplet GGG from the tip of the hexa-nucleotide loop to become partially unexposed in an octa-nucleotide loop which in turn prevents binding to 40S ribosomal subunit. Such mutation was associated with reduced core and E1 protein levels as well as minimal inflammatory activity which subsequently ended up with viral clearance. A viral recurrence was associated with reversion of the mutation to the prototype sequence and elevation of HCV RNA and structural proteins in patient's serum.

 ~ Case Report Top

The patient, who is a clinical laboratory technician, was 16-years-old when he was first tested positive for HCV Abs and HCV RNA by PCR in 2002. Since then, he didn't receive any antiviral therapy for HCV infection, and was diagnosed as a chronic hepatitis C virus patient. The patient had HCV genotype 4 and the viral RNA levels were consistently moderate during the first 9 years of chronic HCV infection ranging from 145,000-290,000 copies/mL (205,000 ± 37,000 copies/mL). Histopathological examination of ultrasound guided liver needle biopsy showed minimal inflammation (A1) and no fibrosis (F0). Ultrasound hepatic investigating did not show any abnormality. Twenty-five patients suffering from chronic hepatitis C were used as positive controls. All patients had elevated ALT 83.8 ± 45.2 IU/mL and AST levels 63.1 ± 26.6 IU/mL. HCV RNA concentrations ranged 738,000 ± 433,000 copies/mL. Histopathological findings of HCV patients were consistent with chronic active hepatitis (A1-A4) and (F1-F3).

After 9 years of chronic infection the patient had managed to resolve the infection completely for a period of 9 months only then the patient experienced a viral recurrence characterized by high viral load and diverse HCV quasispecies. During the 9 month period of viral resolution, HCV +ve (genomic) strand was assessed concomitantly in the patient's serum and in peripheral blood mononuclear cells along with the amplification of the minus (antigenomic) strand in PBMC in a RT-PCR triple test format. [8] The results of the triple test showed a progressive clearance during this period [Table 1]. After these 9 months of viral clearance the patient suffered a viral recurrence and his viral load increased to 1.5 million copies/mL. Both the HCV core and E1 protein levels detected by enzyme linked immunosorbent assay (ELISA) were significantly low in our patient (2.9 and 2.8 O.D.490 nm; respectively) as compared to levels detected in 25 active chronic HCV patients used as positive controls (>4 O.D. 490 nm, respectively) and 25 normal subjects as negative controls (<4 O.D. 490 nm). The core and E1 levels remained within the range of normal subjects during the 9 month period of viral clearance. However, the patient's core and E1 levels have been shifted to 5 O.D. units (490 nm) concomitantly with the beginning of viral recurrence which signifies a much more active HCV infection. The patient was not exposed to any invasive surgical or dental maneuvers and did not receive any blood transfusion during or after the 9 month period of viral clearance. However, this may not exclude infection with wild type HCV by other routes.
Table 1: Results of the triple RT‑PCR Assay during the 9 month period of aviremia

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Total cellular RNA was extracted and reverse transcribed as previously described [8] and the obtained DNA fragment was cloned using cloning kit (Promega, Madison, WI). Seventeen recombinant plasmids were purified and sequenced using the Big Dye terminator method (ABI Foster City, CA). BLAST pair wise alignment was done using the NCBI MEGABLAST and the ClustalW program ( Pair wise alignment against HCV 4a HEMA 51 and multiple alignment of the 5'UTR nucleotide sequence derived from the patient's HCV RNA during the first 9 years of infection against reference sequences of HCV genotypes using BIOEDIT program revealed major similarity with few differences in sub domains IIId and IIIe [Figure 1]. The consensus sequence for all REFSEQ HCV sequences plus our sequence was calculated. Conservation plot revealed overall homology, with few mutations relative to the 4a subtype [Figure 2]. The sequence mutations were investigated against other genotype sequences, after which a double mutation in the IIId subdomain was found to be unique for HCV genome. A BLAST search using the NCBI website tool for our patient's IIId subdomain revealed that a novo mutation at position 254 (in reference to HCV H77 prototype) was not reported before, with another rare mutation at position 253. Determination of suboptimal structures was also done in order to assert more confidence in our analysis. Using RNAFold software (Vienna package) subdomain IIId was found to have an altered secondary structure for the important apical loop from the control 4a. HEMA 51, thus inducing a partial shift of the important triplet GGG from the apical loop towards the stem [Figure 3]a and b]. A transition mutation in subdomain IIIe didn't affect the stem loop structure (data not shown). A similar observation was found for the other optimal structure at 50% sub optimality. Folding using the MFold package, RNA mute, and RDMASS also gave similar results (data not shown). Surprisingly sequence analysis of 5'UTR from the patient's HCV RNA obtained after viral recurrence revealed reversion of the dinucleotide mutation at positions 253 and 254 from AG to the wild type GC. Mutational analysis using recently developed RNA mutational analysis server (RDMASS) indicated the importance of nucleotides 253 and 254 for the stability of the structure as shown in [Figure 4].
Figure 1: Multiple alignments of HCV REFSEQ sequences against our sequence (HCV 4HZ). The 5`UTR region from our patient was cloned and sequenced using standard procedures as described in Materials and Methods. The 4a.Eg.HZ sequence was aligned against the HCV REFSEQ sequences using BIOEDIT program (Hall, 1998). Subdomain IIId here starts from nucleotide 280. Positions 281 and 282 are the locations of the detected dinucleotide mutation

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Figure 2: Part of conservation plot of consensus sequence against all REFSEQ sequences including our sequence (HCV 4a.EG.HZ). The sequence of the IIId subdomain with the dinucleotide mutation uniquely found in our sequence is shown

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Figure 3: HCV subdomain IIId folded using the RNAFOLD software from the Vienna package. (a) HCV HEMA.51 subdomain IIId folded using the RNAFOLD from the Vienna RNA folding server. The apical hexanucleotide loop and the stem before it are correctly predicted, while the internal SRL loop which constituted a tertiary interaction was not predicted. (b) HCV subdomain IIId of HCV-4a.Eg.HZ folded using the RNAFOLD (Vienna RNA folding server). The apical loop is different from the prototype structure and the triplet GGG which have direct contacts to the 40S ribosomal subunit have shifted to the right with the last G becoming part of the stem. Using the MFOLD server and suboptimal folding at 50% gave very similar results

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Figure 4: Deleteriousness profile for subdomain IIId as performed on the RDMAS server. The figure shows the importance of the first two bases (indicated by an arrow) for the stability of the structure of subdomain IIId. Minimum free energy was chosen as the deleteriousness index here, and similar analysis were also done using other topological indices (data not shown)

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 ~ Discussion Top

The 5'UTR was shown to have a quasispecies distribution with minor modification in its sequence and that these changes lead to dramatic variation in translational activity. [9] It was reported that an insertion has lead to enhanced translational activity up to seven folds. [10] It has been repeatedly shown that translational activity is dependent on genotype. Also, translational activities of IRES are dependent on cell types, thus emphasizing the fact that both cis- and trans-acting factors can affect the catabolite activating protein (CAP) independent translation from this region. Domain IIId has been receiving more attention because of its importance in binding to both host and viral proteins. [11] A model in which 40S and the core proteins compete for binding to this domain has been proposed. It has direct binding contacts with the 40S ribosomal subunit via the triplet GGG located in its apical loop. [12],[13] Our logic for the clearance of the HCV infection after 9 years goes as follows: The dinucleotide mutation disrupted the double stranded Watson-Crick pairing of the stem of the IIId sub domain. This is thought to have deleterious effects on translation because of changes to the secondary structure of the helical region. [2],[11] The structure of the apical loop has changed because of this dinucleotide mutation, with the original triplet GGG shifting to the right so that the last GU pair has become part of the stem [Figure 3]b] which reduced the binding affinity to the 40S subunit and caused a gradual decrease in the ability of the virus to effectively translate its proteins and eventual clearance of viremia. Because structure integrity of this loop is crucial for initiation of translation, [2] we conclude that such a change inhibited translation, and thus enabled our patient to gradually clear the first infection as this was the major clone that he initially harbored. Backing up this conclusion is the fact that during the viral recurrence 5'UTR sequence didn't have these mutations, structural protein levels were higher than before, and ALT and AST levels have almost doubled. Our data support a structure-to-function interpretation of the translational misfunction caused by re-conformation of the IIId subdomain. This agrees with the findings of Odreman-Macchioli et al., [6] who concluded that correct folding of the secondary and tertiary elements of the IRES subdomains are critical for efficient binding of subdomain IIId to the 40S subunit and subdomain IIIb to eIF3. Also, our finding are similar to those of El Awady et al., [11] who demonstrated that domain III mutations played important roles in IRES translation activity which inducing significant changes in the course of response to IFN.

Previous studies on the epidemiology of HCV in Egypt indicated a higher HCV clearance rate (50%) as compared to the global rate of viral clearance (~25%). This is the first study on Egyptian viral strains that could help to elucidate some of the viral factors that may be responsible for viral clearance. This case report showed how a genotype variation in an important stem loop of the IRES can induce a direct phenotype change exemplified in the clearance of the virus from the patient. Such critical sequences may be used as therapeutic targets to abolish HCV replication using RNA silencing and/or antisense RNA. Further studies on patients who cleared the infection and analysis of the IRES region is required to assert the conclusion of this report, along with experimental verification of the changes in secondary structure of the IRES's subdomains.

 ~ References Top

Gallego J, Varani G. The hepatitis C virus internal ribosome-entry site: A new target for antiviral research. Biochem Soc Trans 2002;30:140-5.  Back to cited text no. 1
Jubin R, Vantuno NE, Kieft JS, Murray MG, Doudna JA, Lau JY, et al. Hepatitis C virus internal ribosome entry site (IRES) stem loop IIId contains a phylogenetically conserved GGG triplet essential for translation and IRES folding. J Virol 2000;74:10430-7.  Back to cited text no. 2
Wang C, Sarnow P, Siddiqui A. Translation of human hepatitis C virus RNA in cultured cells is mediated by an internal ribosome-binding mechanism. J Virol 1993;67:3338-44.  Back to cited text no. 3
Thurner C, Witwer C, Hofacker IL, Stadler PF. Conserved RNA secondary structures in Flaviviridae genomes. J Gen Virol 2004;85:1113-24.  Back to cited text no. 4
Collier AJ, Gallego J, Klinck R, Cole PT, Harris SJ, Harrison GP, et al. A conserved RNA structure within the HCV IRES eIF3-binding site. Nat Struct Biol 2002;9:375-80.  Back to cited text no. 5
Odreman-Macchioli FE, Tisminetzky SG, Zotti M, Baralle FE, Buratti E. Influence of correct secondary and tertiary RNA folding on the binding of cellular factors to the HCV IRES. Nucleic Acids Res 2000;28:875-85.  Back to cited text no. 6
Gallego J, Varani G.The hepatitis C virus internal ribosome-entry site: A new target for antiviral research. Biochem Soc Trans 2002;30:140-5.  Back to cited text no. 7
El-Awady MK, Ismail SM, El-Sagheer M, Sabour YA, Amr KS, Zaki EA. Assay for hepatitis C virus in peripheral blood mononuclear cells enhances sensitivity of diagnosis and monitoring of HCV-associated hepatitis. Clin Chim Acta 1999;283:1-14.  Back to cited text no. 8
Sergi C, Arnold JC, Rau W, Otto HF, Hofmann WJ. Single nucleotide insertion in the 5'-untranslated region of hepatitis C virus with clearance of the viral RNA in a liver transplant recipient during acute hepatitis B virus superinfection. Liver 2002;22:79-82.  Back to cited text no. 9
Zhang J, Yamada O, Ito T, Akiyama M, Hashimoto Y, Yoshida H, et al. A single nucleotide insertion in the 5'- untranslated region of hepatitis C virus leads to enhanced cap-independent translation. Virology 1999;261:263-70.  Back to cited text no. 10
El Awady MK, Azzazy HM, Fahmy AM, Shawky SM, Badreldin NG, Yossef SS, et al. Positional effect of stem loop III mutations in HCV genotype 4a IRES on initial response of chronic patients to interferon α2a plus ribavirin combined therapy World J Gastroenterol 2009;15:1480-6.  Back to cited text no. 11
Lukavsky PJ, Otto GA, Lancaster AM, Sarnow P, Puglisi JD. Structures of two RNA domains essential for hepatitis C virus internal ribosome entry site function. Nat Struct Biol 2000;7:1105-10.  Back to cited text no. 12
Doshi KJ, Cannone JJ, Cobaugh CW, Gutell RR. Evaluation of the suitability of free-energy minimization using nearest-neighbor energy parameters for RNA secondary structure prediction. BMC Bioinformatics 2004;5:105.  Back to cited text no. 13


  [Figure 1], [Figure 2], [Figure 3], [Figure 4]

  [Table 1]


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