3.2. Mature miRNAs
Comparable to other RNA viruses previously studied, HCV H77 strain miRNAs can be located on either of the two arms in the secondary hairpin structure (see Table 1). Of the ten miRNAs identified in the antigenome of HCV H77 strain, four are located in the 5’-arm of the stem loop hairpin structure, while six are in the 3’-arm (Table 1).
3.3. Mature miRNAs Sequence Conservation among Different HCV Types and Sub-Types
HCV presents a high degree of genetic variability. The high error rate of RNA-dependent RNA polymerase and the pressure exerted by the host immune system, has driven the evolution of HCV into 7 different genotypes and a growing number of subtypes. In order to observe if mature miRNA sequences present in the predicted miRNAs found in the antigenome of HCV H77 strain are conserved among different HCV types and subtypes, we search for the similarity among that sequences and corresponding sequences of HCV strains included in a cu-
Table 1. Predicted hairpin and mature miRNAs sequences within HCV H77 strain antigenome.
aPositions relative to HCV strain H77 antigenome. bThe region of the HCV genome is indicated. cThe VMir score is shown. Mature miRNA sequences are shown in bold.
rated HCV sequence dataset that includes all types and subtypes. The results of these studies revealed that all mature miRNA sequences present in the 10 real pre-miRNA structures found in the antigenome of HCV H77 strain are conserved only among HCV sub-genotype 1a. Interestingly, mature miRNA sequences present in pre-miRNA-MD39 share 100% similarity among HCV strains belonging to genotypes 1a, 1b, 1c, 2a, 2c, 2k, 5, 6 and 7, and 95% similarity to genotype 2a, 3a, 3b, 3k and 4 (data not shown). This revealed a high degree of conservation of the mature miRNA sequences of this predicted miRNA among HCV strains. In order to observe if this pre-miRNA structure is conserved among HCV genotypes, the secondary structure of pre-miRNA-MD39 was predicted using corresponding sequences of pre-miRNA-MD39 of different HCV types and subtypes. The results of these studies are shown in Figure 3.
As it can be seen in the figure, very similar secondary structures are obtained using HCV strains of all types. This revealed that this structure is conserved among HCV genotypes.
3.4. Prediction of the Potential Targets for the Predicted miRNA in the Antigenome of HCV and Functional Annotation
To understand the dynamics between viral miRNAs and their targets is extremely important to understand the complexity of biological regulation and virus-host interaction. In silico prediction of miRNA targets provides a suitable approach for identifying potential target sites based on their complete or partial complementarity with the miRNAs. For these reasons, pairwise comparison of human 3’-UTR gene transcripts involved in six different metabolic pathways and miRNA-MD39 mature sequences were performed. We observed 17 transcript targets for the predicted HCV-miRNA-MD39 (Table 2). These transcripts are mostly involved in apoptosis as well as immune responses pathways of the host cell (Table 2).
Gene ontology is a useful tool for the mining of gene datasets and their functional annotations. In order to gain insight into these matters, we performed a functional enrichment analysis using KEGG pathway maps. A total of 38 genes were identified (Figure 4).
3.5. Effective Hybridization of Predicted HCV H77 miRNA and 3’UTR Gene Transcript Targets
In order to reconfirm effective hybridizations among the identified 3’UTR gene transcript targets and HCV- miR-MD39, we observed their hybridization patterns and calculated the minimum free energy of each hybridization. We have used a MFE of −14 kcal/mol as a cut-off value for potential miRNA pairing. The results of these studies are shown in Table 3. As it can be seen in the table, effective hybridizations were found in all cases included in this analysis.
miRNAs play critical roles in many biological processes, such as cell growth, tissue differentiation, cell proliferation, embryonic development, cell proliferation, and apoptosis. As a consequence, their deregulation perturbs gene expression and can have pathological consequences, as evidenced by their involvement in cancer  . Recent studies revealed that virus genomes from different virus families encode miRNAs. Viral miRNAs have been identified by both traditional cloning strategy from virus-infected cells and computational prediction  . In this study, using bioinformatics approaches, 10 pre-miRNAs structures with high VMir scores  and classified as real pre-miRNA using MiPred  were identified in the antigenome of HCV reference strain H77 (see Table 1). This is in agreement with recent results that identified novel miRNAs in the antigenome of yet another hepatic and cytoplasmaticRNA virus, i.e. Hepatitis A virus  . Moreover, recent studies carried out in Dengue virus revealed that all the predicted miRNAs were encountered in the reverse strand of the viral mRNA, which adds to the importance of the search of structured noncoding RNAs in replication intermediaries  .
Mature miRNA sequences of all these 10 potential miRNAs are conserved among HCV genotype 1a, to which H77 strain belongs. Nevertheless, only mature miRNA sequences of pre-miRNA-MD39 were found to be conserved when all HCV genotypes and sub-types are considered. This predicted miRNA maps at the 5’ UTR of HCV genome, which is the most conserved genomic region among HCV genotypes  (see also Table 1). More-
Figure 3. Predicted secondary structure of pre-miRNA-MD39 in different HCV genotypes. The secondary structures predicted for pre-miRNA-MD39 using corresponding sequences of different HCV genotypes and sub-types is shown. Bars at the bottom of the structures denote base pair probabilities. Only centroid structures are depicted.HCV strains are indicated by name and their genotypes are shown between parentheses next to strain name. Mature miRNA sequences are indicated by a square bracket next to each structure. The minimum free energy (MFE) found for the pre-miRNA structures were as follows: H77(1a) = −35.70 kcal/mol; HC-J4(1b) = −35.60 kcal/mol; HC-J6(2a) = −37.60 kcal/mol; K3A(3a) = −39.90 kcal/mol; ED43(4a) = −39.90 kcal/mol; EUH1480(5a) = −37.90 kcal/mol; EUHK2(6a) = −41.30 kcal/mol and QC69(7) = −35.70 kcal/mol.
Table 2. Predicted HCV H77 antigenome miRNA targets identified by in silico analysis.
Figure 4. Functional enrichment analysis of the predicted targets of HCV-miR-MD39. Metabolic pathways are shown in the left part of the figure. Bars are proportional to the number of gene/terms in the analysis. *means a p-value < 0.05, **means a p-value < 0.01.
over, predictions of the secondary structures of pre-miRNA-MD39 using corresponding sequences of all HCV genotypes and sub-types revealed suitable and similar structures (Figure 3). These studies revealed that this predicted miRNA is conserved among HCV genotypes and may add to the possibility of playing a role in HCV infection.
Recent studies revealed that altered expression of miRNAs is involved in the pathogenesis associated with HCV infection by controlling signaling pathways such as immune response, proliferation and apoptosis  . This is in agreement with the results found in this work, since the predicted targets found for HCV-miR-MD39 are members of metabolic pathways related to apoptosis and immune responses (see Table 2 and Figure 4). Moreover, effective hybridizations among the 3’UTR of target gene transcripts and HCV-miR-MD39 were observed in all cases (Table 3).
Taking all together, the results of this work revealed a candidate miRNAs that should be further confirmed by experimental analysis.
Table 3. Hybridization of predicted HCV H77 miRNA and 3’ UTR transcript gene targets identified by an in silico analysis.
aMFE, minimum free energy.
Figure 5. The structure of HCV reference strain H77 genomic sequences. The scheme shows the genome of HCV H77 strain. Numbers at the top of the scheme shows nucleotide positions. HCV genes are shown at the bottom. Untranslated regions are indicated at the 5’and 3’end of the genome. Red dot indicates location of the pre-miRNA-MD39 mature sequences.
Viral miRNAs can be extraordinary important to modulate cellular gene expression. Their small size, high specificity, and capacity for multiple transcript regulation suggest that they could play an important role in virus- host interactions during infection. Moreover, a more complex scenario can be seen when the search is extended to include viral replication intermediates.
By utilizing a series of bioinformatics tools, we identified a miRNA present in the antigenome of HCV H77 strain (see Figure 5). This miRNA maps in the 5’UTR region of the HCV genome and is found to be conserved among HCV genotypes and sub-types. In silico target prediction generated 17 cellular genes. These potential targets are involved in apoptosis as well as immune response pathways, suggesting that they could play a role in the pathogenesis caused by viral infection. Besides, the results of these studies revealed the presence of a viral miRNA in the negative-sense RNA strand used as a replication template for the HCV genome, as observed for other RNA viruses   . This in silico prediction is a useful guide to experimental design in order to achieve biological validation.
We acknowledge support by Agencia Nacional de Investigación e Innovación (ANII) through project PE_ ALI_ 2009_1_1603, Fondo María Viñas and PhD scholarship program and PEDECIBA, Uruguay.
Conflict of Interest
The authors declare that there is no conflict of interest regarding the publication of this paper.
Cite this paper
Juan Cristina,Ricardo Recarey, (2016) Identification of MicroRNA-Like Molecules Derived from the Antigenome RNA of Hepatitis C Virus: A Bioinformatics Approach. Natural Science,08,180-191. doi: 10.4236/ns.2016.84021
HCV: Hepatitis C Virus