Nada El-Ekiaby

Repressed induction of interferon-related microRNAs miR-146a and miR-155 in peripheral blood mononuclear cells infected with HCV genotype 4

MicroRNAs regulate the expression of many genes and subsequently control various cellular processes, such as the immune response to viral infections mediated by type I interferon (IFN). In this study, the expression pattern of two interferon‐related microRNAs, miR‐146a and miR‐155, was examined in healthy and HCV‐genotype‐4‐infected peripheral blood mononuclear cells (PBMCs) using qRT‐PCR. In contrast to other viral infections, the expression pattern was similar in both healthy and infected PBMCs. This could be attributed to attenuation of IFN pathway by HCV, which was assessed by investigating the expression of MxA, an interferon‐stimulated gene, that showed lower expression in HCV‐infected PBMCs. To determine the site of interference of HCV in the IFN pathway, expression of both microRNAs was examined following stimulation of PBMCs with IFN‐α2a, an activator of the JAK/STAT pathway as well as with imiquimod, a toll‐like receptor‐7 (TLR‐7) agonist that promotes interferon release. IFN stimulation induced the expression of miR‐146a and miR‐155 in HCV‐infected and healthy PBMCs. Stimulation with imiquimod led to a down‐regulation of both microRNAs in infected PBMCs, while it increased their expression in healthy PBMCs, indicating that HCV might interfere with miR‐146a and miR‐155 expression at sites upstream of interferon release, specifically in the TLR‐7 pathway. The pattern of expression of both miR‐146a and miR‐155 was very similar with a strong positive correlation, but showed no correlation to the patients’ clinical or histopathological parameters or response to treatment. In conclusion, HCV infection might repress the induction of miR‐146a and miR‐155 by interfering with TLR‐7 signaling.

Repressing PU.1 by miR-29a∗ in NK cells of HCV patients, diminishes its cytolytic effect on HCV infected cell models.

OBJECTIVES Natural killer cells are immune safeguards against HCV infection. PU.1 is a pivotal transcription factor in the development of NK cells. This study aimed at studying the regulatory effect of miRNAs on both development and function of NK cells isolated from HCV patients. METHODS NK cells were isolated from 17 chronic HCV patients and 12 healthy controls; after which miRNA and mRNA were quantified using qRT-PCR. Manipulating miRNA expression using mimics and antagomirs, was performed followed by investigating downstream targets as well as viral abundance. RESULTS PU.1 expression levels were upregulated in NK cells of HCV patients. In silico analysis revealed PU.1 to be a potential downstream target of miR-29a(∗), where miR-29a(∗) overexpression in NK cells caused a significant downregulation in PU.1 mRNA. Forcing miR-29a(∗) caused a downregulation of the cytotoxicity determinant NK activating receptor (NKG2D) via upregulation of miR-155. Moreover, perforin-1 mRNA was found to be downregulated upon forcing the expression of miR-29a(∗) in NK cells of HCV patients. This decrease in NK cytolytic function was accompanied by an 80% viral load increase in cocultured HCVcc cell models. CONCLUSIONS This study showed that HCV infection might abrogate NK cytotoxic potential through altering PU.1, NKG2D receptor and perforin molecules.

Contradicting roles of miR-182 in both NK cells and their host target hepatocytes in HCV.

BACKGROUND AND AIM Natural killer cells are part of the innate immunity involved in viral eradication and were shown to be greatly affected by HCV infection. Epigenetic regulation of NK cell function by microRNAs was not efficiently studied before and was never studied in HCV infection; therefore the aim of this study was to assess for the first time the role of microRNAs in regulating the function of NK cells of HCV-infected patients and hence viral replication in the target HCV-infected Huh7 cells. METHODOLOGY NK cells were isolated from PBMCs of HCV-infected patients as well as controls, and HCV-infected liver biopsies as well as Huh7 cells infected with the virus were used. For the infection of Huh7 cells, first viral vector was in-vitro transcribed into viral RNA that was then used to infect naїve Huh7 cells. Supernatant from the infected cells was then collected and used for further infection. For manipulation of NK cells or Huh7 cells, miR-182 mimics and inhibitors were transfected via lipofection method. RNA was extracted from each cell population, reverse transcribed. Gene expression as well as viral load was quantified using qPCR. RESULTS Screening of NKG2A and NKG2D between patients and controls showed no difference in expression of NKG2A, while NKG2D was found to be downregulated. In view of that, bioinformatics analysis was performed and showed that miR-182 has potential binding sites on both the inhibitory receptor NKG2A and the activating receptor NKG2D, and on its ligand ULBP2, as well as on the viral genome itself. In NK cells of HCV-infected patients, miR-182 was found to be over-expressed compared to controls; its ectopic expression was found to decrease NKG2D mRNA level, while miR-182 inhibitors were able to decrease NKG2A mRNA compared to untransfected cells. In addition, co-culturing genotype 4 or 2 HCV-infected Huh7 cells with miR-182 mimicked NK cells of HCV-infected patients showed decreased viral replication, suggesting an enhanced NK cell function. On the other hand, miR-182 and ULBP2 were both found to be downregulated in HCV liver tissues and HCV-infected Huh7 cells compared to their controls. miR-182 mimics were found to decrease ULBP2 mRNA and increase viral replication in genotypes 4 and 2 HCV-infected target (Huh7) cells compared to controls, while miR-182 inhibitor decreased viral replication in the cell models. CONCLUSION miR-182 was never investigated before, neither in HCV infection nor in NK cells, and we found it to have dysregulated expression in both liver tissues and NK cells of HCV-infected patients compared to control. In addition to that, miR-182 was found to have a contradicting effect in both effector cell and its HCV-infected target cell regarding HCV replication.

Epigenetic harnessing of HCV via modulating the lipid droplet-protein, TIP47, in HCV cell models

Bioinformatics analysis revealed that miR‐148a and miR‐30a potentially target TIP47. Expression profiling showed that both microRNAs were downregulated, while TIP47 was upregulated in liver biopsies of HCV‐infected patients. Forcing the expression of both microRNAs in JFH‐I infected, oleic acid‐treated Huh7 cells, significantly suppressed TIP47 expression and reduced cellular LDs with marked decrease in viral RNA. This study shows that miR‐148a and miR‐30a, regulate TIP47 expression and LDs in HCV infected cells.

miR-148a and miR-30a limit HCV-dependent suppression of the lipid droplet protein, ADRP, in HCV infected cell models.

Hepatitis C Virus (HCV) promotes lipid droplet (LD) formation and perturbs the expression of the LD associated PAT proteins ADRP and TIP47, to promote its own lifecycle. HCV enhances TIP47 and suppresses ADRP by displacing it from LD surface in infected cell models. We have previously shown that suppression of TIP47 by miR‐148a and miR‐30a decreased intracellular LDs and HCV RNA. Thus, this study aimed at examining whether this microRNA‐mediated suppression of HCV would limit HCV‐dependent displacement of ADRP from LDs. ADRP expression was examined in 21 HCV‐infected liver biopsies and 9 healthy donor liver tissues as well as in HCV‐infected Huh7 cells using qRT‐PCR. miR‐148a and miR‐30a expression was manipulated using specific oligos in JFH‐1 infected, oleic acid treated cells, to study their impact on ADRP expression using qRT‐PCR, and immunofluorescence microscopy. Intracellular HCV RNA was assessed using qRT‐PCR. ADRP is down regulated in patients as well as HCVcc‐JFH‐I infected cell models. Forcing the expression of both miRNAs induced ADRP on the mRNA and protein levels. This study shows that HCV suppresses hepatic ADRP expression in infected patients and cell lines. Forcing the expression of miR‐148a and miR‐30a limits the suppressive effect of HCV on ADRP. J. Med. Virol. 89:653–659, 2017.

Disruption of Claudin-1 Expression by miRNA-182 Alters the Susceptibility to Viral Infectivity in HCV Cell Models

HCV entry involves a complex interplay between viral and host molecules. During post-binding interactions, the viral E2 complexes with CD81 receptor for delivery to the tight junction proteins CLDN1 and OCLN, which aid in viral internalization. Targeting HCV entry receptors represents an appealing approach to inhibit viral infectivity. This study aimed at investigating the impact of targeting CLDN1 by microRNAs on HCV infectivity. miR-155 was previously shown to target the 3′UTR of CLDN1 mRNA. Therefore, miR-155 was used as a control in this study. In-silico analysis and luciferase reporter assay were utilized to identify potential targeting miRNAs. The impact of the identified miRNAs on CLDN1 mRNA and protein expression was examined by qRT-PCR, indirect immunofluorescence and western blotting, respectively. The role of the selected miRNAs on HCV infectivity was assessed by measuring the viral load following the ectopic expression of the selected miRNAs. miR-182 was identified in-silico and by experimental validation to target CLDN1. Both miR-155 and miR-182 inhibited CLDN1 mRNA and protein expression in infected Huh7 cells. Ectopic expression of miR-155 increased, while miR-182 reduced the viral load. In conclusion, despite repressing CLDN1, the impact of miR-155 and miR-182 on HCV infectivity is contradictory. Ectopic miR-182 expression is suggested as an upstream regulator of the entry factor CLDN1, harnessing HCV infection.

Correction to: Ectopic delivery of miR-200c diminishes hepatitis C virus infectivity through transcriptional and translational repression of Occludin

The author would like to correct the errors in the online published article.