Demonstration of the Presence of the “Deleted” MIR122 Gene in HepG2 Cells

Hamad, Ibrahim A. Y.; Fei, Yue; Kalea, Anastasia Z.; Yin, Dan; Smith, Andrew J.; Palmen, Jutta; Humphries, Steve E.; Talmud, Philippa J.; Walker, Ann P.

doi:10.1371/journal.pone.0122471

Cited by 6 publications

(4 citation statements)

References 34 publications

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“…It has been reported that HepG2 cells lack expression of the liver-specific microRNA miR122, necessary for HCV replication. However, a MIR122 gene is present in HepG2 cells, suggesting that under some conditions it may be expressed (28). Our two clones, selected for their polarization properties, could be infected by HCV, but infectivities were lower than for Huh-7 cells.…”

Section: Discussionmentioning

confidence: 93%

Entry and Release of Hepatitis C Virus in Polarized Human Hepatocytes

Belouzard

Danneels

Fénéant

et al. 2017

J Virol

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Hepatitis C virus (HCV) infects primarily hepatocytes that are highly polarized cells. The relevance of cell polarity in the HCV life cycle has only been addressed in distant models and remains poorly understood. Although polarized epithelial cells have a rather simple morphology with a basolateral and an apical domain, hepatocytes exhibit complex polarization structures. However, it has been reported that some selected polarized HepG2 cell clones can exhibit a honeycomb pattern of distribution of the tight-junction proteins, typical of columnar polarized epithelia which can be used as a simple model to study the role of cell polarization in viral infection of hepatocytes. To obtain similar clones, HepG2 cells expressing CD81 (HepG2-CD81) were used and clones were isolated by limiting dilutions. Two clones exhibiting a simple columnar polarization capacity when grown on semi-permeable support were isolated and characterized. To test the polarity of HCV entry and release, our polarized HepG2-CD81 clones were infected with cell culture derived HCV. Our data indicate that HCV binds equally to both sides of the cells, but productive infection occurs mainly when the virus is added at the basolateral domain. Furthermore, we also observed that HCV virions are released from the basolateral domain of the cells. Finally, when polarized cells were treated with oleic acid and U0126, a MEK inhibitor, to promote lipoprotein secretion, a higher proportion of infectious viral particles of lower density was secreted. This cell culture system provides an excellent model to investigate the influence of cell polarization on the HCV life cycle.IMPORTANCE Hepatitis C is a major health burden with approximately 170 million persons infected worldwide. Hepatitis C virus (HCV) infects primarily hepatocytes that are highly polarized cells with a complex organization. The relevance of cell polarity in the HCV life cycle has been addressed in distant models and remains unclear. Hepatocyte organization is complex with multiple apical and basolateral surfaces. A simple culture model of HepG2 cells expressing CD81 that are able to polarize with a unique apical and basolateral domain was developed to study HCV infection. With this model, we demonstrated that HCV enters and exits hepatocytes by the basolateral domain. Furthermore, lower density viral particles were produced in conditions that promote lipoprotein secretion. This cell culture system provides a useful model to study the influence of cell polarization on HCV infection.

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Section: Discussionmentioning

confidence: 93%

Entry and Release of Hepatitis C Virus in Polarized Human Hepatocytes

Belouzard

Danneels

Fénéant

et al. 2017

J Virol

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“…S10-3 cells are known to be the most permissive for HEV-1 among these 3 cell lines (19,20). Previous reports have revealed the presence of higher basal levels of miR-122 in Huh7 cells and lower levels in HepG2 cells, though both are human hepatocarcinoma cell lines (21). The Huh-7 cell line is the parental cell line of the S10-3 clonal cell line, while the HepG2 cell line is the parental cell line of HepG2/C3A clonal cells.…”

Section: Resultsmentioning

confidence: 99%

Positive Regulation of Hepatitis E Virus Replication by MicroRNA-122

Haldipur

Bhukya

Arankalle

et al. 2018

J Virol

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The molecular mechanisms of liver pathology and clinical disease in hepatitis E virus (HEV) infection remain unclear. MicroRNAs (miRNAs) are known to modulate viral pathogenesis either by directly altering viral gene expression or by enhancing cellular antiviral responses. Given the importance of microRNA-122 (miR-122) in liver pathobiology, we investigated possible role of miR-122 in HEV infection. predictions using HEV genotype 1 (HEV-1), HEV-2, HEV-3, and HEV-4 sequences showed that the majority of genomes (203/222) harbor at least one miR-122/microRNA-122-3p (miR-122*) target site. Interestingly, HEV-1 genomes showed a highly (97%) conserved miR-122 target site in the RNA-dependent RNA polymerase (RdRp) region (RdRp). We analyzed the significance of miR-122 target sites in HEV-1/HEV-3 (HEV-1/3) genomes by using a replicon-based cell culture system. HEV infection did not change the basal levels of miR-122 in hepatoma cells. However, transfection of these cells with miR-122 mimics enhanced HEV-1/3 replication and depletion of miR-122 with inhibitors led to suppression of HEV-1/3 replication. Mutant HEV-1 replicons with an altered target RdRp sequence (CACTCC) showed a drastic decrease in virus replication, whereas introduction of alternative miR-122 target sites in mutant replicons rescued viral replication. There was enrichment of HEV-1 RNA and miR-122 molecules in RNA-induced silencing complexes in HEV-infected cells. Furthermore, pulldown of miR-122 molecules from HEV-infected cells resulted in pulldown of HEV genomic RNA along with miR-122 molecules. These observations indicate that miR-122 facilitates HEV-1 replication, probably via direct interaction with a target site in the viral genome. The positive role of miR-122 in viral replication presents novel opportunities for antiviral therapy and management of hepatitis E. Hepatitis E is a problem in both developing and developed countries. HEV infection in most patients follows a self-limited course; however, 20% to 30% mortality is seen in infected pregnant women. HEV superinfections in patients with chronic hepatitis B or hepatitis C virus infections are associated with adverse clinical outcomes, and both conditions warrant therapy. Chronic HEV infections in immunocompromised transplant recipients are known to rapidly progress into cirrhosis. Currently, off-label use of ribavirin (RBV) and polyethylene glycol-interferon (PEG-IFN) as antiviral therapy has shown promising results in both acute and chronic hepatitis E patients; however, the teratogenicity of RBV limits its use during pregnancy, while alpha IFN (IFN-α) increases the risk of transplant rejections. Experimental data determined with genotype 1 virus in the current study show that miR-122 facilitates HEV replication. These observations present novel opportunities for antiviral therapy and management of hepatitis E.

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“…In line with this hypothesis, a recent report suggested that the lack of miR-122 expression in HepG2 cells might be related to a condensed, and hence nonaccessible, chromatin structure within its promoter region. (48) Importantly, we show that miR-122* has a role in the p53-Mdm2 regulatory network. In silico bioinformatic search for genes targeted by miR-122* revealed Mdm2 as a putative target gene.…”

Section: Discussionmentioning

confidence: 68%

“…This result may imply that in cancer, transcriptional silencing of pre‐miR‐122 takes place rather than impaired regulation of the miRNA maturation steps. In line with this hypothesis, a recent report suggested that the lack of miR‐122 expression in HepG2 cells might be related to a condensed, and hence nonaccessible, chromatin structure within its promoter region …”

Section: Discussionmentioning

confidence: 99%