Flow cytometric assay using two fluorescent proteins for the function of the internal ribosome entry site of hepatitis C virus

Abstract

The initiation of translation in hepatitis C virus (HCV) occurs at the internal ribosome entry site (IRES) located at the 5 0 -end of its genomic RNA. To study the function of HCV IRES, we constructed a reporter plasmid that generates a bicistronic mRNA encoding two fluorescent proteins: cap-dependent DsRed2 and IRES-dependent Azami Green (AG). We introduced the plasmid into Huh7.5.1 and HEK293 cells and measured the relative IRES activity from the ratio of AG's signal to DsRed2's in individual cells using flow cytometry. To compare our method and a conventional biochemical method, we constructed a structurally similar reporter in which Renilla and Firefly luciferases replace DsRed2 and AG, respectively. With these systems, we found that the IRES A164G substitution decreased its activity, that interferon alpha affected the IRES activity in a cell type-specific manner, and that a synthetic micro-RNA targeting IRES was able to suppress the gene expression. In conclusion, the two methods were comparable in sensitivity in the studies of IRES mutations and host cell types. We discussed the significance of our findings and potential advantage of the cytometric assay: application to the molecular study of the HCV translation and to screening anti-IRES drugs. ' 2011 International Society for Advancement of Cytometry Key terms hepatitis C virus; internal ribosome entry site; DsRed2; Azami Green; flow cytometry HEPATITIS C virus (HCV) is a major causative agent of liver cirrhosis and hepatocellular carcinoma (1). The HCV genome is a plus-strand RNA consisting of $9,600 nucleotides (nt) and has a single long open reading frame encoding a polyprotein of about 3,000 amino acids (2). Unlike the translation of eukaryotic mRNA starting from the 5 0 -cap structure, the translation of HCV RNA initiates at the internal ribosome entry site (IRES), which encompasses most of the 5 0 -untranslated region (5 0 -UTR) and about 40 nt of the core protein-coding region (3,4). The HCV IRES is highly conserved among HCV isolates in both the primary sequence and secondary structure (5-7). Since the translation is an essential step in the viral replication and possibly in the pathogenic functions, it would be important to evaluate the activity of HCV IRES for such studies.The HCV IRES activity has been studied, by using biochemical methods with luciferase genes as reporters (8-10), which have some flaws resulting from using cell lysates. In this study we applied flow cytometry to the IRES studies, in an attempt to simplify the assay procedure and to expand the scope of the study. Thus, we used a combination of two fluorescent proteins, DsRed2 (11) and Azami-Green (AG) (12), which are excited by the light of the identical wave length and whose emission lights are easily discriminated. We constructed and used a bicistronic reporter plasmid with genes encoding these two fluorescent proteins, and by flow cytometry, quantitatively determined fluorescent signals in individual cells transfected with the plasmid. To

Polypyrimidine-Tract-Binding Protein Isoforms Differentially Regulate the Hepatitis C Virus Internal Ribosome Entry Site

“…Thus, we wondered if our findings could be replicated in a non-hepatic and non-lymphoid cell line such as HEK293T cells. HEK293T cells are not permissive to HCV infection [ 71 ], yet are supportive of HCV IRES function [ 13 , 46 , 72 , 73 ]. Furthermore, a previous report from our laboratory shows that the overexpression of PTB1 enhances HCV IRES activity in HEK293T cells [ 46 ].…”

Current Practice in Bicistronic IRES Reporter Use: A Systematic Review

“…Studies reporting on direct comparison of dual luciferase with dual fluorescence reporters were rare. At least for multiple HCV IRES sequence mutants both reporter configurations provided identical results in HEK293 and Huh-7 cells [ 164 ]. In the same study, stimulation of cells with IFNγ for 36 h demonstrated a higher sensitivity of the dual luciferase system in comparison to the fluorescent reporter.…”

“…Although CAT/βGal reporters see limited use in recent times, their susceptibility to cryptic splicing was shown to be lower than Fluc/Rluc reporters [ 109 ]. Fluorescent reporters can be used for flow cytometric applications; however, reporter protein maturation times are substantially longer [ 222 ], and therefore sensitivity was demonstrated to be lower [ 164 ]. To our knowledge, newer luciferases, such as NanoLuc derived from Oplophorus gracilirostris [ 233 ], have not yet been used in bicistronic reporter configurations.…”

Aptamers: Novel Molecules as Diagnostic Markers in Bacterial and Viral Infections?

“…In this sense the discovery of novel technologies and moreover their subsequent applications have become important in laboratory and field studies. As an example, innovations in high-throughput single cell analysis for diagnostic purposes but also for functional analysis and drug discovery are today available in order to analyze pathogenic organisms such as the viruses Hepatitis C [1], influenza [2], or HIV [3] as well as bacteria like Mycobacterium tuberculosis [4] or Staphylococcus aureus [5]. The SELEX technique (Systematic Evolution of Ligands by Exponential Enrichment) was originally discovered by Gold and Szostak [6, 7].…”