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Abstract
MicroRNAs (miRNAs) may become efficient antiviral agents against the Ebola virus (EBOV) targeting viral genomic RNAs or transcripts. We previously conducted a genome-wide search for differentially expressed miRNAs during viral replication and transcription. In this study, we established a rapid screen for miRNAs with inhibitory effects against EBOV using a tetracistronic transcription- and replication-competent virus-like particle (trVLP) system. This system uses a minigenome comprising an EBOV leader region, luciferase reporter, VP40, GP, VP24, EBOV trailer region, and three noncoding regions from the EBOV genome and can be used to model the life cycle of EBOV under biosafety level (BSL) 2 conditions. Informatic analysis was performed to select up-regulated miRNAs targeting the coding regions of the minigenome with the highest binding energy to perform inhibitory effect screening. Among these miRNAs, miR-150-3p had the most significant inhibitory effect. Reverse transcription polymerase chain reaction (RT-PCR), Western blot, and double fluorescence reporter experiments demonstrated that miR-150-3p inhibited the reproduction of trVLPs via the regulation of GP and VP40 expression by directly targeting the coding regions of GP and VP40. This novel, rapid, and convenient screening method will efficiently facilitate the exploration of miRNAs against EBOV under BSL-2 conditions.
Highlights
MicroRNAs are efficient antiviral agents [1,2], and the interactions between host-encoded miRNAs and viruses during the processes of viral replication and transcription are of interest [3,4]
Reverse transcription polymerase chain reaction (RT-PCR) and sequencing experiments confirmed that the sequences of GP, VP40, and VP24 were the same as those of wild-type EBOV (Zaire Mayinga Ebola virus) (Figure 1b)
We constructed the virus multiplication curve in 293T cells by detecting the TCID50 of the transcription- and replication-competent virus-like particle (trVLP) from the cell culture supernatant (Figure 1d). The results of these analyses confirmed that the trVLPs produced in this study could be used to explore the inhibitory effects of potential anti-Ebola miRNAs
Summary
MicroRNAs (miRNAs) are efficient antiviral agents [1,2], and the interactions between host-encoded miRNAs and viruses during the processes of viral replication and transcription are of interest [3,4]. The cytotoxicity of the Ebola virus (EBOV) glycoprotein can be reduced by inhibiting Hsa-miR-1246, hsa-miR-320a, and hsa-miR-196b-5p [5], and several miRNAs have been identified as potential pathogen-specific diagnostic biomarkers in circulating microRNA profiles of EBOV infection in both nonhuman primates (NHPs) and humans [6,7]. A transcription- and replication-competent virus-like particle (trVLP) system suitable for BSL 2 conditions has been used to study EBOV biology including morphogenesis, budding, entry, genome replication, and transcription. This system employs a tetracistronic minigenome, and its viral components are solely derived from EBOV [10,11]. A rapid screening assay of effective EBOV polymerase inhibitors based on the trVLP system has been performed, and the anti-Ebola effects were confirmed using fully infectious EBOV, confirming the potential utility of this system for identifying anti-Ebola agents [12,13]
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