Establishment of YFV17D Non-infectious Reporter Replicon and Pseudovirus Packaging System

doi:10.13560/j.cnki.biotech.bull.1985.2022-1402

Abstract

Abstract:

The YFV17D noninfectious replicon and pseudovirus packaging system was constructed to further elucidate the molecular mechanism of replication and assembly of yellow fever virus. In this study, the single-copy YFV17D reporter replicon based on the SP6 promoter was transformed into a low-copy YFV17D reporter replicon driven by the CMV promoter using reverse genetics. At the same time, the packaging plasmid expressing YFV17D structural protein and reporter replicon plasmid were co-transfected into BHK-21 cells to establish a YFV17D trans-pseudovirus packaging system. NanoLuc luciferase activity（Nluc）, oxGFP and mCherry fluorescent protein expression, and indirect immunofluorescence assay were used to monitor viral double-stranded RNA to determine the replication and packaging effect of replicon in BHK-21 cells. The results showed that Nluc activity, oxGFP and mCherry fluorescent protein expression increased steadily in wild-type replicon compared with replication-deficient replicon, while dsRNA also increased. After co-transfection of the replicon plasmids and packaging plasmids into BHK-21 cells, the supernatant was collected to re-infect BHK-21 cells. The success of pseudovirus packaging was confirmed by detecting Nluc activity and oxGFP and mCherry protein expression. In conclusion, the YFV17D replicon carrying different reporter genes was successfully constructed in this study to monitor the replication of viral genomic RNA. The structural proteins provided by the packaging plasmid can successfully package the non-infectious YFV17D subgene replicons into a YFV17D pseudovirus system with single round infection ability.

Key words: YFV17D reporter replicon, reverse genetics, trans-complementation, single-round infection virions

HE Yu-hang, HU Tao, WU Zhen, HE Yu, CHENG An-chun, CHEN Shun. Establishment of YFV17D Non-infectious Reporter Replicon and Pseudovirus Packaging System[J]. Biotechnology Bulletin, 2023, 39(8): 165-172.

Figures/Tables 4

Fig. 1 Schematic diagram of the construction of YFV17D reporter replicon

Fig. 2 Fluorescent protein expression of the YFV17D reporter replicon in BHK-21 cells A: Nluc luciferase activity assay, ****P<0.000 1. B: Fluorescent protein expression; （a）YFV17D-mCherry-rep transfection panel.（b）YFV17D-oxGFP-rep transfection panel; replication-defective replicons were used as negative controls

Fig. 3 RNA replication of YFV17D replicon in BHK-21 cells A: YFV17D-mCherry-rep transfection panel; B: YFV17D-oxGFP-rep transfection panel; C: YFV17D-mCherry-rep transfection panel; replication-defective replicons were used as negative controls

Fig. 4 Establishment of the YFV17D packaging system A: Schematic diagram of packaging plasmid construction.B: Schematic presentation of the experimental procedure used for the production of SRIPs. C: Detection of Nluc luciferase activity after SRIPs infection of BHK-21 cells, ****P<0.000 1. D: Fluorescent protein expression after SRIPs infection of BHK-21 cells（a）YFV17D-oxGFP-rep transfection panel;（b）YFV17D-mCherry-rep transfection panel（C: pcDNA3.1-CprME; C28: pcDNA3.1-C28prME）

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