Construction of a Chikungunya Virus, Replicon, and Helper Plasmids for Transfection of Mammalian Cells

Abstract

The genome of Alphaviruses can be modified to produce self-replicating RNAs and virus-like particles, which are useful virological tools. In this work, we generated three plasmids for the transfection of mammalian cells: an infectious clone of Chikungunya virus (CHIKV), one that codes for the structural proteins (helper plasmid), and another one that codes nonstructural proteins (replicon plasmid). All of these plasmids contain a reporter gene (mKate2). The reporter gene in the replicon RNA and the infectious clone are synthesized from subgenomic RNA. Co-transfection with the helper and replicon plasmids has biotechnological/biomedical applications because they allow for the delivery of self-replicating RNA for the transient expression of one or more genes to the target cells.

Keywords: Chikungunya virus; alphavirus DNA vectors; helper plasmid; replicon plasmid.

Figure 1

Construction and evaluation of an expression plasmid for CHIKV. (A) Schematic representation of the plasmids generated here. The SP6-CHIKV plasmid is used for in vitro transcription with SP6 polymerase and contains the full-length genome of the attenuated strain 181/25 of the Chikungunya virus. The genomic CHIKV was inserted into the pVax-CHIKV vector from which the human cytomegalovirus (CMV) immediate early enhancer and promoter sequences and βGH polyA derived from pVAX. The SV40 polyA and hepatitis delta virus (HDV) were inserted, and the genome was moved from the pVAX to the vector pACNR1811. The asterisk means that mKate2 is a reporter gene. (B) Micrographs of HEK-293T cells transfected with the pACNR-CHIKV plasmid in a fluorescent field (mKate2 channel), phase contrast, and a merge of both. (C) Kinetics of the expression of mKate2 from the plasmid pACNR-CHIKV. Bar, 100 µm.

Figure 2

The Chikungunya virus plasmid pACNR-CHIKV produces a viral infection. (A) HEK-293T and Vero E6 cells infected with the supernatant of the producer (transfected) cells in fluorescent-field mKate2 expression (Texas Red filter), nuclei stain (DAPI filter), and merge at MOI of 1 for 24 h.p.i. (B) Cytopathic effects induced by the CHIKV infection in Vero E6 cells at MOI of 1. (C) CHIKV plaques on Vero E6 cells stained with crystal violet and (D) and virus titer measured as plaque-forming units (PFUs). Bar, 200 µm.

Figure 3

Chikungunya virus pACNR-CHIKV plasmid induces replication and assembly of viral infectious particles. Vero E6 cells infected with Chikungunya virus at a MOI of 1 were assessed at 24 h post-infection. TEM images show Chikungunya (A) four extracellular virions (▲), (B) a viral factory, (C) four virions budding from a membrane (*), and (D) type-II cytopathic vacuoles (arrow, CPV-II). Bar, 100 nm.

Figure 4

Generation and evaluation of replicon and helper vector system. (A) Constructions of pACNR-CHIKV, pACNR-Rep, and pVax-Help plasmids. The genomic CHIKV was inserted into the pACNR1811 vector from which the human cytomegalovirus (CMV) immediate early enhancer and promoter sequences, the SV40 polyA, and hepatitis delta virus (HDV) to generate pACNR-CHIKV. The structural proteins were removed to generate pACNR-Rep and derived from pVax-CHIKV and the nonstructural proteins were removed to generate pVax-Help. The asterisk means that mKate2 is a reporter gene. (B) HEK-293T cells transfected with pACNR-Rep, pACNR-Help, and co-transfected with both vectors after 48 h. Fluorescence micrographs with mKate2 expression (mKate2 channel), the morphology of cells in phase contrast, and a merge. Bar, 100 µm.

Figure 5

HEK-293T cells from the entry assay infected with the particles generated with the pACNR-Rep and pVax-Help system in (A) fluorescent-field mKate2 expression (Texas Red filter), nuclei stain (DAPI filter), and (C) overlap of mKate2 and nuclei micrographs, demonstrating that the particles generated by the co-transfection of both plasmids result in the expression of the gene of interest (mKate2) in the target cells. The bar indicates the same scale for all images, 50 μm. HEK-293T cells 48 h post-transfection with replicon and helper system were observed by transmission electron microscopy (TEM). (B) Cytopathic vacuole type 1 (arrow, CPV-I), (C) two virions budding from a membrane (*), and (D) two extracellular virions (▲). Bar B–D, 200 nm.