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.

A novel three‐plasmid packaging system for chimeric SFV/SIN VRPs derived from Semliki Forest virus and Sindbis virus as a candidate gene delivery vector

“…The recombinant virus particles are mostly called virus‐like particles (VRPs). They are also known as virus‐like replicon particles (VLPs) 8–10 . SFV VRPs have been commonly used for cancer therapy and vaccine development in various animal models, and to some extent, in clinical trials 8,11–13 …”

“…They are also known as virus-like replicon particles (VLPs). [8][9][10] SFV VRPs have been commonly used for cancer therapy and vaccine development in various animal models, and to some extent, in clinical trials. 8,[11][12][13] A therapeutic cancer vaccine named Vvax001 was recently tested in a first-in-human phase I trial against human papillomavirus (HPV)-induced cancers.…”

Identification of RACK1 as a novel regulator of non-structural protein 4 of chikungunya virus

“…CHIKV is categorized as a highly pathogenic alphavirus, and live CHIKV should be handled in the P3 laboratory, which hampers the study of CHIKV. Therefore, the CHIKV replicon, virus-like particles (VLPs) and pseudoviruses are alternatives widely used in exploring CHIKV molecular virology and pathogenesis [35][36][37]. In addition, a specific viral protein used as a bait protein for yeast two-hybrid systems, Co-IP or GST pull-down, combined with MS, is commonly used to discover protein-protein interactions and interacting domains [38][39][40].…”

Ultrastructural and Functional Characterization of Mitochondrial Dynamics Induced by Human Respiratory Syncytial Virus Infection in HEp-2 Cells

“…While most studies with hRSV use MOIs between 0.1 and 3.0 [ 24 , 29 , 30 , 31 , 45 , 46 ] we decided to use an MOI of 10 to be able to detect ultrastructural changes, which is one of the limitations of this study. In previous studies with Zika (ZIKV) [ 47 ] and Chikungunya (CHIKV) [ 48 ] virus, we used low MOIs for thin-section TEM. However, preliminary experiments with hRSV at MOIs of 0.1 and 1.0 showed that detecting ultrastructural changes in the mitochondrion was extremely challenging at these MOIs (data not shown).…”

“…However, preliminary experiments with hRSV at MOIs of 0.1 and 1.0 showed that detecting ultrastructural changes in the mitochondrion was extremely challenging at these MOIs (data not shown). Furthermore, in the cases of CHIKV [ 48 ] and ZIKV [ 47 ] infections at an MOI of 1.0 lead to complete destruction of the cellular monolayer within the first 72 h.p.i. ; however, with hRSV, infection at an MOI of 10 does not result in complete disruption of the monolayer even at 96-h.p.i.…”