Dual Promoters Improve the Rescue of Recombinant Measles Virus in Human Cells.

Soroth Chey,Juliane Maria Palmer,Uwe Gerd Liebert,Laura Doerr

doi:10.3390/v13091723

Abstract

Reverse genetics is a technology that allows the production of a virus from its complementary DNA (cDNA). It is a powerful tool for analyzing viral genes, the development of novel vaccines, and gene delivery vectors. The standard reverse genetics protocols are laborious, time-consuming, and inefficient for negative-strand RNA viruses. A new reverse genetics platform was established, which increases the recovery efficiency of the measles virus (MV) in human 293-3-46 cells. The novel features compared with the standard system involving 293-3-46 cells comprise (a) dual promoters containing the RNA polymerase II promoter (CMV) and the bacteriophage T7 promoter placed in uni-direction on the same plasmid to enhance RNA transcription; (b) three G nucleotides added just after the T7 promoter to increase the T7 RNA polymerase activity; and (c) two ribozymes, the hairpin hammerhead ribozyme (HHRz), and the hepatitis delta virus ribozyme (HDVrz), were used to cleavage the exact termini of the antigenome RNA. Full-length antigenome cDNA of MV of the wild type IC323 strain or the vaccine AIK-C strain was inserted into the plasmid backbone. Both virus strains were easily rescued from their respective cloned cDNA. The rescue efficiency increased up to 80% compared with the use of the standard T7 rescue system. We assume that this system might be helpful in the rescue of other human mononegavirales.

Highlights

Published: 30 August 2021The measles virus (MV) is an enveloped, non-segmented, negative-sense, singlestranded RNA virus
The resulting plasmid (Figure 1, step 1) was named pCi-T7-Hammerhead ribozyme (HHrz)-hepatitis delta virus ribozyme (HDVrz) and was used to be subcloned into a low copy plasmid piRFP by digestions of both plasmids with restriction enzymes SalI and SacI
We developed a reverse genetics plasmid platform that follows several improvements to enhance the recovery of MV in the human cells: (1) the RNA polymerase II (CMV)

Summary

Introduction

The measles virus (MV) is an enveloped, non-segmented, negative-sense, singlestranded RNA virus. It carries a single copy of the genome, which is 15,894 bp in length. The genome of MV encodes six major structural proteins, i.e., nucleocapsid protein (N), phosphoprotein (P), matrix protein (M), fusion protein (F), hemagglutinin (H), and the large protein (L). V and C, are produced from the P gene by RNA editing. The viral genome is encapsidated by N, P, and L proteins forming the ribonucleoprotein complex (RNP), surrounded by M protein. H and F proteins mediate virus attachment and fusion, respectively [1]. Attenuated MV is one of the most effective and safe vaccines available.

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Results

Conclusion