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

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.

Keywords: measles virus; mononegavirales; multivalent vaccines; negative-strand RNA virus; oncolytic vectors; recombinant virus; reverse genetics.

Figure 1

Cloning schemata of the vector backbone plasmid pT-HHrz-HDVrz.

Figure 2

Schematic diagrams of construction of full-length antigenome MV plasmids. (A) The vector backbone V was generated from the PCR using shuttle plasmid template and primers MV_Trailer-s and MV_Leader-as. (B) Full-length MV plasmid was cloned by ligation of vector backbone V and three PCRs fragments generated from the donor full-length MV antigenome template plasmid p(+)MV323-eGFP or p(+)MVAIK-eGFP. (C) The plasmid pT(+)MV323-eGFP and pT(+)MVAIK-eGFP clone were analyzed by restriction digestion with BamHI.

Figure 3

Rescuing the MV from cDNA and growth curve kinetic: (a) Schemata of the reverse genetics protocol as described in the method; (b) multiple-step growth kinetics of four recombinant MVs in Vero/hSLAM. pT_IC323 (rescued from plasmids pT(+)MVIC323-eGFP), pT_AIK (rescued from pT(+)MVAIK-eGFP), IC323 (rescued from p(+)MV323-eGFP), and AIK (rescued from p(+)MVAIK-eGFP) were analyzed on Vero/hSLAM at different time points.