An improved reverse genetics system for mammalian orthoreoviruses

Abstract

Mammalian orthoreoviruses (reoviruses) are highly useful models for studies of double-stranded RNA virus replication and pathogenesis. We previously developed a strategy to recover prototype reovirus strain T3D from cloned cDNAs transfected into murine L929 fibroblast cells. Here, we report the development of a second-generation reovirus reverse genetics system featuring several major improvements: (1) the capacity to rescue prototype reovirus strain T1L, (2) reduction of required plasmids from 10 to 4, and (3) isolation of recombinant viruses following transfection of baby hamster kidney cells engineered to express bacteriophage T7 RNA polymerase. The efficiency of virus rescue using the 4-plasmid strategy was substantially increased in comparison to the original 10-plasmid system. We observed full compatibility of T1L and T3D rescue vectors when intermixed to produce a panel of T1LxT3D monoreassortant viruses. Improvements to the reovirus reverse genetics system enhance its applicability for studies of reovirus biology and clinical use.

Fig 1. Experimental strategy to generate reovirus type 1 Lang (T1L) from cloned cDNAs

(A) Rescue plasmids containing T1L gene-segment cDNAs. Vectors contain cDNAs representing each of the ten full-length T1L gene segments. Reovirus cDNAs are flanked by the bacteriophage T7 RNA polymerase promoter (T7P) and the antigenomic hepatitis delta virus (HDV) ribozyme (Rib). (B) Reverse genetics procedure. The ten T1L cDNA constructs are transfected into murine L cells that express T7 RNA polymerase from recombinant vaccinia virus strain DIs-T7pol. Plasmid-derived transcripts correspond to viral mRNAs with native 5’ termini. The 3’ termini of nascent transcripts are fused to HDV ribozyme sequences, which generate authentic 3’ends through autocatalytic cleavage. After five days of incubation, transfected cells are lysed by freeze-thaw, and recombinant-strain (rs) viruses are isolated from lysates by plaque assay using L cells.

Fig 2. Characterization of T1L viruses derived using reverse genetics

(A) Electropherotypes of native T1L and plasmid-derived rsT1L. Purified virions were electrophoresed in an SDS-polyacrylamide gel, followed by ethidium bromide staining to visualize viral gene segments. Size classes of gene segments (L, M, S) are indicated. (B) Novel mutations in the L3 gene of rs viruses. The C→T change at nucleotide 2059 and G→C change at nucleotide 2062 eliminate a unique PmlI site. (C) Digestion of the L3 gene RT-PCR products of T1L and rsT1L with PmlI to confirm the presence or absence of a unique restriction site. Size markers are indicated. (D) Growth of T1L and rsT1L. L cells were adsorbed with T1L or rsT1L at an MOI of 2 PFU/cell, and viral titers in cell lysates were determined by plaque assay at the times shown. Results are presented as mean viral titers for triplicate experiments. Error bars indicate SD. (E) Immunofluorescence analysis of cells infected with T1L and rsT1L. L cells were adsorbed with either T1L or rsT1L and stained at 24 h post-infection with µNS-specific antiserum followed by a fluorophore-conjugated, goat-anti rabbit secondary antibody to visualize reovirus inclusions. Representative images of T1L- and rsT1L-infected cells are shown.

Fig 3. Improved reverse genetics for reovirus strains T1L and T3D

(A, C) Two or four gene transcription cassettes encoding reovirus cDNAs flanked by the T7 RNA polymerase promoter and HDV ribozyme sequences were combined into single plasmids, creating four plasmids for the T1L and T3D reverse genetics systems. (B, D) L cells expressing T7 polymerase were co-transfected with either four or ten rescue plasmids corresponding to the T1L (B) and T3D (D) genomes. Following 24 or 48 h incubation, transfected cells were lysed by freeze-thaw, and viral titers in cell lysates were determined by plaque assay. Results are presented as mean viral titers for triplicate experiments. Error bars indicate SD.

Fig 4. Representative electropherotype of recombinant monoreassortant viruses

Purified virions of rsT1L, rsT3D, and T1L × T3D monoreassortants containing reciprocal exchanges of the M1 gene, rsT1L-T3M1 and rsT3D-T1M1, were electrophoresed in an SDS-polyacrylamide gel, followed by ethidium bromide staining to visualize viral gene segments. Size classes of gene segments (L, M, S) are shown. Positions of the T1L and T3D M1 genes are indicated by red and blue dots, respectively.

Fig 5. Recombinant virus generated using BHK-T7 cells

(A) Schematic of reovirus reverse genetics system using BHK-T7 cells. (B) BHK-T7 cells were co-transfected with four rescue plasmids corresponding to the T1L and T3D genomes. Following 48 h incubation, transfected cells were lysed by freeze-thaw, and viral titers in cell lysates were determined by plaque assay.