Generation of measles virus with a segmented RNA genome

Abstract

Viruses classified in the order Mononegavirales have a single nonsegmented RNA molecule as the genome and employ similar strategies for genome replication and gene expression. Infectious particles of Measles virus (MeV), a member of the family Paramyxoviridae in the order Mononegavirales, with two or three RNA genome segments (2 seg- or 3 seg-MeV) were generated using a highly efficient reverse genetics system. All RNA segments of the viruses were designed to have authentic 3' and 5' self-complementary termini, similar to those of negative-stranded RNA viruses that intrinsically have multiple RNA genome segments. The 2 seg- and 3 seg-MeV were viable and replicated well in cultured cells. 3 seg-MeV could accommodate up to six additional transcriptional units, five of which were shown to be capable of expressing foreign proteins efficiently. These data indicate that the MeV genome can be segmented, providing an experimental insight into the divergence of the negative-stranded RNA viruses with nonsegmented or segmented RNA genomes. They also illustrate a new strategy to develop mononegavirus-derived vectors harboring multiple additional transcriptional units.

FIG. 1.

Diagram of the genome organization of the recombinant viruses. (A) Recombinant MeVs, wt (IC323), IC323-lacZ, IC323-DsRed, and IC323-EGFP with nonsegmented RNA genomes with six or seven transcriptional units. (B) Recombinant MeV with 2 RNA genome segments (2 seg-MeV). One RNA genome segment encodes N, P, M, and F proteins and DsRed2. In addition, the segment has another transcriptional unit at the sixth locus (6) providing a second insertion site for a foreign gene. The sixth transcriptional unit has the authentic GS sequence and the initiation codon of the H gene, followed by two tandem termination codons to stop unwanted translation. After ∼800 base-noncoding sequences, the unit ends with the authentic GE sequence of the L gene. The other RNA genome segment encodes H and L proteins and EGFP. The segment also has another transcriptional unit at the second locus (2′), providing a second insertion site for a foreign gene. The second transcriptional unit starts with the authentic GS sequence of the P gene, combined with the noncoding sequence and the initiation codon of the N gene, and ends with the authentic GE sequence of the F gene after 17 nucleotides. (C) Recombinant MeV with three RNA genome segments (3 seg-MeV). The first RNA genome segment encodes N and P proteins and β-galactosidase. The second RNA genome segment encodes M and F proteins and DsRed2. In addition, the segment has two extra transcriptional units at the second (2′) and fifth (5′) loci, providing additional insertion sites for the foreign genes. The second transcriptional unit has the authentic GS sequence of the P gene combined with the noncoding sequence and the initiation codon of the N gene, followed by tandem termination codons after 24 nucleotides, and ends with the authentic GE sequence of the P gene. The fifth transcriptional unit has the same structure as that of the sixth unit of the first RNA genome segment of 2 seg-MeV. The third RNA genome segment has the same structure as that of the second RNA genome segment of 2 seg-MeV. Filled boxes show ORFs in which encoded proteins are shown by white characters. Open boxes show untranslated regions, and vertical lines with open triangles show the positions of the gene junctions. Panels show light and fluorescence microscopic images of the 2 seg-MeV- or 3 seg-MeV-infected B95a cells. X-Gal staining was performed for 3 seg-MeV-infected cells (results shown in panel C, β-gal).

FIG. 2.

Plaque formation and replication kinetics of recombinant viruses. (A) Neutral red and X-Gal staining of plaques of recombinant MeVs on Vero/hSLAM cells. (B) Plaques and EGFP- and β-galactosidase-expressing individual cells (satellites are indicated by arrows) produced by 3 seg-MeV. (C) Replication kinetics. Vero/hSLAM cells were infected with viruses at a multiplicity of infection of 0.01. At various intervals, cells were harvested with culture medium, and the number of PFU was determined for Vero/hSLAM cells. (D) Production of satellites and plaques by 3 seg-MeV. The number of satellites (green bars) in the samples shown in panel C was counted and compared to the number of plaques (yellow bars).

FIG. 3.

Gene expression of recombinant viruses. (A) Levels of viral mRNAs. Vero/hSLAM cells were infected with IC323-EGFP, 2 seg-MeV, and 3 seg-MeV. At 24 h p.i., viral mRNAs in the 2 seg-MeV- or 3 seg-MeV-infected cells were quantified by reverse transcription and quantitative PCR and compared with those in the IC323-EGFP-infected cells. Relative amounts of them were shown (2 seg-MeV/IC323-EGFP and 3 seg-MeV/IC323-EGFP, respectively). (B) Synthesis of reporter and viral proteins. Cells at 24 h p.i. were pulse-labeled for 1 h, and reporter and viral proteins were immunoprecipitated and analyzed by SDS-PAGE. (C) Quantification of reporter proteins. EGFP autofluorescence and enzymatic activity of β-galactosidase in cells at 24 h p.i. were quantified as described in Materials and Methods. Levels of reporter proteins in the IC323-EGFP- or IC323-lacZ-infected cells were set at 100%.

FIG. 4.

Multiple foreign gene insertion into the 3 seg-MeV genome. (A) Diagram of the 3 seg-MeV-CAT genome. (B) Diagram of the 3 seg-MeV-CAT-SEAP genome. (C) Replication kinetics. Vero/hSLAM cells were infected with IC323-EGFP, 3 seg-MeV, 3 seg-MeV-CAT, and 3 seg-MeV-CAT-SEAP at a multiplicity of infection of 0.005. At various intervals, cells were harvested with culture medium, and the number of PFU was determined with Vero/hSLAM cells. (D and E) Analysis of CAT and SEAP enzymatic activities. In parallel to the results of PFU analysis shown in panel C, CAT activity in cells (D) and SEAP activity in culture medium (E) were quantified as described in Materials and Methods. SEAP activity is expressed in chemiluminescent units per second.