Reovirus reverse genetics: Incorporation of the CAT gene into the reovirus genome

Abstract

We have modified the infectious reovirus RNA system so as to generate a reovirus reverse genetics system. The system consists of (i) the plus strands of nine wild-type reovirus genome segments; (ii) transcripts of the genetically modified cDNA form of the tenth genome segment; and (iii) a cell line transformed so as to express the protein normally encoded by the tenth genome segment. In the work described here, we have generated a serotype 3 reovirus into the S2 double-stranded RNA genome segment of which the CAT gene has been cloned. The virus is stable, replicates in cells that have been transformed (so as to express the S2 gene product, protein final sigma 2), and expresses high levels of CAT activity. This technology can be extended to members of the orbivirus and rotavirus genera. This technology provides a powerful system for basic studies of double-stranded RNA virus replication; a nonpathogenic viral vector that replicates to high titers and could be used for clinical applications; and a system for providing nonselectable viral variants (the result of mutations, insertions, and deletions) that could be valuable for the construction of viral vaccine strains against human and animal pathogens.

Figure 1

(Lane A) ³²P-labeled RNA transcribed by T7 RNA polymerase from the pS2-CAT13 template lacking the CAT gene insert, that is, authentic s2 RNA. (Lane B) Cleavage products after hybridization to the ODRN complementary to residues 937–949 and digestion with RNaseH. Electrophoresis was carried out in an SDS/PAGE.

Figure 2

Demonstration that protein σ2 is expressed in L-ST3.S2 cells. (Lane A) Proteins formed in L929 cells infected with ST3 virus that are precipitated with antiserum against ST3 virus. (Lane B) As in lane A, for mock-infected L929 cells. (Lane C) As in lane A, for uninfected L-ST3.S2 cells. Electrophoresis was carried out in SDS/PAGE.

Figure 3

Growth curves of ST3 virus and ts447 in L929 cells and L-ST3.S2 cells at 39°C. For experimental details, see Materials and Methods and text. (▪) ST3 virus in L929 cells. (▴) ts447 in L929 cells. (□) ST3 virus in L-ST3.S2 cells. (▵) ts447 in L-ST3.S2 cells.

Figure 4

Electropherograms of the genome segments of ST1, ST2, ST3 virus, and ST3.S2.CAT virus. Electrophoresis was carried out in an SDS/PAGE. Lane 1, ST1 virus; lane 2, ST2 virus; lane 3, ST3 virus; lane 4, ST3.S2.CAT virus.

Figure 5

One-step growth curves of ST3 virus and ST3.S2.CAT virus in L929 and L-ST3.S2 cells. For experimental details, see Materials and Methods and text. (▪) ST3 virus in L929 cells. (▴) ST3.S2.CAT virus in L929 cells. (□) ST3 virus in ST3.S2 cells. (▵) L-ST3.S2.CAT virus in L-ST3.S2 cells.

Figure 6

Demonstration of s2.CAT RNA in L-ST3.S2.CAT virus-infected L-ST3.S2 cells. (Lane A) s2 RNA revealed in L929 cells infected with ST3 virus by hybridization with a probe that hybridized to the 18 5′-terminal nucleotides of s2 RNA. (Lane B) s2.CAT RNA revealed in L-ST3.S2 cells infected with ST3.S2.CAT virus by hybridization to a probe that hybridized to the 18 5′-terminal nucleotides of CAT mRNA. For experimental details, see Materials and Methods and text.