Coronavirus derived expression systems

Abstract

Both helper dependent expression systems, based on two components, and single genomes constructed by targeted recombination, or by using infectious cDNA clones, have been developed. The sequences that regulate transcription have been characterized mainly using helper dependent expression systems and it will now be possible to validate them using single genomes. The genome of coronaviruses has been engineered by modification of the infectious cDNA leading to an efficient (>20 microg ml(-1)) and stable (>20 passages) expression of the foreign gene. The possibility of engineering the tissue and species tropism to target expression to different organs and animal species, including humans, increases the potential of coronaviruses as vectors. Thus, coronaviruses are promising virus vectors for vaccine development and, possibly, for gene therapy.

Fig. 1

Coronavirus derived expression systems: A. Helper dependent expression system based on two components, the helper virus and a minigenome carrying the foreign gene (FG). An, poly A. B. Single genome engineered either by targeted recombination or by using an infectious coronavirus cDNA clone (pBAC-TGEV^FL) derived from TGEV genome.

Fig. 2

Structure and genome organization of coronaviruses: A. Schematic diagram of virus structure showing the envelope, the core and the nucleoprotein structure. S, spike protein; M and M′, M proteins with the amino-terminus facing the external surface of the virion and the carboxy-terminus towards the inside or the outside face of the virion, respectively; E, small envelope protein; N, nucleocapsid protein; NC, nucleocapsid. Some coronaviruses of group 2 have an additional protein, the haemagglutinin-esterase (HE) (not shown). B. Representation of a prototype TGEV coronavirus genome and subgenomic RNAs. Beneath the top bar a set of positive- and negative-sense mRNA species synthesized in infected cells is shown. The protein products obtained from each positive-sense RNA are indicated. Two products, polyproteins 1a and 1b, are translated from the genomic RNA by a ribosomal frameshifting mechanism. All other proteins are translated from the first open reading frame of each functionally monocistronic subgenomic RNA (dark lines). Poly(A) and Poly(U) tails are indicated by AAA or UUU. S, spike protein; E, envelope protein; M, membrane protein; N, nucleocapsid protein.

Fig. 3

Summary of helper dependent expression systems based on coronavirus derived minigenomes: A–C. Expression modules based on MHV minigenomes DIssF and DIssE cloned under the control of T7 bacteriophage polymerase (T7), used to express chloramphenicol acetyltransferase (CAT), hemagglutinin-esterase (HE) or interferon-γ using either an IRES (A), or TRSs (B–C). D–E. Expression modules based on the TGEV derived minigenome M39 used to express the GUS. The minigenome was cloned either after T7 (D) or the CMV (E) promoters. F. Expression module based on the IBV derived minigenome CD-61 used to express CAT.

Fig. 4

Single genome expression based on the engineering of coronavirus minigenomes by targeted recombination: A. Basic scheme of targeted recombination in MHV. The black box indicates the approximate location of the N gene region (87 nt) that is deleted in the Alb4 mutant. M, insertion of 5 nt used as a genetic marker (Masters, 1999). B. Targeted recombination within the S gene of TGEV and a minigenome carrying the information for an S gene with three nucleotide mutations (Sdmar) that allow escape from neutralization by two mAbs specific for antigenic sub-sites Ac and Aa of S protein (Sola et al., 2001b).

Fig. 5

Cloning of the TGEV cDNA in BACs and expression of GFP: A. Plasmid pBAC-TGEV^FL (bottom plasmid) was generated using two plasmids, one containing all the virus genome (top plasmid) except a sequence of about 5 kb present between two Cla I sites cloned in a second plasmid (middle plasmid). CMV, cytomegalovirus immediate-early promoter; Poly(A), tail of 24 A residues; HDV, hepatitis delta virus ribozyme; BGH, bovine growth hormone termination and polyadenylation sequences; SC11, S gene of PUR-C11 strain. B. Expression of GFP using an infectious TGEV cDNA clone. Genes 3a and 3b were deleted in the TGEV infectious cDNA, cloned in BAC, leading to a replication competent cDNA (pBAC-TGEV-Δ3ab-GFP). GFP gene (0.72 kb) was inserted within the position of the deleted genes after the TRS of gene 3a. GFP, green fluorescent protein. SC11, S gene of PUR-C11 TGEV strain. An, poly A. HDV, hepatitis delta-virus ribozyme. BGH, bovine growth hormone termination and polyadenylation signals.

Fig. 6

Schematic representation of a coronavirus discontinuous transcription model and the RNA structures involved: A–B. Discontinuous transcription during negative-strand RNA synthesis without (A) and with (B) a schematic representation of protein-RNA complexes potentially involved. During the negative RNA strain synthesis (discontinuous line) the replication complex is detached when the CS sequence is reached, and the complex joins the 3′ end of the leader.

Fig. 7

CSs of coronavirus TRSs: A. Alignment of representative CSs of the three groups of coronaviruses and one arterivirus. B. Relative abundance of the mRNAs produced after the mutagenesis of CSs of different lengths (10, 17 and 18 nt) using the MHV-A59 strain derived minigenomes (modified after van der Most et al., 1994).