Switching species tropism: an effective way to manipulate the feline coronavirus genome - PubMed (original) (raw)

Switching species tropism: an effective way to manipulate the feline coronavirus genome

Bert Jan Haijema et al. J Virol. 2003 Apr.

Abstract

Feline infectious peritonitis virus (FIPV), a coronavirus, is the causative agent of an invariably lethal infection in cats. Like other coronaviruses, FIPV contains an extremely large positive-strand RNA genome of ca. 30 kb. We describe here the development and use of a reverse genetics strategy for FIPV based on targeted RNA recombination that is analogous to what has been described for the mouse hepatitis virus (MHV) (L. Kuo et al., J. Virol. 74:1393-1406, 2000). In this two-step process, we first constructed by targeted recombination a mutant of FIPV, designated mFIPV, in which the ectodomain of the spike glycoprotein was replaced by that of MHV. This switch allowed for the selection of the recombinant virus in murine cells: mFIPV grows to high titers in these cells but has lost the ability to grow in feline cells. In a second, reverse process, mFIPV was used as the recipient, and the reintroduction of the FIPV spike now allowed for selection of candidate recombinants by their regained ability to grow in feline cells. In this fashion, we reconstructed a wild-type recombinant virus (r-wtFIPV) and generated a directed mutant FIPV in which the initiation codon of the nonstructural gene 7b had been disrupted (FIPV Delta 7b). The r-wtFIPV was indistinguishable from its parental virus FIPV 79-1146 not only for its growth characteristics in tissue culture but also in cats, exhibiting a highly lethal phenotype. FIPV Delta 7b had lost the expression of its 7b gene but grew unimpaired in cell culture, confirming that the 7b glycoprotein is not required in vitro. We establish the second targeted RNA recombination system for coronaviruses and provide a powerful tool for the genetic engineering of the FIPV genome.

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Figures

FIG. 1.

FIG. 1.

Overview of the targeted recombination strategy for FIPV. The scheme shows the construction of mFIPV (A) and r-wtFIPV (B) by targeted recombination between FIPV 79-1146 and synthetic donor RNA B and between mFIPV and synthetic donor RNA A, respectively. A single crossover event anywhere within the 3′ domain of the ORF 1b gene present in the donor RNA (indicated by a cross) generates a recombinant genome. In the first step (section A), the recombinant mFIPV acquires the ectodomain-encoding region of the MHV S gene (dotted); in the second step (section B), r-wtFIPV regains the feline S gene. mFIPV should lose the ability to infect feline cells and simultaneously gain the ability to infect murine cells and vice versa for r-wtFIPV.

FIG. 2.

FIG. 2.

Construction and composition of the transcription vectors pBRDI1 and pBRDI2, the templates for the synthetic donor RNA fragments A and B, respectively. (A) Shown at the top is a schematic representation of the FIPV 79-1146 genome. The shaded bars represent the various FIPV cDNA clones used to assemble pBRDI1. For details, see Materials and Methods. Small arrows indicate the primers (see Table 1 for primer numbers, sequences, and locations) used to generate the various fragments (see Materials and Methods). The dotted bars represent sequences encoding the MHV S ectodomain (pTMFS and pTMFS1 are not drawn to scale). The broad line at the left of each vector indicates pBRXN vector sequences. T7, 5′, 3′, and A indicate the T7 RNA polymerase transcription promoter sequence, the 5′- and 3′-untranslated region sequences, and a polyadenylate segment, respectively. Restriction sites relevant to plasmid construction are shown. (B) Some sequences are specified: I, the T7 promoter sequence preceded by a _Xho_I restriction sequence and followed by the sequence of the 5′-terminal region; the transcriptional start is indicated by an asterisk; and II, the in-frame transition of the 5′ ORF 1a and the 3′ ORF 1b sequences. Base pairs changed to create an unique _Sac_I restriction site are in italics and in boldface. Below the nucleotide sequence the amino acid sequence is shown, with the residues that were changed due to the introduction of the _Sac_I site indicated in boldface.

FIG. 3.

FIG. 3.

RT-PCR analysis of recombinant mFIPV. RT-PCR was used to amplify the FIPV-MF S boundaries with RNA isolated from cells infected with mFIPV as a template. (A) Physical map of the genome of the mFIPV recombinant. The dotted area indicates the MF-S gene. The location of the primers used is indicated with an arrow, together with the expected size of the PCR products. (B) RT-PCR with primers PR1254 and PR1255. (C) RT-PCR with primers PR-990 and PR311. See Table 1 for primer sequences. Lanes −RT, no reverse transcriptase added; lanes +RT, reverse transcriptase added; lanes H2O, H2O replacing the template; lanes Contr., pBRDI2 used as a template.

FIG. 4.

FIG. 4.

Viral proteins in mFIPV-infected LR7 cells (A), in r-wtFIPV infected FCWF cells (B), and in FIPVΔ7b-infected FCWF cells (C). In panel A, analyses were done with LR7 cells infected with mFIPV and, for comparison, with MHV-infected LR7 cells and FIPV-infected FCWF cells. In panel B, analyses were done with r-wtFIPV-infected FCWF cells and, for comparison, with FIPV-infected FCWF cells, and in panel C, analyses were done with FIPVΔ7b-infected FCWF cells and, for comparison, with FIPV- and r-wtFIPV-infected FCWF cells. In all cases the cells were labeled for 2 h with 35S-labeled amino acids. Immunoprecipitations were performed on aliquots of cleared lysates of these cells by using the following antibodies: ascitic fluid G73 (αFIPV) from an FIPV-infected cat; K134 (αMHV), a rabbit serum raised against purified MHV strain A59; WA3.10 (αSm), an MAb against an epitope present in the MHV S ectodomain; and 23F4.5 (αSf), an MAb against an epitope in the FIPV S ectodomain. All samples were heated at 95°C prior to electrophoresis, except for the sample analyzed in panel A, lane 4, which was run without heating and analyzed in sodium dodecyl sulfate-12.5% polyacrylamide gels. The positions of the S, N, M, and 7b (C) proteins are indicated at the left for MHV and at the right for FIPV.

FIG. 5.

FIG. 5.

Immunofluorescence analysis of mFIPV infected cells. LR7 cells were infected with mFIPV. For comparison, LR7 cells and FCWF cells were infected with MHV and FIPV, respectively. Infections were visualized at 6 h p.i. by immunofluorescence microscopy on permeabilized cells with the following antibodies: ascitic fluid G73 (αFIPV) from an FIPV-infected cat; 23F4.5 (αSf), an MAb against an epitope in the FIPV S ectodomain; and WA3.10 (αSm), an MAb against an epitope present in the MHV S ectodomain.

FIG. 6.

FIG. 6.

Growth of mFIPV in murine cells (A) and of r-wtFIPV and FIPVΔ7b in feline cells (B). Single-step growth kinetics of mFIPV and MHV in LR7 cells (A) and of r-wtFIPV, FIPVΔ7b, and FIPV in FCWF cells (B). The viral infectivity in the culture medium was determined at different times postinfection by a quantal assay on LR7 cells or FCWF cells, and the 50% tissue culture infective doses (TCID50) were calculated.

FIG. 7.

FIG. 7.

RT-PCR analysis of the r-wtFIPV, FIPVΔ7b, and FIPVΔMlu recombinants. RT-PCR was used to amplify the ORF 7b region (A and B) and the N gene region (C and D) with RNA isolated from cells infected with r-wtFIPV and FIPVΔ7b (A) and with r-wtFIPV and FIPVΔMlu (C) as a template, respectively. A physical map of the FIPV genome is shown in the middle of the figure. The locations of the primers (see Table 1 for sequences) are indicated by arrows, together with the expected sizes of the PCR products predicted for the indicated primer pairs. (B) Sequence of the ORF 7ab region with the gene 7b start codon in boldface and italics (bottom) and with the introduced mutations disrupting this codon and creating a _Pml_I restriction site indicated (top, underlined). (A) RT-PCR products obtained with RNA from four independent FIPVΔ7b mutant viruses with primers PR76 and PR1295. Undigested products are at the top, and products digested with _Pml_I are at the bottom. (D) Partial sequence of the N gene with the _Mlu_I restriction site underlined. (C) RT-PCR products obtained with RNA from four independent FIPVΔMlu mutant viruses with the primers PR1248 and PR1251. Products digested with _Xba_I are at the top, and products digested with _Xba_I and _Mlu_I are at the bottom.

FIG. 8.

FIG. 8.

In vivo survival after infection with FIPV 79-1146 and r-wtFIPV. Kittens (5 months old) were inoculated with 100 PFU of FIPV 79-1146 (n = 4) or r-wtFIPV (n = 5).

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