Generating and evaluating type I interferon receptor-deficient and feline TMPRSS2-expressing cells for propagating serotype I feline infectious peritonitis virus

Abstract

Feline coronavirus infection can progress to a fatal infectious peritonitis, which is a widespread feline disease without an effective vaccine. Generating feline cells with reduced ability to respond to interferon (IFN) is an essential step facilitating isolation of new candidate vaccine strains. Here, we describe the use of Crispr/Cas technology to disrupt type I IFN signaling in two feline cell lines, AK-D and Fcwf-4 CU, and evaluate the replication kinetics of a serotype I feline infectious peritonitis virus (FIPV) within these cells. We report that polyclonal cell populations and a clonal isolate, termed Fcwf-4 IRN, exhibited significantly diminished IFN-responsiveness and allowed FIPV replication kinetics comparable to parental cells. Furthermore, we demonstrate that replication of FIPV is enhanced by ectopic expression of a host serine protease, TMPRSS2, in these cells. We discuss the potential of these cells for isolating new clinical strains and for propagating candidate vaccine strains of FIPV.

Keywords: AK-D cells; Crispr/Cas gene editing; FIPV; Fcwf-4 CU cells; Feline coronavirus; IFNαR-deficient cells; Interferon signaling-deficient cells; TMPRSS2-expressing cells.

Fig. 1

Determining the response of AK-D 2.2 cells to type I interferon and characterizing the replication kinetics and plaque formation of FIPV Black in AK-D 2.2 cells. A) AK-D (black) or AK-D 2.2 (green) cells were treated with increasing concentrations of feline interferon alpha (IFNα). After 6 h, total RNA was extracted and analyzed by qPCR for ISG54 and feline β-actin. ISG54 mRNA expression was normalized to β-actin then presented as average 2^−ΔCt values. B) Growth kinetics of FIPV Black (MOI = 0.1) determined by plaque assay of infected AK-D (black) and AK-D 2.2 (green) cell supernatants. Data representative of two to three independent experiments performed in triplicate and presented mean ± SD. Values were analyzed by unpaired t-tests. ***P < 0.001. C) Fcwf-4 CU cells were infected with MOI = 0.1 serotype I FIPV Black and supernatants were collected at 24 HPI after which plaque assay analysis was performed on AK-D, AK-D 2.2, or Fcwf-4 CU indicator cells. Images were taken at 48 (top) and 72 (bottom) HPI. Images are representative of three independent experiments.

Fig. 2

Evaluating the response to type I interferon and the kinetics of FIPV Black replication in Fcwf-4 parent and IFNαR-deficient cells. A) Fcwf-4 CU, Fcwf-4 CU 2.2 Poly, Fcwf-4 CU 2.2 clones 1, 2 and 3 (IRN cells) were treated with 0 or 1000 U feline IFNα for 6 h and ISG54 transcripts were measured by qPCR. B) FIPV Black growth kinetics (MOI = 0.1) at indicated hours post-infection (HPI) in Fcwf-4 CU and IRN cells. C) Nucleocapsid gene transcript levels measured by qPCR during FIPV Black infection (MOI = 0.1) of Fcwf-4 CU or IRN cells. D) FIPV Black growth kinetics (MOI = 0.01) in indicated cells pre-treated with 0 or 1000 U feline IFNα for 8 h. E) Plaque formation induced by FIPV Black in Fcwf-4 CU or IRN cells at 3–4 days post-infection (DPI). Wells display 10⁻5 virus dilution. F) Plaque sizes (mm²) measured from FIPV Black plaque assays (6-well plates) at 3 and 4 DPI on Fcwf-4 CU and IRN indicator cells at 10⁻5 and 10⁻6 virus dilutions. For mRNA expression, Ct values were normalized to β-actin using the 2^−ΔCt method and presented as fold expression over mock (A) or relative expression (B). Data represent 3 independent experiments in triplicate. Mean ± SD analyzed by unpaired t-tests. Viral titers (B and D) were calculated from triplicate plaque assays per time point on Fcwf-4 CU indicator cells and represent 3 independent experiments. Mean ± SD plaque-forming units (PFU) per mL analyzed by two-way ANOVA by time (D). Plaque sizes (F) were measured using Adobe Photoshop software and mean ± SD values were analyzed using unpaired t-tests. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

Fig. 3

Fcwf-4 IRN cells express an mRNA predicted to generate a truncated, null-mutant IFNαR protein. A) Deduced nucleotide and amino acid sequences of the IFNαR2 region targeted by Crispr/Cas technology. Single-guide RNA sequence target (underlined); protospacer-adjacent motif (PAM) (yellow); STOP codon and asterisk (red) indicate early termination of translation of the IFNαR2 protein in IRN cells. B) Feline IFNαR2 exon 1 expression determined from total RNAs collected from Fcwf-4 CU and Fcwf-4 IRN confluent monolayers. mRNA expression normalized to β-actin and presented as average 2^−ΔCt expression values. Data represent two independent experiments in triplicate. Mean ± SD analyzed by unpaired t-tests. **P < 0.01. C) Melt curves and maximum melt temperatures of IFNαR2 amplicons produced during qPCR of RNAs obtained from Fcwf-4 CU or Fcwf-4 IRN cells. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 4

Expressing TMPRSS2 in feline cells and evaluating the effect on FIPV Black replication. A) Immunofluorescence detection of feline TMPRSS2(V5) in HEK 293T/17 cells. 200 ng of pLVX-fTMPRSS2(V5) or pLVX (mock) was transfected into HEK 293T/17 cells for 18 h. Cells were stained with mouse-anti-V5 (1:500), and 1:1000 Alexa Flor 568-conjugated goat-anti-mouse IgG was used to visualize TMPRSS2(V5); Hoesch 3342 (1:1000) was used to stain nuclei. B) Detection of feline TMPRSS2(V5) by Western blot following transfection of HEK 293T/17 cells (left) or transduction of Fcwf-4 IRN cells with pLVX lentiviruses encoding TMPRSS2(V5) (right). The full length (55 kDa) and cleavage product (>25 kDa) of TMPRSS2 are indicated. Feline β-actin used to visualize protein loading. C-D) The impact of feline TMPRSS2 expression on FIPV replication evaluated in Fcwf-4 IRN cells. Indicated dilutions of pLVX-fTMPRSS2(V5) or Mock (empty) transducing particles were applied to Fcwf-4 IRN cells for 48 h prior to infection with FIPV Black (MOI = 0.1). RNA was isolated after 18 h infection and qPCRs were performed to detect TMPRSS2 (C), N gene (D), and β-actin transcripts. mRNA expression normalized to β-actin and presented as average 2^−ΔCt expression values. Data represent two independent experiments in triplicate. Mean ± SD analyzed by unpaired t-tests. **P < 0.01; ***P < 0.001; not significant (ns).

Fig. 5

Proposed model for IRN cells expressing TMPRSS2 for the recovery of serotype I FIPV from clinical samples. Serotype I FIPV entry likely occurs within endosomes via cathepsin [cyan] cleavage of the CoV spike at S2′ or at the cell surface via protease cleavage of S1/S2 following S engagement of the natural receptor (black) (top). The proposed models for tissue culture-adapted FCoV entry and recovery of serotype I FIPV from clinical samples are depicted in the lower panel. Tissue culture-adapted strains may express spikes (violet) that lose S1/S2 cleavage in favor of heparin (magenta) binding (bottom left). Expressing host factors, such as TMPRSS2 (green), may promote natural receptor usage and cell-surface entry, aiding in recovery of serotype I FCoV strains from clinical samples (bottom right).