Chimeric feline coronaviruses that encode type II spike protein on type I genetic background display accelerated viral growth and altered receptor usage

Abstract

Persistent infection of domestic cats with feline coronaviruses (FCoVs) can lead to a highly lethal, immunopathological disease termed feline infectious peritonitis (FIP). Interestingly, there are two serotypes, type I and type II FCoVs, that can cause both persistent infection and FIP, even though their main determinant of host cell tropism, the spike (S) protein, is of different phylogeny and displays limited sequence identity. In cell culture, however, there are apparent differences. Type II FCoVs can be propagated to high titers by employing feline aminopeptidase N (fAPN) as a cellular receptor, whereas the propagation of type I FCoVs is usually difficult, and the involvement of fAPN as a receptor is controversial. In this study we have analyzed the phenotypes of recombinant FCoVs that are based on the genetic background of type I FCoV strain Black but encode the type II FCoV strain 79-1146 S protein. Our data demonstrate that recombinant FCoVs expressing a type II FCoV S protein acquire the ability to efficiently use fAPN for host cell entry and corroborate the notion that type I FCoVs use another main host cell receptor. We also observed that recombinant FCoVs display a large-plaque phenotype and, unexpectedly, accelerated growth kinetics indistinguishable from that of type II FCoV strain 79-1146. Thus, the main phenotypic differences for type I and type II FCoVs in cell culture, namely, the growth kinetics and the efficient usage of fAPN as a cellular receptor, can be attributed solely to the FCoV S protein.

FIG. 1.

Generation of recombinant FCoVs and phenotypic analysis using FCWF-4 cells. (a) Schematic representation of the genome organization of type II FCoV strain 79-1146, recombinant type I FCoVs recFCoV and recFCoV-GFP, and recombinant chimeric FCoVs recFCoV-GFP-SII and recFCoV-SII. CCoV-derived sequences in the type II FCoV strain 79-1146 genome are indicated in gray, and approximate recombination sites are depicted by arrows. Type I FCoV strain Black-derived sequences are indicated in white, and GFP is indicated in black. Sequences derived from type II FCoV strain 79-1146 in the chimeric viruses recFCoV-GFP-SII and recFCoV-SII are indicated in gray. (b) Plaque morphology of FCoV strain Black, recFCoV, recFCoV-GFP, FCoV strain 79-1146, recFCoV-SII, and recFCoV-GFP-SII 48 h after infection of FCWF-4 cells. (c) Growth kinetics of recFCoV, recFCoV-SII, and FCoV strain 79-1146 after infection of FCWF-4 cells (MOI = 0.1).

FIG. 2.

Generation and infection of fAPN-expressing BHK-21 cells. (a) Immunofluorescence analysis of BHK-21 (left) and BHK-fAPN (right) cells. (b) GFP expression analyzed by fluorescence microscopy of mock-, recFCoV-GFP-, or recFCoV-GFP-SII-infected BHK-21 or BHK-fAPN cells at 36 h p.i. (MOI = 0.1). The white arrow depicts patches of green fluorescent BHK-fAPN cells after recFCoV-GFP infection.

FIG. 3.

Effect of MAb R-G-4 treatment on FCoV infection of FCWF-4 and BHK-fAPN cells. (a) Analysis of GFP expression of recFCoV-GFP (top row)- or recFCoV-GFP-SII (bottom row)-infected FCWF-4 cells by fluorescence microscopy at 36 h p.i. (MOI = 0.1). The leftmost panels show infected FCWF-4 cells without MAb R-G-4 treatment, and the following panels from left to right show FCWF-4 cells that have been treated with increasing amounts of MAb R-G-4 (1, 3, 5, 10, and 50 μl/per well). (b) Fluorescence-activated cell sorter (FACS) analysis of GFP-expressing FCWF-4 cells at 36 h p.i. with recFCoV-GFP (white bars) or recFCoV-GFP-SII (black bars) (MOI = 0.1). Cells were left untreated or were treated with MAb R-G-4 as described above (a). (c) GFP expression analyzed by fluorescence microscopy of recFCoV-GFP-infected BHK-fAPN cells at 36 h p.i. (MOI = 0.1). Cells were either left untreated or were treated with MAb R-G-4 (50 μl/per well). The white arrow depicts patches of green fluorescent cells that were also detectable in the presence of MAb R-G-4.

FIG. 4.

Effect of MAb R-G-4 treatment on FCoV infection of CD14⁺ feline monocytes. (a) Detection of GFP expression in untreated or MAb R-G-4-treated (50 μl/per well) CD14⁺ feline monocytes derived from donor 7 after infection with recFCoV-GFP or recFCoV-GFP-SII (MOI = 0.1) at 36 h p.i. (b) fAPN surface expression by FACS was assessed for FCWF cells and monocytes of donors 4 and 7 as indicated. Cells were incubated with MAb R-G-4, followed by a secondary FITC-labeled goat anti-mouse Ig antibody (green lines) or incubated with the FITC-labeled goat anti-mouse Ig antibody alone (red lines) (control). (c) Growth kinetics of recFCoV-GFP or recFCoV-GFP-SII after infection of untreated or MAb R-G-4-treated (50 μl/per well) CD14⁺ feline monocytes derived from donor 4 or 7 as indicated (MOI = 0.1).