Mutation in Spike Protein Cleavage Site and Pathogenesis of Feline Coronavirus

Beth N. Licitra¹, Jean K. Millet¹, Andrew D. Regan, Brian S. Hamilton, Vera D. Rinaldi, Gerald E. Duhamel, and Gary R. Whittaker

Author affiliations: Cornell University College of Veterinary Medicine, Ithaca, New York, USA

Abstract

Feline coronaviruses (FCoV) exist as 2 biotypes: feline enteric coronavirus (FECV) and feline infectious peritonitis virus (FIPV). FECV causes subclinical infections; FIPV causes feline infectious peritonitis (FIP), a systemic and fatal disease. It is thought that mutations in FECV enable infection of macrophages, causing FIP. However, the molecular basis for this biotype switch is unknown. We examined a furin cleavage site in the region between receptor-binding (S1) and fusion (S2) domains of the spike of serotype 1 FCoV. FECV sequences were compared with FIPV sequences. All FECVs had a conserved furin cleavage motif. For FIPV, there was a correlation with the disease and >1 substitution in the S1/S2 motif. Fluorogenic peptide assays confirmed that the substitutions modulate furin cleavage. We document a functionally relevant S1/S2 mutation that arises when FIP develops in a cat. These insights into FIP pathogenesis may be useful in development of diagnostic, prevention, and treatment measures against coronaviruses.

Feline infectious peritonitis (FIP) is a fatal infection that affects domestic and wild members of the family Felidae and is caused by a feline coronavirus (FCoV) of the family Coronaviridae, subfamily Coronavirinae, genus Alphacoronavirus, species Alphacoronavirus-1 (1). The FCoV genome is ≈29 kB and has 11 open reading frames encoding replicative, structural, and accessory proteins (2). Two serotypes have been identified. Serotype 1 FCoVs are highly prevalent clinically (3–5) but grow poorly in cell culture and are therefore underevaluated when compared with serotype 2 FCoVs, which are easily propagated in vitro but less prevalent.
Within each serotype, there are 2 biotypes, each causing distinct disease outcomes. Feline enteric coronavirus (FECV) of serotypes 1 and 2 infects enterocytes, causing mild and generally self-limiting infections. FECV spreads efficiently through the oral–fecal route, and chronically infected cats can shed infectious virus in feces for a year or longer (6,7). The second biotype found in both serotypes, FIP virus (FIPV) is found less frequently but causes FIP.
The current understanding is that FIPV arises during in vivo infection from a genetic mutation of FECV (8–11). A long-standing hypothesis is that FIP viruses arise from internal mutation of endemic FECVs (12), which is believed to occur in approximately 1%–5% of enteric infections, resulting in the ability of the virus to infect blood monocytes and tissue macrophages. The resulting productive infection of these cells, a hallmark of FIP, enables systemic spread and results in macrophage activation, with concomitant immune-mediated events leading to death. To date, the precise mutation or mutations that cause a shift in FCoV biotype have not been identified.
As with other RNA viruses, coronavirus replication is error-prone; the estimated mutation rate is ≈4 × 10⁻⁴ nucleotide substitutions/site/year (13,14). It has been suggested that mutations in the 3c and 7b genes may be involved in the transition to FIPV (1,12,15). Because FCoV spike protein plays critical roles in receptor binding (S1) and fusion (S2), we focused on structural changes in this protein and potential role in altered cellular tropism. In particular, acquisition of macrophage tropism for a serotype 2 FCoV has previously been mapped to the spike gene (16), further suggesting that key mutations within spike protein may be important for the biotype switch.
The coronavirus spike protein is a class I fusion protein, which typically requires activation by cellular proteases. Mutation of the proteolytic cleavage site often has profound implications for disease progression (17,18). Until recently, FCoVs were thought to have uncleaved spike protein. However, a functional furin cleavage site has been identified in 2 serotype 1 FECVs, located at the shared boundary of the S1 and S2 subunits (19). Furin is a ubiquitous proprotein convertase enriched in the trans-Golgi network and is well-conserved among mammals (20). Furin cleaves a wide range of protein precursors into biologically active products at a consensus motif R-X-K/R-R, where R is the basic arginine residue, X is any residue, and K is the basic lysine residue (21).
In this article, we establish a novel approach to studying FIP that complements previous work. Instead of performing a mutation study based mainly on comparative genetic analysis (15,22–24), we focus on S1/S2, a functionally relevant site, and study variations between the biotypes and their functional effects. This rationale could provide a better means to uncover functionally important mutations that account for FIP.
We considered that mutations at the S1/S2 site could alter proteolytic cleavage and modify S fusogenic properties, leading to tropism expansion, systemic spread and, ultimately, FIP. We investigated genetic variations at the S1/S2 site of serotype 1 FECVs and compared these sequences to those present in viral RNA recovered from tissues of cats with FIP. Fluorogenic peptide cleavage assays were conducted to assess the effects of substitutions found in the S1/S2 site. We document a junction mutation at S1/S2 that arises during development of FIP. Our study has uncovered a molecular basis for FIP that has potential to lead to developments in diagnostics, prevention, and therapies.