Characterization of monoclonal antibodies against feline coronavirus accessory protein 7b

Abstract

Feline coronaviruses (FCoVs) encode five accessory proteins termed 3a, 3b, 3c, 7a and 7b of unknown function. These proteins are dispensable for viral replication in vitro but are supposed to play a role in virulence. In the current study, we produced and characterized 7b-specific monoclonal antibodies (mAbs). A recombinant form of the 7b protein was expressed as a fusion protein in Escherichia coli, purified by immobilized metal affinity chromatography and used as immunogen. Two hybridoma lines, 5B6 and 14D8, were isolated that expressed mAbs that recognized 7b proteins of both FCoV serotypes. Using an extensive set of N- and C-terminally truncated 7b proteins expressed in E. coli and a synthetic peptide, the binding sites of mAbs 5B6 and 14D8 were mapped to an 18-residue region that comprises the only potential N-glycosylation site of the FCoV 7b protein. The two mAbs were suitable to detect a 24-kDa protein, which represents the nonglycosylated form of 7b in FCoV-infected cells. We speculate that glycosylation of 7b is part of the viral evasion strategy to prevent an immune response against this antigenic site.

Identification and characterization of a Golgi retention signal in feline coronavirus accessory protein 7b

“…In a previous study, we reported on the generation of mAbs against the FCoV 7b protein [30]. The mAbs obtained in this study detected the 7b protein in its non-glycosylated precursor form, but unfortunately failed to detect the mature, glycosylated form of the 7b protein.…”

“…Unfortunately, the authentic glycosylated protein was not recognized by the mAbs, but only by its non-glycosylated precursor [30]. Using a recombinant virus that encodes 7b without the single N-glycosylation site, we were able to show that 7b is a non-secreted glycoprotein located in the Golgi apparatus.…”

“…The lysate was incubated on a rotary shaker at 4 C for 30 min and then centrifuged (13 000 r.p.m, 4 C, 30 min). Anti-7b mAb 14D8 [30] was added to the supernatant and incubated on a rotary shaker for 1 h at 4 C. Protein G Sepharose (GE Healthcare) was added to the sample and incubated for another 1 h. Following centrifugation and washing, the pellet was mixed with protein loading buffer (2 % SDS, 10 % glycerol, 5 % b-mercaptoethanol, 0.01 % bromophenol blue) and incubated for 10 min at 96 C. After a brief centrifugation step, proteins in the supernatant were separated in a 10 % tricine-polyacrylamide gel [48]. Following transfer to a PVDF membrane (Roth), proteins were visualized by Ponceau staining and the band corresponding to the non-glycosylated form of 7b (24 kDa) was excised and subjected to amino-terminal sequence analysis by automated Edman degradation on a gas-phase sequencer, Procise model 491 (Applied Biosystems).…”

“…The membrane was blocked, washed with PBST (phosphate-buffered saline containing 0.1 % Tween-20) and incubated with primary antibody for 1 h at room temperature. To detect 7b protein, anti-7b mAb 14D8 [30] or anti-FLAG mAb (Sigma) was used. After washing with PBST, the membrane was incubated with horseradish peroxidase-conjugated goat anti-mouse antibody (Dianova) for 1 h at room temperature.…”

Diagnóstico serológico y molecular del coronavirus felino en las américas

“…El Coronavirus felino (FCoV) pertenece al orden Nidovirales, familia Coronaviridae, subfamilia Coronavirinae, género Alphacoronavirus y especie Alphacoronavirus 1; es un virus RNA de cadena simple con polaridad positiva (ssRNA+), envuelto, su genoma es de acerca 30 kilobases (Kb), el cual tiene 11 marcos de lectura abiertos (ORFs) que codifican para 5 proteínas accesorias: 3a, 3b, 3c, 7a y 7b, 16 proteínas no estructurales que forman el complejo de replicación/transcripción viral que se encuentran codificadas en las ORFs 1a y 1b, y 4 proteínas estructurales: la proteína de la nucleocápside (N), de espícula (S), de envoltura (E) y de matriz (M) (Figura 1) (1). En la envoltura del FCoV se encuentra la proteína de espícula (S), esta glicoproteína media la entrada del virus a la célula huésped uniéndose a los receptores que se encuentran en la membrana celular, como el receptor aminopeptidasa N (APN) o receptores de lectina tipo C de células dendríticas, como el DC-SIGN, estos son usados por el FCoV tipo II.…”