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

Abstract

Feline coronaviruses encode five accessory proteins with largely elusive functions. Here, one of these proteins, called 7b (206 residues), was investigated using a reverse genetic approach established for feline infectious peritonitis virus (FIPV) strain 79-1146. Recombinant FIPVs (rFPIVs) expressing mutant and/or FLAG-tagged forms of 7b were generated and used to investigate the expression, processing, glycosylation, localization and trafficking of the 7b protein in rFIPV-infected cells, focusing on a previously predicted ER retention signal, KTEL, at the C-terminus of 7b. The study revealed that 7b is N-terminally processed by a cellular signalase. The cleavage site, 17-Ala|Thr-18, was unambiguously identified by N-terminal sequence analysis of a 7b processing product purified from rFIPV-infected cells. Based on this information, rFIPVs expressing FLAG-tagged 7b proteins were generated and the effects of substitutions in the C-terminal 202KTEL206 sequence were investigated. The data show that (i) 7b localizes to and is retained in the medial- and/or trans-Golgi compartment, (ii) the C-terminal KTEL sequence acts as a Golgi [rather than an endoplasmic reticulum (ER)] retention signal, (iii) minor changes in the KTEL motif (such as KTE, KTEV, or the addition of a C-terminal tag) abolish Golgi retention, resulting in relocalization and secretion of 7b, and (iv) a KTEL-to-KDEL replacement causes retention of 7b in the ER of rFIPV-infected feline cells. Taken together, this study provides interesting new insights into an efficient Golgi retention signal that controls the cellular localization and trafficking of the FIPV 7b protein in virus-infected feline cells.

Development of HiBiT-Tagged Recombinant Infectious Bronchitis Coronavirus for Efficient in vitro and in vivo Viral Quantification

“…In general, the insertion of short epitope tags in recombinant CoVs was better tolerated. For instance, recombinant CoVs with epitope tags fused with structural, non-structural, or accessory proteins have been described (Yamada and Liu, 2009 ; Venkatagopalan et al, 2015 ; Athmer et al, 2017 ; Florek et al, 2017 ; Kaewborisuth et al, 2018 ; Tan et al, 2018 ). These traditional tags (such as FLAG, HA, Myc) have facilitated the study of protein processing, protein-protein interaction, subcellular localization, membrane topology and so on.…”

“…All three rIBVs replicated similarly to the parental control in cell culture (Yamada and Liu, 2009 ; Tan et al, 2018 ). Short epitope tags have also been successfully incorporated in other coronaviruses, such as a tetracysteine (TC)-tag in the E protein of MHV (Venkatagopalan et al, 2015 ), an HA-tag in the nsp15 of MHV (Athmer et al, 2017 ), a Myc-tag in the ORF3 of porcine epidemic diarrhea virus (PEDV) (Kaewborisuth et al, 2018 ), and a FLAG-tag in the 7b protein of feline infectious peritonitis (FIPV) (Florek et al, 2017 ).…”

Reverse Genetics for Type I Feline Coronavirus Field Isolate To Study the Molecular Pathogenesis of Feline Infectious Peritonitis

“…In total, eight rounds of vaccinia virus-mediated homologous recombination performed with Escherichia coli guanosine-phosphoribosyltransferase (GPT) as a selection marker were used to exchange the serotype II FCoV strain 79-1146 cDNA with the FECV field isolate full-length cDNA in the vaccinia virus backbone. Selection of recombinant vaccinia viruses was performed as described previously ( 40 , 46 , 47 , 68 – 70 ).…”

“…To recover recombinant FCoVs, DNA derived from recombinant vaccinia viruses was extracted in preparative scale, cleaved with ClaI (NEB), and used as a template for in vitro transcription as described previously ( 40 , 46 , 47 , 68 , 70 ). The in vitro -transcribed RNA was electroporated into BHK-Tet/ON-N FECV cells.…”

The alphaherpesvirus conserved pUS10 is important for natural infection and its expression is regulated by the conservedHerpesviridaeprotein kinase (CHPK)

“…and Golgi retention sequence (GRS) were identified based on previous studies [97][98][99] or predicted using NLStradamus [100] and MyHits [101]. PC chickens were infected with vCHPKwt/10HA or vΔCHPK/10HA as described in the Materials & Methods.…”