**1. Introduction**

The co-existence of a host and a virus depends on a subtle balance between the pathogen replication and the host immune response. Virus-derived peptides, originating mainly from the proteasomal degradation, are presented by the major histocompatibility complex class I (MHC I) molecules, leading to the recognition of an infected cell by cytotoxic CD8+ T lymphocytes (CTLs) (reviewed in [1]). The transporter associated with antigen processing (TAP) plays a pivotal role in MHC I-restricted antigen presentation, which makes it an attractive target for viruses that aim to escape the immune system.

TAP is a heterodimer belonging to the ATP-binding cassette (ABC) family transporters. It consists of two subunits, TAP1 (ABCB2) and TAP2 (ABCB3) [2]. The core of each subunit is formed by an N-terminally-located transmembrane domain (TMD), composed of six transmembrane helices

(TMs), responsible for peptide recognition and binding [3], and a highly conserved C-terminal nucleotide-binding domain (NDB), which can bind and hydrolyze ATP [4]. Acquiring both substrates, ATP and the peptide, occurs independently [3]. This induces conformational rearrangements within TAP, resulting in a switch from an inward-open to an outward-facing conformation and release of the peptide into the lumen of endoplasmic reticulum (ER). Afterward, ATP hydrolysis triggers the release of phosphate and restores the resting state of TAP [5]. The presence of core-flanking TMD0 domains (four TMs in TAP1 and three TMs in TAP2) at the N termini of TAP1/TAP2 is not necessary for peptide transport; however, it is crucial for assembly of the peptide-loading complex (PLC) and subsequent exposure of antigenic peptides to CTLs [6].

During co-evolution with their hosts, several herpesviruses and a single known (to date) poxvirus have specialized in TAP inhibition via diverse mechanisms (reviewed in [7]). Herpes simplex virus 1 and 2 (HSV-1 and HSV-2) encode the ICP47 protein, which competes for the peptide-binding site and, through its characteristic structure, tethers the TAP-ICP47 complex in an inward-facing conformation [8–10]. In contrast, the US6 protein of human cytomegalovirus (HCMV) [11–13] and the cowpox virus (CPXV) strain Brighton Red-encoded CPXV012 protein can inhibit ATP binding to NDBs while leaving peptide binding una ffected [14–16]. Mechanisms of TAP inhibition by herpesvirus UL49.5 protein family encoded by members of the *Varicellovirus* genus are still not fully understood and seem to di ffer in detail between virus species. Most of the UL49.5 orthologs inhibit conformational rearrangements within TAP [17]. Bovine herpesvirus 1 (BoHV-1) UL49.5 seems to be unique in its ability to target bovine, human, and murine TAP for proteasomal degradation following the conformational arrest [7,18,19]. Varicella-zoster virus (VZV)-encoded UL49.5 can bind TAP, ye<sup>t</sup> it exhibits no inhibitory properties [20]. TAP degradation activity was also described for the murine gammaherpesvirus-68 MK3 protein [21] and the rodent herpesvirus Peru pK3 ortholog [22], which both carry a cytoplasmic RING (really interesting new gene) finger domain and can act towards the murine transporter. The recently described poxvirus molluscum contagiosum virus MC80 protein can destabilize human TAP; however, in contrast to BoHV-1 UL49.5, the transporter is not the primary target of the inhibitor [23].

Recently, fluorescent tags and gene fusion technology have become indispensable in a wide range of biochemical and cell biology applications, nevertheless in some circumstances designing a functional fluorescent fusion protein remains challenging. Numerous studies have shown that a choice of a linker may have a significant impact on proper folding, yield, and functionality of the fusion protein and its interaction with other proteins. Flexible linkers are usually applied to provide a certain degree of movement, while rigid linkers are preferable to separate two bioactive domains spatially [24].

To investigate the mechanism of TAP inhibition or removal, a TAP-GFP (green fluorescent protein) fusion protein was instrumental, ye<sup>t</sup> GFP-tagging was observed to abolish the susceptibility of TAP to degradation induced by the BoHV-1-encoded UL49.5 [18]. Here, we report the construction of a series of full-length TAP1 and TAP2 variants carrying either N- or C-terminal GFP with di fferent types of linkers and evaluate the impact of the TAP-GFP fusion design on their fluorescence and functionality, as well as susceptibility to virus-induced inhibition and degradation. Such a fluorescent TAP platform may constitute a platform to explain the molecular mechanism of UL49.5 activity and potentially contribute to better characterization of the transporter itself.

### **2. Materials and Methods**

### *2.1. Cells and Viruses*

Madin-Darby bovine kidney (MDBK) cells (ATCC,Manassas, VA, USA, CCL-22), human melanoma Mel JuSo (MJS) cells, MJS TAP1 CRISPR/Cas9 knock-out (TAP1 KO), MJS TAP2 CRISPR/Cas9 knock-out (TAP2 KO) [25], and U937 (ATCC, CRL-1593) were cultured in RPMI 1640 (Corning, Corning, NY, USA) supplemented with 10% fetal bovine serum (FBS, Thermo Scientific (Thermo Scientific, Waltham, MA, USA)) and Antibiotic Antimycotic Solution (Thermo Scientific). Lenti-X HEK293T and GP2-293 cells (both from Takara/Clontech, Kusatsu, Japan) used for lentivirus and retrovirus

production, respectively, were cultured in Iscove's modified Dulbecco's medium (IMDM, Lonza, Basel, Switzerland) supplemented as above. HEK293T (ATCC, CRL-3216) cells were cultured in Dulbecco's modified Eagle's medium (DMEM, high glucose, Lonza) supplemented as above. BoHV-1 field strain Lam (Institute for Animal Health and Science, Lelystad, The Netherlands) was propagated and titrated on MDBK cells.
