*2.2. DNA Constructs*

All TAP constructs were cloned in lentiviral vectors downstream of an EF1 α promoter.

For unmodified (wild-type, wt) TAP1 or TAP2 reconstitution, dual promoter lentiviral vectors described in [25] (pPuroR-GFP-TAP1 and pZeoR-mAmetrine-TAP2) were used. mAmetrine and marker GFP genes were removed from these vectors. Fragments of TAP1 and TAP2 sequences were ordered as synthetic genes designed for cloning in pEGFP-N3 or pEGFP-C1 (Takara/Clontech). For TAP1-N-GFP (TAP1 with the N-terminal GFP, random linker), TAP1-C-GFP (TAP1 with the C-terminal GFP, random linker), TAP2-N-GFP (TAP2 with the N-terminal GFP, random linker), and TAP2-C-GFP (TAP2 with the C-terminal GFP, random linker), fusion genes were re-cloned in the original lentiviral vectors. The amino acid sequences of random linkers resulting from the cloning procedure are depicted in Figure 1A. Fragments coding for TAP1 with helical linker sequences were ordered as synthetic genes designed for cloning in pEGFP-N3 or pEGFP-C1. TAP1-HN-GFP (TAP1 with the N-terminal GFP, helical linker) or TAP1-HC-GFP (TAP1 with the C-terminal GFP, helical linker) were re-cloned in the lentiviral vector pCDH-EF1 α-MCS-(PGK-Puro) (System Biosciences, Palo Alto, CA, USA).

Genes coding for viral TAP inhibitors were cloned in retroviral vectors downstream of a retroviral promoter. The BoHV-1 UL49.5 gene was cloned from pLZRS-BoHV-1 UL49.5-IRES-GFP [18] in BamHI-EcoRI sites of pLZRS-IRES-ΔNGFR [26]. The VZV UL49.5 gene was amplified from the pLZRS-VZV UL49.5-IRES-GFP vector [20] using KOD Hot Start DNA polymerase (Merck, Darmstadt, Germany) and the following primers: forward 5'-CGGGATCCCACCATGGGATCAATTACC-3' and reverse 5'-CCGGAATTCTTACCACGTGCTGCGTAATAC-3'. The PCR product was verified by DNA sequencing and introduced into BamHI and EcoRI sites of the pBABEpuro vector [27]. Synthetic genes encoding: myc-tagged HSV-1 ICP47 (Gene ID: 2703441), myc-tagged HCMV US6 (Gene ID: 3077555), or the FLAG-N-CPXV012 (Gene ID: 1485887) variantlacking six N-terminal amino acid residues [28] were introduced into BamHI and EcoRI restriction sites of pBABEpuro.

### *2.3. Retroviral and Lentiviral Transduction*

For the production of recombinant lentiviruses, third-generation packaging vectors based on the pRSV-Rev and pCgpV plasmids (Cell Biolabs, San Diego, CA, USA), the obtained lentiviral expression vectors, and pCMV-VSV-G (Cell Biolabs) for pseudotyping were co-transfected into Lenti-X HEK293T cells using CalPhos mammalian transfection kit (Takara/Clontech). For recombinant retroviruses, a transfer plasmid (pBABEpuro-based or pLZRS-IRES-ΔNGFR-based) and pCMV-VSV-G were co-transfected into GP2-293 packaging cells as above. Twenty-four hours after transfection the medium was refreshed; for lentiviruses it was supplemented with 1 mM sodium butyrate (Sigma-Aldrich, Saint Louis, MO, USA). Virus-containing supernatants were collected after 48 h, concentrated with PEGit (System Biosciences), and used for transduction in the presence of 0.01 mg mL−<sup>1</sup> polybrene (Sigma-Aldrich). MJS cells with TAP1 or TAP2 knock-outs were stably reconstituted with the wt or fluorescent TAP1 or TAP2 constructs using lentivirus vectors and cell-sorting for GFP- and MHC I-positive cells. The cells were subsequently transduced with a retrovirus coding for BoHV-1 UL49.5 and sorted for nerve growth receptor (NGFR)-positive cells or with a retrovirus coding for HSV-1 ICP47, HCMV US6, VZV UL49.5, or CPXV012, and selected with puromycin (2 μg mL−1) (Sigma-Aldrich).

**Figure 1.** Construction and characterization of fluorescent transporter associated with antigen processing (TAP)-green fluorescent protein (GFP) variants. Mel JuSo (MJS) cells with CRISPR/Cas9 TAP1 or TAP2 knockouts (T1KO, T2KO) were stably reconstituted with fluorescent TAP1 or TAP2 constructs using lentivirus vectors and cell sorting. (**A**) Schematic representation of TAP-GFP constructs. Secondary structures of linkers flanked by ten amino acid residues of fused proteins were determined by the Geneious software; α-helices are depicted in pink, coiled regions in gray, β-strands in yellow, and turns in blue. (**B**) Representative histograms of TAP-GFP fluorescence intensity. (**C**) Comparative TAP-GFP analysis by flow cytometry. The mean fluorescence intensity of three independent measurements is represented as bars with standard deviations. The statistical significance was assessed by *t*-test; *p* ≤ 0.001. (**D**–**F**) Expression of TAP-GFP variants in stable cell lines was determined by SDS-PAGE and immunoblotting using: (**D**) anti-GFP monoclonal antibody (Mab) (**E**) anti-TAP2 MAb (**F**) anti-TAP1 MAb. β-actin was used as a loading control. Abbreviations: T2-C: TAP2-C-GFP (TAP2 with the C-terminal GFP, random linker); T2-N: TAP2-N-GFP (TAP2 with the N-terminal GFP, random linker); T1-C: TAP1-C-GFP (TAP1 with the C-terminal GFP, random linker); T1-HC: TAP1-HC-GFP (TAP1 with the C-terminal GFP, helical linker); T1-N: TAP1-N-GFP (TAP1 with the N-terminal GFP, random linker); T1-HN: TAP1-HN-GFP (TAP1 with the N-terminal GFP, helical linker).

### *2.4. Plasmid Transfection*

HEK293T cells were transfected with plasmids encoding fluorescent TAP variants using JetPRIME (Polyplus-transfection, Illkirch, France) according to the manufacturer's protocol and analyzed after 24 h by flow cytometry.

### *2.5. Generation of A TAP1*/*TAP2 Double Knock-Out U937 Cells for Reconstitution with Fluorescent TAP Variants*

U937 TAP1/TAP2 KO cells were generated with a strategy described for MJS TAP1/TAP2 KO in [25] Briefly, U937 cells were transfected with pSico-CRISPR-PuroR containing the TAP2-targeting crRNA sequence 5'-GGAAGAAGAAGGCGGCAACG-3'. The cells were selected with puromycin (4 μg mL−1), and cloned by limiting dilution. Individual clones were analyzed by flow cytometry to identify clones with low cell surface MHC I expression, followed by immunoblotting and DNA sequencing of the genomic target site. A clone lacking TAP2 was subsequently transfected with a pSicoR-CRISPR-PuroR vector containing the TAP1-targeting crRNA sequence 5'-GGGGTCCTCAGGGCAACGGT-3'. After selection with puromycin and cell cloning, the clones were analyzed for TAP1 expression by immunoblotting and DNA sequencing of the genomic target site. Genomic DNA sequence analysis revealed a 16-bp deletion around the TAP2 gRNA target site and multiple short deletions altering the whole TAP1 gene sequence downstream of the target site. A monoclonal cell line lacking TAP1 and TAP2 was used for reconstitution with a combination of unmodified and fluorescent TAP-encoding sequences delivered by lentivirus vectors. Reconstituted U937 cells were sorted for GFP and high MHC I expression. The cells were subsequently transduced with the BoHV-1 UL49.5-encoding retrovirus and sorted for NGFR.
