Abstract
Transporter associated with antigen processing (TAP), a key player in the major histocompatibility complex class I-restricted antigen presentation, makes an attractive target for viruses that aim to escape the immune system. Mechanisms of TAP inhibition vary among virus species. Bovine herpesvirus 1 (BoHV-1) is unique in its ability to target TAP for proteasomal degradation following conformational arrest by the UL49.5 gene product. The exact mechanism of TAP removal still requires elucidation. For this purpose, a TAP-GFP (green fluorescent protein) fusion protein is instrumental, yet GFP-tagging may affect UL49.5-induced degradation. Therefore, we constructed a series of TAP-GFP variants using various linkers to obtain an optimal cellular fluorescent TAP platform. Mel JuSo (MJS) cells with CRISPR/Cas9 TAP1 or TAP2 knockouts were reconstituted with TAP-GFP constructs. Our results point towards a critical role of GFP localization on fluorescent properties of the fusion proteins and, in concert with the type of a linker, on the susceptibility to virally-induced inhibition and degradation. The fluorescent TAP platform was also used to re-evaluate TAP stability in the presence of other known viral TAP inhibitors, among which only UL49.5 was able to reduce TAP levels. Finally, we provide evidence that BoHV-1 UL49.5-induced TAP removal is p97-dependent, which indicates its degradation via endoplasmic reticulum-associated degradation (ERAD).
Highlights
The co-existence of a host and a virus depends on a subtle balance between the pathogen replication and the host immune response
The random linkers used to join Transporter associated with antigen processing (TAP) and GFP have resulted from the cloning procedure into one of the pEGFP
The statistical significance of differences between Mel JuSo (MJS) TAP2-N-GFP cells and cells with viral inhibitor was assessed by t-test; *** p ≤ 0.001 * p ≤ 0.05. (C) The mean fluorescence intensity of GFP was analyzed by flow cytometry and presented as the percentage of GFP fluorescence of MJS TAP2-N-GFP cells
Summary
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
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