Investigation of the Effects of Primary Structure Modifications within the RRE Motif on the Conformation of Synthetic Bovine Herpesvirus 1‐Encoded UL49.5 Protein Fragments

Abstract

Herpesviruses are the most prevalent viruses that infect the human and animal body. They can escape a host immune response in numerous ways. One way is to block the TAP complex so that viral peptides, originating from proteasomal degradation, cannot be transported to the endoplasmic reticulum. As a result, a reduced number of MHC class I molecules appear on the surface of infected cells and, thus, the immune system is not efficiently activated. BoHV‐1‐encoded UL49.5 protein is one such TAP transporter inhibitor. This protein binds to TAP in such a way that its N‐terminal fragment interacts with the loops of the TAP complex, and the C‐terminus stimulates proteasomal degradation of TAP. Previous studies have indicated certain amino acid residues, especially the RRE(9–11) motif, within the helical structure of the UL49.5 N‐terminal fragment, as being crucial to the protein's activity. In this work, we investigated the effects of modifications within the RRE region on the spatial structure of the UL49.5 N‐terminal fragment. The introduced RRE(9–11) variations were designed to abolish or stabilize the structure of the α‐helix and, consequently, to increase or decrease protein activity compared to the wild type. The terminal structure of the peptides was established using circular dichroism (CD), 2D nuclear magnetic resonance (NMR), and molecular dynamics (MD) in membrane‐mimetic or membrane‐model environments. Our structural results show that in the RRE(9–11)AAA and E11G peptides the helical structure has been stabilized, whereas for the RRE(9–11)GGG peptide, as expected, the helix structure has partially unfolded compared to the native structure. These RRE modifications, in the context of the entire UL49.5 proteins, slightly altered their biological activity in human cells.

Why does the UL49.5 of herpes simplex 1 virus fail to inhibit the TAP-dependent antigen presentation?

“…All the peptides (N.HSV, C.HSV, TMC.HSV and TMC.HSV.KKK) were synthesized using the standard solid-phase synthesis technique (Liberty Blue, CEM Corporation, USA). The synthesis and purification of peptides was carried out according to the procedures described in our previous publications (Karska et al 2019, Karska et al 2021Graul et al 2023).…”

Assessment of the Toxicity of Biocompatible Materials Supporting Bone Regeneration: Impact of the Type of Assay and Used Controls

“…All of the peptides were synthesized using the standard solid-phase synthesis technique (CEM Corporation, Matthews, NC, USA) on Rink Amide ProTide Resin (capacity 0.21 mmol/g, CEM Corporation, Matthews, NC, USA) according to the Fmoc-chemistry strategy. The peptides were cleaved from the resin, and purified according to the procedure described by Karska and colleagues [16]. The identity of peptides was confirmed by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS, Bruker Daltonics, Bremen, Germany), and electrospray ionization mass spectrometry (ESI-MS, Shimadzu, Kyoto, Japan).…”