Henke Wyatt, Kalamvoki Maria, Stephens Edward B
Department of Microbiology, Molecular Genetics, and Immunology, 2000 Hixon Hall, University of Kansas Medical Center, 3901 Rainbow Blvd., Kansas City, Kansas 66160.
bioRxiv. 2023 Jul 24:2023.07.24.550379. doi: 10.1101/2023.07.24.550379.
The open reading frame 3a (ORF3a) is an accessory transmembrane protein that is important to the pathogenicity of SARS-CoV-2. The cytoplasmic domain of ORF3a has three canonical tyrosine-based sorting signals (YxxΦ; where x is any amino acid and Φ is a hydrophobic amino acid with a bulky -R group). They have been implicated in the trafficking of membrane proteins to the cell plasma membrane and to intracellular organelles. Previous studies have indicated that mutation of the YSNV motif abrogated plasma membrane expression and inhibited ORF3a-induced apoptosis. However, two additional canonical tyrosine-based sorting motifs (YYQL, YNKI) exist in the cytoplasmic domain of ORF3a that have not been assessed. We removed all three potential tyrosine-based motifs and systematically restored them to assess the importance of each motif or combination of motifs that restored efficient trafficking to the cell surface and lysosomes. Our results indicate that the YxxΦ motif at position 160 was insufficient for the trafficking of ORF3a to the cell surface. Our studies also showed that ORF3a proteins with an intact YxxΦ at position 211 or at 160 and 211 were most important. We found that ORF3a cell surface expression correlated with the co-localization of ORF3a with LAMP-1 near the cell surface. These results suggest that YxxΦ motifs within the cytoplasmic domain may act cooperatively in ORF3a transport to the plasma membrane and endocytosis to lysosomes. Further, our results indicate that certain tyrosine mutants failed to activate caspase 3 and did not correlate with autophagy functions associated with this protein.
开放阅读框3a（ORF3a）是一种辅助跨膜蛋白，对严重急性呼吸综合征冠状病毒2（SARS-CoV-2）的致病性很重要。ORF3a的细胞质结构域有三个典型的基于酪氨酸的分选信号（YxxΦ；其中x为任意氨基酸，Φ为带有庞大-R基团的疏水氨基酸）。它们与膜蛋白向细胞质膜和细胞内细胞器的运输有关。先前的研究表明，YSNV基序的突变消除了质膜表达并抑制了ORF3a诱导的细胞凋亡。然而，ORF3a的细胞质结构域中还存在另外两个典型的基于酪氨酸的分选基序（YYQL、YNKI），尚未进行评估。我们去除了所有三个潜在的基于酪氨酸的基序，并系统地恢复它们，以评估每个基序或基序组合对恢复向细胞表面和溶酶体的有效运输的重要性。我们的结果表明，第160位的YxxΦ基序不足以使ORF3a运输到细胞表面。我们的研究还表明，在第211位或第（160和211）位具有完整YxxΦ的ORF3a蛋白最为重要。我们发现ORF3a细胞表面表达与ORF3a在细胞表面附近与溶酶体相关膜蛋白1（LAMP-1）的共定位相关。这些结果表明，细胞质结构域内的YxxΦ基序可能在ORF3a向质膜的运输以及向溶酶体的内吞作用中协同发挥作用。此外，我们的结果表明，某些酪氨酸突变体未能激活半胱天冬酶3，并且与该蛋白相关的自噬功能无关。
