Stephens Edward B, Kunec Dusan, Henke Wyatt, Vidal Ricardo Martin, Greishaber Brandon, Saud Rabina, Kalamvoki Maria, Singh Gagandeep, Kafle Sujan, Trujillo Jessie D, Ferreyra Franco Matias, Morozov Igor, Richt Juergen A
Department of Microbiology, Molecular Genetics, and Immunology 2000 Cates Hall, University of Kansas Medical Center, 3901 Rainbow Blvd, Kansas City, KS 66160, USA.
Institut für Virologie, Freie Universität Berlin, Berlin, Germany.
Viruses. 2025 Apr 3;17(4):522. doi: 10.3390/v17040522.
The open reading frame 3a (ORF3a) is a protein important to the pathogenicity of SARS-CoV-2. The cytoplasmic domain of ORF3a has three canonical tyrosine-based sorting signals (160YNSV163, 211YYQL213, and 233YNKI236), and a previous study has indicated that mutation of the 160YNSV163 motif abrogated plasma membrane expression and inhibited ORF3a-induced apoptosis. Here, we have systematically removed all three tyrosine-based motifs and assessed the importance of each motif or combination of motifs in trafficking to the cell surface. Our results indicate that the 160YNSV163 motif alone was insufficient for ORF3a cell-surface trafficking, while the 211YYQL213 motif was the most important. Additionally, an ORF3a with all three YxxΦ motifs disrupted (ORF3a-[ΔYxxΦ]) was not transported to the cell surface, and LysoIP studies indicate that ORF3a but not ORF3a-[ΔYxxΦ] was present in late endosome/lysosome fractions. A growth-curve analysis of different SARS-CoV-2 viruses expressing the different mutant ORF3a proteins revealed no significant differences in virus replication. Finally, the inoculation of K18hACE-2 mice indicated that the SARS-CoV-2 lacking the three YxxΦ motifs was less pathogenic than the unmodified SARS-CoV-2. These results indicate that the tyrosine motifs of ORF3a contribute to cell-surface expression and SARS-CoV-2 pathogenesis.
开放阅读框3a（ORF3a）是一种对严重急性呼吸综合征冠状病毒2（SARS-CoV-2）致病性很重要的蛋白质。ORF3a的胞质结构域有三个典型的基于酪氨酸的分选信号（160YNSV163、211YYQL213和233YNKI236），先前的一项研究表明，160YNSV163基序的突变消除了质膜表达并抑制了ORF3a诱导的细胞凋亡。在此，我们系统地去除了所有三个基于酪氨酸的基序，并评估了每个基序或基序组合在转运至细胞表面中的重要性。我们的结果表明，仅160YNSV163基序不足以实现ORF3a的细胞表面转运，而211YYQL213基序是最重要的。此外，所有三个YxxΦ基序均被破坏的ORF3a（ORF3a-[ΔYxxΦ]）未转运至细胞表面，溶酶体免疫沉淀（LysoIP）研究表明，晚期内体/溶酶体组分中存在ORF3a而非ORF3a-[ΔYxxΦ]。对表达不同突变ORF3a蛋白的不同SARS-CoV-2病毒进行的生长曲线分析显示，病毒复制没有显著差异。最后，对K18hACE-2小鼠的接种表明，缺乏三个YxxΦ基序的SARS-CoV-2的致病性低于未修饰的SARS-CoV-2。这些结果表明，ORF3a的酪氨酸基序有助于细胞表面表达和SARS-CoV-2的发病机制。
