Mishra Seema
Department of Biochemistry, School of Life Sciences, University of Hyderabad, Hyderabad, Telangana, India.
Evol Bioinform Online. 2022 Jul 7;18:11769343221108218. doi: 10.1177/11769343221108218. eCollection 2022.
In an effort to combat SARS-CoV-2 through multi-subunit vaccine design, during studies using whole genome and immunome, ORF10, located at the 3' end of the genome, displayed unique features. It showed no homology to any known protein in other organisms, including SARS-CoV. It was observed that its nucleotide sequence is 100% identical in the SARS-CoV-2 genomes sourced worldwide, even in the recent-most VoCs and VoIs of B.1.1.529 (Omicron), B.1.617 (Delta), B.1.1.7 (Alpha), B.1.351 (Beta), and P.1 (Gamma) lineages, implicating its constant nature throughout the evolution of deadly variants.
The structure and function of SARS-CoV-2 ORF10 and the role it may play in the viral evolution is yet to be understood clearly. The aim of this study is to predict its structure, function, and understand evolutionary dynamics on the basis of mutations and likely heightened immune responses in the immunopathogenesis of this deadly virus.
Sequence analysis, ab-initio structure modeling and an understanding of the impact of likely substitutions in key regions of protein was carried out. Analyses of viral T cell epitopes and primary anchor residue mutations was done to understand the role it may play in the evolution as a molecule with likely enhanced immune response and consequent immunopathogenesis.
Few amino acid substitution mutations are observed, most probably due to the ribosomal frameshifting, and these mutations may not be detrimental to its functioning. As ORF10 is observed to be an expressed protein, ab-initio structure modeling shows that it comprises mainly an α-helical region and maybe an ER-targeted membrane mini-protein. Analyzing the whole proteome, it is observed that ORF10 presents amongst the highest number of likely promiscuous and immunogenic CTL epitopes, specifically 11 out of 30 promiscuous ones and 9 out of these 11, immunogenic CTL epitopes. Reactive T cells to these epitopes have been uncovered in independent studies. Majority of these epitopes are located on the α-helix region of its structure, and the substitution mutations of primary anchor residues in these epitopes do not affect immunogenicity. Its conserved nucleotide sequence throughout the evolution and diversification of virus into several variants is a puzzle yet to be solved.
On the basis of its sequence, structure, and epitope mapping, it is concluded that it may function like those mini-proteins used to boost immune responses in medical applications. Due to the complete nucleotide sequence conservation even a few years after SARS-CoV-2 genome was first sequenced, it poses a unique puzzle to be solved, in view of the evolutionary dynamics of variants emerging in the populations worldwide.
为了通过多亚基疫苗设计对抗严重急性呼吸综合征冠状病毒2（SARS-CoV-2），在使用全基因组和免疫组的研究过程中，位于基因组3'端的开放阅读框10（ORF10）展现出独特特征。它与包括SARS-CoV在内的其他生物体中的任何已知蛋白质均无同源性。据观察，其核苷酸序列在全球范围内来源的SARS-CoV-2基因组中100%相同，即使在最新出现的B.1.1.529（奥密克戎）、B.1.617（德尔塔）、B.1.1.7（阿尔法）、B.1.351（贝塔）和P.1（伽马）谱系的变异株和感兴趣的变异株中也是如此，这表明其在致命变异株的整个进化过程中具有稳定性。
SARS-CoV-2 ORF10的结构和功能以及它在病毒进化中可能发挥的作用尚未完全明确。本研究的目的是基于突变情况以及这种致命病毒免疫发病机制中可能增强的免疫反应，预测其结构、功能并了解进化动态。
进行了序列分析、从头开始的结构建模以及对蛋白质关键区域可能的替换影响的理解。对病毒T细胞表位和主要锚定残基突变进行分析，以了解它作为一种可能具有增强免疫反应及随之而来的免疫发病机制的分子在进化中可能发挥的作用。
观察到少数氨基酸替换突变，很可能是由于核糖体移码造成 的，并且这些突变可能对其功能无害。由于观察到ORF10是一种表达蛋白，从头开始的结构建模显示它主要包含一个α螺旋区域，可能是一种靶向内质网的膜小蛋白。分析整个蛋白质组发现，ORF10呈现出数量众多的可能具有多反应性和免疫原性的细胞毒性T淋巴细胞（CTL）表位，具体而言，在30个多反应性表位中有11个，在这11个中有9个是免疫原性CTL表位。在独立研究中已发现针对这些表位的反应性T细胞。这些表位中的大多数位于其结构的α螺旋区域，并且这些表位中主要锚定残基的替换突变不影响免疫原性。其在病毒进化和多样化为多种变异株的过程中核苷酸序列保持保守，这仍是一个有待解决的谜题。
基于其序列、结构和表位定位，得出结论认为它可能像那些在医学应用中用于增强免疫反应的小蛋白一样发挥作用。鉴于在SARS-CoV-2基因组首次测序后的数年里其核苷酸序列仍完全保守，考虑到全球人群中出现的变异株的进化动态，这构成了一个有待解决的独特谜题。
