Hoad Mikayla, Nematollahzadeh Sepehr, Petersen Gayle F, Roby Justin A, Alvisi Gualtiero, Forwood Jade K
School of Dentistry and Medical Sciences, Charles Sturt University, Wagga Wagga, New South Wales, Australia.
Gulbali Institute, Charles Sturt University, Wagga Wagga, New South Wales, Australia.
J Virol. 2025 Jan 31;99(1):e0134524. doi: 10.1128/jvi.01345-24. Epub 2024 Dec 18.
Adeno-associated viruses (AAVs) are the most extensively researched viral vectors for gene therapy globally. The AAV viral protein 1 (VP1) N-terminus controls the capsid's ability to translocate into the cell nucleus; however, the exact mechanism of this process is largely unknown. In this study, we sought to elucidate the precise interactions between AAV serotype 6 (AAV6), a promising vector for immune disorders, and host transport receptors responsible for vector nuclear localization. Focusing on the positively charged basic areas within the N-terminus of AAV6 VP1, we identified a 53-amino acid region that interacts with nuclear import receptors. We measured the binding affinities between this region and various nuclear import receptors, discovering a notably strong interaction with IMPα5 and IMPα7 in the low nanomolar range. We also elucidated the X-ray crystal structure of this region in complex with an importin alpha (IMPα) isoform, uncovering its binding as a bipartite nuclear localization signal (NLS). Furthermore, we show that using this bipartite NLS, AAV6 VP1 capsid protein can localize to the nucleus of mammalian cells in a manner dependent on the IMPα/IMPβ nuclear import pathway. This study provides detailed insights into the interaction between the AAV6 VP1 capsid protein and nuclear import receptors, deepening our knowledge of AAV nuclear import mechanisms and establishing a basis for the improvement of AAV6-based gene therapy vectors.IMPORTANCEAAVs, recognized as the most extensively researched viral vectors for gene therapy globally, offer significant advantages over alternatives due to their small size, non-pathogenic nature, and innate ability for tissue-specific targeting. AAVs are required to localize to the nucleus to perform their role as a gene therapy vector; however, the precise mechanisms that facilitate this process remain unknown. Despite sharing overt genomic similarities with AAV1 and AAV2, AAV6 is a unique serotype. It is currently recognized for its ability to effectively transduce hematopoietic cell lineages and, consequently, is considered promising for the treatment of immune disorders. Identifying the exact mechanisms that permit AAV6 to access the nucleus can open up new avenues for gene therapy vector engineering, which can ultimately lead to increased therapeutic benefits.
腺相关病毒（AAV）是全球范围内基因治疗研究最为广泛的病毒载体。AAV病毒蛋白1（VP1）的N端控制着衣壳转运至细胞核的能力；然而，这一过程的确切机制在很大程度上尚不清楚。在本研究中，我们试图阐明AAV6（一种有望用于免疫疾病治疗的载体）与负责载体核定位的宿主转运受体之间的确切相互作用。聚焦于AAV6 VP1 N端带正电荷的碱性区域，我们鉴定出一个与核输入受体相互作用的53个氨基酸的区域。我们测量了该区域与各种核输入受体之间的结合亲和力，发现在低纳摩尔范围内与IMPα5和IMPα7有显著的强相互作用。我们还阐明了该区域与一种输入蛋白α（IMPα）亚型形成复合物的X射线晶体结构，发现其作为双分型核定位信号（NLS）的结合方式。此外，我们表明利用这种双分型NLS，AAV6 VP1衣壳蛋白能够以依赖于IMPα/IMPβ核输入途径的方式定位于哺乳动物细胞的细胞核。本研究为AAV6 VP1衣壳蛋白与核输入受体之间的相互作用提供了详细的见解，加深了我们对AAV核输入机制的了解，并为改进基于AAV6的基因治疗载体奠定了基础。
重要性
AAV被认为是全球范围内基因治疗研究最为广泛的病毒载体，由于其体积小、无致病性以及具有组织特异性靶向的固有能力，与其他载体相比具有显著优势。AAV需要定位于细胞核才能发挥其作为基因治疗载体的作用；然而，促进这一过程的确切机制仍然未知。尽管与AAV1和AAV2在基因组上有明显的相似性，但AAV6是一种独特的血清型。它目前因其有效转导造血细胞谱系的能力而受到认可，因此被认为在免疫疾病治疗方面很有前景。确定允许AAV6进入细胞核的确切机制可以为基因治疗载体工程开辟新途径，最终带来更大的治疗益处。
