Marsupials are one of three major mammalian lineages that include the placental eutherians and the egg-laying monotremes. The marsupial brushtail possum is an important protected species in the Australian forest ecosystem. Molecules encoded by the MHC genes are essential mediators of adaptive immune responses in virus–host interactions. Yet, nothing is known about the peptide presentation features of any marsupial MHC class I (MHC I). This study identified a series of possum MHC I Trvu-UB*01:01 binding peptides derived from wobbly possum disease virus (WPDV), a lethal virus of both captive and feral possum populations, and unveiled the structure of marsupial peptide/MHC I complex. Notably, we found the two brushtail possum–specific insertions, the 3-aa Ile⁵²Glu⁵³Arg⁵⁴ and 1-aa Arg¹⁵⁴ insertions are located in the Trvu-UB*01:01 peptide binding groove (PBG). The 3-aa insertion plays a pivotal role in maintaining the stability of the N terminus of Trvu-UB*01:01 PBG. This aspect of marsupial PBG is unexpectedly similar to the bat MHC I Ptal-N*01:01 and is shared with lower vertebrates from elasmobranch to monotreme, indicating an evolution hotspot that may have emerged from the pathogen–host interactions. Residue Arg¹⁵⁴ insertion, located in the α2 helix, is available for TCR recognition, and it has a particular influence on promoting the anchoring of peptide WPDV-12. These findings add significantly to our understanding of adaptive immunity in marsupials and its evolution in vertebrates. Our findings have the potential to impact the conservation of the protected species brushtail possum and other marsupial species.

有袋动物是包括胎盘真兽类和产卵单孔目动物在内的三大哺乳动物谱系之一。有袋类刷尾负鼠是澳大利亚森林生态系统中的重要保护物种。MHC 基因编码的分子是病毒-宿主相互作用中适应性免疫反应的重要介质。然而，对任何有袋动物 MHC I 类 (MHC I) 的肽呈递特征一无所知。本研究鉴定了一系列源自摇摆负鼠病病毒 (WPDV) 的负鼠 MHC I Trvu-UB*01:01 结合肽，WPDV 是一种圈养和野生负鼠种群的致死病毒，并揭示了有袋动物肽/MHC I 复合物的结构. 值得注意的是，我们发现了两个刷尾负鼠特异性插入，即 3-aa Ile ⁵² Glu ⁵³ Arg ⁵⁴和 1-aa Arg ¹⁵⁴插入位于 Trvu-UB*01:01 肽结合沟 (PBG) 中。3-aa 插入在维持 Trvu-UB*01:01 PBG N 末端的稳定性方面起着关键作用。有袋动物 PBG 的这一方面出人意料地与蝙蝠 MHC I Ptal-N*01:01 相似，并且与从弹鳃到单孔目动物的低等脊椎动物共享，表明可能是病原体-宿主相互作用产生的进化热点。残基精氨酸¹⁵⁴位于 α2 螺旋中的插入可用于 TCR 识别，它对促进肽 WPDV-12 的锚定有特殊影响。这些发现极大地加深了我们对有袋动物适应性免疫及其在脊椎动物中的进化的理解。我们的发现有可能影响受保护物种刷尾负鼠和其他有袋动物的保护。