The RGD-binding Integrins αvβ6 and αvβ8 are Receptors for Mouse Adenovirus-1 and -3 Infection

Mammalian AdVs comprise more than ~350 types including over 100 human adenoviruses (HAdVs) and just three mouse AdV (MAdV). While most HAdVs initiate infection by high affinity/avidity binding of their fiber knob (FK) protein to either coxsackievirus AdV receptor (CAR), CD46 or desmoglein (DSG)-2, MAdV-1 (M1) infection requires arginine-glycine-aspartate (RGD) binding integrins. To identify the receptors mediating MAdV infection we generated five novel reporter viruses for MAdV-1/-2/-3 (M1, M2, M3) transducing permissive murine (m) CMT-93 cells, but not B16 mouse melanoma cells expressing mCAR, human (h) CD46 or hDSG-2. Recombinant M1 or M3 FKs cross-blocked M1 and M3 but not M2 infections. Profiling of murine and human cells expressing RGD-binding integrins suggested that αvβ6 and αvβ8 heterodimers are associated with M1 and M3 infections. Ectopic expression of mβ6 in B16 cells strongly enhanced M1 and M3 binding, infections and progeny productions comparable with mαvβ6-positive CMT-93 cells, whereas mβ8 expressing cells were more permissive to M1 than M3. Anti-integrin antibodies potently blocked M1 and M3 binding and infections of CMT-93 cells and hαvβ8-positive M000216 cells. Soluble integrin αvβ6, and synthetic peptides containing the RGDLXXL sequence derived from FK-M1, FK-M3 and foot and mouth disease virus coat protein potently interfered with M1/M3 infections, in agreement with high affinity interactions of FK-M1/FK-M3 with αvβ6/αvβ8, determined by surface plasmon resonance measurements. Molecular docking simulations of ternary complexes revealed a bent conformation of RGDLXXL-containing FK-M3 peptides on the subunit interface of αvβ6/β8, where the distal leucine residue dips into a hydrophobic pocket of β6/8, the arginine residue ionically engages αv aspartate215, and the aspartate residue coordinates a divalent cation in αvβ6/β8. Together, the RGDLXXL-bearing FKs are part of an essential mechanism for M1/M3 infection engaging murine and human αvβ6/8 integrins, which are also highly conserved in other mammals, and thus may favour cross-species virus transmission.

哺乳动物腺病毒（Mammalian AdVs）包含逾350种亚型，其中涵盖100余种人腺病毒（human adenoviruses, HAdVs）以及仅3种小鼠腺病毒（mouse AdV, MAdV）。大多数人腺病毒通过其纤突头部（fiber knob, FK）蛋白以高亲和力/亲合力结合柯萨奇病毒腺病毒受体（coxsackievirus AdV receptor, CAR）、CD46或桥粒芯糖蛋白（desmoglein, DSG）-2启动感染，而小鼠腺病毒1型（MAdV-1, M1）的感染则依赖精氨酸-甘氨酸-天冬氨酸（arginine-glycine-aspartate, RGD）结合型整合素。为鉴定介导小鼠腺病毒感染的受体，我们构建了5株针对MAdV-1/-2/-3（分别记为M1、M2、M3）的新型报告病毒，该类病毒可转导易感的小鼠（murine, m）CMT-93细胞，但无法转导表达小鼠CAR（mCAR）、人CD46（hCD46）或人DSG-2（hDSG-2）的B16小鼠黑色素瘤细胞。重组M1或M3的FK蛋白可交叉阻断M1和M3的感染，但无法阻断M2的感染。对表达RGD结合型整合素的小鼠和人源细胞的表达谱分析显示，αvβ6和αvβ8异二聚体与M1和M3的感染密切相关。在B16细胞中异位表达小鼠β6（mβ6）可显著增强M1和M3的结合能力、感染效率以及子代病毒增殖，其效果与表达αvβ6的易感细胞CMT-93相当；而表达小鼠β8（mβ8）的细胞对M1感染的易感性高于M3。抗整合素抗体可有效阻断CMT-93细胞以及表达人αvβ8的M000216细胞中M1和M3的结合与感染。可溶性整合素αvβ6，以及源自FK-M1、FK-M3和口蹄疫病毒衣壳蛋白的含RGDLXXL基序的合成肽，均可强效干扰M1/M3的感染，这与通过表面等离子体共振（surface plasmon resonance）测定得到的FK-M1/FK-M3与αvβ6/αvβ8存在高亲和力相互作用的结果一致。三元复合物的分子对接模拟显示，含RGDLXXL基序的FK-M3肽段在αvβ6/β8的亚基界面呈现弯曲构象：其中远端亮氨酸残基嵌入β6/8的疏水口袋，精氨酸残基通过离子键与αv亚基的天冬氨酸215结合，天冬氨酸残基则与αvβ6/β8中的二价阳离子配位。综上，携带RGDLXXL基序的FK蛋白是M1/M3感染过程中结合小鼠和人源αvβ6/8整合素的关键机制之一，而该类整合素在其他哺乳动物中也高度保守，因此可能促进病毒的跨物种传播。