病毒组装是一个高度有序的复杂过程，其中病毒衣壳蛋白的正确折叠和组装对形成完整病毒颗粒至关重要。近年来，冷冻电镜技术的突破让我们能更深入地理解病毒组装的复杂过程。研究者们发现，腺病毒（Adenovirus，AdV）和非洲猪瘟病毒（African Swine Fever Virus, ASFV）同属PRD1/腺病毒样谱系，其特征是具有双果冻卷折叠的衣壳蛋白。AdV和ASFV的双果冻卷折叠衣壳蛋白hexon和p72的正确折叠和组装分别依赖于病毒自身编码的分子伴侣蛋白100K和B602L，然而其中的分子机制尚不完全清楚。此外，ASFV除了具有双果冻卷折叠的外衣壳外，还存在一层内衣壳，其组装机制有待阐明。本研究以AdV和ASFV为研究对象，探究100K介导的腺病毒衣壳组装机制、B602L介导的ASFV外衣壳组装机制以及ASFV内衣壳的组装机制。AdV是一种无囊膜病毒，其衣壳主要由hexon蛋白组成。利用冷冻电镜三维重构技术，我们解析了未成熟的hexon和其分子伴侣100K的复合物的结构，结果显示未成熟hexon利用其N端片段和双果冻卷结构域与100K二聚体互作。100K的结合能够防止hexon过早聚集并帮助其正确折叠进而组装成稳定的三聚体。突变研究表明，100K与hexon的N端和双果冻卷结构域的相互作用对于hexon的正确折叠和组装至关重要。 ASFV具有复杂的多层结构，其外衣壳主要由p72蛋白构成；内衣壳据报道是由pp220和pp62的水解产物组装而成。利用冷冻电镜三维重构技术，我们解析了未成熟p72与B602L复合物结构，未成熟p72依赖其N端片段和双果冻卷结构域与B602L互作，这与AdV中hexon与100K的互作类似。 B602L帮助p72正确地折叠，进而组装成稳定的三聚体。此外，我们解析了pp220水解产物p150的晶体结构，发现p150为内衣壳主要结构蛋白，负责形成内衣壳六边形和五边形壳粒。 本研究系统阐明了在分子伴侣辅助下的AdV和ASFV具有双果冻卷折叠的衣壳组装机制，揭示了病毒编分子伴侣的功能机制，为靶向分子伴侣-衣壳蛋白互作界面的抗病毒药物研发提供了指导。特别地，本研究还揭示了ASFV内衣壳的组装特征，为理解核质大DNA病毒内衣壳的组装机制提供了新的视角，有助于针对此类复杂病毒的新型疫苗和抗病毒药物的开发。
Viral assembly is a highly ordered, complex process requiring correct folding and assembly of capsid proteins to form functional virions. Advances in cryo-electron microscopy enhanced our comprehension of viral assembly mechanisms. Researchers have identified Adenovirus (AdV) and African swine fever virus (ASFV) both belong to the PRD1/adenovirus-like lineage, characterized by capsid proteins with the double jelly-roll fold. The correct folding and assembly of the double jelly-roll capsid proteins hexon (in AdV) and p72 (in ASFV) depend on their respective virus-encoded chaperones, 100K and B602L. However, the underlying molecular mechanisms remain incompletely understood. Furthermore, in addition to its double jelly-roll outer capsid, ASFV has an inner capsid whose assembly mechanism is yet to be elucidated. This study investigates the capsid assembly mechanisms in AdV and ASFV, focusing on: 100K-mediated hexon assembly in AdV, B602L- mediated outer capsid formation in ASFV, and the inner capsid assembly mechanism of ASFV.AdV is an icosahedral, non-enveloped DNA virus whose capsid is primarily composed of hexon. Using cryo-electron microscopy(cryo-EM) three-dimensional reconstruction, we determined the structure of the pre-mature hexon in complex with 100K. The structure shows the pre-mature hexon interacts with the 100K dimer via its N-terminal fragment and double jelly-roll domain. The binding of 100K prevents pre-mature aggregation of hexon and aids its proper folding, leading to the assembly of stable trimers. ASFV has a complex multilayered structure: its outer capsid is primarily composed of p72 protein, while the inner capsid is reportedly assembled by proteolytic products from the viral-encoded pp220 and pp62. Using cryo-EM three-dimensional reconstruction, we resolved the structure of the pre-mature p72 in complex with its chaperone B602L, revealing that pre-mature p72 interacts with B602L dimer mainly via its N-terminal fragment and the double jelly-roll domain, which resembles the interactions between hexon and 100K in AdV. The binding of B602L aids the correct folding of p72, facilitating its assembly into stable structures. We further determined the crystal structure of p150 (a proteolytic product of pp220) and identified p150 as the major structural protein of the inner capsid, responsible for forming both hexagonal and pentagonal capsomers.This study systematically elucidates the chaperone-assisted assembly mechanisms of double jelly-roll capsids in both AdV and ASFV, revealing the functional mechanisms of virus-encoded chaperones and contribute to the development of antiviral drugs targeting the chaperone-capsid protein interaction interfaces. Notably, this study provides the structural insights into the assembly of the ASFV inner capsid and provide new insights into the assembly mechanisms of the inner capsids of nucleocytoplasmic large DNA viruses (NCLDVs), which would facilitate the development of vaccines and antivirus drugs against these complex viruses.
