Department of Microbiology, Immunology and Molecular Genetics, University of California, Los Angeles (UCLA), Los Angeles, California 90095, USA.
The California NanoSystems Institute (CNSI), University of California, Los Angeles (UCLA), Los Angeles, California 90095, USA.
Nature. 2018 Jan 25;553(7689):521-525. doi: 10.1038/nature25438. Epub 2018 Jan 17.
Kaposi's sarcoma-associated herpesvirus (KSHV) causes Kaposi's sarcoma, a cancer that commonly affects patients with AIDS and which is endemic in sub-Saharan Africa. The KSHV capsid is highly pressurized by its double-stranded DNA genome, as are the capsids of the eight other human herpesviruses. Capsid assembly and genome packaging of herpesviruses are prone to interruption and can therefore be targeted for the structure-guided development of antiviral agents. However, herpesvirus capsids-comprising nearly 3,000 proteins and over 1,300 Å in diameter-present a formidable challenge to atomic structure determination and functional mapping of molecular interactions. Here we report a 4.2 Å resolution structure of the KSHV capsid, determined by electron-counting cryo-electron microscopy, and its atomic model, which contains 46 unique conformers of the major capsid protein (MCP), the smallest capsid protein (SCP) and the triplex proteins Tri1 and Tri2. Our structure and mutagenesis results reveal a groove in the upper domain of the MCP that contains hydrophobic residues that interact with the SCP, which in turn crosslinks with neighbouring MCPs in the same hexon to stabilize the capsid. Multiple levels of MCP-MCP interaction-including six sets of stacked hairpins lining the hexon channel, disulfide bonds across channel and buttress domains in neighbouring MCPs, and an interaction network forged by the N-lasso domain and secured by the dimerization domain-define a robust capsid that is resistant to the pressure exerted by the enclosed genome. The triplexes, each composed of two Tri2 molecules and a Tri1 molecule, anchor to the capsid floor via a Tri1 N-anchor to plug holes in the MCP network and rivet the capsid floor. These essential roles of the MCP N-lasso and Tri1 N-anchor are verified by serial-truncation mutageneses. Our proof-of-concept demonstration of the use of polypeptides that mimic the smallest capsid protein to inhibit KSHV lytic replication highlights the potential for exploiting the interaction hotspots revealed in our atomic structure to develop antiviral agents.
卡波济肉瘤相关疱疹病毒（KSHV）可导致卡波济肉瘤，这种癌症常见于艾滋病患者，且在撒哈拉以南非洲流行。KSHV 衣壳受到其双链 DNA 基因组的高度加压，就像其他八种人类疱疹病毒的衣壳一样。疱疹病毒的衣壳组装和基因组包装容易受到干扰，因此可以针对衣壳结构来开发抗病毒药物。然而，疱疹病毒衣壳——由近 3000 个蛋白质和超过 1300 Å 直径组成——对原子结构测定和分子相互作用的功能映射构成了巨大的挑战。在这里，我们通过电子计数冷冻电子显微镜报告了 KSHV 衣壳的 4.2 Å 分辨率结构及其原子模型，其中包含主要衣壳蛋白（MCP）、最小衣壳蛋白（SCP）和三聚体蛋白 Tri1 和 Tri2 的 46 个独特构象。我们的结构和突变结果揭示了 MCP 上部结构域中的一个凹槽，其中包含与 SCP 相互作用的疏水性残基，而 SCP 反过来又与同一六聚体中的相邻 MCP 交联，从而稳定衣壳。MCP-MCP 相互作用的多个层次——包括沿着六聚体通道排列的六组堆叠发夹、通道和相邻 MCP 中支撑域的二硫键，以及由 N 套索域形成并由二聚化域固定的相互作用网络——定义了一个坚固的衣壳，能够抵抗封闭基因组所施加的压力。三聚体由两个 Tri2 分子和一个 Tri1 分子组成，通过 Tri1 N 锚定在衣壳底部，填补 MCP 网络中的孔，并将衣壳底部铆接在一起。MCP N 套索和 Tri1 N 锚定的这些基本作用通过串联截断突变得到验证。我们使用模拟最小衣壳蛋白的多肽来抑制 KSHV 裂解复制的概念验证证明，利用我们的原子结构揭示的相互作用热点来开发抗病毒药物具有潜力。
