Zoonotic arenaviruses can lead to life-threating diseases in humans. These viruses encode a large (L) polymerase that transcribes and replicates the viral genome. At the late stage of replication, the multifunctional Z protein interacts with the L polymerase to shut down RNA synthesis and initiate virion assembly. However, the mechanism by which the Z protein regulates the activity of L polymerase is unclear. Here, we used cryo-electron microscopy to resolve the structures of both Lassa and Machupo virus L polymerases in complex with their cognate Z proteins, and viral RNA, to 3.1–3.9 Å resolutions. These structures reveal that Z protein binding induces conformational changes in two catalytic motifs of the L polymerase, and restrains their conformational dynamics to inhibit RNA synthesis, which is supported by hydrogen–deuterium exchange mass spectrometry analysis. Importantly, we show, by in vitro polymerase reactions, that Z proteins of Lassa and Machupo viruses can cross-inhibit their L polymerases, albeit with decreased inhibition efficiencies. This cross-reactivity results from a highly conserved determinant motif at the contacting interface, but is affected by other variable auxiliary motifs due to the divergent evolution of Old World and New World arenaviruses. These findings could provide promising targets for developing broad-spectrum antiviral drugs.

人畜共患沙粒病毒可导致危及人类生命的疾病。这些病毒编码转录和复制病毒基因组的大 (L) 聚合酶。在复制后期，多功能 Z 蛋白与 L 聚合酶相互作用以关闭 RNA 合成并启动病毒粒子组装。然而，Z蛋白调节L聚合酶活性的机制尚不清楚。在这里，我们使用冷冻电子显微镜将拉沙热和 Machupo 病毒 L 聚合酶与其同源 Z 蛋白和病毒 RNA 的复合物结构解析为 3.1–3.9 Å 分辨率。这些结构表明 Z 蛋白结合诱导 L 聚合酶的两个催化基序的构象变化，并限制它们的构象动力学以抑制 RNA 合成，这是由氢 - 氘交换质谱分析支持的。重要的是，我们通过体外聚合酶反应表明，拉沙病毒和马丘波病毒的 Z 蛋白可以交叉抑制它们的 L 聚合酶，尽管抑制效率会降低。这种交叉反应性是由接触界面处高度保守的决定子基序引起的，但由于旧世界和新世界沙粒病毒的不同进化而受到其他可变辅助基序的影响。这些发现可以为开发广谱抗病毒药物提供有希望的目标。这种交叉反应性是由接触界面处高度保守的决定子基序引起的，但由于旧世界和新世界沙粒病毒的不同进化而受到其他可变辅助基序的影响。这些发现可以为开发广谱抗病毒药物提供有希望的目标。这种交叉反应性是由接触界面处高度保守的决定子基序引起的，但由于旧世界和新世界沙粒病毒的不同进化而受到其他可变辅助基序的影响。这些发现可以为开发广谱抗病毒药物提供有希望的目标。