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在早期感染过程中,一个转录活跃的脂质囊泡包裹着注入的基因组。

A transcriptionally active lipid vesicle encloses the injected genome in early infection.

作者信息

Armbruster Emily G, Rani Phoolwanti, Lee Jina, Klusch Niklas, Hutchings Joshua, Hoffman Lizbeth Y, Buschkaemper Hannah, Enustun Eray, Adler Benjamin A, Inlow Koe, VanderWal Arica R, Hoffman Madelynn Y, Daksh Daksh, Aindow Ann, Deep Amar, Rodriguez Zaida K, Morgan Chase J, Ghassemian Majid, Laughlin Thomas G, Charles Emeric, Cress Brady F, Savage David F, Doudna Jennifer A, Pogliano Kit, Corbett Kevin D, Villa Elizabeth, Pogliano Joe

机构信息

School of Biological Sciences, University of California San Diego, La Jolla, CA 92093, USA.

These authors contributed equally: Emily G. Armbruster and Phoolwanti Rani.

出版信息

bioRxiv. 2024 Oct 22:2023.09.20.558163. doi: 10.1101/2023.09.20.558163.

Abstract

Many eukaryotic viruses require membrane-bound compartments for replication, but no such organelles are known to be formed by prokaryotic viruses. Bacteriophages of the family sequester their genomes within a phage-generated organelle, the phage nucleus, which is enclosed by a lattice of the viral protein ChmA. Previously, we observed lipid membrane-bound vesicles in cells infected by , but due to the paucity of genetics tools for these viruses it was unknown if these vesicles represented unproductive, abortive infections or a stage in the phage life cycle. Using the recently-developed dRfxCas13d-based knockdown system CRISPRi-ART in combination with fluorescence microscopy and cryo-electron tomography, we show that inhibiting phage nucleus formation arrests infections at an early stage in which the injected phage genome is enclosed within a membrane-bound early phage infection (EPI) vesicle. We demonstrate that early phage genes are transcribed by the virion-associated RNA polymerase from the genome within the compartment, making the EPI vesicle the first known example of a lipid membrane-bound organelle that separates transcription from translation in prokaryotes. Further, we show that the phage nucleus is essential for the phage life cycle, with genome replication only beginning after the injected DNA is transferred from the EPI vesicle to the newly assembled phage nucleus. Our results show that require two sophisticated subcellular compartments of distinct compositions and functions that facilitate successive stages of the viral life cycle.

摘要

许多真核病毒需要膜结合区室进行复制,但尚无已知原核病毒会形成此类细胞器。 科的噬菌体将其基因组隔离在噬菌体产生的细胞器——噬菌体核内,该细胞器被病毒蛋白ChmA的晶格所包围。此前,我们在受 感染的细胞中观察到脂质膜结合的囊泡,但由于这些病毒的遗传学工具匮乏,尚不清楚这些囊泡是代表无 productive、流产性感染,还是噬菌体生命周期中的一个阶段。使用最近开发的基于dRfxCas13d的敲低系统CRISPRi-ART,结合荧光显微镜和冷冻电子断层扫描,我们表明抑制噬菌体核形成会在早期阶段阻止感染,在此阶段注入的噬菌体基因组被包裹在膜结合的早期噬菌体感染(EPI)囊泡中。我们证明早期噬菌体基因由病毒体相关的RNA聚合酶从区室内的基因组转录而来,使EPI囊泡成为原核生物中第一个已知的将转录与翻译分开的脂质膜结合细胞器的例子。此外,我们表明噬菌体核对噬菌体生命周期至关重要,只有在注入的DNA从EPI囊泡转移到新组装的噬菌体核后,基因组复制才开始。我们的结果表明, 需要两个具有不同组成和功能的复杂亚细胞区室,以促进病毒生命周期的连续阶段。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/7315/11528503/47f3000effd8/nihpp-2023.09.20.558163v2-f0006.jpg

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