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

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

Many eukaryotic viruses require membrane-bound compartments for replication, but no such organelles are known to be formed by prokaryotic viruses1-3. Bacteriophages of the Chimalliviridae family sequester their genomes within a phage-generated organelle, the phage nucleus, which is enclosed by a lattice of the viral protein ChmA4-10. Previously, we observed lipid membrane-bound vesicles in cells infected by Chimalliviridae, but due to the paucity of genetics tools for these viruses it was unknown if these vesicles represented unproductive, abortive infections or a bona fide stage in the phage life cycle. Using the recently-developed dRfxCas13d-based knockdown system CRISPRi-ART11 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 Chimalliviridae require two sophisticated subcellular compartments of distinct compositions and functions that facilitate successive stages of the viral life cycle.

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在噬菌体感染过程中,膜和蛋白质结合的细胞器顺序划分基因组。
真核病毒组装基因组复制所需的隔间,但目前还不知道这种细胞器对原核病毒是必不可少的。Chimalliviridae家族的噬菌体将其基因组隔离在噬菌体产生的细胞器中,噬菌体核被病毒蛋白ChmA的晶格包围。使用基于dRfxCas13d的敲除系统CRISPRi ART,我们表明ChmA对大肠杆菌噬菌体Goslar的生命周期至关重要。在没有ChmA的情况下,感染在早期阶段被阻止,在早期阶段,注射的噬菌体基因组被封闭在能够表达基因但不能复制DNA的膜结合囊泡中。我们不仅证明了噬菌体核对基因组复制至关重要,而且还证明了黑猩猩科早期噬菌体感染(EPI)囊泡是一种转录活性的、噬菌体产生的细胞器。
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