School of Biological Sciences, Division of Molecular Biology, University of California San Diego, La Jolla, CA 92093.
Department of Biochemistry, Faculty of Science, Chulalongkorn University, Bangkok, Thailand 10330.
Proc Natl Acad Sci U S A. 2024 May 7;121(19):e2321190121. doi: 10.1073/pnas.2321190121. Epub 2024 Apr 30.
Targeting proteins to specific subcellular destinations is essential in prokaryotes, eukaryotes, and the viruses that infect them. Chimalliviridae phages encapsulate their genomes in a nucleus-like replication compartment composed of the protein chimallin (ChmA) that excludes ribosomes and decouples transcription from translation. These phages selectively partition proteins between the phage nucleus and the bacterial cytoplasm. Currently, the genes and signals that govern selective protein import into the phage nucleus are unknown. Here, we identify two components of this protein import pathway: a species-specific surface-exposed region of a phage intranuclear protein required for nuclear entry and a conserved protein, PicA (Protein importer of chimalliviruses A), that facilitates cargo protein trafficking across the phage nuclear shell. We also identify a defective cargo protein that is targeted to PicA on the nuclear periphery but fails to enter the nucleus, providing insight into the mechanism of nuclear protein trafficking. Using CRISPRi-ART protein expression knockdown of PicA, we show that PicA is essential early in the chimallivirus replication cycle. Together, our results allow us to propose a multistep model for the Protein Import Chimallivirus pathway, where proteins are targeted to PicA by amino acids on their surface and then licensed by PicA for nuclear entry. The divergence in the selectivity of this pathway between closely related chimalliviruses implicates its role as a key player in the evolutionary arms race between competing phages and their hosts.
靶向特定亚细胞靶标对于原核生物、真核生物和感染它们的病毒至关重要。Chimalliviridae 噬菌体将其基因组包裹在类似于细胞核的复制隔室中，该隔室由 chimallin（ChmA）蛋白组成，该蛋白排除核糖体并使转录与翻译解耦。这些噬菌体在噬菌体核和细菌细胞质之间选择性地分配蛋白质。目前，指导选择性蛋白质导入噬菌体核的基因和信号尚不清楚。在这里，我们确定了这个蛋白质导入途径的两个组成部分：一种噬菌体核内蛋白的物种特异性表面暴露区域，该区域对于核进入是必需的，以及一种保守的蛋白 PicA（Chimalliviruses A 的蛋白质导入蛋白），它促进货物蛋白在噬菌体核壳中的运输。我们还鉴定了一种有缺陷的货物蛋白，该蛋白靶向核周的 PicA，但未能进入核内，这为核蛋白运输的机制提供了深入了解。使用 CRISPRi-ART 蛋白表达敲低 PicA，我们表明 PicA 在 chimallivirus 复制周期的早期是必不可少的。总之，我们的结果使我们能够提出一个 Protein Import Chimallivirus 途径的多步模型，其中蛋白质通过其表面上的氨基酸靶向 PicA，然后由 PicA 许可进入核内。在密切相关的 chimalliviruses 之间，这种途径的选择性分歧暗示了它在竞争噬菌体与其宿主之间的进化军备竞赛中的关键作用。
