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The Phage Nucleus and Phuz Spindle Establish and Maintain Complex Subcellular Organization During the Jumbo Phage Life Cycle

Here we review the current understanding of the phage nucleus and spindle including their roles in jumbo phage replication and evolution. Studies of 201Phi2-1, PhiKZ, and PhiPA3 demonstrated that the tubulin-like protein PhuZ assembles filaments that organize a replication factory contained within a nucleus-like structure (termed the phage nucleus) during infection in the bacterial host cell. Some of these mega phages (>500 kb in size) also encode tubulin homologs. Once thought to be a rarity, metagenomics studies have revealed that jumbo phages are widespread across Earth’s ecosystems, with the largest known phage genome reaching 735 kb ( Al-Shayeb et al., 2020). These three Pseudomonas phages have genomes larger than 200 kb, qualifying them as jumbo phages. The best characterized of these phages are 201Phi2-1, PhiKZ, and PhiPA3. Some phages are now known to encode homologs of the cytoskeletal protein tubulin. Bacillus subtilis phage Phi29 and Escherichia coli phage PRD1 organize their replication machinery close to the host cell membrane by an MreB cytoskeleton-dependent mechanism ( Muñoz-Espín et al., 2009, 2010). Some phages have also been shown to rely on bacterial host cytoskeletal proteins. Even though there is as yet no direct evidence showing the molecular interaction of viractins and nucleocytoplasmic formation, the presence of viractin sequences across the family suggests that they play a role in the NCLDV replication cycle.

Viractins are conserved in the nucleocytoplasmic large DNA viruses (NCLDVs) that assemble a replication factory during its replication in the cell cytoplasm ( Schmid et al., 2014 Cunha et al., 2020). Cunha et al., 2020 recently revealed that 19 viral genomes of viruses in the family Mimiviridae contain actin-related genes, called viractins ( Cunha et al., 2020). While most eukaryotic viruses are thought to utilize the host cytoskeleton, a few are known to encode their own cytoskeletal protein. Eukaryotic viruses also rely upon cytoskeletal proteins to travel to assembly sites before egress ( Greber and Way, 2006 Taylor et al., 2011 Ward, 2011 Simpson and Yamauchi, 2020). In eukaryotic cells, host cytoskeletal structures are utilized as a highway for viruses to travel through the cell cytoplasm in order to reach sites where they will replicate ( Greber and Way, 2006 Cohen et al., 2011 Taylor et al., 2011 Portilho et al., 2016 Simpson and Yamauchi, 2020). Thus, the phage nucleus and PhuZ spindle are defining cell biological structures that serve roles in both the life cycle of nucleus-forming jumbo phages and phage speciation.Ĭytoskeletal structures are well known to facilitate viral replication. During the co-infection of two different nucleus-forming jumbo phages in a bacterial cell, the phage nucleus establishes Subcellular Genetic Isolation that limits the potential for viral genetic exchange by physically separating co-infection genomes, and the PhuZ spindle causes Virogenesis Incompatibility, whereby interacting components from two diverging phages negatively affect phage reproduction. Early during infection, the PhuZ spindle positions the newly formed phage nucleus at midcell and, later in the infection cycle, the spindle rotates the nucleus while delivering capsids and distributing them uniformly on the nuclear surface, where they dock for DNA packaging. Throughout the lytic cycle, the phage nucleus compartmentalizes proteins according to function and protects the phage genome from host defense mechanisms. This subcellular architecture serves as a replication factory for jumbo Pseudomonas phages and provides a selective advantage when these replicate in some host strains. The phage nucleus is made of a proteinaceous shell that surrounds replicating phage DNA and imparts a unique subcellular organization that is temporally and spatially controlled within bacterial host cells by a phage-encoded tubulin (PhuZ)-based spindle. Here, we review our current understanding of jumbo phages that form a nucleus-like structure, or “Phage Nucleus,” during replication. Some jumbo phages, those with a genome size larger than 200 kilobases, have recently been discovered to establish complex subcellular organization during replication. 2Department of Biochemistry, Faculty of Science, Chulalongkorn University, Bangkok, Thailandīacteriophages and their bacterial hosts are ancient organisms that have been co-evolving for billions of years.1Division of Biological Sciences, University of California San Diego, La Jolla, CA, United States.