Genomic transfer via membrane vesicle: a strategy of giant phage phiKZ for early infection.

During infection, the giant phiKZ phage forms a specialized structure at the center of the host cell called the phage nucleus. This structure is crucial for safeguarding viral DNA against bacterial nucleases and for segregating the transcriptional activities of late genes. Here, we describe a morphological entity, the early phage infection (EPI) vesicle, which appears to be responsible for earlier gene segregation at the beginning of the infection process. Using cryo-electron microscopy, electron tomography (ET), and fluorescence microscopy with membrane-specific dyes, we demonstrated that the EPI vesicle is enclosed in a lipid bilayer originating, apparently, from the inner membrane of the bacterial cell. Our investigations further disclose that the phiKZ EPI vesicle contains both viral DNA and viral RNA polymerase (vRNAP). We have observed that the EPI vesicle migrates from the cell pole to the center of the bacterial cell together with ChmA, the primary protein of the phage nucleus. The phage DNA is transported into the phage nucleus after phage maturation, but the EPI vesicle remains outside. We hypothesized that the EPI vesicle acts as a membrane transport agent, efficiently delivering phage DNA to the phage nucleus while protecting it from the nucleases of the bacterium. IMPORTANCE: Our study shed light on the processes of phage phiKZ early infection stage, expanding our understanding of possible strategies for the development of phage infection. We show that phiKZ virion content during injection is packed inside special membrane structures called early phage infection (EPI) membrane vesicles originating from the bacterial inner cell membrane. We demonstrated the EPI vesicle fulfilled the role of the safety transport unit for the phage genome to the phage nucleus, where the phage DNA would be replicated and protected from bacterial immune systems.

Characterization of a lipid-based jumbo phage compartment as a hub for early phage infection.

Viral genomes are most vulnerable to cellular defenses at the start of the infection. A family of jumbo phages related to phage ΦKZ, which infects Pseudomonas aeruginosa, assembles a protein-based phage nucleus to protect replicating phage DNA, but how it is protected prior to phage nucleus assembly is unclear. We find that host proteins related to membrane and lipid biology interact with injected phage protein, clustering in an early phage infection (EPI) vesicle. The injected virion RNA polymerase (vRNAP) executes early gene expression until phage genome separation from the vRNAP and the EPI vesicle, moving into the nascent proteinaceous phage nucleus. Enzymes involved in DNA replication and CRISPR/restriction immune nucleases are excluded by the EPI vesicle. We propose that the EPI vesicle is rapidly constructed with injected phage proteins, phage DNA, host lipids, and host membrane proteins to enable genome protection, early transcription, localized translation, and to ensure faithful genome transfer to the proteinaceous nucleus.

An essential and highly selective protein import pathway encoded by nucleus-forming phage.

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.

Assembly of phiKZ bacteriophage Inner Body during infection.

The giant myovirus phiKZ is characterised by an Inner Body (IB) structure within its capsid, crucial for orderly DNA packaging. The IB is composed of six phiKZ-specific proteins. Notably, four of these IB proteins are co-injected with DNA into the host cell, where they potentially play a role in attacking the bacterial cell. The dynamics of IB assembling within the phiKZ capsid during infection remain poorly understood. In this study, we used fluorescent microscopy to track the localisation of IB proteins fused to fluorescent proteins within the cell throughout the infection process. Our findings reveal that the proteins Gp97 and Gp162 are incorporated into new virion heads during phage head maturation. In contrast, proteins Gp90, Gp93, and Gp95 are likely integrated into the virion shortly before the DNA packaging.

The Dynamics of Synthesis and Localization of Jumbo Phage RNA Polymerases inside Infected Cells.

A nucleus-like structure composed of phage-encoded proteins and containing replicating viral DNA is formed in Pseudomonas aeruginosa cells infected by jumbo bacteriophage phiKZ. The PhiKZ genes are transcribed independently from host RNA polymerase (RNAP) by two RNAPs encoded by the phage. The virion RNAP (vRNAP) transcribes early viral genes and must be injected into the cell with phage DNA. The non-virion RNAP (nvRNAP) is composed of early gene products and transcribes late viral genes. In this work, the dynamics of phage RNAPs localization during phage phiKZ infection were studied. We provide direct evidence of PhiKZ vRNAP injection in infected cells and show that it is excluded from the phage nucleus. The nvRNAP is synthesized shortly after the onset of infection and localizes in the nucleus. We propose that spatial separation of two phage RNAPs allows coordinated expression of phage genes belonging to different temporal classes.

Identifying the core genome of the nucleus-forming bacteriophage family and characterization of Erwinia phage RAY.

We recently discovered that some bacteriophages establish a nucleus-like replication compartment (phage nucleus), but the core genes that define nucleus-based phage replication and their phylogenetic distribution were still to be determined. Here, we show that phages encoding the major phage nucleus protein chimallin share 72 conserved genes encoded within seven gene blocks. Of these, 21 core genes are unique to nucleus-forming phage, and all but one of these genes encode proteins of unknown function. We propose that these phages comprise a novel viral family we term Chimalliviridae. Fluorescence microscopy and cryoelectron tomography studies of Erwinia phage vB_EamM_RAY confirm that many of the key steps of nucleus-based replication are conserved among diverse chimalliviruses and reveal variations on this replication mechanism. This work expands our understanding of phage nucleus and PhuZ spindle diversity and function, providing a roadmap for identifying key mechanisms underlying nucleus-based phage replication.

Replication Compartments of Eukaryotic and Bacterial DNA Viruses: Common Themes Between Different Domains of Host Cells.

Subcellular organization is essential for life. Cells organize their functions into organelles to concentrate their machinery and supplies for optimal efficiency. Likewise, viruses organize their replication machinery into compartments or factories within their host cells for optimal replicative efficiency. In this review, we discuss how DNA viruses that infect both eukaryotic cells and bacteria assemble replication compartments for synthesis of progeny viral DNA and transcription of the viral genome. Eukaryotic DNA viruses assemble replication compartments in the nucleus of the host cell while DNA bacteriophages assemble compartments called phage nuclei in the bacterial cytoplasm. Thus, DNA viruses infecting host cells from different domains of life share common replication strategies.

A cytoskeletal vortex drives phage nucleus rotation during jumbo phage replication in E. coli.

Nucleus-forming jumbo phages establish an intricate subcellular organization, enclosing phage genomes within a proteinaceous shell called the phage nucleus. During infection in Pseudomonas, some jumbo phages assemble a bipolar spindle of tubulin-like PhuZ filaments that positions the phage nucleus at midcell and drives its intracellular rotation. This facilitates the distribution of capsids on its surface for genome packaging. Here we show that the Escherichia coli jumbo phage Goslar assembles a phage nucleus surrounded by an array of PhuZ filaments resembling a vortex instead of a bipolar spindle. Expression of a mutant PhuZ protein strongly reduces Goslar phage nucleus rotation, demonstrating that the PhuZ cytoskeletal vortex is necessary for rotating the phage nucleus. While vortex-like cytoskeletal arrays are important in eukaryotes for cytoplasmic streaming and nucleus alignment, this work identifies a coherent assembly of filaments into a vortex-like structure driving intracellular rotation within the prokaryotic cytoplasm.

The Phage Nucleus and PhuZ Spindle: Defining Features of the Subcellular Organization and Speciation of Nucleus-Forming Jumbo Phages.

Bacteriophages and their bacterial hosts are ancient organisms that have been co-evolving for billions of years. Some jumbo phages, those with a genome size larger than 200 kilobases, have recently been discovered to establish complex subcellular organization during replication. Here, we review our current understanding of jumbo phages that form a nucleus-like structure, or "Phage Nucleus," during replication. 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. This subcellular architecture serves as a replication factory for jumbo Pseudomonas phages and provides a selective advantage when these replicate in some host strains. Throughout the lytic cycle, the phage nucleus compartmentalizes proteins according to function and protects the phage genome from host defense mechanisms. 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. 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. 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.

Polyhedra, spindles, phage nucleus and pyramids: Structural biology of viral superstructures.

Viral infection causes comprehensive rearrangements of the cell that reflect as much host defense mechanisms as virus-induced structures assembled to facilitate infection. Regardless of their pro- or antiviral role, large intracellular structures are readily detectable by microscopy and often provide a signature characteristic of a specific viral infection. The structural features and localization of these assemblies have thus been commonly used for the diagnostic and classification of viruses since the early days of virology. More recently, characterization of viral superstructures using molecular and structural approaches have revealed very diverse organizations and roles, ranging from dynamic viral factories behaving like liquid organelles to ultra-stable crystals embedding and protecting virions. This chapter reviews the structures, functions and biotechnological applications of virus-induced superstructures with a focus on assemblies that have a regular organization, for which detailed structural descriptions are available. Examples span viruses infecting all domains of life including the assembly of virions into crystalline arrays in eukaryotic and bacterial viruses, nucleus-like compartments involved in the replication of large bacteriophages, and pyramid-like structures mediating the egress of archaeal viruses. Among these superstructures, high-resolution structures are available for crystalline objects produced by insect viruses: viral polyhedra which function as the infectious form of occluded viruses, and spindles which are potent virulence factors of entomopoxviruses. In turn, some of these highly symmetrical objects have been used to develop and validate advanced structural approaches, pushing the boundary of structural biology.

Viral Capsid Trafficking along Treadmilling Tubulin Filaments in Bacteria.

Cargo trafficking along microtubules is exploited by eukaryotic viruses, but no such examples have been reported in bacteria. Several large Pseudomonas phages assemble a dynamic, tubulin-based (PhuZ) spindle that centers replicating phage DNA sequestered within a nucleus-like structure. Here, we show that capsids assemble on the membrane and then move rapidly along PhuZ filaments toward the phage nucleus for DNA packaging. The spindle rotates the phage nucleus, distributing capsids around its surface. PhuZ filaments treadmill toward the nucleus at a constant rate similar to the rate of capsid movement and the linear velocity of nucleus rotation. Capsids become trapped along mutant static PhuZ filaments that are defective in GTP hydrolysis. Our results suggest a transport and distribution mechanism in which capsids attached to the sides of filaments are trafficked to the nucleus by PhuZ polymerization at the poles, demonstrating that the phage cytoskeleton evolved cargo-trafficking capabilities in bacteria.