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1.
Cell ; 177(7): 1771-1780.e12, 2019 06 13.
Artigo em Inglês | MEDLINE | ID: mdl-31199917

RESUMO

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.


Assuntos
Proteínas de Bactérias , Citoesqueleto , DNA Viral , Fagos de Pseudomonas , Pseudomonas , Tubulina (Proteína) , Vírion , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/metabolismo , Citoesqueleto/genética , Citoesqueleto/metabolismo , DNA Viral/biossíntese , DNA Viral/genética , Pseudomonas/genética , Pseudomonas/metabolismo , Pseudomonas/virologia , Fagos de Pseudomonas/genética , Fagos de Pseudomonas/metabolismo , Tubulina (Proteína)/genética , Tubulina (Proteína)/metabolismo , Vírion/genética , Vírion/metabolismo
2.
Nat Struct Mol Biol ; 30(11): 1653-1662, 2023 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-37667030

RESUMO

In the arms race between bacteria and bacteriophages (phages), some large-genome jumbo phages have evolved a protein shell that encloses their replicating genome to protect it against host immune factors. By segregating the genome from the host cytoplasm, however, the 'phage nucleus' introduces the need to specifically translocate messenger RNA and proteins through the nuclear shell and to dock capsids on the shell for genome packaging. Here, we use proximity labeling and localization mapping to systematically identify proteins associated with the major nuclear shell protein chimallin (ChmA) and other distinctive structures assembled by these phages. We identify six uncharacterized nuclear-shell-associated proteins, one of which directly interacts with self-assembled ChmA. The structure and protein-protein interaction network of this protein, which we term ChmB, suggest that it forms pores in the ChmA lattice that serve as docking sites for capsid genome packaging and may also participate in messenger RNA and/or protein translocation.


Assuntos
Bacteriófagos , Bacteriófagos/genética , Mapas de Interação de Proteínas , Capsídeo/química , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/química , RNA Mensageiro/análise
3.
bioRxiv ; 2023 May 18.
Artigo em Inglês | MEDLINE | ID: mdl-37292858

RESUMO

In the arms race between bacteria and bacteriophages (phages), some large-genome jumbo phages have evolved a protein shell that encloses their replicating genome to protect it against DNA-targeting immune factors. By segregating the genome from the host cytoplasm, however, the "phage nucleus" introduces the need to specifically transport mRNA and proteins through the nuclear shell, and to dock capsids on the shell for genome packaging. Here, we use proximity labeling and localization mapping to systematically identify proteins associated with the major nuclear shell protein chimallin (ChmA) and other distinctive structures assembled by these phages. We identify six uncharacterized nuclear shell-associated proteins, one of which directly interacts with self-assembled ChmA. The structure and protein-protein interaction network of this protein, which we term ChmB, suggests that it forms pores in the ChmA lattice that serve as docking sites for capsid genome packaging, and may also participate in mRNA and/or protein transport.

4.
Sci Adv ; 8(18): eabj9670, 2022 05 06.
Artigo em Inglês | MEDLINE | ID: mdl-35507660

RESUMO

Many eukaryotic viruses assemble mature particles within distinct subcellular compartments, but bacteriophages are generally assumed to assemble randomly throughout the host cell cytoplasm. Here, we show that viral particles of Pseudomonas nucleus-forming jumbo phage PhiPA3 assemble into a unique structure inside cells we term phage bouquets. We show that after capsids complete DNA packaging at the surface of the phage nucleus, tails assemble and attach to capsids, and these particles accumulate over time in a spherical pattern, with tails oriented inward and the heads outward to form bouquets at specific subcellular locations. Bouquets localize at the same fixed distance from the phage nucleus even when it is mispositioned, suggesting an active mechanism for positioning. These results mark the discovery of a pathway for organizing mature viral particles inside bacteria and demonstrate that nucleus-forming jumbo phages, like most eukaryotic viruses, are highly spatially organized during all stages of their lytic cycle.


Assuntos
Bacteriófagos , Bacteriófagos/genética , Capsídeo/ultraestrutura , Núcleo Celular , Genoma Viral , Vírion
5.
PLoS One ; 16(6): e0251429, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34111132

RESUMO

Upon infection of Pseudomonas cells, jumbo phages 201Φ2-1, ΦPA3, and ΦKZ assemble a phage nucleus. Viral DNA is enclosed within the phage-encoded proteinaceous shell along with proteins associated with DNA replication, recombination and transcription. Ribosomes and proteins involved in metabolic processes are excluded from the nucleus. RNA synthesis occurs inside the phage nucleus and messenger RNA is presumably transported into the cytoplasm to be translated. Newly synthesized proteins either remain in the cytoplasm or specifically translocate into the nucleus. The molecular mechanisms governing selective protein sorting and nuclear import in these phage infection systems are currently unclear. To gain insight into this process, we studied the localization of five reporter fluorescent proteins (GFP+, sfGFP, GFPmut1, mCherry, CFP). During infection with ΦPA3 or 201Φ2-1, all five fluorescent proteins were excluded from the nucleus as expected; however, we have discovered an anomaly with the ΦKZ nuclear transport system. The fluorescent protein GFPmut1, expressed by itself, was transported into the ΦKZ phage nucleus. We identified the amino acid residues on the surface of GFPmut1 required for nuclear targeting. Fusing GFPmut1 to any protein, including proteins that normally reside in the cytoplasm, resulted in transport of the fusion into the nucleus. Although the mechanism of transport is still unknown, we demonstrate that GFPmut1 is a useful tool that can be used for fluorescent labelling and targeting of proteins into the ΦKZ phage nucleus.


Assuntos
Núcleo Celular/metabolismo , Proteínas Luminescentes/metabolismo , Fagos de Pseudomonas/fisiologia , Pseudomonas aeruginosa/citologia , Pseudomonas aeruginosa/virologia , Transporte Ativo do Núcleo Celular
6.
Nat Commun ; 12(1): 342, 2021 01 12.
Artigo em Inglês | MEDLINE | ID: mdl-33436625

RESUMO

Understanding how biological species arise is critical for understanding the evolution of life on Earth. Bioinformatic analyses have recently revealed that viruses, like multicellular life, form reproductively isolated biological species. Viruses are known to share high rates of genetic exchange, so how do they evolve genetic isolation? Here, we evaluate two related bacteriophages and describe three factors that limit genetic exchange between them: 1) A nucleus-like compartment that physically separates replicating phage genomes, thereby limiting inter-phage recombination during co-infection; 2) A tubulin-based spindle that orchestrates phage replication and forms nonfunctional hybrid polymers; and 3) A nuclear incompatibility factor that reduces phage fitness. Together, these traits maintain species differences through Subcellular Genetic Isolation where viral genomes are physically separated during co-infection, and Virogenesis Incompatibility in which the interaction of cross-species components interferes with viral production.


Assuntos
Bacteriófagos/genética , Especiação Genética , Núcleo Celular/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Pseudomonas aeruginosa/virologia , Especificidade da Espécie , Frações Subcelulares
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