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A phage nucleus-associated RNA-binding protein is required for jumbo phage infection

ABSTRACT: Large-genome bacteriophages (jumbo phages) of the Chimalliviriadae family assemble a nucleus-like compartment bounded by a protein shell that protects the replicating phage genome from host-encoded restriction enzymes and CRISPR/Cas nucleases. While the nuclear shell provides broad protection against host nucleases, it necessitates transport of mRNA out of the nucleus-like compartment for translation by host ribosomes, and transport of specific proteins into the nucleus-like compartment to support DNA replication and mRNA transcription. Here we identify a conserved phage nuclear shell-associated protein that we term Chimallin C (ChmC), which adopts a nucleic acid-binding fold, binds RNA with high affinity in vitro, and binds phage mRNAs in infected cells. ChmC also forms phase-separated condensates with RNA in vitro. Targeted knockdown of ChmC using mRNA-targeting dCas13d halts infections at an early stage. Taken together, our data suggest that the conserved ChmC protein acts as a chaperone for phage mRNAs, potentially stabilizing these mRNAs and driving their translocation through the nuclear shell to promote translation and infection progression.

INSTRUMENT(S): Orbitrap Fusion Lumos, TripleTOF 5600

ORGANISM(S): Escherichia Phage Vb_ecom_goslar Pseudomonas Phage Phipa3

SUBMITTER: Eray Enustun

LAB HEAD: Kevin D.

PROVIDER: PXD045260 | Pride | 2024-03-25

REPOSITORIES: Pride

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A phage nucleus-associated RNA-binding protein required for jumbo phage infection

Project description:Large-genome bacteriophages (jumbo phages) of the Chimalliviriadae family assemble a nucleus-like compartment bounded by a protein shell that protects the replicating phage genome from host-encoded restriction enzymes and CRISPR/Cas nucleases. While the nuclear shell provides broad protection against host nucleases, it necessitates transport of mRNA out of the nucleus-like compartment for translation by host ribosomes, and transport of specific proteins into the nucleus-like compartment to support DNA replication and mRNA transcription. Here we identify a conserved phage nuclear shell-associated protein that we term chimallin C (ChmC), which adopts a nucleic acid-binding fold, binds RNA with high affinity in vitro and binds phage mRNAs in infected cells. ChmC also forms phase-separated condensates with RNA. Targeted knockdown of ChmC using mRNA-targeting Cas13d halts infections at an early stage. Taken together, our data suggest that the conserved ChmC protein acts as a chaperone for phage mRNAs, potentially stabilizing these mRNAs and driving their translocation through the nuclear shell to promote translation and infection progression.

2024-03-12 | GSE243675 | GEO

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

Project description: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.

2024-11-26 | PXD049415 | Pride

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

2024-11-26 | PXD050275 | Pride

RIPseq of VP882 phage activation in V. cholerae

Project description:Many, if not all, bacteria use quorum sensing (QS) to control gene expression and collective behaviours, and more recently QS has also been discovered in bacteriophages (phages). Phages can produce communication molecules of their own, or “listen in” on the host’s communication processes, in order to switch between lytic and lysogenic modes of infection. In this project, we studied the interaction of Vibrio cholerae, the causative agent of cholera disease, with the lysogenic vibriophage VP882. The lytic cycle of VP882 is induced by the QS molecule DPO (3,5-dimethylpyrazin-2-ol), however, the global regulatory consequences of DPO-mediated VP882 activation have remained unclear. Using a combination of transcriptomic, genetic, and biochemical approaches, we discovered that induction of VP882 results in binding of phage transcripts to the major RNA chaperone Hfq, which in turn outcompete and down-regulate host-derived Hfq-dependent small RNAs (sRNAs). VP882 itself also encodes Hfq-binding sRNAs and we demonstrate that one of these sRNAs, named VpdS, modulates the expression of multiple host and phage mRNAs through a base-pairing mechanism and thereby promotes phage replication. We further show that host-derived sRNAs can affect phage replication by interfering with the translation of phage mRNAs and thus might be part of the phage defence arsenal of the host. Taken together, our data draw a complex picture of post-transcriptional interactions occurring between host- and phage-derived transcripts that together determine the phage-mediated lysis program.

2023-11-23 | GSE247768 | GEO

RILseq of VP882 phage activation in V. cholerae

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Analysis of the role of yeast Puf3p in translation control

Project description:The Puf family of RNA-binding proteins regulate gene expression by controlling protein synthesis or stimulating RNA decay. Puf3p is one of six related proteins in Saccharomyces cerevisiae characterised as targeting and regulating decay of nuclear-encoded mRNAs specifying mitochondrial proteins. Much prior work has examined how Puf3p regulates COX17 mRNA. We undertook genome-wide approaches to define an expanded set of Puf3p target mRNAs. In agreement with prior studies, our sequencing of affinity-purified Puf3p-TAP associated mRNAs (RIP-seq) identified mRNAs encoding mitochondrially-targeted proteins. We also found an additional 720 mRNA targets that predominantly encode proteins that enter the nucleus.

2016-03-31 | E-MTAB-3413 | biostudies-arrayexpress

RNAseq of VP882 and VP882 ∆vpdS phage activation in V. cholerae

2023-11-23 | GSE247769 | GEO

O1->O139 serogroup conversion / phage selected

Project description:These experiments were performed to show a serogroup conversion of Vibrio cholerae from O1 to O139. For this purpose, V. cholerae O1 El Tor (A1552) was grown on crab shell fragments to induce natural competence for transformation. Purified DNA (4 ug each) from strain MO10, an O139 serogroup strain, was added after 24h and the cells were further grown for 24h. After detachment from the crab shell fragments, bacteria were poured into soft-agar and overlaid onto LB plates. Mukerjee's El Tor phage V (a gift of Dr. M.S. Islam) was dropped onto the surface of the bacteria containing soft-agar. The plaques formed by killing non-transformed A1552 cells possessed resistant clones which were picked and further selected for opaque morphotype and agglutination by O139-specific antiserum. Four clones were selected from each independent experiment and analyzed by microarray hybridization (BioPrime. Array CGH Genomic Labeling from Invitrogen). Two microarray replicates were done per clone. Strain Names: ApO139#2 / ApO139#4 / ApO139#6 / ApO139#8 are four clones analyzed after the first experiment; AIIpO139#3 / AIIpO139#4 / AIIpO139#5 / AIIpO139#6 are four clones analyzed after the second independent experiment. Two MA replicates for each clone were done. CGHs of A1552 versus MO10 are provided as control. Keywords: array CGH

2007-04-25 | GSE5302 | GEO

Comparative genomic hybridization of Vibrio cholerae which have converted from serogroup 01 to 0139 (phage selected) - part 2

Project description:These experiments were performed to show a serogroup conversion of Vibrio cholerae from O1 to O139. For this purpose, V. cholerae O1 El Tor (A1552) was grown on crab shell fragments to induce natural competence for transformation. Purified DNA (4 ug each) from strain MO10, an O139 serogroup strain, was added after 24h and the cells were further grown for 24h. After detachment from the crab shell fragments, bacteria were poured into soft-agar and overlaid onto LB plates. Mukerjees El Tor phage V (a gift of Dr. M.S. Islam) was dropped onto the surface of the bacteria containing soft-agar. The plaques formed by killing non-transformed A1552 cells possessed resistant clones which were picked and further selected for opaque morphotype and agglutination by O139-specific antiserum. Four clones were selected from each independent experiment and analyzed by microarray hybridization (BioPrime. Array CGH Genomic Labeling from Invitrogen). Two microarray replicates were done per clone. Strain Names: AIIIpO139#1 / AIIIpO139#3 / AIIIpO139#4 / AIIIpO139#5 are four clones analyzed after the second experiment; AIVpO139#2 / AIVpO139#4 / AIVpO139#5 / AIVpO139#8 are four clones analyzed after the fourth independent experiment. Two MA replicates for each clone were done.

2007-07-25 | E-SMDB-3821 | biostudies-arrayexpress

Comparative genomic hybridization of Vibrio cholerae which have converted from serogroup 01 to 0139 (phage selected)

Project description:These experiments were performed to show a serogroup conversion of Vibrio cholerae from O1 to O139. For this purpose, V. cholerae O1 El Tor (A1552) was grown on crab shell fragments to induce natural competence for transformation. Purified DNA (4 ug each) from strain MO10, an O139 serogroup strain, was added after 24h and the cells were further grown for 24h. After detachment from the crab shell fragments, bacteria were poured into soft-agar and overlaid onto LB plates. Mukerjeee's El Tor phage V (a gift of Dr. M.S. Islam) was dropped onto the surface of the bacteria containing soft-agar. The plaques formed by killing non-transformed A1552 cells possessed resistant clones which were picked and further selected for opaque morphotype and agglutination by O139-specific antiserum. Four clones were selected from each independent experiment and analyzed by microarray hybridization (BioPrime. Array CGH Genomic Labeling from Invitrogen). Two microarray replicates were done per clone. Strain Names: ApO139#2 / ApO139#4 / ApO139#6 / ApO139#8 are four clones analyzed after the first experiment; AIIpO139#3 / AIIpO139#4 / AIIpO139#5 / AIIpO139#6 are four clones analyzed after the second independent experiment. Two MA replicates for each clone were done. CGHs of A1552 versus MO10 are provided as control.

2007-08-01 | E-SMDB-3752 | biostudies-arrayexpress

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