Robinson, C. F. | Search bioRxiv

bioRxiv2025

Understanding the infection mechanism of {Phi}8 using infection method in Pseudomonas syringae pv. Phaseolicola strain: In-situ CryoET analysis

The Cystoviridae family strain {Phi}8 is an ideal model for studying the virus-host interactions and viral assembly mechanisms. In this study, we explored {Phi}8 infection kinetics in P. syringae pv. phaseolicola host cells using a combination of host lysis assays, transmission electron microscopy (TEM), cryo-focused ion beam (Cryo-FIB) milling, and in-situ cryo-electron tomography (Cryo-ET). Purified {Phi}8 was infected with host cells and observed at different time points. The host-pathogen infection was detected using TEM studies. The infected sample underwent Cryo-FIB milling. Our findings indicated that the infected host cell membrane changes its shape during infection. We were able to visualize the various stages of {Phi}8 assembly using subtomogram averaging. These stages included the unpackaged procapsid (PC) having a size of [~]41 nm and the double-stranded RNA (dsRNA)-packaged procapsid having a size of [~]54 nm. The packaged PC represented the most prevalent intermediate stages during viral assembly. Notably, the majority of complete virions isolated from the host cell were found outside the host cell, revealing the extracellular predominance of fully constructed particles. These findings emphasize the importance of stable intermediate stages in viral assembly and maturation of {Phi}8. This work advances our understanding of viral assembly and complex host-cell interactions. HighlightsO_LISuccessfully isolated and infected Pseudomonas syringae pv. phaseolicola (Pph) host cells with {Phi}8. C_LIO_LILysis assay performed to quantify host lysis to evaluate viral infection efficiency. C_LIO_LICryo-focused ion beam milling enabled precise preparation of cellular cross-sections for structural analysis. This allows identification of key stages of the {Phi}8 life cycle during the infection cycle. C_LIO_LIThe majority of complete virions were localized extracellularly, emphasizing the role of extracellular stages in the {Phi}8 lifecycle. C_LI Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=104 SRC="FIGDIR/small/678632v1_ufig1.gif" ALT="Figure 1"> View larger version (43K): org.highwire.dtl.DTLVardef@6ba760org.highwire.dtl.DTLVardef@1b99d2dorg.highwire.dtl.DTLVardef@fdfe72org.highwire.dtl.DTLVardef@14c077e_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2024

Bacteriophages Phi 8 and Phi 12 host infection are inhibited by OMVs and LPS purified from P. pseudoalcaligenes strain: East River Isolate A

Cystoviridae is a family of double stranded RNA (dsRNA) phage that infects various strains of Pseudomonas syringae, a Gram-negative soil bacteria known to infect various crops. Surrounding the icosahedral capsids of these phages is a bacterial derived phospholipid membrane. Embedded within this membrane is a multi-component protein complex, referred to as the spike complex. The spike complex is responsible for host recognition and membrane fusion. We studied the ability of two members of the Cystivirdae family to infect cells in the presence of purified outer membrane vesicles (OMVs) and lipopolysaccharide (LPS) derived from distinct sources. In this study we determined that OMVs from the host Pseudomonas pseudoalcaligenes strain: East River isolate A (ERA) inhibit Phi 8 and Phi 12 host infection. These OMVs range in size from 30 to 60 nm and bind to Phi 8 and Phi 12. However, OMV purified from P. syringae pv. phaseolicola LM2691 and E. coli {Delta}yciB {Delta}dcrB did not inhibit Phi 8 or Phi 12 host infection. However, LPS derived from ERA and LM2691 inhibited Phi 8 and Phi 12 infection, demonstrating that LPS is the receptor for these two viruses, and that OMV biogenesis is selective of LPS. LPS derived from other non-Cystoviridae Gram-negative bacteria, did not inhibit infection. We confirmed that host proteins are not required for Phi 8 or Phi 12 host interaction. Our results also suggest that differences in lipid A and the core polysaccharide in LPS may influence Phi 8 and Phi 12 host binding. IMPORTANCEMost phage families studied to date use a tailed appendage, composed of a multitude of proteins, for cellular recognition, membrane penetration, and genome injection. This contrasts with members of the Cystoviridae family which possess a phospholipid membrane bilayer with embedded proteins responsible for cellular recognition and membrane fusion. Thus, the Cystoviridae are akin to enveloped viruses which also use protein complexes embedded into their membrane for cellular recognition and membrane fusion. Examples of such viruses include the Retroviridae, Coronoviridae, Herpesviridae, and Orthomyxoviridae families. The binding specifics of Cystoviridae to the host outer membrane are unknown. Using Cystoviridae-OMV interaction we began to uncover the host requirements for binding Cystoviridae. The results presented determine that only lipid A and the core polysaccharide of LPS are required for Cystoviridae outer membrane binding.

microbiology↗