Bacteriophages Phi 8 and Phi 12 host infection are inhibited by OMVs and LPS purified from P. pseudoalcaligenes...

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 Δ yciB Δ 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.

Most 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.