Abstract
Bacteriophage P22 has long been considered a hallmark model for virus assembly and maturation. Repurposing of P22 and other similar virus structures for nanotechnology and nanomedicine has reinvigorated the need to further understand the protein-protein interactions that allow for the assembly, as well as the conformational shifts required for maturation. In this work, gp5, the major coat structural protein of P22, has been manipulated in order to examine the mutational effects on procapsid stability and maturation. Insertions to the P22 coat protein A-domain, while widely permissive of procapsid assembly, destabilize the interactions necessary for virus maturation and potentially allow for the tunable adjustment of procapsid stability. Future manipulation of this region of the coat protein subunit can potentially be used to alter the stability of the capsid for controllable disassembly.
Highlights
Bacteriophage P22 is a dsDNA virion of the Podoviridae family that infects Salmonella enterica serovar typhimurium
As a proof of concept for developing the phage display library, the RGD sequence flanked by two tri-glycine linkers (GGGRGDGGG) was inserted at residue T183 into the A-domain of P22 coat protein, and the resulting procapsids were thoroughly characterized, both in vitro through a BL21 expression system that produces non-infectious P22 procapsids, as well as in vivo utilizing a stable lysogen of P22 and lambda-red recombineering
Heat expansion of P22 procapsids has been used previously to mimic the process of maturation in vitro [17]
Summary
Bacteriophage P22 is a dsDNA virion of the Podoviridae family that infects Salmonella enterica serovar typhimurium. To make P22, an effective vector for nanomedicine, we were interested in developing a phage display system on the surface of the P22 capsid by manipulating sequences of the P22 coat protein in vivo. As a proof of concept for developing the phage display library, the RGD sequence flanked by two tri-glycine linkers (GGGRGDGGG) was inserted at residue T183 into the A-domain of P22 coat protein, and the resulting procapsids were thoroughly characterized, both in vitro through a BL21 expression system that produces non-infectious P22 procapsids, as well as in vivo utilizing a stable lysogen of P22 and lambda-red recombineering. The insight gained from this work indicates that structural changes in the A-domain of the P22 coat protein can controllably manipulate the ability of the phage to mature, while allowing for procapsid assembly
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