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Bacteriophage lambda: The path from biology to theranostic agent

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Viral particles provide an attractive platform for the engineering of semisynthetic therapeutic nanoparticles. They can be modified both genetically and chemically in a defined manner to alter their surface characteristics, for targeting specific cell types, to improve their pharmacokinetic features and to attenuate (or enhance) their antigenicity. These advantages derive from a detailed understanding of virus biology, gleaned from decades of fundamental genetic, biochemical, and structural studies that have provided mechanistic insight into virus assembly pathways. In particular, bacteriophages offer significant advantages as nanoparticle platforms and several have been adapted toward the design and engineering of “designer” nanoparticles for therapeutic and diagnostic (theranostic) applications. The present review focuses on one such virus, bacteriophage lambda; I discuss the biology of lambda, the tools developed to faithfully recapitulate the lambda assembly reactions in vitro and the observations that have led to cooptation of the lambda system for nanoparticle design. This discussion illustrates how a fundamental understanding of virus assembly has allowed the rational design and construction of semisynthetic nanoparticles as potential theranostic agents and illustrates the concept of benchtop to bedside translational research. This article is categorized under: Biology‐Inspired Nanomaterials> Protein and Virus‐Based Structures Biology‐Inspired Nanomaterials> Nucleic Acid‐Based Structures
Bacteriophages are composed of a genome (DNA or RNA) encapsidated within a protective protein coat known as the capsid, which can be rod‐like (filamentous) or icosahedral. In case of the later, a “tail” structure situated at a unique vertex of the icosahedron functions to bind to the host cell and “inject” viral DNA into the cytoplasm. (a) Reprinted with permission from Williams and Fisher (). Copyright 1974 Charles C. Thomas. (b and d) Reprinted with permission from Dr. Robert Duda (2006 and 1998). (c) Reprinted with permission from Dr. Sherwood Casjens (1998)
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Electron micrographs of purified lambda capsids decorated with wild‐type gpD decoration protein (a) and with the gpD‐GFP fusion construct (b). Note the “furry” appearance of the GFP‐decorated particles, which represents the protruding protein ligand. (c) SDS‐PAGE of purified particles decorated in vitro with increasing concentrations of gpD‐GFP, relative to gpD‐WT, added to the reaction mixture, as indicated. Migration of the major capsid protein (gpE), wild‐type gpD, and the gpD‐GFP fusion construct is indicated at right (Reprinted with permission from (Chang et al., ). Copyright 2014 American Chemical Society)
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Engineered designer nanoparticles for theranostic applications. (a). PLP assembly in vitro using purified major capsid protein and scaffolding protein. The cargo can in principle include protein‐scaffold fusion constructs and scaffold‐proteins site‐specifically modified with biological, small molecule or synthetic cargos, including nucleic acids (gene delivery/DNA vaccine), proteins (bioreactor), chemical/biological toxins, and/or detection agents. (b) Customizable surface decoration for specific cell targeting, DMPK (Drug Metabolism and Pharmacokinetics) optimization, immune camouflage, or vaccine applications. Capsids or PLPs (isolated in vivo or assembled in vitro) can be decorated using gpD‐fusion proteins and gpD‐S42C site specifically and covalently modified with biological and synthetic molecules
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(a) CryoEM image of gpD trimer spikes assembled at the threefold axes of the expanded lambda capsid shell. Note the density extending from each gpD monomer in the trimeric spike. Density from the major capsid proteins has been removed for clarity. (b) Crystal structure of the gpD trimer spike (PDB #1C5E), modified to display the S42C mutation, is shown in cartoon representation; each cysteine depicted as a red sphere. Note that these residues are well separated, do not interfere with spike assembly at the shell surface and project away from the particle. (a and b) (Reprinted with permission from Chang et al. (). Copyright 2014 American Chemical Society)
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Lambda genome packaging pathway. The terminase enzyme binds to the cos sequence in a multigenome concatemer and cuts the duplex to “mature” the first genome end to be packaged. The binary complex binds to the portal vertex of the procapsid, activating the motor function of terminase. Upon packaging 15 kb, the procapsid shell undergoes an expansion transition and the gpD decoration protein adds as trimeric spikes to each of the 140 threefold axis of the icosahedron. The translocating motor stops upon reaching the next downstream cos site and again cuts the duplex to terminate the packaging reaction. Finishing proteins and a preassembled tail sequentially add to the portal vertex to afford an infectious virus
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Lambda procapsid assembly pathway. The portal protein assembles into a dodecameric ring structure, which nucleates polymerization of the major capsid protein into an icosahedral shell. Both steps are chaperoned by host groELS and the phage encoded scaffolding protein; 70–200 copies of the scaffolding protein are encapsidated within the immature shell. Procapsid assembly also encapsidates 10–12 copies of the phage protease, which degrades the scaffolding protein, modifies the portal structure and is autoproteolytic. The peptide fragments exit the shell to afford the mature procapsid shell
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Lambda infection of the Escherichia coli cell. An infectious lambda particle binds to the surface of the host cell (filled capsid, upper left) and injects the linear genome into the cytosol; the protein “ghost” remains at the cell exterior (empty capsid, upper left). The linear genome circularizes via the complementary single‐stranded DL and DR ends to afford the intact cohesive end site (cos). DNA replication yields “immature” genome concatemers; gene expression and self‐assembly of the protein products affords procapsid and tail structures
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Biology-Inspired Nanomaterials > Protein and Virus-Based Structures

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