Unnatural Amino Acid Incorporation into Virus-Like Particles

Virus-like particles composed of hepatitis B virus (HBV) or bacteriophage Qβ capsid proteins have been labeled with azide- or alkyne-containing unnatural amino acids by expression in a methionine auxotrophic strain of E. coli. The substitution does not affect the ability of the particles to self-assemble into icosahedral structures indistinguishable from native forms. The azide and alkyne groups were addressed by Cu(I)-catalyzed [3 + 2] cycloaddition: HBV particles were decomposed by the formation of more than 120 triazole linkages per capsid in a location-dependent manner, whereas Qβ suffered no such instability. The marriage of these well-known techniques of sense-codon reassignment and bioorthogonal chemical coupling provides the capability to construct polyvalent particles displaying a wide variety of functional groups with near-perfect control of spacing

Crosslinking of and Coupling to Viral Capsid Proteins by Tyrosine Oxidation

AbstractCowpea mosaic virus is composed of 60 identical copies of a two-subunit protein organized in pentameric assemblies around the icosahedral 5-fold symmetry axis. Treatment of the virus with the Ni(II) complex of the tripeptide GGH and a peroxide oxidant, or irradiation in the presence of Ru(bpy)32+ and persulfate generates covalent crosslinks across the pentameric subunit boundaries, effectively stitching the subunits together. Intersubunit crosslinking was found to occur exclusively at adjacent tyrosine residues (Y52-Y103), as predicted from the X-ray crystal structure of the capsid, and to be more extensive with the photochemical ruthenium system. The Ni/GGH oxidative procedure was also used to make covalent attachments to the virion by trapping with a functionalized disulfide reagent

Interspecies transfer of syntenic <i>RAMOSA1</i> orthologs and promoter <i>cis</i> sequences impacts maize inflorescence architecture

ABSTRACTGrass inflorescences support floral structures that each bear a single grain, where variation in branch architecture directly impacts yield. The maize RAMOSA1 (ZmRA1) transcription factor acts as a key regulator of inflorescence development by imposing branch meristem determinacy. Here, we show RA1 transcripts accumulate in boundary domains adjacent to spikelet meristems in Sorghum bicolor (Sb) and Setaria viridis (Sv) inflorescences similar as in the developing maize tassel and ear. To evaluate functional conservation of syntenic RA1 orthologs and promoter cis sequences in maize, sorghum and setaria, we utilized interspecies gene transfer and assayed genetic complementation in a common inbred background by quantifying recovery of normal branching in highly ramified ra1-R mutants. A ZmRA1 transgene that includes endogenous upstream and downstream flanking sequences recovered normal tassel and ear branching in ra1-R. Interspecies expression of two transgene variants of the SbRA1 locus, modeled as the entire endogenous tandem duplication or just the non-frameshifted downstream copy, complemented ra1-R branching defects and induced novel fasciation and branch patterns. The SvRA1 locus lacks conserved, upstream noncoding cis sequences found in maize and sorghum; interspecies expression of an SvRA1 transgene did not or only partially recovered normal inflorescence forms. Driving expression of the SvRA1 coding region by the ZmRA1 upstream region, however, recovered normal inflorescence morphology in ra1-R. These data leveraging interspecies gene transfer suggest that cis-encoded temporal regulation of RA1 expression is a key factor in modulating branch meristem determinacy that ultimately impacts grass inflorescence architecture.</jats:p

Natural Nanochemical Building Blocks: Icosahedral Virus Particles Organized by Attached Oligonucleotides

Virus particles, like metal nanoparticles and organic polymer beads, may be induced to assemble together by virtue of duplex base pairing of attached oligonucleotide chains and to disassemble on heating with unusually sharp temperature profiles. Icosahedral virions of the type used here have the added property of being structurally known to atomic resolution, offering unique opportunities to program assembly characteristics at the level of particle structure

“Click” Chemistry in a Supramolecular Environment: Stabilization of Organogels by Copper(I)-Catalyzed Azide−Alkyne [3 + 2] Cycloaddition

Organogels are thermoreversible, viscoelastic (soft) materials consisting of low molecular weight compounds which self-assemble into fibers, often of micrometer lengths and nanometer diameters. The installation of terminal azide and alkyne functional groups on the end of a standard alkylamide-based organogelator was found to cause a modest disruption in the gelation properties of the molecule. Cross-linking of those groups by the copper(I)-catalyzed azide−alkyne cycloaddition reaction produced thermoreversible materials of substantially greater gelation temperatures and mechanical rigidity. These results highlight the ability of azides and alkynesparticipants in the most commonly used “click” reactionto function as innocuous precursors to meaningful covalent interactions in materials science

Cowpea Mosaic Virus Capsid: A Promising Carrier for the Development of Carbohydrate Based Antitumor Vaccines

Immunotherapy targeting tumor cell surface carbohydrates is a promising approach for cancer treatment. However, the low immunogenecity of carbohydrates presents a formidable challenge. We describe here the enhancement of carbohydrate immunogenicity by an ordered display on the surface of the cowpea mosaic virus (CPMV) capsid. The Tn glycan, which is overexpressed on numerous cancer cell surfaces, was selected as the model antigen for our study. Previously it has been shown that it is difficult to induce a strong T cell-dependent immune response against the monomeric form of Tn presented in several ways on different carriers. In this study, we first synthesized Tn antigens derivatized with either a maleimide or a bromoacetamide moiety that was conjugated selectively to a cysteine mutant of CPMV. The glyco-conjugate was then injected into mice and pre-and post-immune antibody levels in the mice sera were measured by enzyme linked immunosorbant assays. High total antibody titers and, more importantly, high IgG titers specific for Tn were obtained in the post-immune day 35 serum, suggesting the induction of T cell-dependent antibody isotype switching by the glyco-conjugate. The antibodies generated were able to recognize Tn antigens presented in their native conformations on the surfaces of both MCF-7 breast cancer cells and the multi-drug resistant breast cancer cell line NCI-ADR RES. These results suggest that the CPMV capsid can greatly enhance the immunogenicity of weak antigens such as Tn and this can provide a promising tool for the development of carbohydrate based anti-cancer vaccines

Polyvalent Display of Heme on Hepatitis B Virus Capsid Protein through Coordination to Hexahistidine Tags

SummaryThe addition of a hexahistidine tag to the N terminus of the hepatitis B capsid protein gives rise to a self-assembled particle with 80 sites of high local density of histidine side chains. Iron protoporphyrin IX has been found to bind tightly at each of these sites, making a polyvalent system of well-defined spacing between metalloporphyrin complexes. The spectroscopic and redox properties of the resulting particle are consistent with the presence of 80 site-isolated bis(histidine)-bound heme centers, comprising a polyvalent b-type cytochrome mimic