Generalizable assay for virus capsid assembly
AssigneeIndiana University Reasearch and Technology Corporation
InventorsAdam Zlotnick, Stella Vieweger
Life of this patent
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Abstract
Virus capsids protect the viral genome and play roles in its delivery and intracellular transport, making them an attractive target for antiviral therapeutics. The difficulty in targeting capsid assembly is to identify molecules that interfere with the weak protein-protein interactions that drive the reaction. We have developed an in vitro assay for capsid assembly that works on a range of viruses at biologically relevant protein concentrations to facilitate screening large libraries of chemicals for lead compounds.
Claims23
A generalized in vitro viral assembly assay system, comprising:
- a. a concentration of at least one generalized oligonucleotide that is fluorophore labeled, wherein said fluorophore is either a donor (D), an acceptor (A), or both (DA), wherein (a) length of said generalized oligonucleotide, (b) sequence of said generalized oligonucleotide, and (c) D and A ratio are optimized to obtain energy transfer from D to A;
- and
- b. a concentration of unlabeled viral capsid protein titrated relative to the concentration of said fluorophore labeled generalized oligonucleotide, wherein said unlabeled viral capsid protein assembles so that said fluorophore labeled generalized oligonucleotide is associated with the resulting viral nucleocapsid particles and generates a D to A fluorescent signal.
A method to screen molecules that target viral assembly, comprising:
- a. providing a generalized viral assembly assay system that includes:
- (i) a chemically relevant concentration of at least one generalized oligonucleotide that is fluorophore labeled, wherein said fluorophore is either a donor (D), an acceptor (A), or both (DA), wherein a ratio of D and A is optimized to obtain energy transfer from D to A;
- and
- (ii) a titrated concentration of viral capsid protein, wherein said viral capsid protein and said fluorophore labeled generalized oligonucleotide partially assemble into a labile complex;
- b. applying a testing molecule to said viral assembly assay system;
- and
- c. measuring a fluorescent signal to ascertain the effect of said testing molecule on viral assembly.
A method to screen molecules that target alphavirus capsid assembly, comprising:
- a. providing a first fluorophore labeled generalized oligonucleotide, wherein said fluorophore is a donor (D), a second fluorophore labeled generalized oligonucleotide, wherein said second fluorophore is an acceptor (A), and excess unlabeled generalized oligonucleotide;
- b. providing an alphavirus capsid protein or a mutant capsid protein at titrated concentration sufficient to allow said capsid protein to assemble on many generalized oligonucleotides to produce maximum fluorescent signal;
- c. altering solution conditions to attenuate the association of the capsid protein or the mutant for the nucleotide and produce a reduced fluorescent signal;
- and
- d. providing a testing molecule to said labeled generalized oligonucleotide capsid protein mixture, wherein said molecule reduces or enhances fluorescent signal compared to step c.
A method to screen molecules that target HIV Gag assembly, comprising:
- a. providing a fluorophore labeled oligonucleotide DA, wherein said DA has consecutive adenosines;
- b. providing an HIV Gag protein, or a mutant form of Gag protein, at titrated concentration so that an HIV Gag capsid protein or an HIV Gag mutant capsid protein assembles with said fluorophore labeled oligonucleotide DA in a manner of several capsid proteins binding to one oligonucleotide to give maximum fluorescent signal;
- c. contriving solution conditions to attenuate the association of the Gag protein or the mutant Gag for the nucleotide and produce a reduced fluorescent signal;
- and
- d. providing a testing molecule to said fluorophore labeled oligonucleotide DA and Gag protein mixture, wherein said molecule reduces or enhances fluorescent signal compared to step c.
Dependencies are inferred from the claim text.