Viral capsid assembly intermediates and methods of production
AssigneesThe Regents of the University of California, University of Washington
InventorsJaisri R. Lingappa, Kevin C. Klein, Vishwanath R. Lingappa
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Abstract
A cell-free method for translation and assembly of viral capsid and capsid intermediates is disclosed. Also disclosed are novel capsid assembly intermediates and novel host proteins which bind to such assembly intermediates. The invention also includes a screening method for compounds that alter viral capsid assembly, and a method of treating viral infection using compounds which inhibit the capsid assembly pathway.
Claims26
A method for identifying a viral gene required for capsid assembly, said method comprising:
- isolating nucleic acid putatively encoding said viral gene from said viral genome;
- programming a cell free translation system with said nucleic acid;
- and
- determining that capsid assembly has occurred as an indication that said viral gene is required for capsid assembly.
A composition comprising:
- isolated nucleic acid encoding a host protein required for capsid assembly.
A method for identifying a compound that inhibits viral capsid assembly, said method comprising:
- programming a cell free translation system with nucleic acid encoding a protein required for capsid assembly of said virus in the presence and absence of said compound;
- and
- determining whether capsid assembly has occurred as an indication as to whether said compound inhibits capsid assembly, wherein inhibition of capsid assembly is inferred from a change selected from the group consisting of (a) a change in distribution of assembly intermediates in the cell-free system;
- (b) a change in localization of host proteins in the cell-free system on glycerol or sucrose gradients;
- (c) a change in distribution of assembly intermediates in cells;
- (d) a change in level of assembly intermediates produced in cells;
- and (e) a change in co-localization of host protein and capsid protein in cells as observed during viral infection.
A method for obtaining one or more host proteins that interact with one or more viral proteins required for capsid assembly of a virus, said method comprising:
- programming a cell free translation system with nucleic acid encoding one or more protein required for capsid assembly of said virus, whereby translation products for said one or more capsid proteins are produced;
- incubating said translation mixture for a period of time sufficient for said translation products to assemble into one or more capsid intermediates, wherein said one or more capsid intermediates each comprise a complex of polymerized viral capsid protein and a host protein;
- isolating said one or more capsid intermediates;
- and
- dissassociating said one or more capsid intermediates whereby said one or more host proteins are obtained.
A method of obtaining a capsid intermediate involved in assembly of a virus, said method comprising:
- combining a nucleic acid enoding a viral gene required for capsid assembly with a cell-free protein translation mixture;
- incubating said mixture for a period of time sufficient to assemble translation products of said viral gene into viral capsid intermediates;
- separating said translation mixture into fractions of one or more capsid intermediates;
- and
- isolating said one or more capsid intermediates whereby capsid intermediates of said virus are obtained.
A method for identifying compounds that interfere with capsid assembly of a virus, said method comprising:
- expressing a protein required in said capsid assembly in a mammalian cell;
- identifying co-localization of said protein and one or more host protein using immunofluorescence in said mammalian cells;
- and
- screening compounds for those that interfere with co-localization of said protein required in said capsid and said one or more host protein in said mammalian cells, whereby compounds that interfere with capsid assembly are identified by a change from co-localization of said immunofluorescence to a diffuse staining pattern.
A method of identifying a compound that inhibits capsid assembly of a virus, said method comprising:
- adding a test compound to a cell-free translation mixture programmed with nucleic acid from a virus encoding one or more protein required for capsid assembly, whereby capsids are produced;
- comparing assembly in the absence of said test compound to assembly in the presence of said test compound, wherein less assembly measured in the presence of said compound is indicative of a compound that inhibits capsid assembly.
A method for treating symptoms of a viral infection in an animal in need thereof, said method comprising:
- isolating nucleic acid putatively encoding a viral gene involved in capsid assembly from said viral genome;
- programming a cell free translation system with said nucleic acid for a period of time sufficient to assemble translation products of said viral gene into viral capsid intermediates;
- separating said translation products into fractions of one or more capsid intermediates;
- isolating said one or more capsid intermediates;
- separating said capsid intermediates to obtain one or more host proteins that were bound to one or more viral protein in said capsid intermediates;
- comparing a biochemical characteristic of said individual one or more host proteins to a library comprising biochemical characteristics of a plurality of viral capsid assembly chaperones individually cross-referenced with one or more small molecules that inhibit interaction between an individual member of said library and a viral capsid protein;
- and
- providing said animal with a small molecule cross-referenced with an individual member of said library having a biochemical characteristic in common with said one or more host protein, whereby said symptoms are treated.
A method for producing viral capsids, said method comprising:
- programming a cell free translation system with said nucleic acid from said virus;
- and
- incubating said translation mixture for a period of time sufficient for said translation products to assemble into capsids;
- and
- isolating said capsids.
Antibodies to viral capsid assembly intermediates.
Dependencies are inferred from the claim text.