HIV capsid assembly-associated compositions and method
AssigneeThe Regents of the University of California
InventorsJaisri R. Lingappa, Vishwanath R. Lingappa
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Abstract
A cell-free method for translation and assembly of retroviral, particularly HIV, capsid and capsid intermediates is disclosed. Also disclosed are novel HIV capsid assembly intermediates and novel host proteins which bind to such assembly intermediates. The invention also includes a screening method for compounds that alter retrovirus capsid assembly, and a method of treating HIV using compounds which inhibit the HIV capsid assembly pathway.
Claims22
A cell-free system for translation and assembly of an HIV capsid, comprising
- a cell-free translation mixture,
- an mRNA molecule encoding a Gag Pr55 protein derived from human immunodeficiency virus (HIV), and
- myristoyl coenzyme A.
A method of producing an HIV capsid intermediate in a cell-free system, comprising
- adding to a cell-free protein translation mixture which contains a cell-free extract, amino acids, transfer RNA (tRNA), ribosomes and an energy source:
- (i) an mRNA molecule encoding an HIV Pr55 Gag protein, and (ii) a concentration of myristoyl coenzyme A that is greater than about 0.1 micromlar, to form a reaction mixture;
- incubating said reaction mixture for a period of time sufficient to assemble Gag Pr55 mRNA translation products into an immature HIV capsid.
An isolated HIV capsid intermediate selected from a group of HIV capsid intermediates having buoyant densities selected from the group of about 10 S, about 80 S, about 150 S and about 500 S, wherein said buoyant densities are measured by sedimentation in a linear sucrose density gradient ranging from 15% to 60% sucrose.
A method of selecting a compound capable of altering HIV capsid assembly, comprising
- adding a test compound to a reaction mixture which includes (i) a cell-free translation mixture that includes a cell-free extract, tRNA, ribosomes, amino acids and an energy source, (ii) an mRNA molecule encoding HIV Gag Pr55, (iii) myristoyl coenzyme A, present at a concentration greater than about 0.1 micromolar,
- measuring capsid assembly in the presence of said test compound,
- comparing assembly in the absence of said test compound to assembly in the presence of said compound,
- selecting the compound as a compound capable of altering HIV capsid assembly if assembly measured in the absence of said compound is significantly different than assembly measured in the presence of said compound.
A method of inhibiting HIV capsid formation in a cell, comprising
- adding to the cell a compound selected for its ability to inhibit HIV capsid formation in a cell-free translation system consisting essentially of (i) a cell-free translation mixture which contains a cell-free extract, tRNA, ribosomes, amino acids and an energy source, (ii) an mRNA molecule encoding a HIV capsid assembly protein Pr55, and (iii) myristoyl coenzyme A.
A method of selecting a compound capable of altering HIV capsid assembly in cells, comprising
- adding a test compound to cells that are forming retroviral capsids,
- measuring the quantity and nature of capsid assembly intermediates formed within cells in the presence of said test compound,
- comparing the quantity and nature of assembly intermediates formed within cells in the absence of said test compound to said quantity and nature of intermediates formed in the presence of test compound,
- selecting the compound as a compound capable of altering formation of HIV assembly intermediates if the quantity or nature of intermediates measured in the presence of said compound is significantly different than the quantity or nature of intermediates measured in the absence of said compound.
Dependencies are inferred from the claim text.