Viral capsid assembly intermediates and methods of production

Cell Free Protein Synthesis

[0091]1. Transcription

[0092]The plasmid containing the Gag coding region was linearized at the EcoRl site (as described in the NEB catalogue). The linearized plasmid was purified by phenol-chloroform extraction (as described in Sambrook, J., et al., in Molecular Cloning. A Laboratory Manual) and this plasmid was adjusted to a DNA concentration of 2.0 mg / ml. Transcription was carried out using a reaction that contained: 40 mM Tris Ac (7.5), 6 mM Mg Ac, 2 mM Spermidine, 0.5 mM ATP, 0.5 mM CTP, 0.5 mM UTP, 0.1 mM GTP, 0.5 mM diguanosine triphosphate (cap), 10 mM Dithiothreitol, 0.2 mg / ml transfer RNA (Sigma Chemical Co.), 0.8 units / microliter RNAse inhibitor (Promega), 0.4 units per microliter of SP6 Polymerase (NEB). Mutant DNAs were prepared as described by Gottlinger, H. G., et al., Proc. Natl. Acad. Sci. 86:5781-5785 (1989); Jowett, J. B. M., et al., J. Gen. Virol. 73:3079-3086 (1992); Hockley, D. J. et al., J. Gen. Virol. 75:2985-2997 (1994); or Zhao, Y....

Preparation of HSS, HSP, and HSPd

[0103]Where indicated, wheat germ extract prepared as described in Example 1 was centrifuged at either 50,000 rpm for 21 min or 100,000 rpm for 30 min in a TLA 100 rotor (Beckman Instruments, Palo Alto, Calif.). The supernatant (high-speed supernatant, HSS) of the 50,000 rpm spin was used for cell-free translation and assembly reactions. The pellet of the 100,000 rpm spin (high speed pellet, HSP) was resuspended at a 5× concentration in buffer (25 mM Hepes pH 7.4, 4 mM MgAc, 100 mM KAc, 0.25M sucrose). Wheat germ extract adjusted to contain a concentration 0.5% “NIKKOL” was subjected to the same ultracentrifugation in parallel to generate the detergent treated high-speed pellet (HSPd). This pellet was washed twice with 200 RL of the above non-detergent buffer in order to remove traces of detergent, and then resuspended as described above. Following treatment with emetine at 50 mm, 1.8 μL of HSP or HSPd was added to the 18 mL cell-free reactions progr...

Translation of Gag Pr55 Protein in a Cell Free System

[0104]The cell-free translation / assembly system of the invention contains the components described in Part A, above. Example 1 provides details of an exemplary system derived from wheatgerm extract, which is capable of supporting translation and assembly of HIV capsids. Briefly, protein synthesis was initiated in the cell-free translation / assembly system by adding an mRNA that encodes Gag Pr55 protein. Alternatively, when the system includes transcription means, such as SP6 or T7 polymerase, the reaction may be initiated by addition of DNA encoding the protein. Complete synthesis of protein and assembly into capsids is usually achieved within about 150 minutes. FIG. 2 shows that capsids formed in the cell-free system of the invention are substantially the same as those formed in cells. Shown in the Figure is a comparison of migration of the capsids through an isopycnic CsCl gradient, where capsids formed in the cell-free translati...