Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Enzymatic production of a protein or polypeptide
Reexamination Certificate
1998-02-06
2003-07-15
Housel, James (Department: 1648)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Enzymatic production of a protein or polypeptide
C424S058000, C424S074000, C424S093210, C424S093700, C424S132100, C424S725000, C424S750000, C435S006120, C435S069100, C435S070100, C514S783000, C536S023100, C536S023720, C800S320300
Reexamination Certificate
active
06593103
ABSTRACT:
FIELD OF THE INVENTION
The invention is concerned with a method for producing HIV capsids in a cell-free extract. Also described are capsid intermediate compositions, auxiliary proteins, and screening assays that measure the ability of drugs to inhibit this process.
REFERENCES
Bryant, M., et al.,
Proc. Natl. Acad. Sci
. 87:523-527 (1990).
Dubochet, J., et al.,
J. Ultrastruct. Res
. 35:147-167 (1971).
Ericksons, A. H., and Blobel, G.,
Methods Enzymol
. 96:38-50 (1983).
Facke, M., et al.,
J. Virol
. 67:4972-4980 (1993).
Forsayeth, J. R. and Garcia, P. D., Biotechniques 17:354-358 (1994).
Gelderblom, H. R.,
AIDS
5:617-638 (1991).
Gheysen, D., et al.,
Cell
59:103-112 (1989).
Gottlinger, H. G., et al.,
Proc. Natl. Acad. Sci
. 86:5781-5785 (1989).
Hockley, D. J., et al.,
J. Gen. Virol
. 75:2985-2997 (1994).
Hynes, G., et al.,
Electrophoresis
17: 1720-1727 (1996).
Jacobs, E., et al.,
Gene
79:71-81 (1989).
Jowett, J. B. M., et al.,
J. Gen. Virol
. 73:3079-3086 (1992).
Lingappa, J. R., et al.,
J. Cell. Biol
. 125:99-111 (1984).
McEwen, C. R., Anal.
Biochem
. 20:114-149 (1967).
Melton, D. A., et al.,
Nucleic Acids Res
. 12:7035-7056 (1984).
Mergener, K., et al.,
Virology
186:25-39 (1992).
Merrick, W. C.,
Methods Enzymol
. 101:606-615 (1983).
Platt, E. J. and Haffar, O. K.,
Proc. Natl. Acad. Sci
. 91:4594-4598 (1994).
Royer, M., et al.,
Virology
184:417422 (1991).
Sambrook, J., et al., in
MOLECULAR CLONING, A LABORATORY MANUAL
, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989).
Smith, A. J., et al.,
J. Virol
. 67:2266-2275 (1993).
Spearman, P., et al.,
J. Virol
. 68:3232-3242 (1994).
Spirin, A. S., et al.,
Science
, 242: 1162-1164 (1988).
Towler, D. A., et al.,
Ann. Rev. Biochem
. 57:69-99 (1988).
Trono, D., et al.,
Cell
59:113-120 (1989).
Walter, P. and Blobel, G.,
Proc. Natl. Acad. Sci. USA
77:7112-7116 (1980).
Walter, P. and Blobel, G.,
Cell
34:525-533 (1983).
Wang, C.-T. and Barklis, E.,
J. Virol
. 67:4264-4273 (1993).
Wills, J. W. and Craven, R. C.,
AIDS
5:639-654 (1991).
Willison, K., et al.,
Cell
57: 621-632 (1989)
Zhao, Y., et al.,
Virology
199:403-408 (1994).
BACKGROUND OF THE INVENTION
The protein shell of the HIV virion, termed the HIV capsid or core, is composed of approximately 1500 copies of the Pr55 Gag structural protein precursor (Gelderblom, 1991). For proper assembly of the capsid to occur, Pr55 chains must undergo myristoylation (Gheysen, et al., 1989; Gottlinger, et al., 1989), an N-terminal modification thought to occur co-translationally (Towler, et al., 1988). The myristoylated chains are targeted to the host plasma membrane where assembly takes place concomitant with RNA encapsidation. As capsids are formed, they bud into the plasma membrane. This results in envelopment and subsequent release of viral particles from the cell. Coincident with their release, the immature viral particles undergo a maturation process, involving proteolytic processing of the precursor structural proteins and condensation of the capsids into collapsed, electron-dense cores (Gelderblom, 1991; Wills and Craven, 1991).
The manner in which HIV capsids assemble differs from that of many other retroviruses. Other retroviruses of the type B and type D category assemble “preformed” capsids in the cytoplasm of the infected cells. Such preformed capsids are then transported to other areas of the cell, such as the plasma membrane. In contrast, HIV capsids and other type C retroviruses form in intimate association with the plasma membrane, as described above. This important characteristic of HIV capsid formation has been demonstrated through electron microscopic studies (reviewed by Gelderblom, 1991; Wills and Craven, 1991).
Analyses of various mutants of Pr55 have revealed key domains required for efficient capsid assembly and targeting to the plasma membrane (see for example Gheysen, et al., 1989; Gottlinger, et al., 1989; Trono, et al., 1989; Royer, et al., 1991; Jowett, et al., 1992; Facke, et al., 1993; Wang and Barklis, 1993; Spearman, et al., 1994; Hockley, et al., 1994; Zhao, et al., 1994). However, the actual mechanisms involved in coordinating the formation of an HIV capsid from 1500 Gag monomers have not been elucidated. Many important questions about HIV capsid assembly remain unanswered, including whether assembly is an energy-dependent process, whether host proteins are required for assembly to take place, and whether assembly proceeds by way of discrete intermediates.
A major obstacle to addressing these questions experimentally has been the inherent difficulty of studying capsid assembly in cellular systems. In cells, many of the events in question proceed extremely rapidly and are not readily amenable to manipulation, making it difficult to identify trans-acting factors and energy substrates that may be required for assembly.
Development of a cell-free system that recreates capsid biogenesis would greatly facilitate a biochemical dissection and mechanistic understanding of capsid formation. Moreover, such a system would be useful as a screening assay for identifying drugs that interfere with the process.
It is the discovery of the present invention that immature HIV capsids can be assembled in a cell-free protein translation system, when certain key components are added to the reaction. Capsid formation by this method has the same requirement as capsid formation in vivo, including a requirement for myristoylation of Gag and an apparent requirement for membranes. Furthermore, in the present invention, this method for cell-free assembly of HIV capsids is used to reveal the existence of previously unknown steps in HIV virus formation. This system has now been used to demonstrate that capsid formation is dissociable into co- and post-translational phases, each of which has distinct co-factor and/or energy requirements. The reactions that occur during the post-translational phase are dependent on ATP and at least two independent host factors which are distinguished by their differential sensitivities to non-ionic detergents. This system can be used as a screening assay or selection assay for identification of new compounds that interfere with capsid formation, and hence with production of infectious virus.
Included in this invention is the discovery that formation of HIV capsids proceeds by way of a pathway of previously unrecognized assembly intermediates, in both cells and in the cell-free system. Such intermediates have utility, for example, in the design of drugs (including peptides and antibodies) and vaccines that interfere with progression from one intermediate to the next, in the design of drugs that act by inhibiting host cell machinery involved in capsid formation, and in the design of assay systems that examine the efficacy and mechanism of action of drugs that inhibit capsid formation.
In a related aspect, also forming part of the invention are host proteins that are involved in HIV capsid formation. An exemplary host protein, termed HP68, is a 68 kD protein present in a cell-free fraction of wheat germ extract and which forms part of one or more of the intermediate complexes described above. This protein is useful as a component of the cell-free translation systems and methods described above. It has further utility in the design of drugs that block or alter its association with HIV Gag and which therefore prevent formation of immature HIV capsids.
Forming yet another related aspect of the present invention, is the discovery that pieces of genomic HIV RNA can be encapsidated into the HIV capsids produced in the cell-free system by adding such RNA to the system. This feature of the invention can be used to design of drugs that interfere with encapsidation and in the design of assay systems that examine the mechanism of actions of drugs that inhibit encapsidation.
SUMMARY OF THE INVENTION
In one aspect, the present invention is concerned with a cell-free system for translation and assembly of retroviral capsids and capsid intermediates. In particular, the invention includes a cell-free system for translation/asse
Lingappa Jaisri R.
Lingappa Vishwanath R.
Housel James
Rae-Venter Barbara
Rae-Venter Law Group
The Regents of the University of California
Winkler Ulrike
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