Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1999-07-12
2001-11-13
Park, Hankyel T. (Department: 1648)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S069100, C435S069700, C435S320100, C536S023100, C536S023720, C424S160100
Reexamination Certificate
active
06316190
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to the fields of molecular biology, immunology, retrovirus biology, and biochemistry.
BACKGROUND OF THE INVENTION
A single retroviral protein species, the Gag polyprotein, is sufficient for assembly of retrovirus particles. Since this process includes the selective encapsidation of viral RNA, this protein is evidently capable of specific interactions with nucleic acids. The nature of these interactions is not well understood as yet. After the virion is released from the cell, the polyprotein is cleaved by the virus-coded protease; one of the cleavage products, termed the nucleocapsid (NC) protein, then binds to the genomic RNA, forming the ribonucleoprotein core of the mature particle.
The interaction between Gag and genomic RNA is known to involve the NC domain of the polyprotein, since mutants within NC are defective in RNA packaging and since the specificity of encapsulation tends to be determined by the NC domain in chimeric Gag molecules. However, NC is a basic protein and has frequently been described as binding to single-stranded DNA or RNA in a sequence-independent manner. Indeed, it has been hypothesized to be capable of binding to any single-stranded nucleic acid under appropriate conditions. This binding activity appears to be important at several stages of virus replication.
A search of all known retroviruses reveals a highly conserved structure in their NC proteins. All NC proteins of the Oncovirinae and Lentivirinae subfamilies of Retroviridae contain one or two copies of a conserved sequence motif termed the “cysteine array” or “Cys-His box.” This motif can be represented as Cys(X)
2
Cys(X)
4
His(X)
4
Cys (SEQ ID NO:1) (Henderson et al.,
J. Biol. Chem
., 256:8400 (1981)). This motif is also known as the NC zinc finger or, alternatively, as the retroviral CCHC (SEQ ID NO:2) zinc finger because it chelates zinc through histidine imidazole and cysteine thiolates with a K
d
less than 10
−13
(Berg,
Science
, 232:485 (1986); Bess, Jr., et al.,
J. Virol
., 66:840 (1992); Chance, et al.,
Proc. Natl. Acad. Sci. U.S.A
., 89:10041 (1992); South, et al.,
Adv. Inorg. Biochem
., 8:199 (1990); South, et al.,
Biochem. Pharmacol
., 40:123 (1990)). Examples of retroviruses which possess at least one CCHC (SEQ ID NO:2) type zinc finger per nucleocapsid protein include, but are not limited to, HIV-1, HIV-2, SIV, BIV, EIAV, Visna, CaEV, HTLV-1, BLV, MPMV, MMTV, RSV, MuLV, FeLV, BaEV, and SSV.
The function of the NC zinc finger is not yet fully understood. However, all mutations in the zinc-binding residues which been described to date have been lethal for the virus. Virions produced by these mutants are frequently defective with respect to genomic RNA content (Aldovini, et al.,
J. Virol
., 64:1920-1926 (1990); Dorfman, et al.,
J. Virol
., 67:6159-6169 (1993); Dupraz, et al.,
J. Virol
., 64:4978-4987 (1990); Gorelick, et al.,
Proc. Natl. Acad. Sci. USA
, 85:8420-8424 (1988); Gorelick, et al.,
J. Virol
., 46:3207-3211 (1990); Méric, et al.,
J. Virol
., 63:1558-1568 (1989). As such, it is thought that the zinc fingers participate (as part of the Gag polypeptide precursor) in RNA packaging during virion assembly. Significantly, however, the mutant particles are far more defective with respect to infectivity than with respect to genomic RNA content (Gorelick, et al.,
Proc. Natl. Acad. Sci. USA
, 85:8420-8424 (1988)). A new class of zinc-finger mutants of Moloney MuLV (Mo-MuLV) have recently been characterized (Gorelick, et al. (1996)
J. Virol
. 70:2593-2597), and indeed, it has been found that these mutants package normal levels of genomic RNA, but are nevertheless noninfectious. Such observations imply that the zinc fingers play other roles in the viral life cycle in addition to their function in RNA packaging. It has been suggested that NC has an important role in maturation of the released virus particle (Fu, et al.,
J. Virol
., 68:5013-5018 (1994); Fu and Rein,
J. Virol
., 67:5443-5449 (1993)), and performs one or more functions in reverse transcription (Allain, et al.,
EMBO J
., 13:973-981 (1994); Lapadat-Tapolsky, et al.,
Nucleic Acids Res
., 21:831-839 (1993); Nagy, et al.,
J. Virol
., 68:757-765 (1994); Peliska, et al.,
Biochemistry
, 33:13817-13823 (1994); Roberts, et al.,
Biochem. Biophys. Res. Commun
., 160:486-494 (1989); Rodriguez-Rodriguez, et al.,
J. Biol. Chem
., 270:15005-15011 (1995); You, et al.,
J. Biol. Chem
., 269:31491-31495 (1994); Wu et al. (1996)
J. Virol
. 7132-7142). To date, however, the significance of the zinc finger and, in turn, the NC protein is not fully understood, nor is the binding of NC to nucleic acids.
SUMMARY OF THE INVENTION
The present invention stems from the surprising discovery that retroviral nucleocapsid proteins, such as NC and the Gag precursor, bind to specific nucleic acid sequences with high affinity. In the results described herein, the binding of recombinant HIV-1 NC protein to short oligonucleotides is assessed. These studies were typically performed at moderate ionic strengths, at which the nonspecific electrostatic interaction between NC and nucleic acids is minimized. Under these conditions, the protein exhibits profound sequence preferences. This sequence-specific binding is dependent upon the zinc fingers of the NC protein and has a strong hydrophobic component.
In one aspect, specific nucleic acid sequences which bind NC are useful as molecular decoys for retroviral nucleocapsid proteins, for making fusion molecules which inactivate retroviral nucleocapsid proteins, in screening assays for detecting molecules which inactivate retroviral nucleocapsid protein nucleic acid binding, and for purification of retroviral nucleocapsid proteins.
Example oligonucleotides which specifically bind retroviral nucleocapsid protein include oligonucleotides comprising the sequences TGTG (SEQ ID NO:3), TGTGT (SEQ ID NO:4), GTGTG (SEQ ID NO:5), (TG)
4
(SEQ ID NO:6), (TG)
5
(SEQ ID NO:7), (TG)
10
(SEQ ID NO:8), TGTGTG (SEQ ID NO:9), GACTTGTGGA (SEQ ID NO:10), and GACUUGUGG (SEQ ID NO:11). Examples of retroviruses which possess nucleocapsid proteins include, but are not limited to, HIV-1, HIV-2, SIV, BIV, EIAV, Visna, CaEV, HTLV-1, BLV, MPMV, MMTV, RSV, MuLV, FeLV, BaEV, and SSV.
In one embodiment, the invention provides a targeted molecule comprising an oligonucleotide which binds to a retroviral nucleocapsid protein with high affinity, and a fusion partner. The targeted molecule binds to the retroviral nucleocapsid protein with high affinity. Example retroviral nucleocapsid proteins include the Gag and NC proteins from HIV-1, HIV-2, SIV, BIV, EIAV, Visna, CaEV, HTLV-1, BLV, MPMV, MMTV, RSV, MuLV, FeLV, BaEV, and SSV.
The fusion partner optionally reacts with the retroviral nucleocapsid protein, thereby reducing the ability of the nucleocapsid protein to package retroviral RNA. This is accomplished by disrupting the viral nucleic acid binding site through altering amino acid side chains in the nucleocapsid nucleic acid binding site, or by attaching the targeted molecule to the binding site and sterically inhibiting nucleic acid binding of the retroviral nucleocapsid protein.
Alternatively, the fusion partner is optionally a cytotoxic molecule which is sequestered to cells comprising retroviral nucleocapsid proteins (e.g., a retrovirally infected cell), thereby killing the cell and preventing retroviral replication in the cell.
In one embodiment, the fusion partner does not react with the nucleocapsid protein, but is simply a label, such as a biotin or dye molecule. In this embodiment, the targeted molecule is used to visualize nucleocapsid proteins by binding the targeted molecule comprising a label to the nucleocapsid protein. In other embodiments, the targeted molecule comprises an oligonucleotide specific for nucleocapsid proteins, a fusion partner, and a label. In this embodiment, the fusion partner is optionally reactive with the nucleocapsid protein, and the label is available for visualizing nucleocapsid-targeted molecule interactions.
The invention pr
Casas-Finet Jose
Fisher Robert
Fivash Matthew
Henderson Louis E.
Rein Alan
Park Hankyel T.
The United States of America as represented by the Secretary of
Townsend & Townsend & Crew LLP
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