Compositions and methods for providing a protein to a virion

Details Compositions and methods for providing a protein to a virion Compositions and methods for providing a protein to a virion

1999-03-30

2003-04-01

Housel, James (Department: 1648)

Drug, bio-affecting and body treating compositions

Antigen, epitope, or other immunospecific immunoeffector

Amino acid sequence disclosed in whole or in part; or...

C435S005000, C435S007100, C436S501000, C530S300000

Reexamination Certificate

active

06541002

ABSTRACT:

FIELD OF THE INVENTION
The invention relates to antiviral and virucidal compositions and methods and to methods of incorporating a polypeptide into a virus or a virus vector.
BACKGROUND OF THE INVENTION
Human immunodeficiency virus type 1 (HIV-1), a lentivirus, exhibits a complex viral life cycle. Replication of HIV-1 is tightly regulated by numerous cell-and virus-encoded regulatory proteins (Cullen, 1992. Microbiol. Rev. 56:375-394). Essential virus-encoded enzymes, including reverse transcriptase (RT), ribonuclease H (RNaseH), protease (PR), and integrase (IN), do not have cell-encoded counterparts, and for this reason have been used as targets for developing agents which inhibit virus replication without significantly adversely affecting cells (Debouck, 1992, AIDS Res. Hum. Retrovir. 8:153-164; Ridky et al., 1995, J. Biol. Chem. 270:29621-29623; Miller et al., 1996, AIDS Res. Hum. Retrovir. 12:859-865). Although considerable advances have been made in developing agents effective for inhibiting RT and PR, the mutability of HIV-1 has led to strains of the virus which exhibit resistance to such agents (Richman, 1995, Clin. Infect. Dis. 21 (Suppl. 2):S166-S169). Thus, there is a pressing need to develop alternative anti-HIV therapeutic strategies.
Specific cell compartmental localization of therapeutic moieties influences their therapeutic effect. For example, single chain variable region antibody fragments (SFvs) which bind specifically to HIV-1 IN and which are localized in the cytoplasm of a T lymphocyte inhibit infection of the lymphocyte more efficaciously than SFvs which are concentrated in the nucleus of the lymphocyte (Levy-Mintz et al., 1996, J. Virol. 70:8821-8832). Ribozymes have been localized within the virion of murine leukemia virus (MuLV; Rothman et al., 1996, Science 272:227-234). Incorporation of chimeric proteins into retrovirus particles has been reported, wherein the chimeric proteins had amino acid sequences which comprised a portion of the amino acid sequence of a non-viral protein and a portion of either HIV-1 Gag protein or HIV-1 Vpr protein (Wu et al., 1995, J. Virol. 69:3389-3398; Wu et al., 1996, Virology 219:307-313; Jones et al., 1990, J. Virol. 64:2265-2279; Wang et al., 1994, Virology 200:524-534; Weldon et al., 1990, J. Virol. 64:4169-4179).
It has been suggested that sorting of most cellular proteins into specific compartments is determined by protein-protein interactions mediated by specific domains of the proteins involved (Rothman et al., supra). This model of protein sorting is designated the protein-docking model. For example, interaction between a domain of a membrane protein with the signal sequence domain of a cytoplasmic protein can result in export of the cytoplasmic protein into either the Golgi apparatus or a mitochondrion (Rothman et al., supra).
Numerous proteins are packaged into HIV-1 virions, including RT, RNaseH, PR, IN, and proteins designated Gag, Pol, and Env. The genome of HIV-1 encodes other proteins which are packaged into HIV-1 virions, including a protein designated Vpr, which is present in virions of all primate lentivirus (Tristem et al., 1992, EMBO J. 11:3405-3412). The role of Vpr in infection of cells by lentiviruses has been studied extensively. Vpr is expressed relatively late in the lentiviral life cycle and encodes a 14 kilodalton protein which is predominantly localized in the nucleus of an infected cell (Wong-Staal et al., 1987, AIDS Res. Hum. Retrovir. 3:33-39; Lu et al., 1993, J. Virol. 67:6542-6550). Vpr is reported to be incorporated into lentivirus particles in quantities equal to the quantity of Gag protein (Lu et al., Id.; Cohen et al., 1990, J. Virol. 64:3097-3099).
Prior art investigations indicate that the carboxyl terminal domain (p6 region) of the Gag precursor designated p55 is involved in packaging of Vpr into HIV-1 virions (Lavallee et al., 1994, J. Virol. 68:1926-1934; Kondo et al., 1996, J. Virol. 70:159-164; Lu et al., 1995, J. Virol. 69:6873-6879; Paxton et al., 1993, J. Virol. 67:7229-7237). None of these reports identified a site at which p55 and Vpr interact within the amino acid sequence of either Vpr or the p6 region. A direct interaction between Vpr and the nucleocapsid protein designated Ncp7 has been demonstrated (Lim Tung et al., 1997, FEBS Lett. 401:197-201; De Rocquigny et al., 1997, J. Biol. Chem., J. Biol. Chem. 272:30753-30759). This interaction is mediated in vitro by the zinc finger regions of Ncp7 and the sixteen carboxyl terminal amino acids of Vpr (De Rocquigny et al., Id.). It may be that binding of Ncp7 in cooperation with another HIV-1 protein, possibly the p6 region of Gag, induces incorporation of Vpr into mature HIV-1 particles (De Rocquigny et al., Id.).
Several biological functions of Vpr have been defined. For example, Vpr is able to transactivate several heterologous viral promoters which do not share a common DNA sequence element (Cohen et al., 1990, J. Acquir. Immune Defic. Syndr. 3:11-18). Vpr is essential for optimal infection of macrophages by HIV-1 and influences nuclear transport of the HIV-1 pre-integration complex (Balliet et al., 1994, Virology 200:623-631; Connor et al., 1995, Virology 206:935-944; Westervelt et al., 1992, J. Virol. 66:3925-3931; Heinzinger et al., 1994, Proc. Natl. Acad. Sci. USA 91:7311-7315). Vpr also activates transcription from the HIV-1 long terminal repeat (LTR), and influences terminal differentiation of certain cell-types, such as rhabdomyosarcoma cells (Agostini et al., 1996, J. Mol. Biol. 261:599-606; Cohen et al., 1990, J. Acquir. Immune Defic. Syndr. 3:11-18; Wang et al., 1995, J. Biol. Chem. 270:25564-25569; Levy et al., 1993, Cell 72:541-550). Addition of exogenous Vpr to cells latently infected with HIV-1 can reactivate replication of the virus, indicating that Vpr may increase HIV-1 expression by affecting transcriptional or translational events (Levy et al., 1994, Proc. Natl. Acad. Sci. USA 91:10873-10877; Levy et al., 1995, J. Virol. 69:1243-1252). Vpr also causes cell cycle arrest in the G2/M phase and is capable of inducing apoptosis following cell cycle arrest (He et al., 1995, J. Virol. 69:6705-6711; Jowett et al., 1995, J. Virol. 69:6304-6313; Re et al., 1995, J. Virol. 69:6859-6864; Rogel et al., 1995, J. Virol. 69:882-888; Stewart et al., 1997, J. Virol. 71:5579-5592). All of these effects are probably mediated by interactions between Vpr and one or more cellular proteins.
Vpr is able to associate with the major uracil DNA glycosylase (UDG) involved in cellular DNA repair (Slupphaug et al., 1995, Biochemistry 34:128-138; BouHamdan et al., 1996, J. Virol. 70:697-704). The cellular physiological role of UDGs and deoxyuracil triphosphate pyrophosphatases (dUTPases) is believed to be prevention of misincorporation of deoxyuracil into DNA during DNA synthesis. A recent report excludes involvement of UDG in contributing to G2 arrest of cells. Mutational analysis of Vpr has been used to demonstrate that binding of Vpr to UDG is neither necessary nor sufficient to effect cell cycle arrest (Selig et al., 1997, J. Virol. 71:4842-4846). It may be that association of Vpr with UDG permits incorporation of UDG into HIV-1 virions, with the result that, upon subsequent infection of a cell by such a virion, uracil mis-incorporation into DNA transcribed from HIV-1 RNA is reduced. Thus, UDG encoded by a host cell genome may have a physiological role in the infectious cycle of HIV-1 that is similar to the role of dUTPases of non-primate lentiviruses. It has been reported that a strain of caprine arthritis encephalitis virus which is deficient in dUTPase accumulates G-to-A substitutions in its genome in vivo (Turelli et al., 1997, J. Virol. 71:4522-4530). It has also been reported that the vpr gene partially accounts for the lower-than-predicted in vivo mutation rate of HIV-1 (Mansky, 1996, Virology 222:391-400).
The ability of Vpr to associate with other cellular proteins, including glucocorticoid receptors, the basal transcription factor TFIIB, transcription factor Sp1, and the cellular DNA repair protein designated HH23A ha

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