Assays for screening for inhibitors of HIV

Details Assays for screening for inhibitors of HIV Assays for screening for inhibitors of HIV

1997-04-15

2001-05-01

Myers, Carla J. (Department: 1655)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving virus or bacteriophage

C435S006120, C435S007100, C536S023100, C536S024100, C536S024320, C536S024500, C514S04400A

Reexamination Certificate

active

06225045

ABSTRACT:

FIELD OF THE INVENTION
This invention relates to inhibition of replication of human immunodeficiency virus (HIV).
BACKGROUND OF THE INVENTION
HIV is the causative agent of acquired immunodeficiency syndrome (AIDS). The trans-activation region (TAR) and the Rev-response element (RRE) (Rosen et al., 1988; Dayton et al., 1989; Malim et al., 1990) of HIV are found in unspliced or partially spliced HIV mRNA introns. During replication of HIV, the RRE and TAR RNAs interact with specific HIV proteins. The RRE is recognised by the HIV protein Rev (Daly et al., 1989; Zapp & Green, 1989; Cochrane et al., 1990; Heaphy et al., 1990; Malim et al., 1990) which stimulates mRNA export from the nucleus (Emerman et al., 1989; Malim et al., 1990; Malim & Cullen 1993; Fischer et al., 1994; Meyer & Malim, 1994; Bogerd et al., 1995; Stutz et al., 1995) via the formation of a Rev/RRE complex which displays a nuclear export signal that is essential for Rev-mediated export of RNA from the nucleus and also for Rev shuttling (Malim et al., 1991; Fischer et al., 1994; Meyer & Malim, 1994; Fischer et al., 1995; Stutz et al., 1995; Wen et al., 1995; Wolff et al., 1995). The Rev/RRE interaction regulates the cytoplasmic accumulation of HIV genomic and structural mRNAs and is therefore essential if the virus is to propagate.
The RRE contains a series of stem-loop structures protruding from a long central stem, known as Stem I (Dayton et al., 1989; Malim et al., 1989b; Dayton et al., 1992; Mann et al., 1994), as shown in
FIG. 1
(RRE-WT; SEQ ID NO: 1). At the base of Stem IIb is a high-affinity Rev-binding motif which is recognised by a single Rev protein with a K
d
of approximately 1 nM (Bartel et al., 1991; Heaphy et al., 1991; Iwai et al., 1992; Kjems et al., 1992; Tiley et al., 1992). This high-affinity motif is a purine-rich bubble stabilised by non-Watson-Crick G&Circlesolid;A and G&Circlesolid;G base pairs (Heaphy et al., 1991; Bartel et al., 1991; Iwai et al., 1992; Pritchard et al., 1994). Together with a bulged-out uridine nucleotide, these non-Watson-Crick base pairs open the major groove of the mRNA duplex and permit the recognition of functional groups on the two base pairs either side of the bulged region inside the widened major groove. In addition to these base-specific contacts, phosphate contacts are made around the bubble as well as with base-paired nucleotides further away from the bubble (Iwai et al., 1992; Kjems et al., 1992; Pritchard et al., 1994).
Mutational analysis of the RRE has shown that the high-affinity interaction with a single Rev protein is necessary, but not sufficient, for Rev activity in vivo (Dayton et al., 1989; Malim et al., 1989b; Malim et al., 1990; Olsen et al., 1990; Bartel et al., 1991; Huang et al., 1991; Dayton et al., 1992; Holland et al., 1992; Mann et al., 1994). For full activity, further Rev monomers must be able to oligomerize along stem I of the RRE (Heaphy et al., 1990, 1991; Malim & Cullen, 1991; Mann et al., 1994). Truncations of Stem I that do not affect the high-affinity motif reduce Rev responses by removing additional potential binding sites for Rev monomers, with the longest truncations producing the greatest losses of activity (Mann et al., 1994). Similarly, mutations in the Rev protein that block oligomerization along the RNA stem result in an inactive protein (Malim & Cullen, 1991; Zapp et al., 1991).
It has been suggested that up to twelve Rev monomers in total can bind to each wild-type RRE (Mann et al., 1994). The high-affinity motif is not the sole Rev binding site on the RRE, however, unless a monomer is bound to the high-affinity motif, the oligomerization of Rev cannot take place. The binding of a single Rev to the high-affinity motif facilitates the binding and co-operative oligomerization of additional Rev monomers along the RRE (Iwai et al., 1992; Mann et al., 1994), with neighbouring Rev monomers in contact with one another (Mann et al., 1994).
Various models have been proposed as to the mechanism by which Rev oligomerization is achieved. Kjems et al. (1991) suggested that Rev monomers bind to a variety of sequence-specific sites in the RRE. Zapp et al. (1991) argued that Rev binds to the RRE high-affinity site as a pre-existing tetramer. Malim & Cullen (1991) ascribed the oligomerization solely to protein/protein interactions between neighbouring Rev monomers, and Tiley et al. (1992) reached the same conclusion. Powell et al. (1995) refined this view, believing that sequence-specific information in the RNA can exert a subtle influence on higher-order binding, but maintain that protein/protein interactions are the major determinant directing oligomerization.
Disruption of the natural Rev/RRE interaction via mutation of the natural sequences has been explored in the prior art as a potential avenue to the use of altered Rev or RRE molecules in anti-HIV therapy. Transdominant Rev mutants which retain the RRE-binding features of wild-type Rev but which are defective in certain other features have been described(eg. Malim et al., 1989a; Malim et al., 1991; Bogerd et al., 1995).
Harada et al. (1996) relates to in vivo methods for selecting short peptides which bind Rev.
Jensen, K. B. et al. (1995) disclose chemically modified RNA sequences (i.e., containing 5-iodouridine) which bind Rev in vitro with higher affinity than the RRE and which are able to crosslink with Rev at a 1:1 ratio. These are postulated as potential suicide ligands for in vivo disease inhibition, however, non-specific interactions with chemically reactive bases cannot be ruled out in an in vivo situation.
WO92/05195 discloses molecules which mimic the high-affinity binding site of the native RRE in order to act as competitive inhibitors, thus sequestering free Rev protein and preventing it from interacting with those mRNAs which contain the RRE. These molecules contain a greater number of Rev binding sites than are contained in viral RRE-containing mRNAs.
One object of the invention is to provide nucleic acid molecules which inhibit HIV replication.
Another object of the invention is to provide a nucleic acid decoy which binds HIV Rev protein so as to inhibit HIV infection.
Another object of the invention is to provide a nucleic acid decoy which binds HIV Rev protein with greater co-operativity than the wild-type RRE.
SUMMARY OF THE INVENTION
The invention is based on the unexpected discovery that model RREs comprising a high-affinity binding-motif flanked by perfect duplex RNA can only bind a monomer of Rev, and that disruptions to the RNA duplex in the vicinity of the high-affinity motif are necessary to permit the binding of additional Rev monomers. It also has been discovered that each disruption seems to allow the binding of an additional Rev monomer.
Therefore, according to the present invention there is provided an isolated nucleic acid comprising two or more operatively linked binding sites for HIV Rev protein, the sites comprising at least one nucleation motif and at least one oligomerization motif, wherein the nucleic acid binds Rev protein monomers with a higher degree of co-operativity than wild-type RRE.
As used herein, the term “operatively linked” means that oligomerization of a second HIV Rev protein along a nucleic acid molecule of the invention is initiated by the sequence-specific binding of a single Rev monomer at a nucleation motif. Therefore, in order for binding of a second Rev protein to occur, the binding of a first Rev protein at a high affinity site (i.e., a nucleation motif) must occur.
As used herein, the term “nucleation motif” refers to a nucleic acid binding site for Rev protein, wherein the Rev occupancy of the binding site is independent of the presence of any other bound Rev monomers.
The term “oligomerization motif” refers to a nucleic acid binding site for Rev protein, wherein the Rev occupancy of the binding site requires at least one Rev monomer to have already bound to an operatively linked Rev-binding site.
The nucleation motif is recognised by Rev in a sequence-specific manner. The nucleation motif may comprise a sequence of t

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