Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving virus or bacteriophage
Reexamination Certificate
1998-07-30
2001-08-07
Park, Hankyel (Department: 1645)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving virus or bacteriophage
C530S324000, C530S350000, C530S388350
Reexamination Certificate
active
06270956
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to compounds useful for the modulation of Tat transactivation, methods for the modulation of Tat transactivation and methods for the identification of such compounds.
BACKGROUND OF THE INVENTION
The human immunodeficiency virus (HIV) encodes a nuclear transcriptional activator, Tat, which acts to enhance the processivity of RNA polymerase II (RNAPII) complexes that would otherwise terminate transcription prematurely at random locations downstream of the viral RNA start site. The mechanism of Tat transactivation is unique in that the cis-acting transactivation response element (TAR) is a stable RNA stem-loop structure that forms at the 5′ end of nascent viral transcripts. Transcriptional activation by Tat through TAR requires proper folding of the RNA as well as specific bases in the bulge and apical loop of the TAR RNA hairpin structure (for review, see Cullen, B. (1993)
Cell
73:417-420; Jones and Peterlin (1994)
Annu Rev Biochem
63:717-743).
The interaction of Tat with TAR RNA is mediated through an arginine-rich motif (ARM) that is characteristic of a family of sequence-specific RNA-binding proteins (Gait and Karn (1993)
Trends Biochem Sci
18:255-259). However, several lines of evidence suggest that the ARM of Tat is not an independent domain. First, the transactivation domain of Tat cannot be substituted by the activation domains of other transcription factors, such as the herpes virus VP16 protein, even though the VP16 activation domain is capable of activating transcription when tethered to RNA through a different RNA-binding domain (Tiley et al. (1992)
Genes Dev
6:2077-2087; Ghosh et al. (1993)
J Mol Biol
234:610-619). Second, the full-length Tat-1 protein, but not a mutant Tat protein that retains the ARM but lacks the transactivation domain, is able to target a heterologous protein to TAR RNA in vivo (Luo et al. (1993)
J Virol
67:5617-5622), indicating that the activation domain is required to target Tat to TAR in the cell. Third, amino acid insertions that separate the Tat activation domain from the ARM strongly reduce transactivation through TAR, but do not affect TAR-independent transactivation by chimeric Tat proteins (Luo and Peterlin (1993)
J Virol
67:3441-3445). Fourth, over-expression of mutant Tat proteins that contain the ARM does not block transactivation by the wild-type Tat protein in vivo (Madore and Cullen (1993)
J Virol
67:3703-3711). In addition, residues in the core of the transactivation domain have been found to enhance the affinity and specificity of the Tat:TAR interaction in vitro (Churcher et al. (1993)
J Mol Biol
230:90-110). Taken together, these studies strongly suggest that amino acid residues within the transactivation domain are required, directly or indirectly, for efficient binding of Tat to TAR RNA in vivo.
Tat recognizes a specific sequence in TAR that forms between the bulge and the upper stem, but does not require sequences in the loop of the hairpin that are essential for transactivation both in vivo and in vitro (for review, see Gait and Karn (1993)
Trends Biochem Sci
18:255-259). Based on these findings, it has been postulated that Tat must interact with a host cell RNA-binding cofactor in order to recognize TAR RNA with high affinity and in a sequence-appropriate manner. Consistent with this possibility, it has been shown that high levels of Tat cannot overcome the specific inhibition of transactivation that occurs when cells are exposed to high levels of exogenous synthetic TAR “decoy” RNAs (Sullenger et al. (1990)
Cell
63:601-608, Sullenger et al. (1991)
J Virol
65:6811-6816; Bohjanen et al. (1996)
Nucl Acids Res
24:3733-3738). Thus exogenous TAR RNAs appear to sequester a cellular cofactor in addition to Tat. Moreover, genetic studies indicate that a species-specific host cell factor is necessary for Tat to activate transcription through TAR in vivo. In particular, it has been found that murine and Chinese hamster ovary (CHO) cell lines do not support efficient transcription by Tat through TAR RNA (Hart et al. (1989)
Science
246:488-491; Newstein et al. (1990)
J Virol
64:4565-4567), whereas these same cell lines can support TAR-independent transactivation by chimeric Tat proteins (e.g., GAL4-Tat, Rev-Tat, MS2CP- Tat) that are targeted to their responsive promoters through a heterologous DNA- or RNA-binding domain (Alonso et al. (1992)
J Virol
66:4617-4621; Newstein et al. (1993)
Virol
197:825-828). Therefore the defect in nonpermissive rodent cells appears to be due to a problem of TAR RNA recognition.
Analysis of human:CHO hybrid cell clones reveals that a factor encoded on human chromosome 12 (Chr 12) can support a modest level of Tat activity in rodent cells (Hart et al. (1989)
Science
246:488-491; Newstein et al. (1990)
J Virol
64:4565-4567), and, most importantly, that the chromosome 12-encoded factor confers a specific requirement for sequences in the loop of TAR RNA that are otherwise dispensable for the residual low-level Tat activity that is observed in rodent cells (Alonso et al. (1994)
J Virol
66:6505-6513; Hart et al. (1993)
J Virol
67:5020-5024; Sutton et al. (1995)
Virol
206:690-694). UV cross-linking studies have identified a cellular 83 kDa RNA-binding protein that is present in human and CHO-Chr12 cells, but not in CHO cells, which binds to TAR RNA in a loop-dependent manner (Hart et al. (1995)
J Virol
69:6593-6599). Taken together, these results suggest that a human species-specific factor mediates the high-affinity, loop-specific binding of Tat to TAR RNA in vivo.
It has been generally presumed that the TAR RNA-binding cofactor would be distinct from the transcriptional coactivator for Tat. By contrast with the ARM, the N-terminal half of Tat can function autonomously as a transcriptional activation domain when fused to the DNA- or RNA-binding domain of a heterologous protein and targeted to an appropriate promoter. Truncated Tat-1 proteins that contain only the transactivation domain (aa 1-48) also act as potent dominant negative inhibitors of the wild-type HIV-1, HIV-2 and EIAV (equine infectious anemia virus) Tat proteins, suggesting that this region of Tat can sequester a limiting host cell transcription factor(s) that is necessary for Tat transactivation. Tat controls an early step in transcription elongation that is sensitive to inhibition by protein kinase inhibitors such as 5,6-dichloro-1-b-D-ribofuranosylbenzimidazole (DRB) (Kao et al. (1987)
Nature
330:489-493; Laspia et al. (1993)
J Mol Biol
232:732-746; Marciniak et al. (1990)
Cell
63:791-802; Marciniak and Sharp (1991)
EMBO J
10:4189-4196), and Tat transactivation in vivo and in vitro requires the carboxyl-terminal domain (CTD) of the largest subunit of RNA polymerase II (Chun and Jeang (1996)
J Biol Chem
271:27888-27894; Okamoto et al. (1996)
Proc Natl Acad Sci USA
93:11575-11579; Parada and Roeder (1996)
Nature
384:375-378; Yang et al. (1996)
J Virol
70:4576-4584).
The RNAPII carboxyl-terminal domain is predominantly unphosphorylated in assembled RNAPII preinitiation complexes and in complexes that pause shortly after initiation, but becomes heavily phosphorylated upon entry into productive elongation (for review, see Dahmus, M. (1996)
J Biol Chem
271:19009-19012). Although the carboxyl-terminal domain is critical for gene expression in vivo and for regulated transcription in crude extracts, it is not required for basal promoter activity in purified reconstituted transcription systems (Serizawa et al. (1993)
Nature
363:371-374). For many genes, the carboxyl-terminal domain has been found to be significantly more important for elongation than for initiation, which provides further support for the notion that carboxyl-terminal domain hyperphosphorylation may be an important step marking the transition of RNAPII molecules to forms that are competent for elongation (for review see Maldonado and Reinberg (1995)
Curr Opin Cell Biol
7:352-361; Shilatifard et al. (1997)
Curr Opin Genet Dev
7:199-204). The available evidence
Fang Shi-Min
Garber Mitchell
Jones Katherine A.
Wei Ping
Gray Cary Ware & Freidenrich LLC
Park Hankyel
Reiter Stephen E.
The Salk Institute for Biological Studies
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