Chemistry: molecular biology and microbiology – Animal cell – per se ; composition thereof; process of... – Animal cell – per se – expressing immunoglobulin – antibody – or...
Reexamination Certificate
2002-06-12
2004-11-02
Park, Hankyel T. (Department: 1648)
Chemistry: molecular biology and microbiology
Animal cell, per se ; composition thereof; process of...
Animal cell, per se, expressing immunoglobulin, antibody, or...
C435S005000, C435S006120, C435S069700
Reexamination Certificate
active
06812025
ABSTRACT:
Throughout this application, various publications are referenced within parentheses. Disclosures of these publications in their entireties are hereby incorporated by reference into this application to more fully describe the state of the art to which this invention pertains. Full bibliographic citations for these references may be found immediately preceding the claims.
BACKGROUND OF THE INVENTION
HIV-1 reverse transcriptase (RT) catalyzes the conversion of genomic RNA into cDNA. The enzyme is a heterodimer of p66 and p51 subunits, and the dimerization of these subunits is required for optimal enzyme activity. To analyze this process at the genetic level we developed constructs that permit the detection of the interaction between these subunits in the yeast two-hybrid system. Genetic analysis of RT subdomains required for heterodimerization revealed that the fingers and palm of p66 were dispensable for p51 interaction. However, as little as a 26-amino acid deletion at the C terminus of p51 prevented dimerization with p66. A primer grip mutation, L234A, previously shown to inhibit RT dimerization by biochemical assays, also prevented RT dimerization in the yeast two-hybrid system. Second-site mutations that restored RT dimerization in yeast to the L234A parent were recovered in the tryptophan repeat region at the dimer interface and at the polymerase active site, suggesting the involvement of these sites in RT dimerization. In vitro binding experiments confirmed the effects of the L234A mutation and the suppressor mutations on the interaction of the two subunits. The RT two-hybrid assay should facilitate the extensive genetic analysis of RT dimerization and should make possible the rapid screening of potential inhibitors of this essential process.
The HIV type 1 (HIV-1) reverse transcriptase (RT) is required for the conversion of genomic RNA into double-stranded proviral DNA, catalyzed by the RNA- and DNA-dependent polymerase and ribonuclease H activities of the enzyme. HIV-1 RT is an asymmetric dimer formed by the association of p66 and p51 polypeptides, which are cleaved from a large Pr160
GagPol
precursor by the viral protease during virion assembly p51 contains identical N-terminal sequences as p66, but lacks the C-terminal ribonuclease H (RNase H) domain (1). The structure of HIV-1 RT has been elucidated by x-ray crystallography in a variety of configurations, including unliganded (2), complexed to nonnucleoside RT inhibitors (3), or complexed with double-stranded DNA either with (4) or without deoxynucleotide triphosphate (5, 6). Such analyses have shown that p66 can be divided structurally into the polymerase and RNase H domains, with the polymerase domain further divided into the fingers, palm, thumb and connections subdomains (6). Although p51 has the same polymerase domains as p66, the relative orientations of these individual domains differ markedly, resulting in p51 assuming a closed structure.
The RT heterodimer represents the biologically relevant form of the enzyme; the monomeric subunits have only low catalytic activity (7). Structural analysis reveals three major contacts between p66 and p51, with most of the interaction surfaces being largely hydrophobic (8, 9). The three contacts comprise an extensive dimer interface that includes the fingers subdomain of p51 with the palm of p66, the connection subdomains of both subunits, and the thumb subdomain of p51 with the RNase H domain of p66 (9). Several single amino acid substitutions in HIV-1 RT have been shown to inhibit heterodimer association (10-12). These include the mutations L234A (10, 11), G231A (11) and W229A (11), all located in the primer grip region of the p66 subunit, and L289K (12) in the thumb subdomain. Remarkably, these mutations are not located at the dimer interface and probably mediate their effects indirectly through conformational changes in the p66 subunit.
Several biochemical assays have been used previously to specifically measure RT dimerization. Some are based on the physical separation of monomers and dimers as determined by analytical ultracentrifugation (8) and gel filtration (7). Other assays include intrinsic tryptophan fluorescence (13) chemical crosslinking (14), the use of affinity tags (15) and polymerase activity itself (7). Although these methods detect dimerization, they either lack specificity or are not easy to perform. Moreover, these assays do not facilitate the rapid genetic analysis of protein-protein interactions under physiological conditions nor are they suitable for high throughput screening for RT dimerization inhibitors.
The yeast two-hybrid (Y2H) system (16) has been exploited to study the homomeric interactions of several retroviral proteins (see, e.g., ref. 17), and heteromeric interactions between viral proteins and various cellular partners (see, e.g., ref. 18). We have used this system to perform a genetic analysis of the determinants of RT dimerization. In addition, we have identified second-site mutations that restore heterodimerization to a noninteracting mutant p66.
SUMMARY OF THE INVENTION
This invention provides a method of determining whether a compound inhibits HIV-1 reverse transcriptase which comprises:
a) contacting a yeast cell with the compound, which cell comprises (i) a first plasmid which expresses a fusion protein comprising a p66 subunit polypeptide of HIV-1 reverse transcriptase, (ii) a second plasmid which expresses a fusion protein comprising a p51 subunit polypeptide of HIV-1 reverse transcriptase, and (iii) a reporter gene which is activated in the presence of a complex between the p66 subunit polypeptide and the p51 subunit polypeptide, and determining the level of activity of the reporter gene in the cell in the presence of the compound; and
b) comparing the level of activity of the reporter gene determined in step (a) with a level of activity of the reporter gene determined in the absence of the compound, wherein a decreased level of activity of the reporter gene in step (a) indicates that the compound inhibits formation of a complex between the p51 subunit polypeptide of HIV-1 reverse transcriptase and the p66 subunit polypeptide of HIV-1 reverse transcriptase, thereby indicating that the compound inhibits HIV-1 reverse transcriptase.
This invention provides a method of determining whether a compound inhibits formation of a complex between a p66 subunit polypeptide of HIV-1 reverse transcriptase and a p51 subunit polypeptide of HIV-1 reverse transcriptase which comprises:
a) contacting a yeast cell with the compound, which cell comprises (i) a first plasmid which expresses a fusion protein comprising a p66 subunit polypeptide of HIV-1 reverse transcriptase, (ii) a second plasmid which expresses a fusion protein comprising a p51 subunit polypeptide of HIV-1 reverse transcriptase, and (iii) a reporter gene which is activated in the presence of a complex between the p66 subunit polypeptide and the p51 subunit polypeptide, and determining the level of activity of the reporter gene in the cell in the presence of the compound; and
b) comparing the level of activity of the reporter gene determined in step (a) with a level of activity of the reporter gene determined in the absence of the compound, wherein a decreased level of activity of the reporter gene in step (a) indicates that the compound inhibits formation of a complex between the p51 subunit polypeptide of HIV-1 reverse transcriptase and the p66 subunit polypeptide of HIV-1 reverse transcriptase.
This invention provides a method of determining whether a compound inhibits HIV-1 reverse transcriptase which comprises:
a) contacting a yeast cell with the compound, which cell comprises (i) a first plasmid which expresses a fusion protein comprising a p66 subunit polypeptide of HIV-1 reverse transcriptase, (ii) a second plasmid which expresses a fusion protein comprising a p51 subunit polypeptide of HIV-1 reverse transcriptase, and (iii) a reporter gene which is activated in the presence of a complex between the p66 subunit polypeptide and the p51 subunit polypeptide, and determining t
Goff Stephen P.
Tachedjian Gilda
Cooper & Dunham LLP
Park Hankyel T.
The Trustees of Columbia University in the City of New York
White John P.
LandOfFree
Two hybrid assay that detects HIV-1 reverse transcriptase... does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Two hybrid assay that detects HIV-1 reverse transcriptase..., we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Two hybrid assay that detects HIV-1 reverse transcriptase... will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-3347990