Proteins are involved in various equilibria that play a major role in their activity or regulation. The design of molecules that shift such equilibria is of great therapeutic potential. This fact was demonstrated in the cases of allosteric inhibitors, which shift the equilibrium between active and inactive (R and T) states, and chemical chaperones, which shift folding equilibrium of proteins. Here, we expand these concepts and propose the shifting of oligomerization equilibrium of proteins as a general methodology for drug design. We present a strategy for inhibiting proteins by ''shiftides'': ligands that specifically bind to an inactive oligomeric state of a disease-related protein and modulate its activity by shifting the oligomerization equilibrium of the protein toward it. We demonstrate the feasibility of our approach for the inhibition of the HIV-1 integrase (IN) protein by using peptides derived from its cellularbinding protein, LEDGF/p75, which specifically inhibit IN activity by a noncompetitive mechanism. The peptides inhibit the DNA-binding of IN by shifting the IN oligomerization equilibrium from the active dimer toward the inactive tetramer, which is unable to catalyze the first integration step of 3 end processing. The LEDGF/ p75-derived peptides inhibit the enzymatic activity of IN in vitro and consequently block HIV-1 replication in cells because of the lack of integration. These peptides are promising anti-HIV lead compounds that modulate oligomerization of IN via a previously uncharacterized mechanism, which bears advantages over the conventional interface dimerization inhibitors.allostery ͉ protein equilibrium ͉ shiftides ͉ peptides ͉ drug design

We have constructed a plasmid that, when introduced into Escherichia coil, induces the synthesis of large quantities of a protein with an apparent molecular mass of 66 kDa that differs from human immunodeficiency virus (HIV) RNA-dependent DNA polymerase (deoxynucleoside-triphosphate:DNA deoxynucleotidyltransferase or reverse transcriptase, EC 2.7.7.49) only in that it has two additional aminoterminal amino acids. This protein is soluble in E. coli extracts, is active in reverse transcriptase assays, and shows inhibition profiles with dideoxy-TTP and dideoxy-GTP that are indistinguishable from the viral enzyme. The deletion of 23 amino-terminal or carboxyl-terminal amino acids or the insertion of 5 amino acids at position 143 substantially decreases the polymerizing activity of the HIV reverse transcriptase made in E. coli. The properties of a 51-kDa reverse transcriptaserelated protein made in E. coli suggests that the p51 found in the virion probably does not have substantial polymerizing activity. The full-length HIV reverse transcriptase and the various mutant proteins produced in E. coli should be quite useful for structural and biochemical analyses as well as for the production of antibodies. In the virion, the reverse transcriptase is released from a larger polypeptide precursor by proteolytic cleavage (1). To permit the expression of the free HIV reverse transcriptase, we have taken the viral segment encoding the reverse transcriptase and modified the region at the ends of this segment so that there are initiation and termination codons at the positions where proteolytic cleavage takes place. The modified segment was introduced into an Escherichia coli expression plasmid. When introduced into E. coli, the plasmid with the HIV reverse transcriptase insert induces the synthesis of a large amount (several percent of the total E. coli protein) of soluble, enzymatically active, HIV reverse transcriptase. We show here that the ability of the HIV reverse transcriptase produced in E. coli to be inhibited by the dideoxy analogs of thymidine and guanosine is identical to the inhibition of virion reverse transcriptase. To study the catalytic domains of the HIV reverse transcriptase, we have begun a mutagenic analysis of this enzyme using the E. coli system. The E. coli-produced HIV polymerase can be used to search for inhibitors, to facilitate structural and biochemical studies, and to produce both monoclonal and polyclonal antibodies.

AIDS, caused by human immunodeficiency virus (HIV), is one of the world's most serious health problems, with current protocols being inadequate for either prevention or successful long-term treatment. In retroviruses such as HIV, the enzyme reverse transcriptase copies the single-stranded RNA genome into double-stranded DNA that is then integrated into the chromosomes of infected cells. Reverse transcriptase is the target of the most widely used treatments for AIDS, 3'-azido-3'-deoxythymidine (AZT) and 2',3'-dideoxyinosine (ddI), but resistant strains of HIV-1 arise in patients after a relatively short time. There are several nonnucleoside inhibitors of HIV-1 reverse transcriptase, but resistance to such agents also develops rapidly. We report here the structure at 7 A resolution of a ternary complex of the HIV-1 reverse transcriptase heterodimer, a monoclonal antibody Fab fragment, and a duplex DNA template-primer. The double-stranded DNA binds in a groove on the surface of the enzyme. The electron density near one end of the DNA matches well with the known structure of the HIV-1 reverse transcriptase RNase H domain. At the opposite end of the DNA, a mercurated derivative of UTP has been localized by difference Fourier methods, allowing tentative identification of the polymerase nucleoside triphosphate binding site. We also determined the structure of the reverse transcriptase/Fab complex in the absence of template-primer to compare the bound and free forms of the enzyme. The presence of DNA correlates with movement of protein electron density in the vicinity of the putative template-primer binding groove. These results have important implications for developing improved inhibitors of reverse transcriptase for the treatment of AIDS.

Reverse transcription is a critical step in the life cycle of all retroviruses and related retrotransposons. This complex process is performed exclusively by the retroviral reverse transcriptase (RT) enzyme that converts the viral single-stranded RNA into integration-competent double-stranded DNA. Although all RTs have similar catalytic activities, they significantly differ in several aspects of their catalytic properties, their structures and subunit composition. The RT of human immunodeficiency virus type-1 (HIV-1), the virus causing acquired immunodeficiency syndrome (AIDS), is a prime target for the development of antiretroviral drug therapy of HIV-1/AIDS carriers. Therefore, despite the fundamental contributions of other RTs to the understanding of RTs and retrovirology, most recent RT studies are related to HIV-1 RT. In this review we summarize the basic properties of different RTs. These include, among other topics, their structures, enzymatic activities, interactions with both viral and host proteins, RT inhibition and resistance to antiretroviral drugs.

A reverse transcriptase (RT) cDNA, designated HERV-K-T47D-RT, was isolated from a hormonally treated human breast cancer cell line. The protein product putative sequence is 97% identical to the human endogenous HERV-K retroviral sequences. Recombinant T47D-RT protein was used to generate polyclonal antibodies. The expression of HERV-K-T47D-RT protein increased in T47D cells after treatment with estrogen and progesterone. The RT-associated DNA polymerase activity was substantially increased after over-expressing a chimeric YFP-HERV-K-T47D-RT protein in cells. This RT-associated polymerase activity was significantly reduced by mutating the active site sequence YIDD to SIAA. Moreover, the endogenous RT activity observed in T47D cells was decreased by HERV-K-T47D-RT-specific siRNA, confirming the dependence of the endogenous enzymatic activity. To assess HERV-K-T47D-RT expression in human breast tumors, 110 paraffin sections of breast carcinoma biopsies were stained and subjected to confocal analysis. Twenty-six percent (28/110) of the tumor tissues and 18% (15/85) of the adjacent normal tissue, from the same patients, expressed the RT. HERV-K-T47D-RT expression significantly correlates with poor prognosis for disease-free patients and their overall survival. These results imply that HERV-K-T47D-RT might be expressed in early malignancy and might serve as a novel prognostic marker for breast cancer. Furthermore, these results provide evidence for the possible involvement of endogenous retrovirus in human breast carcinoma.

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We studied the effect of the natural marine substance mimaquinone on the catalytic activities of reverse transcriptase from human immunodeficiency virus type 1. Illimaquinone inhibited the RNase H activity of the enzyme at concentrations of 5 to 10 ,ug/ml, whereas RNA-dependent DNA polymerase and DNA-dependent DNA polymerase activities were considerably less susceptible to this inhibition. Two synthetic derivatives of illimaquinone, in which the 6'-hydroxyl group at the ortho position to one of the carbonyl groups of the quinone ring was modified, proved ineffective in inhibiting the human immunodeficiency virus type 1 reverse transcriptase RNase H function, suggesting involvement of the 6'-hydroxyl group in blocking the enzymatic activity.Reverse transcriptases (RTs) are key enzymes in the life cycle of retroviruses, since they are responsible for the transcription of the viral RNA into the provirus DNA that is subsequently integrated into the host cell DNA (34). RT is a multifunctional enzyme; the same protein molecules exhibit both RNA-dependent DNA polymerase (RDDP) and DNAdependent DNA polymerase (DDDP) activities as well as an inherent RNase H activity (32). Drugs acting at the reverse transcription level block specifically the replication of human immunodeficiency virus (HIV), the human retrovirus that causes acquired immunodeficiency syndrome, by preventing the unique transcription of genomic viral RNA. Several compounds with diverse molecular structures, including various 2',3'-dideoxynucleosides (14, 21, 22) and 3'-azidothymidine (9, 23, 35) that inhibit the viral RT in the form of 5'-triphosphates, foscarnet (28, 33), suramine (2, 7), rifabutin (1), and HPA 23 (26), have been shown to be active against HIV RT in vitro. In contrast to the relatively large volume of research on the inhibition of the DNA polymerase activities associated with various retroviral RTs, there is a paucity of information on the inhibition of the RT-associated RNase H function. RNase H specifically degrades the RNA in the RNA-DNA heteroduplex after the minus-strand DNA has been synthesized. Removal of the RNA strand enables the synthesis of the plus strand of DNA and the formation of the proviral double-stranded DNA, which is subsequently inserted into the host cellular genome (34). The fact that there is a distinction between the DNA polymerase and RNase H activities of retroviral RTs, including that of HIV (11,25,30,31), could be utilized to develop drugs that will specifically inhibit the RNase H function without appreciably affecting the DNA-polymerizing function. Since the physiological significance of RNase H in mammalian cells is far from clear (6), it is possible that even a partial inhibition of this activity would have minimal effects on the metabolism of normal cells but a substantial inhibitory effect on the virus. The availability of large amounts of nearly authentic (560 amino acids long with an apparent molecular weight of * Corresponding author. 66,000) enzymatically active and soluble recombinant HIV type 1 (HI...