Amnon Hizi

Inhibiting HIV-1 integrase by shifting its oligomerization equilibrium

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 cellular-binding 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.

Retroviral reverse transcriptases.

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.

Incorporation of the Guanosine Triphosphate Analogs 8-Oxo-dGTP and 8-NH2-dGTP by Reverse Transcriptases and Mammalian DNA Polymerases

We have measured the efficiencies of utilization of 8-oxo-dGTP and 8-NH2-dGTP by human immunodeficiency virus type 1 and murine leukemia virus reverse transcriptases and compared them to those of DNA polymerases α and β. Initially, we carried out primer extension reactions in the presence of dGTP or a dGTP analog and the remaining three dNTPs using synthetic DNA and RNA templates. These assays revealed that, in general, 8-NH2-dGTP is incorporated and extended more efficiently than 8-oxo-dGTP by all enzymes tested. Second, we determined rate constants for the incorporation of each analog opposite a template cytidine residue using steady state single nucleotide extension kinetics. Our results demonstrated the following. 1) Both reverse transcriptases incorporate the nucleotide analogs; discrimination against their incorporation is a function primarily of Km or Vmax depending on the analog and the enzyme. 2) Discrimination against the analogs is more stringent with the DNA template than with a homologous RNA template. 3) Polymerase α exhibits a mixed kinetic phenotype, with a large discrimination against 8-oxo-dGTP but a comparatively higher preference for 8-NH2-dGTP. 4) Polymerase β incorporates both analogs efficiently; there is no discrimination with respect to Km and a significantly lower discrimination with respect to Vmax when compared with the other polymerases.

Mutational analysis of the ribonuclease H activity of human immunodeficiency virus 1 reverse transcriptase.

We have constructed a series of plasmids that, when introduced into Escherichia coli, induce the expression of high levels of either wild-type or mutated forms of the reverse transcriptase (RT) of human immunodeficiency virus type 1 (HIV-1). Mutant forms of RT that had been previously analyzed for their RNA-dependent DNA polymerase activity were tested for RNase H activity using an in situ polyacrylamide gel assay. Mutations affecting the RNase H are not clustered in a single region of the 66-kDa RT molecule. With only few exceptions, mutations that affect the RNase H activity also cause a substantial decrease in the DNA polymerase function. This suggests that, unlike the RT from murine leukemia virus (MuLV), it is difficult to genetically separate the catalytic domains responsible for the RNase H and DNA polymerase functions of HIV-1 RT. Those few mutations that differentially affect the RNase H and the polymerase activities of HIV-1 RT suggest that, as in MuLV, the polymerase domain is in the amino-terminus and the RNase H domain is in the carboxy-terminus. We have also generated chimeric molecules that are composed of sequences from the RT of HIV-1 and MuLV and these hybrid RTs were analyzed for their enzymatic properties. Two of these chimeric RTs possess RNase H activity but lack detectable DNA polymerase activity.

The inhibition of human immunodeficiency virus type 1 reverse transcriptase by avarol and avarone derivatives

We have analyzed the effects of several natural compounds related to avarols and avarones on the catalytic functions of human immunodeficiency virus type 1 (HIV‐1) reverse transcriptase (RT). The most potent substances, designated as avarone A,B and E and avarol F, inhibited indiscriminately the enzymatic activities of HIV‐1 RT, namely the RNA‐dependent and DNA‐dependent DNA polymerase as well as the ribonuclease H. The inhibition of the DNA polymerase activity was found to be non‐competitive with respect to either the template‐primer or the deoxynucleotide‐triphosphate. These studies suggest that the hydroxyl group at the ortho position to the carbonyl group at the quinone ring is involved in blocking the RT activity. The identification of the active site of the inhibitors will hopefully lead to the rational design of new potent anti‐HIV drugs.

Three new sesquiterpene hydroquinones from marine origin

Abstract Three new sesquiterpene hydroquinones peyssonol A and B (5 and 6 and hyatellaquinone (7) have been isolated from the alga Peyssonnelia sp. and the sponge Hyatella intestinalis. The structure of the three compounds was determined mainly by NMR measurements. Peyssonol A (5) is the first reported bromo sesquiterpene hydroquinone. Several of the new compounds were found in a preliminary test to inhibit the reverse transcriptase of human immunodeficiency virus (HIV).

Mutational analysis of the DNA polymerase and ribonuclease H activities of human immunodeficiency virus type 2 reverse transcriptase expressed in Escherichia coli.

We have constructed a plasmid that, when introduced into Escherichia coli, induces the synthesis of large quantities of a polypeptide with an apparent molecular weight of 68 kDa. The HIV-2 reverse transcriptase (RT) made in E. coli is soluble in bacterial extracts and possesses both RNA-dependent DNA polymerase and ribonuclease H (RNase H) activities typical of retroviral RTs. The HIV-2 RT expression clone was used to generate mutations in HIV-2 RT. There is a strong correlation between the effects of individual mutations on the DNA polymerase and RNase H activities. Mutations that profoundly affect the two catalytic functions are not clustered in any particular region of the polypeptide. Those few mutations that selectively affect either the RNase H or the DNA polymerase suggest that, like other retroviral RTs, the DNA polymerase is associated with the amino-terminal portion of HIV-2 RT and the RNase H with the carboxy-terminal portion. Genetically, the HIV-2 RT resembles the HIV-1 RT more closely than it resembles Moloney murine leukemia virus RT. The two catalytic functions of Moloney murine leukemia virus RT can be separately expressed in active form by molecular cloning; those of HIV-1 and HIV-2 RT cannot.

Interaction between HIV-1 Rev and integrase proteins: a basis for the development of anti-HIV peptides.

Human immunodeficiency virus 1 (HIV-1) Rev and integrase (IN) proteins are required within the nuclei of infected cells in the late and early phases of the viral replication cycle, respectively. Here we show using various biochemical methods, that these two proteins interact with each other in vitro and in vivo. Peptide mapping and fluorescence anisotropy showed that IN binds residues 1-30 and 49-74 of Rev. Following this observation, we identified two short Rev-derived peptides that inhibit the 3′-end processing and strand-transfer enzymatic activities of IN in vitro. The peptides bound IN in vitro, penetrated into cultured cells, and significantly inhibited HIV-1 in multinuclear activation of a galactosidase indicator (MAGI) and lymphoid cultured cells. Real time PCR analysis revealed that the inhibition of HIV-1 multiplication is due to inhibition of the catalytic activity of the viral IN. The present work describes novel anti-HIV-1 lead peptides that inhibit viral replication in cultured cells by blocking DNA integration in vivo.

Fidelity of the reverse transcriptase of human immunodeficiency virus type 2

The relatively low fidelity of human immunodeficiency virus type 1 reverse transcriptase (HIV‐1 RT) was implicated as a major factor that contributes to the genetic variability of the virus. Extension of mismatched 3′ termini of the primer DNA was shown to be a major determinant of the infidelity of HIV‐1 RT. Human immunodeficiency virus type 2 (HIV‐2) also shows extensive genetic variations. Therefore, we have analyzed the fidelity of the DNA‐dependent DNA polymerase activity of HIV‐2 RT and compared it with those of RTs of HIV‐1 and murine leukemia virus (MLV). Like other retroviral RTs, the HIV‐2 RT was shown to lack a 3′→5′ exonuclease activity. The ability of HIV‐2 RT to extend preformed 3′‐terminal A:A, A:C and A:G mispairs was examined by quantitating the amount and length of extended primers. The results demonstrate a relatively efficient mispair extension by HIV‐2 RT with a specificity of A:C⪢A:A>A:G. The mispair extension appears to be affected mainly by the increase of apparent K m values rather than by the change in V max values. The relative extension frequencies from all mispairs with HIV‐1 and HIV‐2 RTs was 6‐ to 9‐fold greater than that of MLV RT, suggesting that the HIV enzymes are substantially more error‐prone than MLV RT.

The fidelity of the reverse transcriptases of human immunodeficiency viruses and murine leukemia virus, exhibited by the mispair extension frequencies, is sequence dependent and enzyme related

Sequence variations in HIV‐1 and HIV‐2 probably result in part from inaccurate DNA synthesis by viral reverse transcriptases (RTs). We have studied in vitro the fidelity of both the DNA‐ and RNA‐dependent DNA polymerization functions of the two HIV RTs, as compared to that of murine leukemia virus (MLV) RT. The two HIV RTs were less accurate than MLV RT. The mispair extension frequencies observed previously with ribosomal RNA (rRNA) template were higher than those detected with ØX174am3 DNA template with all three RTs. In the current study we have investigated whether the nature of the copied nucleic acid (RNA vs. DNA) or the template nucleotide sequences affect the accuracy of DNA synthesis. We have analyzed the fidelity of DNA synthesis with DNA sequences identical to those of the rRNA sequences previously employed for reverse transcription. The results indicate that the fidelity of DNA synthesis depends mainly on the nucleotide sequences copied by every given RT. Yet, fidelity of DNA synthesis depends not only on the sequences copied but also on the nature of the enzymes per se. It is possible that these factors are major contributors to the high mutation rates of the two human immunodeficiency viruses.