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.     

Correlation between shiftide activity and HIV-1 integrase inhibition by a peptide selected from a combinatorial library

The human immunodeficiency virus type 1 (HIV-1) integrase (IN) protein is an emerging target for the development of anti-HIV drugs. We recently described a new approach for inhibiting IN by “shiftides”—peptides that inhibit the protein by shifting its oligomerization equilibrium from the active dimer to the inactive tetramer. In this study, we used the yeast two-hybrid system with the HIV-1 IN as a bait and a combinatorial peptide aptamer library as a prey to select peptides of 20 amino acids that specifically bind IN. Five non-homologous peptides, designated as IN-1 to IN-5, were selected. ELISA studies confirmed that IN binds the free peptides. All the five peptides interact with IN with comparable affinity (K_d≈10 μM), as was revealed by fluorescence anisotropy studies. Only one peptide, IN-1, inhibited the enzymatic activity of IN in vitro and the HIV-1 replication in cultured cells. In correlation, fluorescence anisotropy binding experiments revealed that of the five peptides, only the inhibitory IN-1 inhibited the DNA binding of IN. Analytical gel filtration experiments revealed that only the IN-1 and not the four other peptides shifted the oligomerization equilibrium of IN towards the tetramer. Thus, the results show a distinct correlation between the ability of the selected peptides to inhibit IN activity and that to shift its oligomerization equilibrium.     

A novel short peptide is a specific inhibitor of the human immunodeficiency virus type 1 integrase

The retroviral encoded protein integrase (IN) is required for the insertion of the human immunodeficiency virus type 1 (HIV-1) proviral DNA into the host genome. In spite of the crucial role played by IN in the retroviral life cycle, which makes this enzyme an attractive target for the development of new anti-AIDS agents, very few inhibitors have been described and none seems to have a potential use in anti-HIV therapy. To obtain potent and specific IN inhibitors, we used the two-hybrid system to isolate short peptides. Using HIV-1 IN as a bait and a yeast genomic library as the source of inhibitory peptides (prey), we isolated a 33-mer peptide (I33) that bound tightly to the enzyme. I33 inhibited both in vitro IN activities, i.e. 3' end processing and strand transfer. Further analysis led us to select a shorter peptide, EBR28, corresponding to the N-terminal region of I33. Truncated variants showed that EBR28 interacted with the catalytic domain of IN interfering with the binding of the DNA substrate. Alanine single substitution of each EBR28 residue (alanine scanning) allowed the identification of essential amino acids involved in the inhibition. The EBR28 NMR structure shows that this peptide adopts an alpha-helical conformation with amphipathic properties. Additionally, EBR28 showed a significant antiviral effect when assayed on HIV-1 infected human cells. Thus, this potentially important short lead peptide may not only be helpful to design new anti-HIV agents, but also could prove very useful in further studies of the structural and functional characteristics of HIV-1 IN.     

A comparative study of backbone versus side chain peptide cyclization: application for HIV-1 integrase inhibitors

Peptide cyclization is an important tool for overcoming the limitations of linear peptides as drugs. Backbone cyclization (BC) has advantages over side chain (SC) cyclization because it combines N-alkylation for extra peptide stability. However, the appropriate building blocks for BC are not yet commercially available. This problem can be overcome by preparing SC cyclic peptide analogs of the most active BC peptide using commercially available building blocks. We have recently developed BC peptides that inhibit the HIV-1 integrase enzyme (IN) activity and HIV-1 replication in infected cells. Here we used this system as a model for systematically comparing the BC and SC cyclization modes using biophysical, biochemical and structural methods. The most potent SC cyclic peptide was active almost as the BC peptide and inhibited IN activity in vitro and blocked IN activity in cells even after 6 days. We conclude that both cyclization types have their respective advantages: The BC peptide is more active and stable, probably due to the N-alkylation, while SC cyclic peptides are easier to synthesize. Due to the high costs and efforts involved in preparing BC peptides, SC may be a more approachable method in many cases. We suggest that both methods are interchangeable.     

Mechanism of action of the HIV-1 integrase inhibitory peptide LEDGF 361-370

The HIV-1 integrase protein (IN) mediates integration of the viral cDNA into the host genome and is a target for anti-HIV drugs. We have recently described a peptide derived from residues 361-370 of the IN cellular partner protein LEDGF/p75, which inhibited IN catalytic activity in vitro and HIV-1 replication in cells. Here we performed a comprehensive study of the LEDGF 361-370 mechanism of action in vitro, in cells and in vivo. Alanine scan, fluorescence anisotropy binding studies, homology modeling and NMR studies demonstrated that all residues in LEDGF 361-370 contribute to IN binding and inhibition. Kinetic studies in cells showed that LEDGF 361-370 specifically inhibited integration of viral cDNA. Thus, the full peptide was chosen for in vivo studies, in which it inhibited the production of HIV-1 RNA in mouse model. We conclude that the full LEDGF 361-370 peptide is a potent HIV-1 inhibitor and may be used for further development as an anti-HIV lead compound.     

Study on the molecular mechanism of inhibiting HIV-1 integrase by EBR28 peptide via molecular modeling approach

Human immunodeficiency virus type 1 (HIV-1) integrase (IN) is an essential enzyme in the HIV-1 lifecycle which aids the integration of viral DNA into the host chromosome. Recently synthesized 12-mer peptide EBR28, which can strongly bind to IN, is one of the most potential small peptide leading compounds inhibiting IN binding with viral DNA. However, the binding mode between EBR28 peptide with HIV-1 IN and the inhibition mechanism remain uncertain. In this paper, the binding modes of EBR28 with HIV-1 IN monomer core domain (IN(1)) and dimmer core domain (IN(2)) were investigated by using molecular docking and molecular dynamics (MD) simulation methods. The results indicated that EBR28 bound to the interfaces of the IN(1) and IN(2) systems mainly through the hydrophobic interactions with the beta3, alpha1 and alpha5 regions of the proteins. The binding free energies for IN(1) with a series of EBR28 mutated peptides were calculated with the MM/GBSA model, and the correlation between the calculated and experimental binding free energies is very good (r=0.88). Thus, the validity of the binding mode of IN(1) with EBR28 was confirmed. Based on the binding modes, the inhibition mechanism of EBR28 was explored by analyzing the essential dynamics (ED), energy decomposition and the mobility of EBR28 in the two docked complexes. The proposed inhibition mechanism is represented that EBR28 binds to the interface of IN(1) to form the IN(1)_EBR28 complex and preventes the formation of IN dimmer, finally leads to the partial loss of binding potency for IN with viral DNA. All of the above simulation results agree well with experimental data, which provide us with some helpful information for designing anti-HIV small peptide drugs.     

Peptides derived from HIV-1 Rev inhibit HIV-1 integrase in a shiftide mechanism

The HIV-1 Integrase protein (IN) mediates the integration of the viral cDNA into the host genome. IN is an emerging target for anti-HIV drug design, and the first IN-inhibitor was recently approved by the FDA. We have developed a new approach for inhibiting IN by "shiftides": peptides derived from its cellular binding protein LEDGF/p75 that inhibit IN by shifting its oligomerization equilibrium from the active dimer to an inactive tetramer. In addition, we described two peptides derived from the HIV-1 Rev protein that interact with IN and inhibit its activity in vitro and in cells. In the current study, we show that the Rev-derived peptides also act as shiftides. Analytical gel filtration and cross-linking experiments showed that IN was dimeric when bound to the viral DNA, but tetrameric in the presence of the Rev-derived peptides. Fluorescence anisotropy studies revealed that the Rev-derived peptides inhibited the DNA binding of IN. The Rev-derived peptides inhibited IN catalytic activity in vitro in a concentration-dependent manner. Inhibition was much more significant when the peptides were added to free IN before it bound the viral DNA than when the peptides were added to a preformed IN-DNA complex. This confirms that the inhibition is due to the ability of the peptides to shift the oligomerization equilibrium of the free IN toward a tetramer that binds much weaker to the viral DNA. We conclude that protein-protein interactions of IN may serve as a general valuable source for shiftide design.     

Identification by phage display selection of a short peptide able to inhibit only the strand transfer reaction catalyzed by human immunodeficiency virus type 1 integrase

Human immunodeficiency virus type 1 integrase catalyzes the integration of proviral DNA into the infected cell genome, so it is an important potential target for antiviral drug design. In an attempt to search for peptides that specifically interact with integrase (IN) and inhibit its function, we used an in vitro selection procedure, the phage display technique. A phage display library of random heptapeptides was used to screen for potential peptide ligands of HIV-1 IN. Several phage clones were identified that specifically bound IN. Two of the selected peptides (FHNHGKQ and HLEHLLF) exhibited a high affinity for IN and were chemically synthesized. High affinity was confirmed by a displacement assay which showed that these two synthetic peptides were able to compete with the phages expressing the corresponding peptide. These agents were assayed on the in vitro IN activities. While none of them inhibited the 3'-processing reaction, the FHNHGKQ peptide was found to be an inhibitor of the strand transfer reaction. Despite its high affinity for IN, the HLEHLLF peptide selected and assayed under the same conditions was unable to inhibit this reaction. We showed that the FHNHGKQ peptide inhibits specifically the strand transfer activity by competing with the target DNA for binding to IN. These IN-binding agents could be used as a base for developing new anti-integrase compounds as well as for structural studies of the still unknown three-dimensional structure of the entire integrase molecule.     

Immune responses to the 18-kDa protein of Mycobacterium leprae. Similar B cell epitopes but different T cell epitopes seen by inbred strains of mice.

Antibody responses to the 18-kDa protein of Mycobacterium leprae have been analyzed in different strains of mice. High, intermediate, and low responder strains have been identified and these response patterns show clear linkage to genes encoded in the H-2 complex. Three peptides, residues 1-50, 51-100, and 101-148 have been synthesized, as well as a series of 20-mer peptides, which span the entire 18-kDa protein. Repeated immunization of different strains of mice with the 18-kDa protein resulted in IgG responses to epitopes found on all three synthetic peptides. Immunization of BALB/cJ and B10.BR mice, two high responder strains, with 18-kDa protein resulted in high levels of IgG antibody to epitopes found on peptides 1-20, 16-35, 31-50, 46-65, and 76-95. B10.BR mice also contained IgG that bound peptide 61-80 and BALB/cJ mice produced IgG that bound peptide 91-110. Although B10.BR mice produced IgG that bound the 50-mer peptide 101-148, this IgG was not detected by binding to peptides 91-110, 106-125, 121-140, and 131-148. Immunization of B10.BR mice with individual overlapping 20-mer peptides as Ag revealed that peptides 1-20, 16-35, 31-50, and 76-95 elicited high titers of IgG that bound both the immunizing peptide as well as 18-kDa protein. As these peptides induce antibody synthesis they must contain both B cell and T cell epitopes. By contrast, immunization of BALB/cJ mice with the same 20-mer peptides, all of which contain B cell epitopes for this strain, failed to elicit IgG responses with one exception. Peptide 91-110 induced IgG that bound peptide 91-110, but not the intact 18-kDa protein. We conclude that peptides 1-20, 16-35, 31-50, and 76-95 either lack T cell epitopes for BALB/cJ mice, or activate different T cell subpopulations in the two strains. We suggest that the induction of IgG responses to small peptide Ag is an in vivo assay of the activity of Th2 cell subpopulations.     

Host protein Ku70 binds and protects HIV-1 integrase from proteasomal degradation and is required for HIV replication

HIV-1 integrase (IN) is a key viral enzymatic protein acting in several viral replication steps, including integration. IN has been shown to be an unstable protein degraded by the N-end rule pathway through the host ubiquitin-proteasome machinery. However, it is still not fully understood how this viral protein is protected from the host ubiquitin-proteasome system within cells during HIV replication. In the present study, we provide evidence that the host protein Ku70 interacts with HIV-1 IN and protects it from the Lys(48)-linked polyubiquitination proteasomal pathway. Moreover, Ku70 is able to down-regulate the overall protein polyubiquitination level within the host cells and to specifically deubiquitinate IN through their interaction. Mutagenic studies revealed that the C terminus of IN (residues 230-288) is required for IN binding to the N-terminal part of Ku70 (Ku70(1-430)), and their interaction is independent of Ku70/80 heterodimerization. Finally, knockdown of Ku70 expression in both virus-producing and target CD4(+) T cells significantly disrupted HIV-1 replication and rendered two-long terminal repeat circles and integration undetectable, indicating that Ku70 is required for both the early and the late stages of the HIV-1 life cycle. Interestingly, Ku70 was incorporated into the progeny virus in an IN-dependent way. We proposed that Ku70 may interact with IN during viral assembly and accompany HIV-1 IN upon entry into the new target cells, acting to 1) protect IN from the host defense system and 2) assist IN integration activity. Overall, this report provides another example of how HIV-1 hijacks host cellular machinery to protect the virus itself and to facilitate its replication.     

Contribution of host nucleoporin 62 in HIV-1 integrase chromatin association and viral DNA integration

HIV-1 integration is promoted by viral integrase (IN) and its cellular cofactors. The lens epithelium-derived growth factor (LEDGF/p75), an IN interacting cellular cofactor, has been shown to play an important role in HIV-1 chromatin targeting and integration. However, whether other cellular cofactors are also involved in viral replication steps is still elusive. Here, we show that nucleoporin 62 (Nup62) is a chromatin-bound protein and can specifically interact with HIV-1 IN in both soluble nuclear extract and chromatin-bound fractions. The knockdown of Nup62 by shRNA reduced the association of IN with host chromatin and significantly impaired viral integration and replication in HIV-1-susceptible cells. Furthermore, the expression of the IN-binding region of Nup62 in CD4(+) T cells significantly inhibited HIV-1 infection. Taken together, these results indicate that the cellular Nup62 is specifically recruited by HIV-1 IN and contribute to an efficient viral DNA integration.     

A New Class of Multimerization Selective Inhibitors of HIV-1 Integrase

The quinoline-based allosteric HIV-1 integrase (IN) inhibitors (ALLINIs) are promising candidates for clinically useful antiviral agents. Studies using these compounds have highlighted the role of IN in both early and late stages of virus replication. However, dissecting the exact mechanism of action of the quinoline-based ALLINIs has been complicated by the multifunctional nature of these inhibitors because they both inhibit IN binding with its cofactor LEDGF/p75 and promote aberrant IN multimerization with similar potencies in vitro. Here we report design of small molecules that allowed us to probe the role of HIV-1 IN multimerization independently from IN-LEDGF/p75 interactions in infected cells. We altered the rigid quinoline moiety in ALLINIs and designed pyridine-based molecules with a rotatable single bond to allow these compounds to bridge between interacting IN subunits optimally and promote oligomerization. The most potent pyridine-based inhibitor, KF116, potently (EC₅₀ of 0.024 µM) blocked HIV-1 replication by inducing aberrant IN multimerization in virus particles, whereas it was not effective when added to target cells. Furthermore, KF116 inhibited the HIV-1 IN variant with the A128T substitution, which confers resistance to the majority of quinoline-based ALLINIs. A genome-wide HIV-1 integration site analysis demonstrated that addition of KF116 to target or producer cells did not affect LEDGF/p75-dependent HIV-1 integration in host chromosomes, indicating that this compound is not detectably inhibiting IN-LEDGF/p75 binding. These findings delineate the significance of correctly ordered IN structure for HIV-1 particle morphogenesis and demonstrate feasibility of exploiting IN multimerization as a therapeutic target. Furthermore, pyridine-based compounds present a novel class of multimerization selective IN inhibitors as investigational probes for HIV-1 molecular biology.     

Huwe1, a novel cellular interactor of Gag-Pol through integrase binding, negatively influences HIV-1 infectivity

Integration, an indispensable step for retrovirus replication, is executed by integrase (IN), which is expressed as a part of a Gag-Pol precursor. Although mechanistic detail of the IN-catalyzed integration reaction is well defined, numerous evidence have demonstrated that IN is involved in multiple steps of retrovirus replication other than integration. In this study, Huwe1, a HECT-type E3 ubiquitin ligase, was identified as a new cellular interactor of human immunodeficiency virus type 1 (HIV-1) IN. The interaction was mediated through the catalytic core domain of IN and a wide-range region of Huwe1. Interestingly, although depletion of Huwe1 in target cells did not affect the early phase of HIV-1 infection in a human T cell line, we found that infectivity of HIV-1 released from the Huwe1 knockdown cells was significantly augmented more than that of virus produced from control cells. The increase in infectivity occurred in proviral DNA synthesis. Further analysis revealed that Huwe1 interacted with HIV-1 Gag-Pol precursor protein through an IN domain. Our results suggest that Huwe1 in HIV-1 producer cells has a negative impact on early post-entry events during the next round of virus infection via association with an IN region of Gag-Pol.     

Effective recognition of HIV-1-infected cells by HIV-1 integrase-specific HLA-B∗4002-restricted T cells

HLA-B?4002 is one of the common HLA-B alleles in the world. All 7 reported HLA-B?4002-restricted HIV epitopes are derived from Gag, Nef, and Vpr. In the present study we sought to identify novel HLA-B?4002-restricted HIV epitopes by using overlapping 11-mer peptides of HIV-1 Nef, Gag, and Pol, and found that 6 of these 11-mer Pol peptides included HLA-B?4002-restricted epitopes. Analysis using truncated peptides of these 6 peptides defined 4 optimal Pol (integrase) epitopes. All epitopes previously reported had Glu at position 2 (P2), suggesting that Glu at P2 is the anchor residue for HLA-B?4002; whereas only 2 of the integrase epitopes that we here identified had Glu at P2. CTL clones specific for the 2 epitopes effectively recognized HIV-1-infected cells whereas those for other 2 epitopes only weakly recognized them. The antigen sensitivity of the former clones for the epitope peptide was much higher than that of the latter clones, suggesting 2 possibilities: 1) the former T cells have high-affinity TCRs and/or 2) the epitope peptides recognized by the former T cells are highly presented by HLA-B?4002 in HIV-1-infected cells. These integrase-specific T cells with high antigen sensitivity may contribute to the suppression of HIV-1 replication in HIV-1-infected HLA-B?4002+ individuals.     

Carbonyl J Derivatives: A New Class of HIV-1 Integrase Inhibitors

Integration of a DNA copy of the HIV-1 genome is required for viral replication and pathogenicity, and this highly specific molecular process is mediated by the virus-encoded integrase protein. The requirement for integration, combined with the lack of a known analogous process in mammalian cells, makes integrase an attractive target for therapeutic inhibitors of HIV-1 replication. While many reports of HIV-1 IN inhibitors exist, no such compounds have yet emerged to treat HIV-1 infection. As such, new classes of integrase inhibitors are needed. We have combined molecular modeling and combinatorial chemistry to identify and develop a new class of HIV-1 integrase inhibitors, the Carbonyl J [N,N'-bis(2-(5-hydroxy-7-naphthalenesulfonic acid)urea] derivatives. This new class includes a number of compounds with sub-micromolar IC(50) values for inhibiting purified HIV-1 integrase in vitro. Herein we describe the chemical characteristics that are important for integrase inhibition and cell toxicity within the Carbonyl J derivatives. Copyright 2000 Academic Press.     

Lys-34, dispensable for integrase catalysis, is required for preintegration complex function and human immunodeficiency virus type 1 replication

Retroviral integrases (INs) function in the context of preintegration complexes (PICs). Two conserved Lys residues in the N-terminal domain of human immunodeficiency virus type 1 (HIV-1) IN were analyzed here for their roles in integration and virus replication. Whereas HIV-1(K46A) grew like the wild type, HIV-1(K34A) was dead. Yet recombinant IN(K34A) protein functioned in in vitro integration assays, and Vpr-IN(K34A) efficiently transcomplemented the infectivity defect of an IN active site mutant virus in cells. HIV-1(K34A) was therefore similar to a number of previously characterized mutant viruses that failed to replicate despite encoding catalytically competent IN. To directly analyze mutant PIC function, a sensitive PCR-based integration assay was developed. HIV-1(K34A) and related mutants failed to support detectable levels (<1% of wild type) of integration. We therefore concluded that mutations like K34A disrupted higher-order interactions important for PIC function/maturation compared to the innate catalytic activity of IN enzyme.     

Complementation of integrase function in HIV-1 virions.

Proviral integration is essential for HIV-1 replication and represents an important potential target for antiviral drug design. Although much is known about the integration process from studies of purified integrase (IN) protein and synthetic target DNA, provirus formation in virally infected cells remains incompletely understood since reconstituted in vitro assays do not fully reproduce in vivo integration events. We have developed a novel experimental system in which IN-mutant HIV-1 molecular clones are complemented in trans by Vpr-IN fusion proteins, thereby enabling the study of IN function in replicating viruses. Using this approach we found that (i) Vpr-linked IN is efficiently packaged into virions independent of the Gag-Pol polyprotein, (ii) fusion proteins containing a natural RT/IN processing site are cleaved by the viral protease and (iii) only the cleaved IN protein complements IN-defective HIV-1 efficiently. Vpr-mediated packaging restored IN function to a wide variety of IN-deficient HIV-1 strains including zinc finger, catalytic core and C-terminal domain mutants as well as viruses from which IN was completely deleted. Furthermore, trans complemented IN protein mediated a bona fide integration reaction, as demonstrated by the precise processing of proviral ends (5'-TG...CA-3') and the generation of an HIV-1-specific (5 bp) duplication of adjoining host sequences. Intragenic complementation between IN mutants defective in different protein domains was also observed, thereby providing the first evidence for IN multimerization in vivo.     

Nuclear localization of human immunodeficiency virus type 1 preintegration complexes (PICs): V165A and R166A are pleiotropic integrase mutants primarily defective for integration,not PIC nuclear import

Retroviral replication requires the integration of reverse-transcribed viral cDNA into a cell chromosome. A key barrier to forming the integrated provirus is the nuclear envelope, and numerous regions in human immunodeficiency virus type 1 (HIV-1) have been shown to aid the nuclear localization of viral preintegration complexes (PICs) in infected cells. One region in integrase (IN), composed of Val-165 and Arg-166, was reportedly essential for HIV-1 replication and nuclear localization in all cell types. In this study we confirmed that HIV-1(V165A) and HIV-1(R166A) were replication defective and that less mutant viral cDNA localized to infected cell nuclei. However, we present three lines of evidence that argue against a specific role for Val-165 and Arg-166 in PIC nuclear import. First, results of transient transfections revealed that V165A FLAG-tagged IN and green fluorescent protein-IN fusions carrying either V165A or R166A predominantly localized to cell nuclei. Second, two different strains of previously described class II IN mutant viruses displayed similar nuclear entry profiles to those observed for HIV-1(V165A) and HIV-1(R166A), suggesting that defective nuclear import may be a common phenotype of replication-defective IN mutant viruses. Third, V165A and R166A mutants were defective for in vitro integration activity, when assayed both as PICs isolated from infected T-cells and as recombinant IN proteins purified from Escherichia coli. Based on these results, we conclude that HIV-1(V165A) and HIV-1(R166A) are pleiotropic mutants primarily defective for IN catalysis and that Val-165 and Arg-166 do not play a specific role in the nuclear localization of HIV-1 PICs in infected cells.