Characterization of the nuclear import pathway for HIV-1 integrase

The karyophilic properties of the human immunodeficiency virus, type I (HIV-1) pre-integration complex (PIC) allow the virus to infect non-dividing cells. To better understand the mechanisms responsible for nuclear translocation of the PIC, we investigated nuclear import of HIV-1 integrase (IN), a PIC-associated viral enzyme involved in the integration of the viral genome in the host cell DNA. Accumulation of HIV-1 IN into nuclei of digitonin-permeabilized cells does not result from passive diffusion but rather from an active transport that occurs through the nuclear pore complexes. HIV-1 IN is imported by a saturable mechanism, implying that a limiting cellular factor is responsible for this process. Although IN has been previously proposed to contain classical basic nuclear localization signals, we found that nuclear accumulation of IN does not involve karyopherins alpha, beta1, and beta2-mediated pathways. Neither the non-hydrolyzable GTP analog, guanosine 5'-O-(thiotriphosphate), nor the GTP hydrolysis-deficient Ran mutant, RanQ69L, significantly affects nuclear import of IN, which depends instead on ATP hydrolysis. Therefore these results support the idea that IN import is not mediated by members of the karyopherin beta family. More generally, in vitro nuclear import of IN does not require addition of cytosolic factors, suggesting that cellular factor(s) involved in this active but atypical pathway process probably remain associated with the nuclear compartment or the nuclear pore complexes from permeabilized cells.     

Two nuclear localization signals in the HIV-1 matrix protein regulate nuclear import of the HIV-1 pre-integration complex

Replication of HIV-1 in non-dividing and slowly proliferating cell populations depends on active import of the viral pre-integration complex (PIC) into the cell nucleus. While it is commonly accepted that this process is mediated by an interaction between the HIV-1 PIC and the cellular nuclear import machinery, controversial results have been reported concerning the mechanisms of this interaction. Here, we demonstrate that a recently identified nuclear localization signal within the HIV-1 matrix protein (MA), MA NLS-2, together with previously described MA NLS-1, mediates nuclear import of the HIV-1 PIC. Inactivation of both MA NLSs precluded nuclear translocation of MA and rendered the virus defective in nuclear import and replication in non-dividing macrophage cultures, even when functional Vpr and integrase (IN), two more viral proteins implicated in HIV-1 nuclear import, were present. Taken together, these results indicate that Vpr does not function as an independent nuclear import factor and demonstrate that HIV-1 MA, by virtue of its two nuclear localization signals, regulates HIV-1 nuclear import.     

Inhibition of HIV-1 nuclear import via schiff base formation with arylene bis(methylketone) compounds

Arylene bis(methylketone) compounds specifically block nuclear translocation of the HIV-1 pre-integration complex by forming Schiff-base adducts with contiguous lysines within nuclear localization signal.     

Inhibition of nuclear import by the proapoptotic protein CC3

We report here that the normal cellular protein CC3/TIP30, when in excess, inhibits nuclear import in vitro and in vivo. CC3 binds directly to the karyopherins of the importin beta family in a RanGTP-insensitive manner and associates with nucleoporins in vivo. CC3 inhibits the nuclear import of proteins possessing either the classical nuclear localization signal or the M9 signal recognized by transportin. CC3 also inhibits nuclear translocation of transportin itself. Cells modified to express higher levels of CC3 have a slower rate of nuclear import and, as described earlier, show an increased sensitivity to death signals. A mutant CC3 protein lacking proapoptotic activity has a lower affinity for transportin, is displaced from it by RanGTP, and fails to inhibit nuclear import in vitro and in vivo. Together, our results support a correlation between the ability of CC3 to form a RanGTP-resistant complex with importins, inhibit nuclear import, and induce apoptosis. Significantly, a dominant-negative form of importin beta1 shown previously to inhibit multiple transport pathways induces rapid cell death, strongly indicating that inhibition of nuclear transport serves as a potent apoptotic signal.     

Potent inhibition of HIV-1 replication by backbone cyclic peptides bearing the Rev arginine rich motif

Summary Due to its essential role in the virus life cycle, the viral regulatory protein Rev constitutes an attractive target for the development of new antiviral molecules. In this work, a series of Backbone Cyclic Peptide (BCP) analogs that bear a conformationally constrained arginine rich motif (ARM) of Rev were tested for in vitro inhibition of HIV-1 replication. We observed a potent suppression of HIV-1 replication in chronically infected T lymphocytic cells treated with Rev-BCPs. We further investigated possible mechanisms of HIV-1 inhibition and showed that Rev-BCPs interfere slightly with the nuclear import process and are very efficient in blocking a mechanism that controls Pr55^gag and gp160^env synthesis. Interestingly, these protein precursors are known to be encoded by mRNAs that require Rev-binding for nuclear export. In situ hybridization using a Cy-3 conjugated HIV-1 gag oligonucleotide probe indicated that Rev-BCPs prevent the intracellular accumulation of unspliced viral RNA. As a model, the most promising analog, Rev-BCP 14, was studied by molecular modeling and dynamics in order to identify its binding site on the Rev Response Element (RRE). The annealing simulation suggests that upon binding on the RRE, Rev-BCP 14 widens the distorted major groove of the viral RNA. Numerous contacts between peptide and RNA were found within the complex and some were identified as key components for the interactions. Altogether, our data indicate that the use of conformationally constrained Rev-BCPs represents a promising strategy for the development of new peptide-based therapeutic agents against HIV-1.     

Inhibition of HIV-1 protease by short peptides derived from the terminal segments of the protease.

The active HIV-1 protease is a homodimeric enzyme. A beta-sheet consisting of N- and C-terminal segments provides the main driving force for dimerization of the inactive protomers. Several short peptides with sequences derived from the N- and C-termini of the protease were tested for inhibition of protease activity and for inhibition of HIV-1 replication in lymphocytes. Medium inhibitory activity was found with each of the peptides in the enzyme test and no inhibition of the lymphocytes was found up to 200 micrograms/ml. The enzyme tests indicate that HIV-1 protease is the target of the inhibitory action. Synergistic action could not be found with pairs of the peptides derived from the two different termini. Prolonged incubation with one of the peptides increased inhibition indicating a slow dissociation of the protease dimers. No cytotoxic effect of the inhibitors could be found below 200 micrograms/ml.     

Inhibition of HIV-1 integrase by modified oligonucleotides derived from U5' LTR.

Retroviral integrase catalyzes integration of double-stranded viral DNA into the host chromosome by a process that has become an attractive target for drug design. In the 3' processing reaction, two nucleotides are specifically cleaved from both 3' ends of viral DNA yielding a 5' phosphorylated dimer (pGT). The resulting recessed 3' hydroxy groups of adenosine provide the attachment sites to the host DNA in the strand transfer reaction. Here, we studied the effect of modified double-stranded oligonucleotides mimicking both the unprocessed (21-mer oligonucleotides) and 3' processed (19-mer oligonucleotides) U5 termini of proviral DNA on activities of HIV-1 integrase in vitro. The inhibitions of 3' processing and strand transfer reactions were studied using 21-mer oligonucleotides containing isopolar, nonisosteric, both conformationally flexible and restricted phosphonate internucleotide linkages between the conservative AG of the sequence CAGT, and using a 21-mer oligonucleotide containing 2'-fluoroarabinofuranosyladenine. All modified 21-mer oligonucleotides competitively inhibited both reactions mediated by HIV-1 integrase with nanomolar IC50 values. Our studies with 19-mer oligonucleotides showed that modifications of the 3' hydroxyl significantly reduced the strand transfer reaction. The inhibition of integrase with 19-mer oligonucleotides terminated by (S)-9-(3-hydroxy-2-phosphonomethoxypropyl)adenine, 9-(2-phosphonomethoxyethyl)adenine, and adenosine showed that proper orientation of the 3' OH group and the presence of the furanose ring of adenosine significantly influence the strand transfer reaction.     

Development of a functional backbone cyclic mimetic of the HIV-1 Tat arginine-rich motif

We have used the backbone cyclic proteinomimetics approach to develop peptides that functionally mimic the arginine-rich motif (ARM) of the HIV-1 Tat protein. This consensus sequence serves both as a nuclear localization signal (NLS) and as an RNA binding domain. Based on the NMR structure of Tat, we have designed and synthesized a backbone cyclic ARM mimetic peptide library. The peptides were screened for their ability to mediate nuclear import of the corresponding BSA conjugates in permeabilized cells. One peptide, designated "Tat11," displayed active NLS properties. Nuclear import of Tat11-BSA was found to proceed by the same distinct pathway used by the Tat-NLS and not by the common importin alpha pathway, which is used by the SV40-NLS. Most of the Tat-derived backbone cyclic peptides display selective inhibitory activity as demonstrated by the inhibition of the nuclear import mediated by the Tat-NLS and not by the SV40-NLS. The Tat-ARM-derived peptides, including Tat-11, also inhibited binding of the HIV-1 Rev-ARM to its corresponding RNA element (Rev response element) with inhibition constants of 5 nm. Here we have shown for the first time (a) a functional mimetic of a protein sequence, which activates a nuclear import receptor and (b) a mimetic of a protein sequence with a dual functionality. Tat11 is a lead compound which can potentially inhibit the HIV-1 life cycle by a dual mechanism: inhibition of nuclear import and of RNA binding.     

Inhibition of HIV-1 infection by synthetic peptides derived CCR5 fragments

HIV-1 infection requires interaction of viral envelope protein gp160 with CD4 and a chemokine receptor, CCR5 or CXCR4 as entry coreceptor. We designed HIV-inhibitory peptides targeted to CCR5 using a novel computer program (ANTIS), which searched all possible sense-antisense amino acid pairs between proteins. Seven AHBs were found in CCR5 receptor. All AHB peptides were synthesized and tested for their ability to prevent HIV-1 infection to human T cells. A peptide fragment (LC5) which is a part of the CCR5 receptor corresponding to the loop between the fifth and sixth transmembrane regions (amino acids 222-240) proved to inhibit HIV-1IIIB infection of MT-4 cells. Interaction of these antisense peptides could be involved in sustaining HIV-1 infectivity. LC5 effectively indicated dose-dependent manner, and the suppression was enhanced additively by T20 peptide, which inhibits infection in vitro by disrupting the gp41 conformational changes necessary for membrane fusion. Thus, these results indicate that CCR5-derived AHB peptides could provide a useful tool to define the mechanism(s) of HIV infection, and may provide insight which will contribute to the development of an anti-HIV-1 reagent.     

Viral protein R regulates nuclear import of the HIV-1 pre-integration complex.

Replication of human immunodeficiency virus type 1 (HIV-1) in non-dividing cells critically depends on import of the viral pre-integration complex into the nucleus. Genetic evidence suggests that viral protein R (Vpr) and matrix antigen (MA) are directly involved in the import process. An in vitro assay that reconstitutes nuclear import of HIV-1 pre-integration complexes in digitonin-permeabilized cells was used to demonstrate that Vpr is the key regulator of the viral nuclear import process. Mutant HIV-1 pre-integration complexes that lack Vpr failed to be imported in vitro, whereas mutants that lack a functional MA nuclear localization sequence (NLS) were only partially defective. Strikingly, the import defect of the Vpr- mutant was rescued when recombinant Vpr was re-added. In addition, import of Vpr- virus was rescued by adding the cytosol of HeLa cells, where HIV-1 replication had been shown to be Vpr-independent. In a solution binding assay, Vpr associated with karyopherin alpha, a cellular receptor for NLSs. This association increased the affinity of karyopherin alpha for basic-type NLSs, including that of MA, thus explaining the positive effect of Vpr on nuclear import of the HIV-1 pre-integration complex and BSA-NLS conjugates. These results identify the biochemical mechanism of Vpr function in transport of the viral pre-integration complex to, and across, the nuclear membrane.     

Nuclear exportin receptor CAS regulates the NPI-1-mediated nuclear import of HIV-1 Vpr

Vpr, an accessory protein of human immunodeficiency virus type 1, is a multifunctional protein that plays an important role in viral replication. We have previously shown that the region between residues 17 and 74 of Vpr (Vpr(N17C74)) contained a bona fide nuclear localization signal and it is targeted Vpr(N17C74) to the nuclear envelope and then imported into the nucleus by importin α (Impα) alone. The interaction between Impα and Vpr is important not only for the nuclear import of Vpr but also for HIV-1 replication in macrophages; however, it was unclear whether full-length Vpr enters the nucleus in a manner similar to Vpr(N17C74). This study investigated the nuclear import of full-length Vpr using the three typical Impα isoforms, Rch1, Qip1 and NPI-1, and revealed that full-length Vpr is selectively imported by NPI-1, but not Rch1 and Qip1, after it makes contact with the perinuclear region in digitonin-permeabilized cells. A binding assay using the three Impα isoforms showed that Vpr bound preferentially to the ninth armadillo repeat (ARM) region (which is also essential for the binding of CAS, the export receptor for Impα) in all three isoforms. Comparison of biochemical binding affinities between Vpr and the Impα isoforms using surface plasmon resonance analysis demonstrated almost identical values for the binding of Vpr to the full-length isoforms and to their C-terminal domains. By contrast, the data showed that, in the presence of CAS, Vpr was released from the Vpr/NPI-1 complex but was not released from Rch1 or Qip1. Finally, the NPI-1-mediated nuclear import of Vpr was greatly reduced in semi-intact CAS knocked-down cells and was recovered by the addition of exogenous CAS. This report is the first to show the requirement for and the regulation of CAS in the functioning of the Vpr-Impα complex.     

Inhibition of nuclear import and alteration of nuclear pore complex composition by rhinovirus

Nucleocytoplasmic trafficking pathways and the status of nuclear pore complex (NPC) components were examined in cells infected with rhinovirus type 14. A variety of shuttling and nonshuttling nuclear proteins, using multiple nuclear import pathways, accumulated in the cytoplasm of cells infected with rhinovirus. An in vitro nuclear import assay with semipermeabilized infected cells confirmed that nuclear import was inhibited and that docking of nuclear import receptor-cargo complexes at the cytoplasmic face of the NPC was prevented in rhinovirus-infected cells. The relocation of cellular proteins and inhibition of nuclear import correlated with the degradation of two NPC components, Nup153 and p62. The degradation of Nup153 and p62 was not due to induction of apoptosis, because p62 was not proteolyzed in apoptotic HeLa cells, and Nup153 was cleaved to produce a 130-kDa cleavage product that was not observed in cells infected with poliovirus or rhinovirus. The finding that both poliovirus and rhinovirus cause inhibition of nuclear import and degradation of NPC components suggests that this may be a common feature of the replicative cycle of picornaviruses. Inhibition of nuclear import is predicted to result in the cytoplasmic accumulation of a large number of nuclear proteins that could have functions in viral translation, RNA synthesis, packaging, or assembly. Additionally, inhibition of nuclear import also presents a novel strategy whereby cytoplasmic RNA viruses can evade host immune defenses by preventing signal transduction into the nucleus.     

Inhibition of nuclear import mediated by the Rev-arginine rich motif by RNA molecules

The HIV-1 Rev protein plays a pivotal role in viral replication, and therefore, inhibition of its function should block the progression of the virus-induced immune deficiency syndrome (AIDS). Here, RNA molecules have been shown to inhibit import of the HIV-1 Rev protein into nuclei of permeabilized cells. Nuclear uptake of biotinylated recombinant His-tagged Rev-GFP was assessed in nuclear extracts from digitonin-permeabilized cells by binding to either importin beta-receptors or nickel molecules immobilized on a microtiter plate. Using this method together with fluorescence microscopy, we determined that nuclear import of Rev is inhibited by the addition of a reticulocyte lysate which routinely is used as a source of nuclear import receptors. This inhibition was released by treatment with the RNase enzyme. Also t-RNA molecules and the oligoribonucleotide RRE IIB, namely, the second stem structure of the Rev responsive element (RRE) of the viral RNA, inhibit Rev nuclear import. Similar results were obtained when BSA molecules with covalently attached Rev-arginine rich motif (ARM) peptides were used as a nuclear transport substrate, indicating that the nuclear import inhibition of the Rev protein is due to the presence of the ARM domain. Binding experiments revealed that the RNA molecules inhibit the interaction between the ARM region and importin beta, implying that the RNA prevents the formation of the import complex. The implication of our results for the regulation of the nuclear import of Rev as well as for the use of RNA molecules as antiviral drugs is discussed.     

Therapeutic potential of HIV-1 entry inhibitor peptidomimetics

Human immunodeficiency virus 1 (HIV-1) infection remains a public health concern globally. Although great strides in the management of HIV-1 have been achieved, current highly active antiretroviral therapy is limited by multidrug resistance, prolonged use-related effects, and inability to purge the HIV-1 latent pool. Even though novel therapeutic options with HIV-1 broadly neutralizing antibodies (bNAbs) are being explored, the scalability of bNAbs is limited by economic cost of production and obligatory requirement for parenteral administration. However, these limitations can be addressed by antibody mimetics/peptidomimetics of HIV-1 bNAbs. In this review we discuss the limitations of HIV-1 bNAbs as HIV-1 entry inhibitors and explore the potential therapeutic use of antibody mimetics/peptidomimetics of HIV-1 entry inhibitors as an alternative for HIV-1 bNAbs. We highlight the reduced cost of production, high specificity, and oral bioavailability of peptidomimetics compared to bNAbs to demonstrate their suitability as candidates for novel HIV-1 therapy and conclude with some perspectives on future research toward HIV-1 novel drug discovery.     

Inhibition of Candida albicans secreted aspartic protease by a novel series of peptidomimetics,also active on the HIV-1 protease

Nineteen reduced amide, monohydroxy- or dihydroxyethylene-based transition-state peptidomimetics, known to be good inhibitors of the aspartic protease of HIV-1, were tested against a secreted aspartic protease (Sap2), purified from the culture medium of a virulent strain of Candida albicans. Ten of these compounds exhibited IC(50)s against Sap2 lower than 15 microM; the best inhibitor, Kyn-Val-Phe-Psi[OH-OH]-Phe-Val-Kyn, when added to the C. albicans culture, repressed the hydrolysis of bovine serum albumin (BSA), contained in the culture medium, and inhibited the growth of the fungus.