Strategies to inhibit viral protein nuclear import: HIV1 as a target

Strategies to inhibit viral protein nuclear import: HIV-1 as atarget

Aviad Levin1, Abraham Loyter1, and Michael Bukrinsky2,*

1 Department of Biological Chemistry, The Alexander Silberman Institute of Life Sciences, TheHebrew University of Jerusalem, Jerusalem 91904, Israel2 The George Washington University, Washington, DC 20037, USA

AbstractNuclear import is a critical step in the life cycle of HIV-1. During the early (pre-integration) stagesof infection, HIV-1 has to transport its pre-integration complex into the nucleus for integrationinto the host cell chromatin, while at the later (post-integration) stages viral regulatory proteins Tatand Rev need to get into the nucleus to stimulate transcription and regulate splicing and nuclearexport of subgenomic and genomic RNAs. Given such important role of nuclear import in HIV-1life cycle, this step presents an attractive target for anti-viral therapeutic intervention. In thisreview, we describe the current state of our understanding of the interactions regulating nuclearimport of the HIV-1 pre-integration complex and describe current approaches to inhibit it.

1. IntroductionViruses are intracellular parasites that commandeer cellular processes, such as nuclearimport and export machineries, to perform virus-specific functions. For this purpose, manyviral proteins and nucleoprotein complexes shuttle between the nuclear and cytoplasmiccompartments, and this process is especially critical for viruses with a distinct replicationstep occurring in the nucleus. Among many different families of these viruses, retroviruses,and lentiviruses in particular, are of major interest both because of their clinical importance(HIV-1 is the most notable lentiviral pathogenic agent [103]) and their potential relevance asvectors for gene therapy [17]. While replication in the host cell’s nucleus provides clearbenefits for the virus, such as ready access to cellular transcription and splicing apparatus, itimposes the barrier of the nuclear envelope that has to be overcome. During the early (pre-integration) stages of infection, lentiviruses have to transport their genome from the site ofpenetration to the nucleus and then through the nuclear membrane, while at the later (post-integration) stages they need to import regulatory proteins (Tat and Rev in the case ofHIV-1) to stimulate transcription and regulate splicing and nuclear export of subgenomicand genomic RNAs. Given such important role that nuclear import plays in HIV-1 life cycle,it presents an attractive target for anti-viral therapeutic intervention. In this review, we willdescribe the current state of our understanding of the interactions regulating nuclear importof the HIV-1 pre-integration complex (PIC) and HIV-1 proteins critical for viral replication,focusing on potential targets for therapeutic interventions. In contrast to a recent article by

*Address correspondence to: Dr. Michael Bukrinsky, 2300 I St. NW, Washington, DC 20037, USA. [email protected]'s Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to ourcustomers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review ofthe resulting proof before it is published in its final citable form. Please note that during the production process errors may bediscovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

NIH Public AccessAuthor ManuscriptBiochim Biophys Acta. Author manuscript; available in PMC 2012 September 1.

Published in final edited form as:Biochim Biophys Acta. 2011 September ; 1813(9): 1646–1653. doi:10.1016/j.bbamcr.2010.07.010.

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Zhan et al. [111] covering similar topics, we will attempt to provide a critical analysis of thepublished data.

2. Nuclear import in the HIV-1 life cycleThe idea that nuclear import may play a special role in lentiviral life cycle [13] wasprompted by the finding that HIV-1 can infect non-dividing cells such as terminallydifferentiated macrophages [67,100]. This ability implied that HIV-1, unlikegammaretroviruses which critically depend on nuclear envelope disassembly during mitosisfor nuclear entry [87], acquired the ability to pass through the intact nuclear envelope.Because mitosis constitutes only a small part of the cell cycle, the ability to enter nuclearcompartment during the interphase provides a huge benefit to HIV-1 and may account for itsvery high replication rate observed in HIV-infected patients [53,99]. Initially, it waspresumed that HIV can get into the nucleus both during the interphase (through the nuclearenvelope) and during the mitosis (by-passing the nuclear envelope), prompting the idea thatinfection of dividing and non-dividing cells by HIV occurs through different pathways andthus should involve different mechanisms [105]. However, a number of studiesdemonstrated that passage through the nuclear envelope is a necessary step in HIV infectionof both dividing and non-proliferating cells [54,57,85], underscored by the requirement ofthe nuclear import factors transportin-SR2 (TNPO3/TRN-SR2) [22,39,61] and importin 7[108] for infection of both cell types. Such solution appears evolutionary sensible, asevolution usually leads to development of the new pathways at the expense of the old ones,and it would be wasteful for a virus to preserve two replication mechanisms (one fordividing and one for non-dividing cells), when one is so much superior to the other. Therequirement of active nuclear import for HIV infection of both dividing and non-dividingcells would imply that differences between these cell types in HIV replication cannot beused for conclusions regarding the role of HIV nuclear import in the viral life cycle[61,105,106]. Indeed, it is very difficult to make reliable conclusions from comparison ofnon-dividing cells growth arrested by potent anti-mitotic agents such as aphidicolin tountreated controls. In addition, recent studies demonstrated profound differences betweendividng and non-dividing, post-mitotic cells in the genesis of nuclear pore complexes(NPC), the multiprotein assemblies that regulate exchange between the nucleoplasm andcytoplasm. In dividing cells, NPCs disassemble during mitosis and reassemble into thenewly forming nuclei. In contrast, the renewal of NPCs is absent in post-mitotic cells, andcertain nucleoporins, such as Nup107/160 complex, remain incorporated in the nuclearmembrane during the entire lifespan of a cell [24,27]. Lack of NPC turnover in post-mitoticcells was associated with aging-related deterioration of NPCs, a loss of the nuclearpermeability barrier and the leaking of cytoplasmic proteins into the nuclear compartment[52]. Such functional changes may greatly affect the NPC activity during nuclear import ofviral PICs in non-dividing cells.

Being cellular parasites, viruses hijack cellular machinery to accomplish the necessary tasksin their replication. In the case of nuclear import, this paradigm implies that the HIV-1 PICand the viral proteins needed in the nucleus (Tat and Rev) interact with the cellular nuclearimport machinery that normally mediates transport of certain cellular proteins carryingspecific nuclear localization signals (NLSs) from the cytoplasm into the nucleus. Thismachinery involves a number of nuclear transport proteins, importins, that bind andtranslocate NLS-containing proteins through the NPC via a complex series of interactionswith NPC proteins nucleoporins (reviewed in [92]).

To use this machinery, the HIV-1 PIC and the viral proteins needed in the nucleus (Tat andRev) have to express NLSs that could be recognized by importins. The questions concerningthe nature of these NLSs, the PIC proteins expressing them, and the importins involved in

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translocation of the HIV-1 PIC remain the most controversial issue in HIV research. HIV-1PIC contains at least four karyophilic proteins of both viral (MA, IN, Vpr) and host cell(LEDGF/p75) origin which have been shown to interact with importins. Some of theexisting controversy comes from this unusually high number of potential factors regulatingHIV-1 nuclear import. Indeed, nuclear import of the HIV-1 PIC cannot be completelysuppressed by inactivation of NLSs in any one, or even simultaneously in several,karyophilic proteins within the PIC [104]. However, this finding can simply indicate that thenuclear import of the HIV PIC results from a cumulative activity of several karyophilicfactors, or that different factors work in different circumstances (e.g., different cell types,different activation conditions, etc.). This would be a logical solution on the part of the virusto maximize efficiency of its nuclear import. Consistent with this interpretation, deletion orinactivation of known nuclear import signals in the HIV-1 PIC leads to about a 4 logdecrease in infectivity [104]. Importantly, consistent with the critical role that nuclear importplays in HIV-1 infection of dividing and non-dividing cells, inactivation of NLSs equallyaffected viral replication in both cell types [105].

Similar considerations concern importins. A number of these nuclear import receptors,including importins α, β, 7 and TNPO3/TRN-SR2 have been proposed as the keycomponents of the HIV nuclear import system in some reports, while other studiesquestioned their role. For example, using an in vitro model of nuclear import, Fassati andcolleagues identified importin 7 as the critical component of PIC nuclear import and showedthat RNAi-mediated knockdown of Imp7 in macrophages inhibits viral nuclear import andreplication [31]. However, later study by Zielske and Stevenson [113] questioned this resultby showing that knocking down Imp7 in macrophages did not impair infection by HIV.Potential resolution of this controversy may come from a recent report by Zaitseva and co-authors [108] which suggested that inactivation of Imp7 affects kinetics of the PIC nuclearimport but not the final accumulation of PICs in the nuclear compartment. This scenario issimilar to the situation with PIC NLSs (see below), whose role might be to accelerate theprocess of PIC nuclear import.

Interestingly, different lentiviruses appear to use alternative nuclear import pathways toaccess the nucleus. For example, Imp7 is required for rapid nuclear import of HIV-1, but notfor HIV-2 or SIV [108]; TNPO3 is required for infection by HIV, SIV and BIV, but isdispensable for FIV [59]; nuclear import of primate lentiviruses can be inhibited by afragment of cleavage and polyadenylation factor 6, CPSF6-358, whereas FIV is insensitiveto this factor [61]. The reason for such diversity may lie in differences between the hostspecies in the level of immune cell activation associated with lentiviral infection or thepredominance of a certain target cell type. Indeed, hyperactivation of the immune systemaccompanies HIV infection of humans, whereas no such prolonged activation is observed insooty mangabeys or African green monkeys naturally infected with SIV [89]. The primarytarget and reservoir of HIV infection are CD4+ T lymphocytes, whereas maedi-visna virustargets preferentially macrophages and does not infect T lymphocytes [93].

In addition to classical NLS-dependent pathways, HIV may use alternative nuclear importpathways that appear to be NLS-independent. One such pathway was identified by analyzingHeLa cytosolic fractions for the ability to support nuclear import of purified HIV-1 PICsinto permeabilized nuclei. Surprisingly, the functional component of the active fractionturned out to be enriched in tRNA molecules, mostly with defective 3’ CCA ends [109].Synthetic tRNAs also promoted nuclear import of the PIC. Intriguingly, retrograde tRNAtransport in Saccharomyces cerevisiae is mediated by MTR10, a yeast orthologue ofTNPO3/Transportin-SR2 which has been implicated in HIV nuclear import [22].Association of tRNAs with PIC appears to depend on the Gag protein, at least replacementof HIV gag with gag of MLV blocked incorporation of tRNA species into the PIC. It

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remains to be determined whether this unusual pathway contributes to nuclear import ofHIV in natural target cells.

Historically, MA was the first protein implicated in HIV-1 nuclear import [12,98]. Whilemost of MA in the virion localizes between the viral core and the envelope, some MAmolecules are found in tight association with the HIV core and the PIC [14,76]. TwoNLSs, 26KKKYK and 110KSKKK, were identified in MA and shown to contribute to thenuclear localization of MA and PIC via interaction with importin α [29,43,78]. Interestingly,the basic aminoacids in the aminoterminal portion of MA, together with the N-terminalmyristoyl moiety, also function as the membranotropic signal during viral assembly[40,112]. Switch of the MA tropism from membrane during viral assembly to nucleus duringthe early steps of infection remains poorly understood but is accomplished most likely byposttranslational modifications such as phosphorylation [10]. Such dual tropism of MA maybe a reason that nuclear import activity of MA has not been detected in some reports[25,50]. Alternatively, the MA basic domain may function at a post-nuclear import pre-integration step, as reported by Mannioui and colleagues who found that mutation of MAresidues Lys26Lys27 impaired viral infection and 2-LTR circle formation but did not reduceHIV-1 DNA docking to chromatin [73]. Overall, MA, while contributing to PIC nuclearimport, appears to be non-essential as it is likely only one of several factors regulating thisprocess (see below). This consideration explains reports showing that the virus with MAdeletions was still capable of replication, albeit with a greatly reduced efficiency [32,84].Nevertheless, NLSs within the MA may provide a good target for therapeutic approachesaimed at reducing HIV replication.

Vpr is another HIV-derived protein that has been implicated in nuclear import. Vprsignificantly stimulates HIV-1 replication, in particular in macrophages, presumably byenhancing the nuclear import of the PIC [23,51,79]. Vpr has been shown to interact withimportin α [83,97] using amino acid residues 17–34 [80], providing a mechanism for itsnuclear localization. The increased activity of Vpr in macrophages versus CD4+ T cells wasinitially explained by the non-dividing state of the former, but in view of the requirement fornuclear import in both cell types it appears more likely that the PIC nuclear import pathwayin macrophages is different from that in T cells and needs Vpr. Such flexibility of nuclearimport pathways is consistent with a recent report showing that HIV-1 can use differentnucleoporins and importins for PIC import [61]. Similar to MA, the role of Vpr is notstrictly essential, as viruses lacking Vpr can still replicate in macrophages, albeit withreduced efficiency [51]. The importin-interacting domain of Vpr presents another target forpotential therapeutic interventions (see below).

A role for IN in HIV-1 nuclear import was originally proposed by Gallay and co-workers[36], who demonstrated that IN interacts with importin α and can target a fusion GST-INprotein into the nucleus of microinjected cells. They also identified a basic-type NLSspanning amino acids 186–189, but this claim was later refuted by Tsurutani and colleagues[95] who demonstrated that this region is involved in reverse transcription rather thannuclear import. A more recent report [9] identified an unusual NLS spanning residues 161–173 within the central core domain of IN. IN is highly karyophilic and has been shown tointeract with different importins using different domains, including importin α1 via thecentral core domain [5,9,36], importin α3 via the C-terminal domain (amino acids 250–270)[3], importin 7 via two regions, 235WKGPAKLLWKG and 262RRKAK, in the C-terminaldomain [2] and TNPO3/transportin-SR2 via the catalytic core domain close to its N terminus[22]. These importin-interacting domains of IN present an attractive target for therapeuticinterventions.

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In addition to being karyophilic itself, IN was shown to interact with a host cell karyophilicprotein lens epithelium-derived growth factor/transcriptional co-activator p75 (LEDGF/p75)[20,71]. LEDGF/p75 protein markedly stimulated HIV-1 IN activity in vitro and wasreported to be associated with functional HIV-1 PICs [68]. The normal cellular function ofLEDGF/p75 is still poorly understood, but appears to involve chromatin tethering of variouscellular proteins; a similar function was proposed for LEDGF/p75 in HIV replication [74].Interestingly, despite the presence of a canonical basic-type NLS responsible for nuclearlocalization of LEDGF/p75, disruption of this NLS did not abolish chromatin association ofectopically expressed IN and LEDGF/p75 [96]. This result suggests that LEDGF/p75, in anNLS-independent manner, may tether IN to chromatin during mitosis and prevent nuclearexclusion of this complex after completion of mitosis. This interpretation does not negatethe possibility that NLS of LEDGF/p75 may facilitate the nuclear import of HIV-1 PIC [70],although solid experimental support for such prospect is lacking. Interaction between IN andLEDGF/p75 is determined by Trp131/132 and residues 161–170 of the HIV-1 IN [15,30].These regions of IN present another target for potential interventions.

Recent reports suggested that HIV-1 nuclear import is regulated by the capsid protein CAp24 [59,61,107]. Indeed, mutations in CA changed the importins and nucleoporins used forPIC nuclear import, and C-terminally truncated cleavage and polyadenylation factor 6(CPSF6-358) bound CA and inhibited HIV-1 nuclear import [61]. However, no interactionbetween CA and importins or nucleoporins has been reported, suggesting that the role of CAmay simply reflect dependence of the PIC nuclear import on uncoating [26]. CA associatedwith the incompletely uncoated PIC may alter accessibility of NLSs to importins and/orinteraction between the PIC and nucleoporins (Fig. 1), and by targeting CA CPSF6-358 mayaffect uncoating similar to the effect of TRIM5α. Blocking uncoating of HIV-1 by targetingCA may be another approach to indirectly inhibit HIV nuclear import. A similarconsideration applies to another factor associated with HIV-1 nuclear import, the HIV-1DNA Flap. This structure formed during HIV-1 reverse transcription has been implicated inHIV-1 nuclear import [110]. The mechanism of DNA Flap activity is most likely associatedwith facilitation of PIC uncoating at the nuclear pore [4]. Another possibility is that Flapprovides a proper structure to the PIC allowing accurate exposure of the NLSs. Again, drugstargeting Flap may inhibit PIC nuclear import by affecting uncoating or disturbing the PICconformation. A model presenting the currently known factors playing role in HIV-1 PICnuclear import is shown in Fig. 1. In addition to nuclear import of PIC, successful HIVreplication also depends on nuclear import of regulatory proteins Tat and Rev. Tat and Revpossess arginine-rich NLSs (49RKKRRQRRR57 in Tat and 35RQARRNRRRRWR46 inRev), which, unlike most basic-type NLSs, interact with importin β but not with importin α[94]. Drugs inhibiting interaction between the HIV-1 regulatory proteins and importin β,presumably by targeting the NLS sequences, are expected to curtail viral replication.

3. Peptides as a tool to inhibit nuclear importThe process of HIV-1 nuclear import depends on protein-protein interactions between thePIC components or individual HIV-1 proteins and the cellular nuclear import factors. Inmany cases, the domains responsible for these interactions have been characterized (seeabove). Although some small molecules have been discovered that can modulate suchinteractions (see below), the design of these agents purely from the knowledge of the detailsof a given protein-protein interaction, or through screening, remains highly difficult [101].Therefore, using peptides derived from either protein binding partner directly, or aftermodification to improve affinity and physicochemical properties, continues to be veryattractive and resulted in obtaining peptides that can be used to interfere with protein-proteininteractions as well as to obtain anti-viral drugs [60].

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Short peptides have a low metabolic stability since linear peptides are rapidly degraded byintracellular proteolytic activities [8,102]. Structural modifications such as cyclization havebeen shown to increase metabolic stability by making the modified peptide un-available tointracellular proteolysis [38]. Side chain to side chain cyclization (SC) utilizes functionalgroups like amine, carboxylic acid or thiol, that naturally exist on the side chains or at theamino or carboxy ends of the peptide, to form the cyclization through amide or disulfidebonds. Another type of cyclization is the backbone cyclization (BC) that is preformed by acovalent interconnection of two backbone amides by artificial spacer. This strategysignificantly increases metabolic stability of the resulted BC peptides [16,37]. Below, weprovide a short overview of the use of synthetic peptides to inhibit nuclear import of theHIV-1 PIC and its karyophilic proteins.

There are two possible mechanisms by which peptides can inhibit NLS-dependent nuclearimport: they can either specifically interact with the NLS domain of a karyophilic proteinmasking this domain and thus preventing its interaction with the appropriate importin, orthey can mimic the domain competing with the NLS-carrying protein for interaction withimportin (Fig.2). So far, most nuclear import-inhibiting peptides were designed to functionthrough the latter mechanism. Peptides derived from SV40 large T-antigen NLS, which issimilar to the NLS of MA and mediates interaction with importin α, were shown to inhibitnuclear import of the HIV-1 in CD4+ MT4 cells [41]. Furthermore, screening of the libraryof BC peptides bearing the NLS of the MA protein identified a BC peptide, designated asBCvir, which inhibited infection of HIV-1 [35,45].

One of importins shown to interact with HIV-1 IN is importin α [36,49], so peptidesmimicking importin α-interacting NLS on IN were expected to inhibit HIV-1 infection. Thisassumption was confirmed by studies using the peptide containing residues 161–173 of INdesignated as NLS-IN [5,65]. Both a cell permeable peptide bearing the SV40 NLS and theNLS-IN peptide inhibited nuclear accumulation of IN in transfected cells and integration ofviral cDNA as well as HIV-1 replication in infected cells [65]. As expected, both peptidesdisrupted the IN-importin α complex. A similar inhibitory effect was observed with IN-derived peptide inhibiting IN-TNPO3 interaction (unpublished observation), consistent withproposed flexible use of nuclear import pathways by HIV-1 PIC [61].

The cellular protein LEDGF/p75, a binding partner of IN, interacts with IN primarilythrough a domain termed the Integrase Binding Domain (IBD) [19]. Based on the crystalstructure and solution structure analysis of the complex between the LEDGF/p75 IBD and adimer of the IN catalytic core domain it can be inferred that residues 361–370 of the IBDinteract with the putative IN-NLS (residues 161–173) [18,21]. Support for this view comesfrom results showing that interaction between a nonkaryophilic version of LEDGF/p75,mutated at its NLS, and IN blocked nuclear import of the IN [70]. Therefore, a peptidebearing residues 361–370 of LEDGF/p75 was a good candidate as an inhibitor of IN NLS.This notion was tested by Hayouka et.al. who demonstrated that a cell permeable peptidebearing LEDGF/p75 residues 361–370 inhibits HIV-1 replication and infection of culturedcells [46,47]. The LEDGF/p75 361–370 peptide may function as an “anti IN-NLS” (Fig. 2),at the same time preventing interaction of IN with LEDGF/p75 through direct competitionwith IBD. Interestingly, the LEDGF/p75-derived peptide was found to interact also with theNLS sequence of the HIV-1 Rev protein, but this interaction did not lead to inhibition of theRev nuclear import [66].

Recently, an interaction between the HIV-1 Rev and IN proteins has been reported [66,88].Surprisingly, this interaction inhibited IN nuclear import [63]. Two Rev-derived peptides,comprising Rev residues 13–23 and 53–67, were found to specifically interact with IN andto promote dissociation of the Rev-IN complex in HIV-infected cells, supposedly via

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competition with Rev for binding to IN [66,88] (Fig. 3). Binding to IN of both peptidesblocked nuclear import of the viral IN and inhibited viral replication ([63,72,86,88] and Fig.3). Interestingly, in contrast to the SV40 and NLS-IN peptides, the Rev-derived peptides didnot disrupt the intracellular IN-importin α complex in virus-infected cells, but causeddissociation of the IN-TNPO3 complex (A.L., unpublished observation). In addition, thepeptides promoted a shift in the oligomerization state of IN to predominantly tetramericform [48]. It remains to be determined whether inhibition of the IN nuclear import by thesepeptides is related to their ability to disrupt the IN-TNPO3 interaction or to promote INtetramerization. A similar nuclear import inhibitory effect and promotion of INtetramerization was observed with another IN interacting peptide selected from a peptidelibrary using a yeast 2 hybrid (Y2H) screen [72,86].

Interaction between IN and Rev implies a possibility that IN-derived peptides may be usedto block nuclear import of Rev. To identify the IN domains responsible for interaction withRev, screening of an IN-derived peptide library was performed and revealed two specificpeptides bearing IN residues 66–80 and 118–128 (designated INr-1 and INr-2, respectively[64]). Binding of the INr peptides to Rev in HIV-infected cells disrupted the Rev-INcomplex, similar to the Rev-derived peptides, and thus released the IN from the Revinhibitory effect [62,64] (Fig. 3). At the same time, the INr peptides blocked Rev nuclearimport [63]. An ELISA based screening assay indicated that the INr peptides interact withthe oligomerization domains of the Rev protein [55,58,64] and thus may inhibit Rev nuclearimport by blocking Rev oligomerization required for this step [91].

Tat is translocated into nuclei of infected cells by arginine rich motif (ARM) sequencesinteracting with importin β [94]. Import of Tat-NLS-BSA conjugate into nuclei ofpermeabilized cells was inhibited by several BC peptides bearing the Tat ARM but not—asexpected—by a peptide containing the SV40 NLS [34].

Taken together, the studies described above provide a strong rationale for furtherdevelopment of peptide-based inhibitors of HIV-1 nuclear import.

4. Small molecular weight inhibitors of HIV-1 nuclear importMA was the first HIV-1 protein implicated in PIC nuclear import, and the first efforts toidentify inhibitors of HIV-1 nuclear translocation focused on MA NLS. We approached thisgoal by looking for compounds that could neutralize the positive charge on the basic typeNLS of MA. The first such compound was ITI-002 {2-amino-4-(3,5,diacetylphenyl)-amino-1,6-dimethyl pyrimidine}, which reduced HIV-1 nuclear import and inhibited viralreplication [28,44]. Biochemical analysis showed that ITI-002 associated with HIV-1 MAprotein via the carbonyl residues on the phenol ring of the compound by forming Schiff baseadducts with lysine residues, likely in the first NLS of MA [1]. Such mechanism suggeststhat the compound may interact with all lysine-rich sequences and thus may be very toxic.However, this was not the case. The reason for the ITI-002 specificity for MA lies in theinitial interaction between the pyrimidine side chain of ITI-002 and the HIV-1 RT, which isrequired for accumulation of the drug in the PIC and shifting the Schiff reaction towards theadducts [82]. The interaction with RT appears to be outside the enzyme active site sincegeneration of the full-length nascent viral cDNA was not affected by the drug. Thisinteraction with RT is critical for activity of ITI-002, as the drug did not inhibit nuclearimport of model substrates carrying basic-type NLS, and modified drugs unable to bind RTdid not inhibit HIV-1 replication [82]. It is likely that interaction with RT is required tocreate high concentration of the drug in the vicinity of MA NLS, thus shifting theequilibrium of the reaction towards Schiff base formation. This interesting property ofarylene bis(methyl ketone) compounds explains their low toxicity and high specificity.

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ITI-002 inhibited HIV-1 replication in both macrophages and CD4+ T cells, as well as in exvivo cultured lymphoid tissue [44]. Given that MA appears to be only one of severalinterchangeable factors mediating HIV-1 nuclear import, such potent inhibition of HIV-1replication by MA NLS-targeting compound came as a surprise. A likely explanation is thatITI-002 may inhibit also later (post-integration) steps of viral replication which aredependent on the basic region of MA. One such step is viral assembly, as the basic region ofMA within the Gag precursor is a key membranotropic signal [81].

Another class of compounds targeting the MA NLS, oxadiazols, was selected usingcomputer aided drug design (CADD) by modeling a 250,000 compound library on thecrystal structure of the HIV-1 MA [42]. These compounds were predicted to bind to MAwithin a structural groove in the N-terminal NLS generated by the inward orientation of thetyrosine residue (amino acid #29). ITI-367 [3-(2-methoxyphenyl)-4-[3-(trifluoromethyl)phenyl-1,2,4-oxadiazol-5(4H)-one], a lead compound from this group,inhibited the binding of the PIC to importin α, PIC nuclear import, and HIV replication inmacrophages and CD4+ T cells [42]. Importantly, ITI-367 demonstrated at least an additiveinhibitory effect when combined in vitro with nucleoside reverse transcriptase inhibitors(AZT, d4T), non-nucleoside reverse transcriptase inhibitors (efavirenz), or proteaseinhibitors (saquinavir, nelfinavir). Following culture of HIV-infected cells in the presence ofincreasing concentrations of ITI-367, a drug-resistant mutant was produced. Surprisingly,mutations identified in this virus localized to the cytoplasmic domain of gp41, but not toMA (unpublished results). This result suggests that the main step of HIV replication affectedby ITI-367 is the step of virus assembly, rather than PIC nuclear import. Indeed, interactionbetween the MA-corresponding part of Gag and the cytoplasmic tail of gp41 is critical forproduction of infectious virions [6,7,69], and the L30E mutation located within the MA NLSimpairs this interaction [7]. This result, consistent with flexible use of nuclear importpathways by HIV-1 PIC, underscores attractiveness of the MA protein as a target fortherapeutic interventions.

Vpr is another HIV-1 protein implicated in PIC nuclear import. Although a number ofmechanisms were proposed to explain nuclear localization of Vpr [11], association of Vprwith importin α has been reported by several independent groups [56,80,83,97]. Searchingfor compounds that can specifically inhibit the interaction between importin α and aminoacids 17–34 of Vpr, shown to be indispensable for nuclear import of Vpr and for HIV-1replication in macrophages [80], identified hematoxylin [90]. Hematoxilin inhibited specificbinding of Vpr to importin α in a dose-dependent manner, it also inhibited HIV-1 replicationin macrophages and nuclear import of HIV-1 PIC at concentrations 20–40 μM [90].Importantly, hematoxylin did not impair the ability of Vpr to induce G2 cell cycle arrest,suggesting a specific effect on Vpr-mediated nuclear import. Surprisingly, the authorsreported that the drug did not inhibit HIV-1 replication in PBMC. This result may reflectdifferent requirements for PIC nuclear import in different cell types; nuclear import inPBMC may not require Vpr.

Styrylquinolines have been recognized as potent HIV-1 IN inhibitors that block HIV-1replication in cell-based assays [75]. This activity may be determined, at least in part, by aspecific inhibition of IN nuclear import [77]. It should be noted here that the effect on INnuclear import was only seen at relatively high concentrations of the drug (30 μM).However, nuclear import of karyophiles carrying basic-type NLSs similar to the NLS ofSV40 T antigen was not affected by these concentrations of styrylquinolines, suggesting thatthe drug inhibited IN interaction with importins that are not involved in transport of SV40 Tantigen-type NLSs, e.g., importin 7 or TNPO3. It seems likely that binding of thecompounds to IN impair both the integration activity and nuclear import of the protein.These functions appear to be regulated by adjacent or even overlapping sites on IN.

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5. ConclusionsDevelopment of HIV-specific nuclear import inhibitors critically depends on the progress ofbasic research aimed at understanding the molecular mechanisms of this process. This fieldis rapidly evolving. At present, it appears that nuclear import of HIV-1 PIC is a highlyflexible process involving a number of interchangeable pathways. It is possible that thesepathways are specific for certain cell types (e.g., macrophages or CD4+ T cells).Alternatively, several pathways may be used by the virus in each cell type, with selectiondepending on a cell status, such as activation or cell cycle progression. The cell statusparameters may influence HIV nuclear import through an effect on viral uncoating:accelerated or delayed uncoating may change the import pathway used by the virus throughaltering accessibility of NLSs on PIC proteins. Therefore, the optimal approach to designinga universal inhibitor of HIV-1 PIC nuclear import is to target a step in viral replicationpreceding the nuclear import pathway bifurcation (Fig. 1). A good candidate for thisapproach is the step of HIV uncoating regulated by the CA protein. Although uncoating isnot formally a step of nuclear import, CA appears to determine the nuclear import pathwayused by the virus [61], so drugs blocking or modifying uncoating may also block viralnuclear import. Targeting nuclear import of individual viral proteins, Tat and Rev, may bean easier task, although such approach has the danger of allowing viral integration.

It can be expected that new findings will provide the basis for the design of compoundstargeting not only viral proteins, but also their cellular counterparts, such as importins andnucleoporins. While targeting cellular proteins with drugs carries a danger of cytotoxicity,some compromises may be worked out. For example, multiple isoforms of proteins involvedin nuclear import are expressed in eukaryotic cells [33]. These isoforms are specific fortransporting certain substrates, and it is possible that isoforms may be identified that areessential for survival of the virus but not of the host cell.

A major concern with any new anti-HIV therapy is the development of drug-resistantmutants. Future studies will likely identify mutations that provide some resistance to nuclearimport inhibitors. It would be interesting to find out whether resistant virus can emergewhen drugs targeting several viral nuclear import factors are used simultaneously. Giventhat viral proteins need to interact with very conserved cellular nuclear import factors, it canbe expected that the fitness of such resistant virus will be greatly compromised. This wouldbe good news for anti-HIV therapy, as unfit HIV may be well controlled by the immunesystem and other drugs in the HAART cocktail.

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Figure 1. Nuclear import of the HIV-1 PICCA protein covers PIC NLSs preventing their interaction with importins. Partial uncoatingin the perinuclear area frees some NLSs. Availability of a particular NLS determinesbinding of an importin (one or several). The figure shows PICs binding only one ofimportins (importin α/β, importin 7 or TNPO3). The importins, probably via interaction withan importin-specific nucleoporin, target the PIC to the nuclear pore complex and into thenucleus.

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Figure 2. Peptides targeting NLS of INNLS bearing peptides—such as NLS-IN—compete with IN for binding to nuclear importreceptor (like importin α) and thus block nuclear import. On the other hand anti-NLSpeptides—such as LEDGF 361–370—bind the NLS of the karyophilic protein (IN) thuspreventing its binding to the nuclear import receptor (importin α). See text for details.

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Figure 3. Peptides that disrupt Rev-IN interactionRev derived peptides (Rev 13–23 and Rev 53–67) promote dissociation of the Rev-INcomplex allowing nuclear import of Rev oligomers while shifting the IN oligomeric state totetramer and consequently blocking its nuclear import. On the other hand, the INr peptides(INr-1 and INr-2), which also dissociate the Rev-IN complex, allow nuclear import of INbut block Rev nuclear import, probably by preventing Rev from forming the oligomericstate required for its nuclear import. See text for details.

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Strategies to inhibit viral protein nuclear import: HIV1 as a target

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