INTRODUCTION

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Current AIDS therapies use inhibitors of reverse transcriptase (RT) and protease (PR), two of the human immunodeficiency virus (HIV) enzymes. Maximum efficacy occurs when these molecules are used in double or triple therapies which can lead to long-term decreases in viral load to below detectable levels (19). However, patient compliance with highly active antiviral therapies (HAART) is low, and there is evidence that all virus is not eliminated from the body. Virus survival of HAART is thought to be due to (i) outgrowth of viral strains with lower sensitivity to one or more of the inhibitors, (ii) latency in the form of integrated and nonintegrated DNA in lymphocytes and monocytes, (iii) very high levels of replication in lymphoid tissues, and (iv) replication in some organs (e.g., brain) that have reduced accessibility to inhibitors or the immune system (21, 23, 25, 31, 50). At the molecular level, replication features of HIV with high levels of mutation and recombination probably account for the rapid selection and propagation of inhibitor-resistant viral species (12). Mutations conferring increased resistance occur at the level of the target enzymes and, in the case of protease inhibitors, at the level of both the enzyme and its viral substrates (50).

Thus, there is still need for novel HIV inhibitors. Strategies employed so far in the development of molecules used in patients involve substrate analogs (RT and PR) or conformational ligands (RT). However, virtually all of the other viral components (including genomic RNA) can be viewed as potential therapeutic targets. Special emphasis has been placed on the nucleocapsid protein NCp7, the smallest of the three main structural proteins. NCp7 is present at approximately 2,500 copies per virion and coats the genomic RNA to form the nucleocore. NCp7, probably as part of its Gag precursor, directs genomic RNA dimerization and packaging in particles through specific interactions and participates in virus assembly through protein-protein interactions (reviewed in references 9 and 13). NCp7 also functions during early steps of the infection process, such as viral DNA synthesis and protection (4, 34, 43).

NCp7 has two zinc fingers of the form C-X₂-C-X₄-H-X₄-C in which the zinc ion is coordinated to three cysteine residues and one histidine residue. All retroviral nucleocapsid proteins have either one or two zinc fingers of this structure, and they are of crucial importance since in all documented cases, mutating cysteine or histidine residues leads to a total loss of virus infectivity (2, 11, 26). The fact that this motif is highly conserved among retroviruses makes it an interesting target for inhibitors. Zinc coordination is the main effector of the zinc finger folding, and the two zinc fingers correspond to the only domain with a defined three-dimensional structure in NCp7 (10, 27, 28, 42). Therefore, it can be postulated that there may be little possibility of viral escape from inhibitors directed against HIV NCp7 zinc fingers.

NCp7 inhibitors were first characterized after a random screening of a molecular library for anti-HIV type 1 (HIV-1) activity. Molecules found (NOBA and DIBA) were shown to irreversibly displace zinc ions from virus-associated as well as recombinant NCp7 (37, 38, 44). This was associated with time and concentration-dependent inactivation of cell-free viral preparations. However, replication events targeted by the inhibitor were not characterized in these studies. In other investigations, inhibitors of the DIBA type were shown to inhibit viral protein maturation in chronically infected cells. This suggests that these drugs act intracellularly and interfere with late steps of viral replication (46). Moreover, inhibition of maturation was accompanied by the establishment of Gag-Gag intermolecular disulfide bonds, presumably resulting from the effect of DIBA on NCp7 cysteine residues and concomitant zinc displacement.

We investigated the effects of DIBA-1 on HIV-1 structure and replication and compared it with a protease inhibitor. Results presented here show that DIBA-1 alters virion core structure and subsequently inhibits proviral DNA synthesis. In addition, DIBA-1 possibly affects viral DNA stability in infected cells, a unique property that highlights zinc ejector compounds as molecules potentially able to destroy certain viral reservoirs in infected individuals.

	MATERIALS AND METHODS

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Cell lines and DNA. SupT1, a T-cell line highly sensitive to HIV infection (41), and HeLa cells were used. Two reporter cell lines were employed to monitor HIV infection: (i) P4 cells that are HeLa cells constitutively expressing CD4 and LacZ but under the control of the HIV long terminal repeat (LTR) (6) and (ii) CEMgfp, a T-cell line expressing the green fluorescent protein (GFP) under the control of the HIV LTR (17). HeLa and P4 cells were maintained in Dulbecco modified Eagle medium; CEMgfp and SupT1 cells were maintained in RPMI. All media were supplemented with 10% fetal calf serum and antibiotics. P4 and CEMgfp were cultured in the presence of 500 µg of G418 per ml.

Virus production and infection. Viruses were produced upon transfection of HeLa cells by using the calcium phosphate method (5). First, 2 × 10⁶ cells were resuspended by trypsinization and mixed with the DNA preparation in 10 ml of medium. The DNA preparation contained 10 to 20 µg of pNL4-3 or a mixture of 10 µg of pNL-EN and 2 µg of pCEL/E160 DNA. Except where indicated, transfected cell pools were distributed in 10-cm plates. The following day, cells were rinsed and fresh medium was added with or without the inhibitor. Two days later supernatants were clarified by low-speed centrifugation and used to infect cells or to carry out biochemical analyses. Since p24 monitoring by our enzyme-linked immunosorbent assay (ELISA) assay was found to largely depend upon its level of maturation, this method was not used for viral normalization, instead we simply used identical volumes of samples derived from a single transfection pool. Western blot analysis indicated that the amount of particles was indeed similar in the samples. As a negative control, we used diluted supernatant from mock-transfected cells. All experiments involving a virus produced in the presence of inhibitor included a parallel titration of these viruses on P4 cells. To this end, P4 cells were plated in 24-well plates at 10⁵ cells per well in 0.5 ml. Medium was removed the day after and replaced by 0.5 ml of the pure or 10-fold-diluted viral preparation. After 24 h cells were fixed and processed for X-Gal (5-bromo-4-chloro-3-indolyl--D-galactopyranoside) staining as described previously (4). Blue cells, corresponding to a single round of replication, were counted.

Inhibitors. Palinavir (22) and DIBA-1 (36) were gifts from Biomega and Rhone-Poulenc Rorer, respectively. Both molecules are hydrophobic and were stored as solutions in dimethyl sulfoxide (DMSO) at a concentration of 100 mM. Before use, molecules were diluted to final concentrations and were added to cells so that DMSO did not exceed 1/250 of the culture volume to avoid toxic effects. Effects of these inhibitors on cell viability were measured by using the XTT assay (48). For this, 50 µl of a solution of 1 mg of 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)carbonyl]-2H-tetrazolium hydroxide (XTT) and 0.01 mM of N-methylphenazonium methosulfate were added to the cells and 3 h later 0.2 ml of supernatant was collected. Relative levels of soluble formazan in the supernatants were measured by spectrophotometry at 450 nm (by using a 650-nm wavelength reference). Cell expression of GFP in response to HIV infection was monitored immediately after the XTT assay.

Biochemical characterization of viruses. Quantitation of RT activity has been extensively described (18, 29). For analysis of virion proteins, supernatants from transfected cells were concentrated by ultracentrifugation through a 20% sucrose cushion and resuspended in 100 µl of TNE (25 mM Tris, pH 7.5; 100 mM NaCl; 1 mM EDTA) containing 1% sodium dodecyl sulfate (SDS). Samples (20 µl) with or without 5% -mercaptoethanol were heated at 95°C and loaded onto a 5 to 20% gradient polyacrylamide gel. After electrophoresis, proteins were transferred onto a polyvinylidene difluoride (PVDF) membrane and immunostained by using polyclonal anti-NCp7 and anti-RT antibodies (4, 43). For virion genomic RNA content, RNA was extracted from viral pellets as described previously (14). RNA was resuspended in water, denatured for 10 min at 70°C, and blotted onto a nylon membrane by using a Gibco slot blot apparatus. Unspliced HIV RNAs were probed by using a radiolabelled DNA corresponding to the matrix protein sequence.

PCR detection of viral DNA. Prior to infection, viral supernatant was subjected to DNase digestion with 0.1 mg of DNase I (Boehringer) per ml in the presence of 10 mM MgCl₂ for 30 min at 20°C. To monitor HIV-1 DNA synthesized in SupT1 cells, 2 × 10⁵ cells were infected with 0.5 ml of viral supernatant for 5 h at 37°C; cells were then rinsed twice in PBS, and half of the cells were used to extract DNA while the other half was maintained for an additional 15 h. To analyze viral DNA synthesis in CEMgfp cells, 5 × 10⁵ cells were infected for 3 days; one-third of the culture was used to extract DNA, and the rest was used to monitor GFP expression (see above). Cells were lysed in an SDS-2-mercaptoethanol solution (4), and nucleic acids were extracted once with phenol-chloroform and once with chloroform. DNA was ethanol precipitated, pelleted, and resuspended in water. PCR reactions were performed by using different primer sets. The PC03-PC04 set amplifies a 112-bp region of the human -globin gene (40). SU₃-ASU₅ and SU₃-ASPBS sets (43) are specific for early and late events of HIV-1 DNA synthesis, respectively. PCR with these primer sets will amplify DNA synthesized after the first (SU₃-ASU₅) or the second (SU₃-ASPBS) strand transfer. It is important to point out that, because there are sequence differences between the 5' and the 3' LTRs of pNL4-3, amplification with these primers will be specific for the newly made cDNA as opposed to pNL4-3 DNA. This minimizes further the risk of transfected DNA contamination in PCR reactions. Quantitative PCRs were performed by using the following procedure: DNA from 10,000 to 20,000 cells were subjected to 25-cycle amplification under reaction conditions previously described (4) except that the reaction mixture contained 0.8 mM dCTP and 2 mM dTTP, dATP, and dGTP. Each reaction mixture contained 1 µCi of [^-32P]dCTP (Amersham). PCR products were resolved on a 8% polyacrylamide gel electrophoresis (PAGE) gel; bands were revealed by autoradiography (1- to 3-day exposure) and quantified by phosphorimaging.

Electron microscopy analyses. HeLa cells were transfected with pNL4-3 and exposed to inhibitor as described above. At 3 days after infection, cells were fixed and prepared for electron microscopy as described earlier (4) (CMEABG, Villeurbanne, France). For in situ hybridization and CAp24 immunodetection, 5 × 10⁶ SupT1 cells were infected for 10 h with 5 ml of supernatant from transfected cells that had been exposed or not to inhibitor at the desired concentration. Cells were fixed in 4% paraformaldehyde in phosphate buffer, dehydrated in ethanol, embedded in LR White, then applied onto 400 mesh, Formvar-coated nickel grids (32). LR White sections were stored at room temperature until use. All subsequent incubations were performed by floating the grids on drops of the different solutions. For in situ hybridization, sections were subjected to 1 mg of RNase A per ml digestion (1 h at 37°C in 2× SSC buffer [1× SSC is 0.15 M NaCl plus 0.015 M sodium citrate]) and alkaline treatment (0.5 N NaOH at 20°C for 4 min), followed by three washes in water and 10 min of air drying. In situ hybridization was performed by using a 30-base synthetic nucleotide complementary to bases 555 to 585 of pNL4-3 (5'-ACC AGA GTC ACA CAA CAG ACG GGC ACA CAC). 3'-terminal labelling of the oligonucleotide was performed by using digoxigenin-11-dUTP (Boehringer-Mannheim) (32). The hybridization solution contained 30% deionized formamide, 4× SSC, 1 nM digoxigenin oligonucleotide probe (extemporaneously denatured), 2× Denhardt's solution (0.44% bovine serum albumin, 0.44% Ficoll 400, 0.44% polyvinylpyrrolidone), and 1 mg of denatured salmon sperm DNA per ml. After overnight incubation at 37°C, grids were washed once in 4× SSC, once in 2× SSC, and twice in 1× SSC; grids were then postfixed in 4% paraformaldehyde and washed in TBS+ (20 mM Tris, pH 8.2; 150 mM NaCl, pH 8.2; 0.1% ovalbumine; 0.1% Tween 20; 1% horse serum). Hybridized digoxigenin probes were reacted for 1 h at 37°C with sheep antidigoxigenin (1:50) conjugated to 10-nm gold particles. Finally, grids were washed twice in TBS, once in 2× SSC, and then fixed in 2.5% glutaraldehyde and washed with distilled water. Sections were examined after uranyl acetate staining on a Philips CM120 electron microscope (CMEABG, Villeurbanne, France). For statistical analysis of viral DNA distribution, cytoplasmic and nuclear surfaces of 30 randomly chosen cells from each sample were monitored by using a Quantimeter 570, and gold beads in each compartment were visually counted. The number of gold particles bound to 30 cells was between 300 to 650 except for the negative control.

Immunocytochemistry. Monoclonal anti-capsid p24 (CAp24) antibodies were purchased from Dako. LR White sections (see electron microscopy section) were incubated in anti-CAp24 monoclonal antibody diluted 1/5 in TBS+ for 60 min and washed in the same buffer, and the antigen-antibody complexes were revealed by incubation for 60 min with a goat anti-mouse antibody (5 mU/ml) conjugated to 10-nm gold particles (BioCell, Cardiff, United Kingdom) diluted 1/50 in TBS+. Grids were washed again, fixed in 2.5% glutaraldehyde in 2× SSC, washed in 2× SSC and then in distilled water, counterstained in saturated aqueous uranyl acetate (10 min), and air dried. Controls were carried out without the primary antiserum or by using nontransfected cells and led to no detection in both cases. Gold particles were counted in 25 to 30 randomly chosen cells, and the number of total gold particles per sample was between 80 to 100 except for the negative control.

	RESULTS

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DIBA-1 inhibits the early and late phases of HIV-1 replication. A number of studies indicate that NCp7 is involved in both the early (from cell entry to proviral DNA integration) and late (viral protein synthesis and virion formation) phases of HIV-1 replication (2, 4, 34, 43). Therefore, NCp7 inhibitors might be expected to act at either or both these stages of virus replication. We investigated this possibility by using single-round replicating HIV-1 and NCp7 inhibitor DIBA-1, in parallel with a protease inhibitor (Palinavir) on target and/or producer cells (Fig. 1). In the absence of inhibitor, about 5% of the cells produced high levels of GFP in response to HIV infection. As expected, Palinavir had little or no effect on the infection process when added to target cells only (Fig. 1C), whereas an 80% inhibition of infectivity was observed when it was added to both virus producer and target cells (Fig. 1A). Interestingly, DIBA-1 caused a significant (30 to 70%) inhibition when added to the target cells only (Fig. 1C). This inhibitory effect was higher when DIBA-1 was also added to producer cells and nearly complete at 10 µM (Fig 1A). Cell viability was not significantly impaired by DIBA-1 or Palinavir at 1 µM but started to be impaired at 10 µM DIBA-1 (Fig. 1; see also Fig. 7), while toxic effects were seen at 10 µM Palinavir (Fig. 1).

View larger version (34K): [in this window] [in a new window]   FIG. 1.   DIBA-1 and Palinavir act via distinct mechanisms. HeLa cells (3 × 106) were transfected with pNL.EN and pCEL-E160 distributed into nine wells and processed as indicated in Materials and Methods. DIBA-1 or Palinavir was added at the indicated concentration 48 h prior to transfection or/and infection with HeLa-virus producer cells and CEMgfp target cells, or on CEMgfp target cells only, or with no inhibitor (control). (A and C) At 3 days after infection 100,000 CEMgfp cells were analyzed by FACS and GFP-positive cells were counted. (B and D) The viability of infected cells was determined prior to FACS analysis by XTT assay. Values correspond to an absorbance at 450 nm (after background substraction and with 650 nm as a reference) of 200 µl of cell supernatant, positively correlated to cell number and their metabolic activity. All results are given as the mean values of triplicates with the standard deviation.

DIBA-1 affects virion protein content. In an attempt to understand the mechanism by which DIBA-1 inhibits virus infectivity, virions produced in the presence of DIBA-1 (1 or 10 µM) or Palinavir (1 µM) were analyzed. As expected (Fig. 1), DIBA-1 and Palinavir were able to strongly inhibit a single round of HIV-1 replication, especially at 10 µM DIBA-1 (Fig. 2A, d10). Virion proteins were analyzed by Western blotting with anti-NCp7 (Fig. 2B) or anti-RT antibodies (Fig. 2C). In the absence of inhibitor, NCp7 was predominantly in the mature forms of 71 and 55 amino acids (Fig. 2B, lane 5) (33). In presence of 10 µM DIBA-1 (lane 4, d10) or 1 µM palinavir (lane 2, pal1), Pr55^gag precursor was the predominant form, indicating a gag maturation defect. Palinavir corresponds to a substrate analog and thus acts by directly interacting with the protease; therefore a maturation defect is expected to take place (compare lanes 2 and 4). DIBA-1 also appears to cause defects of Gag processing, especially at 10 µM (Fig. 2B, lanes 3 and 4). Note that the maturation intermediate (resulting from a cleavage between NCp7 and p6) indicated by an asterisk is absent when virus is produced in the presence of DIBA-1. Thus, the maturation defect caused by DIBA-1 is both quantitative and qualitative.

View larger version (32K): [in this window] [in a new window]   FIG. 2.   Characterization of HIV-1 produced in the presence of DIBA-1 or Palinavir. HeLa cells were transfected with pNL4-3 and then distributed into 10-cm2 plates (see Materials and Methods). The next day, DIBA-1 or Palinavir was added, and 2 days later the supernatants were clarified. (A) Infectious titer. A 500-µl portion of supernatant was added to 105 HeLa and P4 cells plated the day before X-Gal staining was performed 24 h later. Values represent the total of blue cells per well. Columns: 1, negative control (supernatant from nontransfected cells); 2, 1 µM Palinavir; 3, 1 µM DIBA-1; 4, 10 µM DIBA-1; 5, untreated (positive control). (B) Maturation of virion proteins in the presence of DIBA-1 or Palinavir. Virions from 2 ml of supernatant were treated with SDS and 2-mercaptoethanol; viral proteins were then separated onto a 5 to 15% gradient acrylamide gel, blotted, and probed with an anti-NCp7 polyclonal antibody. Molecular weight markers are shown on the left, and viral proteins with the NC domain are indicated on the right. Lane 1, negative control Ct; lane 2, 1 µM Palinavir; lane 3, 1 µM DIBA-1; lane 4, 10 µM DIBA-1; lane 5, untreated virus. (C) Presence of HIV-1 RT in DIBA-1- or Palinavir-treated virions. Samples were processed as in panel B, viral proteins were probed with polyclonal anti-NCp7 antibody, and membrane was stripped (but trace amounts of Pr55gag can still be seen) and reprobed with a polyclonal anti-RT antibody. Lane 1, negative control Ct; lane 2, 1 µM Palinavir; lane 3, 1 µM DIBA-1; lane 4, 10 µM DIBA-1; lane 5, wild-type virus sample; lane 6, 25% of wild-type virus. (D) Virion genomic RNA content. Virion RNA corresponding to 5% of the supernatant was extracted, slot blotted, and probed with a DNA corresponding to a sequence in MA (see Materials and Methods). Slots were revealed by autoradiography, quantified by phosphorimaging, and expressed as arbitrary units (see Materials and Methods). Lane 1, negative control Ct; lane 2, 1 µM Palinavir; lane 3, 1 µM DIBA-1; lane 4, 10 µM DIBA-1; lane 5, wild-type virus sample; lane 6, 25% of wild-type virus; lane 7, 15% of wild-type virus. (E) RT activity of DIBA-1- or Palinavir-treated virions. Portions (30 µl) of supernatant were subjected to exogenous RT assay as described before (4). Dots were revealed by autoradiography, quantified by phosphorimaging, and expressed as arbitrary units (see text). Lane 1, negative control Ct; lane 2, 1 µM Palinavir; lane 3, 1 µM DIBA-1; lane 4, 10 µM DIBA-1; lane 5, wild-type virus sample; lane 6, 50% of wild-type virus.

DIBA-1 promotes formation of NC multimers. DIBA-1 was reported to establish disulfide linkages between nucleocapsid protein or/and Gag precursor molecules, thus promoting the formation of NC multimers (46). In order to investigate the extent of NC multimer formation in our experimental system, we performed Western blot analysis of virions produced in the presence of drug and in the absence of 2-mercaptoethanol (Fig. 3; note that all bands contain the NCp7 domain). In the presence of the reducing agent, results were similar to those of Fig. 2B, showing a maturation defect caused by Palinavir and DIBA-1, as evidenced by large amounts of partially or unprocessed Gag precursor (lanes 1 to 3). It should be taken into account that 10-fold more virions were used in these experiments than in Fig. 2B, explaining why most if not all Gag species are apparent (lanes 1 to 4). Western patterns of wild-type and Palinavir-treated virions were similar in the presence or absence of 2-mercaptoethanol (compare lanes 4 and 9 for untreated virions and lanes 1 and 6 for Palinavir-treated virions). Interestingly enough, DIBA-1-treated virions exhibited significantly altered patterns at both 1 and 10 µM (compare lanes 2 and 7 and lanes 3 and 8). At 1 µM DIBA-1, a diffuse smear is apparent in the 20- to 50-kDa size range, as well as two discrete species indicated with an asterisk (lane 7). At 10 µM DIBA-1, most partially processed gag molecules and NCp7 have disappeared, but high-molecular-weight multimers were visible (lane 8). These results suggest that cysteine residues of nucleocapsid zinc fingers may undergo conformational changes caused by DIBA-1 with the concomitant establishment of inter- and possibly intramolecular disulfide bonds, resulting in the formation of gag and NCp7 multimers, representing about 50 and 100% of all NCp7 and gag molecules at 1 and 10 µM DIBA-1, respectively (lanes 7 and 8).

View larger version (66K): [in this window] [in a new window]   FIG. 3.   Formation of disulfide bonds is induced by DIBA-1. Viruses were produced in the presence of inhibitor as described in Fig. 2. Proteins corresponding to 10 ml of supernatant were prepared for SDS-PAGE in reducing or nonreducing conditions and then resolved on a 5 to 15% polyacrylamide gel. Proteins were transferred to PVDF membranes and probed with a polyclonal anti-NCp7 antibody. Molecular weight markers are shown on the left. Lanes 1 and 6, 1 µM Palinavir; lanes 2 and 7, 1 µM DIBA-1; lanes 3 and 8, 10 µM DIBA-1; lanes 4 and 9, wild-type virus sample; lane 5, negative control. Note the formation of viral protein multimers containing NCp7 (the asterisks indicate discrete species).

Modifications of virion ultrastructure caused by DIBA-1. The morphology of virions produced in presence of DIBA-1 or Palinavir was investigated by electron microscopy. Most wild-type viral particles had a mature morphology with sometimes a canonical conical core (Fig. 4). Palinavir-treated virions were typically immature, as evidenced by a broad periphery with no clear core structure. Viral particles produced in presence of DIBA-1 showed a composite structure since some particles had a mature morphology, while others were predominantly immature (Fig. 4). Moreover, the periphery of immature particles was often discontinuous, and mature particles had unexpected highly condensed cores. Interestingly, DIBA-1-treated virion cores were generally localized at the periphery, in close contact with the envelope (arrows). It is unclear whether these highly condensed nucleoid structures contain capsid or matrix protein in addition to nucleocapsid protein molecules, and this is presently under investigation. Finally, measurement of virion size by previously described methods (4) showed that particles produced in presence of 10 µM DIBA-1 were strikingly small (93 ± 9 nm, n = 61) compared to wild-type particles (114 ± 12 nm, n = 52) or to virions produced in the presence of 1 µM Palinavir (103 ± 15 nm, n = 26).

View larger version (70K): [in this window] [in a new window]   FIG. 4.   Structure of HIV-1 virions produced in the presence of DIBA-1 or Palinavir. HeLa cells transfected with pNL4.3 were cultured in the presence of DIBA-1 or Palinavir. After 2 days the cells were prepared for electron microscopy. Representative viral particles, from left to right, included wild-type virus, Palinavir (1 µM), and DIBA-1 (10 µM). For DIBA-1-treated HIV-1, note the two virion structures corresponding to an immature morphology with frequent discontinuous peripheral rings (small arrows) or highly condensed and excentric cores (the freezing-core effect of DIBA-1; see large arrows). Also note that virions produced in the presence of DIBA-1 are much smaller (see text). The bar is 100 nm for all pictures.

Inhibition of proviral DNA synthesis by palinavir and DIBA-1. To analyze proviral DNA synthesis in newly infected cells, virus-producing cells were treated with inhibitor, and infectivity was monitored on HeLa P4 cells after one round of replication (see Materials and Methods and Fig. 5A). Virus titers were similar to those reported above, with more than a 10-fold reduction at 1 µM Palinavir or 10 µM DIBA-1 and a threefold decrease in presence of 1 µM DIBA-1. These virus preparations were used to infect SupT1 cells for 5 h, and then half of the cells were frozen, while the other half were maintained in culture for an additional 15 h. DNA extracted from all cell samples was subjected to amplification with primer pairs specific for the early or late steps of proviral DNA synthesis (4, 43).

View larger version (32K): [in this window] [in a new window]   FIG. 5.   Fate of HIV-1 cDNA synthesized in the presence of DIBA-1 or Palinavir. For pNL4.3 transfection and HeLa cell culture, see Materials and Methods. One day after transfection, inhibitor was added, and 2 days later supernatants were clarified and DNase treated. (A) Infectious titers. Portions (500 µl) of supernatant were added to 105 HeLa and P4 cells, and 24 h later X-Gal staining was carried out. Results are expressed as the number of blue cells per well. (B) PCR amplification of viral cDNA. Portions (500 µl) of supernatant were used to infect 2 × 105 SupT1 cells for 5 h. Infections were also done with two dilutions (1/5 and 1/25) of untreated HIV-1. Cells were rinsed, and half were cultured for an additional 20 h. Nucleic acids were extracted, and [32P]dCTP-labelled PCR amplifications were carried out on 5% of each sample with primer pairs specific for early (corresponding to minus strand cDNA after the first transfer) and late (corresponding to double-stranded DNA after the second transfer) stages of the reverse transcription process (see panels). PCR DNA products were analyzed and quantified as indicated in Materials and Methods. Gels were autoradiographed (see results under the graphs) and submitted to phosphorimaging quantification (arbitrary units). Lane 1, negative control; lane 2, 1 µM Palinavir; lane 3, 1 µM DIBA-1; lane 4, 10 µM DIBA-1; lane 5, wild-type virus; lane 6, 20% of wild-type virus; lane 7, 4% of wild-type virus.

DIBA-1 modifies the cellular localization of reverse transcription complexes. To gain some insight into the fate of reverse transcription complexes in newly infected cells, CAp24 antigen and viral cDNA products were visualized by means of canonical immunodetection and in situ hybridization protocols (see Materials and Methods and Fig. 6). Similar densities of CAp24-bound gold particles were observed in the cytoplasm of cells infected with virus produced in the presence or absence of Palinavir or DIBA-1, confirming that these inhibitors did not affect viral entry (data not shown). Also and as expected, no antigen labelling was found in nuclei, since CAp24 protein is most probably not part of the preintegration complex (data not shown; reference 16). The density of virus DNA-bound gold particles was similar in the cytoplasm and nucleus area (Fig. 6A), but there was a large increase in the level of nuclear-envelope-associated viral DNA and CAp24 upon treatment with DIBA-1 or Palinavir (Fig. 6B). This is probably linked to a defect in preintegration complex formation due to inhibition of virion core maturation after virus treatment with Palinavir or DIBA-1.

View larger version (23K): [in this window] [in a new window]   FIG. 6.   Effect of DIBA-1 or Palinavir on the localization of HIV-1 reverse transcription complexes. SupT1 cells were infected with HIV-1 produced in the presence of 1 µM palinavir or 10 µM DIBA-1. After 10 h cells were fixed and prepared for electron microscopy. The negative control is made up of noninfected cells. (A) Detection of early viral cDNA products. Sections were probed with an oligonucleotide complementary to the U5 sequence of genomic RNA. Gold particles were counted in the cytoplasm and nucleus areas of 30 randomly selected cells, and the densities are reported as numbers of gold particles per 100 µm2. (B) Nuclear membrane-associated viral cDNA and CAp24. Bars represent the density of gold particles corresponding to viral cDNA or CAp24 found at the nuclear envelope per 100 µm.

Proviral DNA synthesis is impaired by DIBA-1 in newly infected cells. As reported above, proviral DNA synthesis can be inhibited in newly infected cells, probably due to severe structural changes of the virion core caused by DIBA-1 (Fig. 4 and 5). Interestingly, DIBA-1 was also able to attenuate HIV infectivity when added only on the target cells (Fig. 1). To investigate this effect, we performed an analysis of the dose-response of the inhibitory effect of DIBA-1 on virus infectivity and viral DNA synthesis. Reporter T cells were infected with pseudotyped HIV (single cycle infection) according to the protocols described above. The percentage of infected cells and possible toxic effects were determined, and after 3 days no toxic effect was seen in the 1 to 10 µM DIBA-1 concentration range. DNA was then extracted from cells treated and submitted to PCR amplification by using primer pairs specific for proviral DNA or the -globin gene. Results with -globin confirmed that cell growth was not or was poorly affected by 10 µM DIBA-1 (Fig. 7B) since the curve parallels that of the XTT assay. Interestingly, analysis of early and late HIV DNA products shows a clear DIBA-1 concentration-dependent decrease of PCR-amplified proviral DNA (Fig. 7C and D). This finding shows that DIBA-1 can inhibit proviral DNA synthesis and/or impair its stability in newly infected cells. In agreement with the latter possibility, the DIBA-1-induced decrease of viral DNA level appears to be more pronounced than the reduction of infectivity, suggesting that DIBA-1 can promote degradation of unintegrated HIV-1 DNA. Very similar results were obtained when HIV was pseudotyped with the VSV-G envelope, instead of the HIV-1 envelope, implying that early effects of DIBA-1 are independent from the virus entry route (data not shown).

View larger version (33K): [in this window] [in a new window]   FIG. 7.   Inhibition of proviral DNA synthesis by DIBA-1. Single-cycle HIV-1 was produced by cotransfecting pNL4-3.EN and pCEL/E160 in HeLa cells. The inhibitor-free virus preparation was used to infect CEMgfp cells cultured for 2 days in DIBA-1 (from 0 to 400 µM), and cells were cultured for 3 more days. (A) CEMgfp cell viability was monitored by use of the XTT assay (), and the number of GFP-positive cells was determined (; values represent the number of positive cells of 106 cells) for different concentrations of DIBA-1 (x axis). (B to D) Nucleic acids were extracted from one-third of the cells and subjected to radiolabelled PCR amplification in the presence of [32P]dCTP with primers specific for the -globin gene (B), early (C), and late (D) cDNA products. Autoradiographs are shown on the top of each panel. Lane 1, control HIV-1; lanes 2 to 13, twofold increase from 0.01 to 20 µM DIBA-1. Quantifications by phosphorimaging are reported in arbitrary units.

	DISCUSSION

Top Abstract Introduction Materials and Methods Results Discussion References

In this study, we explored the mechanism by which DIBA-1, an inhibitor of nucleocapsid protein NCp7, inhibits HIV replication. Our aim was to study its characteristics as a model for zinc ejector compounds, assuming that more potent and specific molecules are yet to be characterized. In the present experimental system, DIBA-1 was able to inhibit viral replication in single-cycle assays at levels comparable to that of Palinavir, a potent HIV-1 protease inhibitor. DIBA-1 was able to interfere with both early and late steps of HIV-1 replication, a property not shared by Palinavir and the RT inhibitor 3TC (data not shown). At the molecular level, DIBA-1 ejects zinc ions from zinc fingers, and this results in the formation of covalent disulfide bonds between the freed cysteine lateral chains. Our results suggest that intermolecular bonds are formed, leading to oligomerization of NCp7 or of its precursors (Fig. 3). We propose that DIBA-induced modifications of the virion core result in a freezing of the virion core structure that might be called a "core-freezing effect." This is illustrated in Fig. 4, which shows highly condensed viral cores, presumably due to the narrowing of intermolecular distances between NCp7 molecules after establishment of disulfide bonds. The core freezing effect may well cause a defect in RT activity, probably resulting from the inability of RT molecules to escape modified viral cores in the presence of detergent (Fig. 2E). However, we cannot completely rule out the possibility that modified NCp7 can directly inhibit RT in virions since NCp7 and RT were found to interact in an RNA-independent fashion in vitro (24).

In an attempt to understand the maturation defects of HIV-1 induced by DIBA-1, one should consider that virion protein maturation is most probably a multistep process with first dimerization of Gag-Pol, giving rise to PR maturation and its release in a dimeric active form. Next, PR needs to gain access to the cleavage sites to process the Gag and Gag-Pol polyproteins. Since DIBA-1 can modify the structure of the NC domain of Gag and Gag-Pol, this is expected to have an impact on both the dimerization and cleavage reactions. DIBA-1 was found to have a small effect on genomic RNA packaging (Fig. 2D), implying that NCp7 or Gag binding to viral RNA is not subsequently disrupted by the ejection of zinc from the zinc fingers, thus underlying the role of NCp7 positively charged residues in the binding and packaging of the genomic RNA (4, 9, 34, 49). As part of the reverse transcription and preintegration complexes (9, 15, 16), NCp7 appears to greatly facilitate viral DNA synthesis and probably integration (3, 9). Recent results have indicated the importance of the zinc fingers in these processes (24). In addition, mutations in NCp7 which lead to an NCp7 maturation defect enhance viral DNA degradation in infected cells (4), probably due to the fact that Gag binds less tightly to nucleic acids than does NCp7 (7a, 9). Also, this may account for the instability of viral DNA generated by viruses formed in presence of Palinavir (Fig. 5).

Virus produced in the presence of DIBA-1 appears to behave very differently, since cDNA synthesis proceeds slowly, whereas its stability is increased. These two characteristics may result from the core-freezing effect, which may slow down the reverse transcription process while preventing the access of cellular DNases to the viral DNA. In cells infected by HIV-1 virus formed in the presence of either DIBA-1 or Palinavir, reverse transcription complexes appear to accumulate at the nuclear envelope (Fig. 6). This may be the result of a covalent association of p24 with reverse transcription-preintegration complexes slowing down their nuclear translocation.

In addition to interfering with viral core maturation, DIBA-1 can impair viral DNA synthesis (Fig. 5 and 7). DIBA-1 was also found to inactivate cell-free virus in a time-dependent manner (38). We were able to repeat these assays, and the results show that viral suspensions were inactivated by more than 95% after a couple of hours of exposure to DIBA-1, whereas free RT remained fully active under the same conditions (data not shown). Therefore, it would appear that when added after viral particle production, DIBA-1 does not trigger a viral core-freezing effect. While infection of CEMgfp cells with single-cycle viruses was inhibited by 30 to 70% in the presence of 1 to 10 µM DIBA-1, PCR results showed a surprisingly strong inhibition of both early and late viral DNAs with DIBA-1. This probably does not arise from a sensitivity problem inherent in the PCR assay, since Fig. 5 shows a good correlation between levels of late viral DNAs (detected with this assay) and viral infectivity. Therefore, these results suggest that unintegrated viral DNA is actively degraded in the presence of DIBA-1, at concentrations far below the occurrence of drug-associated toxic effects. Although more exhaustive studies will be needed to confirm this observation, this raises the exciting possibility that zinc ejectors are able to target viral reservoirs present as unintegrated labile DNA in quiescent cells (25). Studies of the effect of DIBA-1 on HIV-1 infection of primary cells such as macrophages may support this hypothesis.

The strongest argument against the development of zinc ejector NC inhibitors is their probable poor specificity. In this respect it is confusing that many zinc ejectors harboring quite different structures all seem to be specific for HIV NCp7 versus cellular proteins which contain zinc fingers (36-39). We believe that some of these data require further investigation; for example, in our hands azodicarbonamide (39, 47) did not inhibit HIV infectivity at nontoxic concentrations (not shown). The cellular tolerance for DIBA-1 and related compounds may be due to the fact that they cannot access cellular compartments where families of zinc finger-containing cellular proteins, e.g., transcription factors, are located. However, it is clear that DIBA-1 is able to enter cells and interfere with cell-associated viral maturation (46). Thus, the effects observed are not due only to virucidal action. Current development of hydrophilic zinc ejectors (45) may help clarify this important question.