HIV-1 is the causative agent of AIDS, currently one of the world's foremost health problems. In the USA alone there are over 40 000 new HIV infections per year. More globally, the established market economies have over 1.2 million current infections, while in developing nations close to 40 million infections are estimated [1,2]. Currently 54 drugs have Food and Drugs Administration (FDA) approval for HIV and AIDS-related ailments and in 1987, the FDA established priority categories for the analysis and approval of AIDS drugs . However all of the compounds, except for the recent fusion inhibitors, target HIV genes within the viral pol gene complex. It is frustrating that, in all cases, these approaches appear to be associated with viral escape. Furthermore, there are toxicities associated with long-term use of many combinations of these agents. As it is unlikely that a vaccine will be available for several years there is a great need for additional therapeutic agents for this infection. It appears that even heavily treated and monitored individuals are not able to clear the underlying HIV viral infection, or prevent low level viral replication supporting the belief that over time the vast majority of treated individuals will require additional therapies [4–6]. In theory, developing new therapies, which target host cellular pathways that are utilized by the virus, could impose additional important and valuable obstacles for viral replication. Such therapies might be expected to be less susceptible viral escape during therapy [1,7–9].
One such important host pathway is the mitogen activated protein kinase (MAPK) signaling pathway [10–12] that regulates cellular responses to various environmental stimuli. MAPK can be grouped into three structural families: extracellular signal related kinases, c-Jun N-terminal kinase (JNK) and p38 kinases [13,14]. In mammalian cells, p38 MAPK can be activated by multiple stimuli, including physical–chemical stress, proinflammatory cytokines, and growth factors . The p38 MAPK can influence cell signaling pathways and play a role in viral infection. Several steps in the HIV-1 virus life cycle appear to depend on cellular activation by mitogenic stimuli [12,15]. If activation by mitogenic stimulation does not occur, virus replication is thought to be blocked due to incomplete reverse transcription and a lack of proviral DNA integration . However, upon stimulation with mitogens, such as phytohemagglutinin or interleukin (IL)-2, reverse transcription proceeds to completion and allows integration and virus production to occur. Accordingly, p38 inhibitors may interfere with HIV replication. Recently, this hypothesis was tested on a single HIV-1 laboratory isolate. Specifically, in the presence of a selective p38 inhibitor (SB203580), IL-1β-induced HIV production was suppressed . A separate study showed that p38 MAPK is a key modulator of HIV gene expression in response to UV radiation that acts independently of nuclear factor-κB . These initial data suggest a role for p38 MAPK during HIV-1 infection. However, additional studies are needed to clarify the utility of inhibiting these pathways and importantly to demonstrate that p38 pathway inhibitors can impact on primary patient-derived isolates.
To test this hypothesis, we have analyzed a novel compound (RWJ67657), which selectively inhibits the serine-threonine protein kinases p38 and JNK2 with high bioactivity [17,18]. We analyzed the ability of this compound to inhibit both primary and T-cell HIV-1 mediated infection. Our data show that RWJ67657 was highly effective at inhibiting HIV replication in both T-cell and monocyte cell lines, irrespective of the coreceptor used by the virus for entry into the cell. Importantly, both reverse transcriptase and protease resistant escape mutant viruses were effectively suppressed by RWJ67657. In addition, the tested compounds block HIV-induced T-cell apoptosis, a critical means of T-cell depletion linked to AIDS progression. These findings support the further evaluation of p38 MAPK/JNK inhibitors as unique anti-HIV therapeutics.
Materials and methods
MAPK inhibitor (p38 inhibitors)
The p38 inhibitors RWJ67657 and SB203580 were dissolved in dimethylsulfoxide and diluted in RPMI medium (GibcoBRL, Carlsbad, California, USA) to final concentrations as indicated. As a control an equal concentration of dimethylsulfoxide was added to the experimental control cells .
Jurkat, U937 and Monomac6 cells were obtained from the American Type Culture Collection (Rockville, Maryland, USA). Jurkat cells were maintained in RPMI 1640 medium (GibcoBRL, Gaithersburg, Maryland, USA) supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 100 U/ml penicillin G, and 100 μg/ml streptomycin maintained at 37°C and 5% CO2. Human primary blood mononuclear cells (PBMC) were isolated from healthy HIV-seronegative donors by Ficoll-Paque separation (Pharmacia Biotech AB, Uppsala, Sweden). For PBMC culture, cells were incubated for 2 days in the presence of 5 μg/ml phytohemagglutinin (Sigma, St. Louis, Missouri, USA) followed by the addition of 5 U/ml human recombinant IL-2 (hrIL-2; R&D Systems, Minneapolis Minnesota, USA), and cultured as described by previously .
Viral infection and p24 assay
Viruses that were assayed in these studies included the pNL4-3 (dual tropic) virus, which uses CCR5 and CxCR4 receptors, 89.6 (dual tropic), which uses CCR5 and CxCR4 receptors, and Bal-mac (m-tropic) virus, which uses CCR5 only [7,20,21]. For infection studies, human PBMC were isolated from normal, seronegative donors as mentioned above, and infection was performed by incubating target cells with appropriate virus at a concentration of 100 50% tissue culture infectious doses (TCID50)/1 × 106 cells/ml . Following infection, the cells were washed three times with phosphate-buffered saline and resuspended in growth medium. Six hours post infection, cells were washed three times with 1 × PBS and re-suspended either in medium alone or with the specific compound. Culture supernatant was collected as indicated and assayed for virus production by measuring the p24 antigen released into the medium by ELISA (Coulter, Fullerton, California, USA) according to the manufacturer's instructions. Data are presented as mean ± SEM. Also, for data presented as percent change, the baseline (medium alone) value was subtracted from the value of each experimental condition as described in the legend.
Real-time quantitative reverse transcriptase (RT)–PCR
We evaluated the viral replication by means of quantitative real-time PCR by using the ABI Prism 7700 sequence detector (Applied Biosystems, Foster City, California, USA). Cells were collected after infection and treatment, lysed at a density of 1 × 106/ml in the following buffer: 10 nM Tris–HCl pH 8.0, 1 mM EDTA, 0.2 mM CaCl2, 0.001% SDS, 0.001% Triton X-100, 1 mg proteinase K/ml. Lysates were digested overnight at 58°C, and then the protease was heat inactivated for 15 min at 95°C. Two-step PCR amplification was performed as described . HIV-1 Gag specific probe, labeled at its 5′ terminus with the reporter fluorophore 6-carboxyfluorescein (6FAM) and at its 3′ terminus with the quencher 4-(4′-dimethylamino-phenylazo)-benzene (DABCYL), had the following sequence: (6FAM)-5′-GCGAGTCACACAACA GACGGGCACACACTACTCGC-(DABCYL)-3′ sequence were selected according to Applied Biosystems guidelines. A reference standard curve was obtained from serially diluted plasmids containing the target genes. Using this system, we measured each viral load. We analyzed the dissociation curve for each amplification to confirm that there were no non-specific PCR products.
Drug resistant virus
The drug resistant viruses used in this study were obtained from the NIH AIDS Research and Reference Reagent Program. These are patient-derived viruses that contain escape mutations in specific gene sequences [7,21]. HIV-174V/MT-2 contains mutations in RT and is resistant to didanosine and zalcitabine drug treatments. This virus utilizes CxCR4 for entry. The HIV-1 nevirapine resistant virus (N119) and the zidovudine resistant strain (A012) utilize CxCR4 for entry into the host cell. The saquinavir protease inhibitor resistant virus (Ro31-8959) utilizes R5X4.
FACS analysis was performed to identify cells undergoing apoptosis. Equal numbers of cells from each test group were collected for analysis . Apoptosis in experimental cells was analyzed by using an Annexin-V assay kit from PharMingen (San Diego, California, USA). Data was analyzed by the CELL Quest program (Beckton-Dickinson, San Diego, California, USA).
All of the experiments were performed at least three times. Results are expressed as mean ± SE. Statistical comparisons were made by ANOVA followed by an unpaired two-tailed Student's t test. P values < 0.05 were considered significant.
Activity of p38 inhibitors
We evaluated two different p38 kinase inhibitors in these studies: the p38 kinase inhibitor SB203580 [4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl) imidazole], a selective p38 kinase inhibitor  (Fig. 1a), and a new novel p38/JNK2 inhibitor RWJ67657 (4-[4-(4-fluorophenyl)-1-(3-phenylpropyl)-5-(4-pyridyl)-1H-imidazol-2-yl]-3-butyn-1-ol)  (Fig. 1b). RWJ67657 potently inhibits lipopolysaccharide (LPS)-induced tumor necrosis factor (TNF)-α production from human PBMC and inhibits LPS-induced TNF-α production in vivo, in mice when dosed orally. This compound has been evaluated for safety and is being evaluated clinically in the treatment of autoimmune disease [23,24]. It is also a potent inhibitor of p38 kinase activity [50% inhibitory concentration (IC50) 20–30 nM] and is reported to be approximately 10-fold less potent against JNK2 [17,18].
HIV infection increases p38 phosphorylation
Activation of p38 kinase following viral infection has been demonstrated previously [12,25,26]. Activation was not a result of virus binding to the cell surface, but occurred after virus internalization. We examined the effect of HIV-1 infection on p38 phosphorylation, a major indicator of p38 kinase activity. HIV-1 infected Jurkat cells were harvested at various times following infection. Western blot analysis was carried out using antibodies specific for both Thr180 and Thr182 phosphorylated forms of p38. We observed that p38 was rapidly phosphorylated following infection. Both SB203580 and RWJ67657 exhibited significant activity against p38 phosphorylation (Fig. 1c) supporting the theory that these compounds interfere with the viral activated host pathway of p38 MAPK phosphorylation.
Viral infection and p38 inhibitor titration
To test whether p38 inhibitors have an effect on HIV-1 replication, human Jurkat cells (1 × 106) were infected with pNL4-3 virus at a concentration of 100 TCID50/1 × 106 cells/ml . After 6 h cells were washed free of virus and incubated with different concentrations of either the RWJ67657 or SB203580 inhibitors. For these experiments, cells without inhibitor but with the carrier dimethylsulfoxide were used as controls. As shown in Fig. 2, both p38 inhibitors significantly reduced p24 production from infected culture in a dose-dependent manner. The maximal inhibition (80 ± 5%) was seen at an inhibitor concentration of 1 μM for RWJ67657 and 10 μM for SB203580 (50 ± 5%) (Fig. 2a). For subsequent assays 1 μM was used. The results support the notion that p38 inhibitor broadly impacts HIV replication. As p38 inhibition should not overlap with other established anti-HIV pathways we sought evidence to support that p38 inhibition was similar to that observed with tradition HIV antiviral agents. We compared HIV suppression by the p38 inhibitor with that of the protease inhibitor saquinavir (Invirase, Hoffman-LaRoche, Nutley, New Jersey, USA) in vitro. pNL4-3 is an HxB2 virus backbone with changes in the envelope region. This backbone is sensitive to saquinavir as reported by Klabe et al. . Replication of the pNL4-3 strain in Jurkat T cells was inhibited by both compounds (Fig. 2b).
p38 inhibitor suppresses HIV-1 replication
Activation of target cells by HIV during infection is important in the infection process of both T cells and monocytes [7,10,20,21,28]. Therefore we next compared the antiviral effects of 1 μM RWJ67657 and SB203580 in several different cell lines of both T-cell (Jurkat) and macrophage (U937 and Monomac 6) lineages. We examined three different dual or R5 tropic viral strains, pNL4-3, 89.6 and Bal, to determine if viral tropism was relevant for the p38 inhibitors under examination. p24 antigen was used as the indicator of viral replication. Cells were infected for 6 h as described, washed free of virus with PBS, resuspended in medium containing the test compounds or controls, and then assayed as described.
pNL4-3 replication in Jurkat T cells was inhibited by both compounds. Inhibition was 75% with RWJ67657 (Fig. 3a) and 45% with SB203580 (Fig. 3b) at day 10 post-treatment. A similar pattern was observed for the p38 inhibitors when the activity was examined in macrophage cell lines (Fig. 3a and b). At 1 μM RWJ67657 suppressed HIV-1 replication twice as effectively as SB203580. Importantly, these data show that the p38 inhibitors were effective in both cell lineages tested.
While these data suggest that the inhibitors could impact viral replication in both T cells as well as monocytes, it was important to examine this activity in primary human cells. Accordingly, we next examined viral inhibition in human PBMC by means of a p24 assay and by quantitative real-time PCR. In these studies both compounds were highly effective at blocking viral replication; viral suppression was greater than that observed in the cell line studies. This probably reflects a greater dependency of HIV on metabolic activities in primary cells during the infection process. In contrast tumor lines are actively cycling and therefore there is a lower dependency. Inhibition of HIV-1 replication was 45% following SB203580 treatment and 75%, following RWJ67657 treatment at 1 μM. In the RWJ67657 treated group there was almost no viral replication observed during the assay period (Fig. 4a). Further evaluation of viral replication by means of quantitative real-time PCR again confirmed the suppression of viral replication (Fig. 4b) again RWJ67657 was more effect than SB203580 in this assay.
The question of viral tropism was addressed in these PBMC studies. First we examined the inhibitory activity of the compounds using the dual tropic virus 89.6. This virus has a significantly broader tropism than the pNL4-3 virus. For both the T-cell line (Jurkat) and the myeloid line (monomac 6), both compounds were effective at inhibiting 89.6 viral replication (Fig. 5a and b). RWJ67657 was the more effective compound in both cell lines. Inhibition of 89.6 replication in human PBMC was similar to inhibition of pNL4-3. The compounds were more effective at suppressing viral replication in PBMC than in cell lines (Fig. 5c).
We examined viral suppression of these compounds on Bal (m-tropic) virus replication. This viral isolate is unique in that utilizes only the CCR5 coreceptor for entry into the host cell. Since most early stage viruses are restricted to cell entry through the CCR5 receptor, this virus probably represents an earlier stage isolate. Bal viral replication in Jurkat cells was lower than in the monomac 6 cell lines as expected. Interestingly, the inhibition of viral replication was significantly greater for the Bal virus grown in monomac 6 cells. These results support a requirement of the virus for the p38 pathway to support high levels of replication in the monocyte line. RWJ67657 was again more effective at suppression of Bal viral replication in both the T-cell and monocyte cell line (Fig. 6a and b) and in PBMC (Fig. 6c).
These results show that for three different viral strains, in two different T-cell lines, two myeloid lines and in human PBMC, two different p38/JNK inhibitors exhibited a consistent ability to limit HIV replication. Furthermore, RWJ67657 was highly potent in each system, consistent with its high potency for inhibition of p38/JNK, further suggesting that inhibition of replication was due to inhibition of p38 kinase and/or JNK2 activity. An important question that remained was whether these results could be translated to more primary patient-derived isolates as well as to the traditional drug resistant viral isolates.
Inhibitors of drug-resistant patient-derived isolates
The results thus far support the notion that inhibition of p38/JNK appears to broadly impact HIV replication. As these kinases represent non-viral targets for HIV inhibition, viral escape from suppression by these compounds might be more difficult. Furthermore, the p38/JNK pathways should not overlap with traditional anti-HIV targets. We next sought to confirm the independence of these pathways by testing the susceptibility of patient-derived drug escape mutant HIV viruses to the inhibitors. We first examined the susceptibility of the HIV-1 isolate A012 (zidovudine resistant HIV-1) . This patient-derived isolate is mutated in the RT portion of pol, which renders it resistant to zidovudine. Replication of this strain in PBMC was significantly inhibited in the presence of either SB203580 or RWJ67657, illustrating the independence of the p38/JNK inhibitors for RT resistant strains (Fig. 7a). We examined patient-derived isolates HIV-174V/MT-2 (didanosine and zalcitabine resistant) and the nevirapine resistant HIV-1 isolates (N119). We also examined the HIV-174V/MT-2 (didanosine and zalcitabine resistant) virus which is resistant to both didanosine and zalcitabine therapy due to pol escape mutations [29,30]. These patient-derived isolates were highly susceptible to both p38 inhibitors (Fig. 7b). We also evaluated a triple RT escape mutant viral isolate (Fig. 7c). This isolate was again susceptible to suppression by both inhibitors, supporting the independence of the p38 and these traditional therapeutic pathways.
We sought to evaluate a drug resistant escape virus that was mutated in a non-RT–Pol region. Accordingly, a saquinavir-resistant (HIV-1 protease inhibitor resistant)  patient isolate was next evaluated. The p38 inhibitors suppressed its replication as well (Fig. 7d). Collectively, these data illustrate the potential of the p38/JNK pathways as intervention targets for inhibition of the replication of diverse HIV isolates. It is also noteworthy that for all four escape mutant viruses studied, RWJ67657 was at least twofold more effective then SB203580, consistent with its greater potency for inhibition of p38/JNK activity. The greatest degree of viral inhibition was observed in treatment of the protease-resistant viral isolate whose replication was almost completely suppressed for unknown reasons. This results again suggests that both primary as well as late HIV isolates are susceptible to p38/JNK blockade.
Blockade of HIV-induced apoptosis by RWJ67657
One concern with inhibition of cell pathways is that such inhibition could result in cellular toxicity via induction of apoptosis of host cells [20,28]. As HIV infection is associated with increased host cell apoptosis it was possible that the inhibitors induced apoptotic destruction of host cells. However, we did not detect a loss of cell viability or morphological changes induced by infection in the presence of RWJ67657 (data not shown). This suggested that this compound may actually prevent HIV-induced apoptosis. To verify this observation we sought to test apoptotic effects directly. Equal numbers of Jurkat or PBMC were infected with pNL4-3 in the presence or absence of RWJ67657 inhibitor (1 μM), or SB203580 (1 μM) (Fig. 8a and b). As shown in Fig. 8, virus infected cells exhibited strong apoptosis 5 days post infection (68.3 ± 5%), whereas RWJ67657 strongly blocked virus induced apoptosis compared to the SB203580 treated group. The results indicate that RWJ67657 treated cells exhibited a considerable decrease in apoptosis as compared with the SB203580 treated cells and there was a significant protective effect or anti-apoptotic effect resulting from the treatment with the p38 inhibitor RWJ67657.
Many new antiviral HIV drugs have been licensed in recent years, and are effective in specific combinations as treatment for HIV infection. Antiviral drug design could, in principle, be targeted at either viral gene products or cellular proteins. One advantage of targeting host cell pathways that are essential for HIV replication is that it is likely that such inhibitors would be less susceptible to viral escape. In this regard, proteins involved in signal transduction pathways regulating transcription factors important in immune activation represent another class of host protein targets. The serine/threonine kinase p38 MAPK, a member of the mitogen-activated protein (MAP) kinase superfamily of signal transduction molecules probably represents one such class of molecules [10,12,32–34]. Recent studies have reported that a p38 inhibitor could block HIV reactivation in regard to cytokine stimulation, suggesting a role for anti-p38 compounds in HIV therapy .
The p38 MAPK pathway regulates the production of several cytokines and chemokines, the most studied of which are TNFα and IL-1β [35,36]. p38 MAPK inhibitors SB203580 and RWJ67657, selectively inhibit the production of IL-4 in human Th2 cells without affecting the production of Th1 cytokines [37,38]. In another study, SB203580 inhibited CD28-dependent T-cell proliferation and IL-2 in vitro . The activation of the immune system and HIV infection and replication are believed to be linked as activated CD4 cells are the preferred target of HIV [4,20,40]. It has been suggested the controlling HIV driven immune activation could impact HIV infection . Activation of p38 kinase on by TNF-α is associated with HIV-1 production in primary infected human T cells .
We observed that incubation of HIV-infected cells (T cells, macrophages or primary cells) with RWJ67657 results in a significant decrease in viral production. Moreover, when added at the time of infection, RWJ67657 reduces p24 production in a dose-dependent manner (data not shown). In addition to RWJ67657, we have confirmed that another inhibitor of the p38 kinase pathway, SB203580, also suppresses HIV replication, as measured by the production of the viral p24 protein. These data extend the observations of others, which have implicated the host p38 kinase activity as a requirement for optimal HIV replication in vitro [12,25]. For example, Shapiro et al.  showed that treatment of cells with SB203580 at the time of HIV infection inhibited p24 antigen expression. Interestingly, inhibition was not enhanced by prolonged treatment with SB203580, suggesting that SB203580-mediated inhibition occurred at a relatively early stage in the viral lifecycle. In the present study we extend these prior observations in several ways. We show that these inhibitors are effective on diverse viral isolates irrespective of viral phenotype. Furthermore prior drug resistance profiles were not effective at providing viral resistance to p38 inhibitors, suggesting an important role for these compounds in preventing viral escape. p38 blockade was also effective at interfering with viral replication irrespective of viral coreceptor usage.
The inhibitors used in the present study also inhibit the related JNK2 enzyme at the concentrations required for maximal inhibition of HIV replication [10,12,41–43]. Cohen et al.  reported that inhibition of p38 expression by antisense construct severely limited replication of HIV, and sustained p38 kinase activation is observed following HIV infection (data not shown), but not as a consequence of virus binding to the cell surface. Furthermore, the fold difference in potency between RWJ67657 and SB203580 for inhibition of HIV replication closely matches their different potencies for inhibition of p38 but not JNK. These data suggest an important role for the p38 kinase pathway at some point in the HIV lifecycle after viral entry. We hypothesize that cellular and/or viral factors which are required for productive HIV-1 infection or replication are regulated at least in part by p38, or are downstream protein(s)/factors activated by p38. In addition the possibility that the inhibitors act at least in part via binding to an HIV protein is not formally excluded.
Finally these compounds may also provide the additional benefit of reducing the HIV-induced aberrant immune activation phenotype. In addition to current HIV antiviral compounds, this avenue of drug development probably represents a potentially important therapeutic strategy that deserves further attention.
The authors thank F. Shaheen, Dept of Medicine, CFAR, University of Pennsylvania for excellent technical assistance with the real-time PCR assay. We also thank M. A. Chattergoon for constant advice and help in this work and M. J. Merva for administrative assistance.
Sponsorship: Supported by Johnson & Johnson Pharmaceutical Research and Development grant to D.B.W and K.M. Support from the NIH AIDS Research and Reference Reagents program and CFAR, University of Pennsylvania, is acknowledged.
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