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Infection of CD4⁺ Memory T Cells by HIV-1 Requires Expression of Phosphodiesterase 4¹

Yu Sun^2,*, Linsong Li^3,, Fion Lau^*, Joseph A. Beavo and Edward A. Clark^4,*,

^* Regional Primate Research Center, Department of Pharmacology and Molecular, and Department of Microbiology, Cellular Biology Program, University of Washington, Seattle, WA 98195

	Abstract

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Using PCR to monitor HIV-1 RNA genome reverse transcription and nuclear import of preintegration complexes, we found that memory, but not naive, CD4⁺ T cells could support transport of HIV-1 DNA to nuclei upon TCR/CD3 and IL-2 stimulation. Moreover, memory CD4⁺ T cells, unlike naive CD4⁺ T cells, express high levels of phosphodiesterase 4 (PDE4) constitutively. Selective blocking of PDE4 activity inhibited IL-2R expression and thereby led to abolishing HIV-1 DNA nuclear import in memory T cells; however, full-length viral DNA synthesis was not affected. Thus, blocking PDE4 prevents initiation of HIV-1 DNA circle formation in T cells. The fact that PDE4 is expressed constitutively at higher levels in memory vs naive CD4⁺ T cells may help HIV-1 readily infect memory T cells.

	Introduction

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Cyclic AMP is a key second messenger that plays a pivotal role in regulation of a wide range of cellular functions. The intracellular level of cAMP is controlled by two distinct enzyme superfamilies: the adenylyl cyclases, which use ATP as a substrate to synthesize cAMP; and the cAMP-specific phosphodiesterases (PDEs),⁵ which catalytically hydrolyze cAMP to its biologically inactive adenosine 5'-monophosphate. Increasing cAMP levels by cAMP analogues, cAMP-elevating drugs, or PDE inhibitors results in inhibition of T cell activation, proliferation, and cytokine production (1, 2, 3, 4, 5, 6). Currently, PDEs are grouped into 11 broad families; each PDE differs from the others in its substrate specificity, inhibitor sensitivity, tissue distribution, and regulation of activity (7, 8, 9, 10, 11, 12). So far, at least three cAMP-specific PDEs have been reported in T cells: PDE3, PDE4, and PDE7 (13, 14). Among them, PDE4 is thought to be the predominant regulator of degradation of cAMP (10). Selectively blocking PDE4 activity has been shown to inhibit inflammatory responses and HIV-1 replication in CD4⁺ T cells (15, 16, 17).

According to their activation stage and biological function, CD4⁺ T cells can be divided into CD45RA⁺ naive and CD45RO⁺ memory cell subsets (18, 19). CD4⁺ memory T cells differ from naive T cells in their responses to SIV and HIV-1 infection. Compared with naive T cells, memory T cells are more easily targeted and their functions are impaired earlier and more severely during SIV and HIV infection (20, 21, 22, 23, 24, 25). There is substantial evidence that turnover of CD4⁺ memory T cells is faster than naive T cells (26, 27, 28, 29), suggesting that HIV-1 replicates optimally in memory T cells. Indeed, highly purified CD4⁺ memory T cells from HIV-infected individuals harbored 4- to 10-fold higher viral DNA than in naive T cells (24). Furthermore, recent studies have shown that temporally labile postfusion HIV-1 complexes exist in memory T cells longer than in naive T cells (30). These observations indicate that memory T cells may serve as a principal reservoir in HIV-1 infection.

It is well known that establishment of SIV/HIV infection requires T cell activation (31, 32, 33, 34, 35, 36, 37, 38, 39). After virus entry, the processes required for completion of SIV or HIV-1 reverse transcription and nuclear import of preintegration complexes (PICs) are not active in resting T cells (35, 36). Two T cell activation signals are required to overcome these blockages: one signal through the TCR, which regulates cell passage through G0 to G1, induces the completion of reverse transcription of SIV or HIV-1 RNAs; the second signal, through CD28 or the IL-2 receptor complex (IL-2R), controls the entry of virus PICs to the nuclei (35, 36). Moreover, the c-Myc oncoprotein plays a key role in regulation of this process (39). Given the fact that the activation requirements are less stringent for memory T cells than for naive T cells (40, 41, 42, 43), it is perhaps not surprising that CD45RO⁺ memory are more susceptible to HIV-1 infection than CD45RA⁺ naive T cells (24, 44, 45, 46). However, the mechanisms by which HIV-1 preferentially infects CD4⁺ memory T cells are unknown.

Here we show that CD4⁺ memory T cells produce more HIV-1 DNA than CD4⁺ naive T cells in response to CD3 mAb together with IL-2. Most strikingly, translocation of HIV-1 DNA to the nuclei occurred only in CD4⁺ memory T cells, but not in CD4⁺ naive T cells under the same stimulation. High levels of PDE4 were constitutively present in CD4⁺ memory T cells. Selectively blocking PDE4 activity resulted in inhibition of nuclear import of HIV-1 DNA in memory T cells, whereas full-length viral DNA synthesis was not affected. In addition, blocking PDE4 activity abolished induction of IL-2R-chain and c-Myc expression, suggesting that PDE4 may regulate HIV-1 infection by interfering with IL-2R.

	Materials and Methods

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Reagents

Purified mAbs to human CD8 (G10-1, IgG2a), CD16 (FC-2, IgG2b), CD20 (1F5, IgG2a), and HLA-DR (HB10a, IgG2a) were produced by us and used with negative selection to purify human primary CD4⁺ T cells as described (36, 39). Biotin-conjugated CD45RA (3AC5, IgG2a) or CD45RO (UCHL-1, IgG2a) Abs were also prepared by us and used to isolate CD4⁺ naive and memory T cells. Streptavidin-magnetic microbeads and goat anti-mouse IgG conjugated to magnetic microbeads were purchased from Miltenyi Biotec (Sunnyvale, CA). mAbs to human CD3 (64.1, IgG2a), CD28 (9.3, IgG2a), and IL-2 (Cetus) were used to activate CD4⁺ T cells. Dr. M. D. Houslay (University of Glasgow, Scotland, U.K.) kindly provided the affinity-purified rabbit antiserum (651) against PDE4A. Phospho-c-Myc (Thr^-58 and Ser^-62) polyclonal Ab was purchased from New England BioLabs (Beverly, MA). Anti-human c-Myc mAb (9E10, IgG1) and rabbit polyclonal anti-ERK1 (c-16) serum were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). FITC-conjugated CD25 mAb was purchased from Dako (Carpinteria, CA). Rolipram and forskolin were obtained from Biomol (Plymouth Meeting, PA). Protein kinase A (PKA) inhibitors, rabbit peptide (Rp)-cAMP and Rp, were obtained from BioLog (La Jolla, CA) and Sigma (St. Louis, MO), respectively.

Purification of CD45RA⁺ and CD45RO⁺ T cell subsets

CD4⁺ T cells were prepared from peripheral blood samples from healthy, HIV-seronegative donors as described (36, 39). Highly purified CD4 T cells were further fractionated by negative selection using magnetic beads. The purity of isolated CD45RA⁺ and CD45RO⁺ T cells was 90–95% and 95–98%, respectively, as monitored by flow cytometry. In some experiments, more than one treatment with negative selection was required to remove contaminating cells. Cells were cultured in RPMI 1640 medium supplemented with 5% heat-inactivated human AB serum, 2 mM glutamine, 10 U/ml penicillin, 10 mg/ml streptomycin, 1 mM pyruvate, and nonessential amino acids.

HIV-1 infection and PCR assays

HIV-1_Lai strain was prepared as described (36). Cells were infected with HIV-1 at a multiplicity of infection of 0.01 per cell. DNA was extracted from HIV-1-infected cells as described (36). Primers and the thermal cycling reaction for monitoring initiation, elongation of HIV-1 DNA synthesis, and virus DNA nuclear import have been described (36, 39).

RT-PCR assay for PDE4 mRNA expression

Total RNA was extracted using TRIzol (Life Technologies, Grand Island, NY) according to the manufacturer’s instructions. Primers used to amplify PDE4A were as described (6). Reverse transcription was performed as follows: 1 µg of RNA with 1 µl of random primer (1 µg/µl) (Promega, Madison, WI) were incubated at 70°C for 10 min. After incubation, the mixture was immediately cooled on ice and the following reagents added: 23 U avian myeloblastosis virus reverse transcriptase (Promega), 40 U RNAsin (Promega), 500 µM dNTP, 500 µM DTT, and 4 µl 5x avian myeloblastosis virus buffer. The final volume was 20 µl. The cDNA synthesis reactions were conducted at 37°C for 1 h, following by heating of the samples at 95°C for 5 min to stop the reactions. Thermal cycling reaction conditions were the same as described (39).

Western blotting

The cell lysates (equivalent to 1x10⁶ cells) were electrophoresed by 8% SDS-polyacrylamide gel and then transferred to nitrocellulose membranes (Schreicher & Schuell, Dases, Germany). Western blot analysis was performed as described (39).

PDE4 activity assay

Ten to 20 million cells were homogenized as described (6) and sonicated. Freshly prepared lysates were processed as described (47) with some modifications: cell lysates were incubated with 1 µM [³H]cAMP in the presence or absence of 10 µM rolipram, a PDE4 inhibitor, at 30°C for 15 min. The reactions were stopped by placing tubes in a boiling water bath for 1 min. After the addition of snake venom, the samples were incubated at 30°C for 5 min and then applied to DEAE A-25 ion exchange resin columns. Eluted [³H]adenosine from the resin was measured by a beta counter.

PKA activity assay

PKA activity was measured using a PKA assay system (Life Technologies) according to manufacturer’s instruction. Briefly, 2 x 10⁶/ml CD4⁺ T cells were incubated in the presence or absence of PKA rabbit peptide inhibitor, Rp (5 µM), and stimulated with 1 µg/ml CD3 mAb and 10 U/ml IL-2 for 24 and 48 h. Cellular protein was extracted by repeated freeze/thaw cycles in extraction buffer provided by the company. PKA activity results in the transfer of ³²P-labeled phosphate from [-³²P]ATP to the substrate-biotinylated Kemptide (Biomol, Plymouth Meeting, PA). The phosphorylated Kemptide was then captured on a streptavidin matrix, and incorporated radioactivity was determined by a beta counter.

	Results

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CD4⁺CD45RO⁺ memory T cells are more readily infected by HIV-1 compared with CD4⁺CD45RA⁺ naive T cells

To study whether CD4⁺ naive vs memory T cells are equally susceptible to HIV-1 infection upon TCR ligation in combination with either IL-2R or CD28 cross-linking, we infected these two subsets with HIV-1_Lai for 3 days. PCR was performed to monitor early (LTR/LTR) and late (LTR/gag) products of virus reverse transcription (36, 39), which represent initiation and completion of HIV-1 RNA genome reverse transcription, respectively, and also to monitor formation of the HIV-1 LTR circle (indicating nuclear import of HIV-1 PICs). Initiation of reverse transcription occurred in both T cell subsets at equivalent levels without any stimulation, suggesting that HIV-1 can enter both T cell subsets equally well (Fig. 1, A and B, LTR/LTR). CD3 and CD28 costimulation in both naive and memory T cells produced similar amounts of full-length HIV-1 DNA and circular forms of viral DNA (Fig. 1B, LTR/gag and LTR circle). In contrast, TCR and IL-2R ligation induced 2- to 5-fold more full-length viral DNA synthesis (LTR/gag) in memory cells as quantified by densitometry from three independent experiments (Fig. 1A, LTR/gag). Moreover, LTR circle formation was detected only in CD4⁺ memory T cells upon activation through the combination of TCR and IL-2R (Fig. 1A, LTR circle). When PMA and ionomycin were used as stimuli, nuclear import of viral DNA could be induced in both subsets. Taken together, these data indicate that T cell activation requirements for supporting translocation of HIV-1 DNA to nuclei are less stringent for memory T cells than for naive T cells.

View larger version (35K): [in this window] [in a new window]   FIGURE 1. CD3 and IL-2R ligation induce nuclear import of HIV-1 DNA in CD4+ memory T cells but not in naive CD4+ T cells. A, Freshly isolated human CD4+CD45RA+ naive and CD4+CD45RO+ memory T lymphocyte subsets were infected with HIV-1Lai and were either unstimulated (Media) or activated with soluble anti-CD3 (1 µg/ml) plus IL-2 (10 U/ml) (CD3+IL-2) or PMA (10 ng/ml) plus ionomycin (50 ng/ml) (PMA+Iono). Heat-inactivated HIV-1 controls were infected in parallel. At day 3, DNA was extracted and the levels of initiation of reverse transcription (LTR/LTR), full-length virus DNA (LTR/gag), and formation of LTR circular DNA (LTR circle) were detected by PCR, as described (36 39 ). Serial diluted HIV-1-infected CEM DNA was used to monitor the PCR condition under the linear ranger of amplification. ß-globin was used to standardize input of DNA. Similar results were obtained in three additional experiments. B, Naive and memory T cells were infected with HIV-1 in the absence or presence of CD3 and CD28 mAbs. At day 3, DNA was extracted and subjected to PCR to monitor LTR/LTR, LTR/gag, and LTR circles. One of three experiments.

Although the molecular basis of the regulation of memory T cell activation is largely unknown (18, 19), some studies suggest that increasing PDE activity, thereby decreasing intracellular cAMP levels, may account for some of the distinct features of CD4⁺ memory T cells (2) (48, 49). In addition, rolipram, a PDE4-specific inhibitor, can inhibit T cell activation and infectious HIV-1 virion production in T cells (16, 17). Based on these studies, we measured PDE4 expression in naive vs memory T cells. RT-PCR and Western blot analysis showed that PDE4A mRNA and protein are constitutively expressed in memory T cells (Fig. 2, A and B), while little or no PDE4 is expressed in naive T cells. The levels of PDE4 mRNA and protein in CD4⁺ memory T cells were, respectively, 51- and 6-fold higher than in naive CD4⁺ T cells as measured by densitometry. Stimulation of naive CD4⁺ T cells with CD3 and CD28 mAbs could induce expression of PDE4 mRNA and protein (Fig. 2, C and D). There are several PDE4 family members (PDE4A, 4B, 4C, and 4D), and it was not clear how much PDE activity was attributable to PDE4A. Therefore, we estimated total PDE4 activity in both CD4⁺ T cell subsets using the selective inhibitor, rolipram. We next performed an activity assay to detect total PDE4 activity in naive vs memory CD4⁺ T cells. To pick an appropriate time point to investigate PDE4 activity in both CD4⁺ T subsets, time course experiments in CD4⁺ T cells was performed first (Fig. 2E). Total PDE4 activity was rapidly induced within 1 h upon CD3 mAb and IL-2 stimulation and sustained at the same level for 18 h. Consistent with RT-PCR and Western blot results, the enzymatic activity of PDE4 in memory T cells was 4-fold higher than in CD4⁺ naive T cells (Fig. 2F). In general, the same set of signals that efficiently stimulated HIV-1 circle formation also efficiently increased PDE4 activity. For naive T cells, CD3 mAb and IL-2 stimulation was not as efficient at inducing increases in PDE4 activity as either a nonspecific positive control stimulation, PMA and ionomycin (Fig. 2G), or a combination of CD3 and CD28 mAb (Fig. 2H), which both induce HIV-1 circles in naive T cells (Fig. 1, A and B). For memory T cells, all three stimulatory conditions that could induce circles, CD3 mAb plus IL-2, PMA plus ionomycin, or CD3 and CD28 mAbs (Fig. 1), also consistently elevated PDE4 levels (Fig. 2, G and H).

View larger version (31K): [in this window] [in a new window]   FIGURE 2. PDE4 is constitutively expressed at high levels in CD4+ memory T cells but at low levels in naive T cells. A, RT-PCR analysis of PDE4A gene expression in memory and naive CD4+ T cells. One microgram of total RNA was subjected to reverse transcription. The reverse transcription product was diluted as indicated, and PDE4A was quantified by PCR. Data are from one of three representative experiments. B, Western blot analysis of PDE4A protein. The same blot was probed with an anti-extracellular signal-related kinase serum as a protein loading control. Induction of both PDE4A mRNA (C) and protein (D) in CD4+ naive T cells after costimulation with soluble CD3 (1 µg/ml) and soluble CD28 (10 µg/ml) mAbs. The cells were stimulated for the indicated times. E, Time course of PDE4 activity (1 µM cAMP as substrate). Total CD4+ T cells were stimulated by soluble CD3 mAb (1 µg/ml) and IL-2 (10 U/ml) for the indicated times. Total PDE activity was measured in the presence or absence of 10 µM rolipram. The numbers in the open bars represent rolipram-sensitive activity (PDE4), and the dotted area indicates rolipram-resistant PDE activity. F, PDE4 activity in CD4+ naive vs memory T cells. Two CD4+ T cell subsets from three different donors were subjected to PDE4 activity analysis as described in Materials and Methods. Induction of PDE4 activity in naive and memory T cells by different stimulation. Naive and memory T cells were stimulated as indicated for 1 h (G) or for 4 h (H). PDE4 activity was measured as described above in E. The numbers in the open bars represent activity of rolipram-sensitive PDE4. Data are from one of three representative experiments.

The correlative results above suggested that PDE4 may play a role in the formation of HIV-1 circles. To test whether expression of PDE4 is required for infection of CD4⁺ memory T cells, we examined whether selective blockage of PDE4 activity could inhibit nuclear import of HIV-1 DNA in CD4⁺ memory T cells. As shown in Fig. 3, blocking PDE4 activity by rolipram, a PDE4-specific inhibitor, abolished HIV-1 DNA nuclear import in memory T cells, but full-length viral DNA synthesis was not affected. The same result was obtained using forskolin, a cAMP-elevating drug (Fig. 3). Thus, PDE4 appears to be required for a step after viral DNA synthesis leading to translocation of HIV-1 PICs to the nuclei.

View larger version (43K): [in this window] [in a new window]   FIGURE 3. Blocking PDE4 activity leads to abolition of nuclear import of HIV-1 DNA in CD4+ memory T cells. Isolated naive and memory CD4+ T cells were infected by HIV-1 in the presence or absence of 10 µM rolipram or 20 µM of forskolin, a cAMP-elevating drug, for 3 days. DNA was prepared and subjected to PCR to monitor HIV-1 DNA synthesis (LTR/gag) and LTR circle formation (LTR/LTR). One of three representative experiments.

IL-2/IL-2R interaction provides necessary signaling for translocation of SIV PICs to the nucleus of macaque CD4⁺ T cells (35). Moreover, increasing cAMP levels by either cAMP-elevating drugs or with a PDE4 inhibitor can suppress IL-2R signals (6, 7, 50). Thus, it seemed appropriate to test whether preferential infection of memory T cells by HIV-1 upon TCR and IL-2R cross-linking results from differential expression of IL-2R. IL-2R is composed of three subunits, , ß, and c. Of these, IL-2R (CD25) is required for high-affinity IL-2 binding and is not expressed on resting T cells, but is potently induced after T cell activation (51, 52). Stimulation of naive T cells with soluble CD3 mAb and IL-2 failed to induce IL-2R expression (Fig. 4). However, the same stimulation could up-regulate IL-2R-chain expression in memory T cells (Fig. 4). Selectively blocking PDE4 activity by rolipram or increasing cAMP by forskolin inhibited the induction of IL-2R expression (Fig. 4).

View larger version (22K): [in this window] [in a new window]   FIGURE 4. Expression of IL-2R-chain in CD4+CD45RO+ memory T cells requires PDE4. Two CD4+ T cell subsets were either cultured in the media alone or stimulated with CD3 mAb together with IL-2 in the presence 10 µM rolipram, 20 µM forskolin, or DMSO (drug solvent) for 3 days. The samples were stained with CD25-FITC or isotype control and subjected to FACScan. Similar results were obtained in three additional experiments.

Elevation of cAMP results in activation of cAMP-dependent protein kinase, PKA. In addition, the cAMP-PKA pathway is involved in regulation of T cell proliferation (5, 53). To explore the involvement of PKA in the regulation of HIV-1 DNA nuclear import, we preincubated CD4⁺ T cells with rolipram in the presence or absence of the PKA inhibitors, Rp or Rp-cAMP, followed by TCR and CD28 ligation. Selectively inhibiting PDE4 activity led to a block of HIV-1 DNA nuclear import in CD4⁺ T cells upon costimulation with CD3 and CD28 mAbs (Fig. 5A). However, neither PKA inhibitor restored nuclear import of HIV-1 DNA in CD4⁺ T cells exposed to rolipram. In parallel experiments, the same dose of Rp-cAMP restored proliferation of CD4⁺ memory T cells blocked by PDE7 antisense oligonucleotides (6).⁶

View larger version (30K): [in this window] [in a new window]   FIGURE 5. A cAMP-PKA-dependent pathway does not regulate movement of HIV-1 DNA to the nucleus. A, CD4+ T cells were activated by CD3 and CD28 mAbs and infected with HIV. Either rolipram or a combination of rolipram and a PKA inhibitor, Rp (20 µM) or Rp-cAMP (500 µM), was added in the defined groups. LTR circle formation was detected by PCR. Induction of c-Myc expression requires PDE4 (B) but is independent of the cAMP-PKA pathway (C). CD4+ T cells were activated by CD3 and CD28 mAbs as described in Fig. 2D in the presence of different doses of rolipram for 24 h (B) or the PKA inhibitor Rp (5 µM) was added. In C, 10 µM of rolipram and 20 µM of Rp were used. Expression of c-Myc was detected by Western blot. Data are from one of three representative experiments.

	Discussion

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Nuclear import of virus PIC is a key step for establishment of HIV-1 infection in target cells. In this study, we found that both CD4⁺ naive and memory T cells are equally susceptible to HIV-1 infection after both TCR and CD28 ligation at the preintegrated stage of the HIV-1 life cycle. However CD4⁺ memory T cells, but not naive T cells, can support the process of HIV-1 DNA nuclear translocation after TCR/CD3 and IL-2 stimulation (Fig. 1). These data indicate that the activation requirements for establishment of HIV-1 infection are less stringent for memory T cells than for naive T cells. Upon entry into the nuclei of the target memory cells, some linear HIV-1 DNA integrates into the host genome to form proviruses. Therefore, these results may explain, at least in part, why CD4⁺ memory T cells serve as a principal reservoir of HIV-1 in infected individuals.

Although T cell activation is essential for establishment of HIV-1 infection in CD4⁺ T cells, the molecular basis of why memory T cells are infected readily by HIV-1 is not well understood. All RT-PCR, Western blot, and enzymatic activity assays clearly showed the constitutive presence of high levels of PDE4 mRNA, protein, and activity in CD4⁺ memory T cells. More importantly, we found that expression of PDE4 is essential for the nuclear import of HIV-1 DNA in memory T cells. To date, the only known function of PDE4 is to hydrolyze cAMP in cells and thereby eliminate cAMP’s effects. It is established that cAMP elevating agents, such as forskolin, PGE2, or dibutyryl cAMP, can block IL-2-dependent signaling at multiple levels, including down-regulation of IL-2 and IL-2R (6, 7), reducing JAK3 levels (50) and c-Myc oncogene expression (50, 54). Presumably, the presence of high levels of PDE4 activity in memory T cells and a subsequent decrease in a pool of intracellular cAMP facilitates IL-2R expression in this subset upon TCR/CD3 ligation (Fig. 4). When IL-2 binds to IL-2R, c-Myc expression is induced, and this is required for translocation of HIV-1 PIC to the nuclei (39). Because blocking PDE4 did not affect synthesis of viral DNA, it is likely that IL-2 signals are not required for initial HIV-1 DNA synthesis. The completion of HIV-1 DNA synthesis does require a TCR/CD3 signal, while CD28 or IL-2 signals are responsible for transport of viral DNA to nuclei (35, 36). Inhibiting IL-2 signaling (35) blocks CD28-dependent viral DNA circle formation. Thus, we hypothesis that when memory T cells are activated by CD3 mAb and IL-2, the TCR/CD3 signal induces HIV-1 DNA synthesis, as well as IL-2R expression via a PDE4-dependent pathway. Then IL-2/IL-2R signaling initiates viral DNA transport to nuclei via a c-Myc-dependent pathway. Consistent with this model, after TCR ligation, naive T cells fail to express IL-2R (Fig. 4), probably due to a lack of PDE4, and therefore cannot support transport of HIV-1 DNA to nuclei. Although this model suggests that induction of PDE4 is essential for HIV-1 DNA circle formation, it by no means suggests that an increase in PDE4 alone is sufficient for this process. In fact, unstimulated CD4⁺ memory T cells do not support transport of HIV-1 DNA to nuclei even though PDE4 is constitutively expressed in these cells.

Most recently, two subsets of memory T cells with distinct homing potentials and effector functions were identified by their differential expression of CCR7, a chemokine receptor (55). CCR7^- memory T cells display immediate effector function and are consistently more sensitive to anti-CD3 in the presence or absence of costimulation. CCR7⁺ memory T cells can efficiently stimulate dendritic cells and differentiate into CCR7^- T cells. The susceptibility of the two memory T cell subsets to HIV-1 infection is not known; further studies are also required to define the relationship between susceptibility to HIV-1 infection and PDE4 expression in these two memory subsets. As mentioned earlier, besides PDE4, other two cAMP-specific PDEs, PDE3 and PDE7, are also present in peripheral T cells (13). We will address the constitutive expression of PDE7 in CD4⁺ memory T cells and its role in the regulation of this subset’s functions elsewhere.⁶ Investigating the key role of different PDE families in HIV-1-infected T cells, or other HIV-1 targeted cells, such as monocytes/macrophages or dendritic cells, may provide potential intracellular targets for the treatment of AIDS.

Both PKA-dependent and -independent pathways are reported to be involved in cAMP-mediated signaling (6, 50, 56, 57, 58). In our experiments, PKA inhibitors had no effect on restoration of c-Myc expression and HIV-1 LTR circle formation in CD4⁺ T cells exposed to rolipram. Thus, the data suggest that cAMP-mediated suppression of HIV-1 DNA translocation to the nuclei may be PKA independent.

Previous studies have shown that specific blockade of PDE4 by rolipram inhibits provirus transcription and p24 Ag release from both acutely and chronically HIV-1-infected T cells (16, 17). The mechanism leading to these effects was first hypothesized to be due to prevention of TNF- production by rolipram (16). However, later studies conducted by Navarvo et al. (17) did not support this model, because treatment of HIV-1-infected T cells with TNF- could not restore rolipram inhibition of p24 production. Moreover, a PKA inhibitor prevented the inhibition of TNF- secretion but not that of HIV-1 replication caused by rolipram. Instead, inhibition of NF-B and NF-AT activation is more likely to contribute to blockage of HIV-1 replication by rolipram (17, 59, 60). Taken together, these results suggest PDE4 may affect HIV-1 infection at both pro- and postintegration stages.

	Acknowledgments

 
We gratefully thank Marj Domenowske for preparation of some of the figures and Kate Elias for editorial assistance.

	Footnotes

 
¹ This work was supported by National Institutes of Health Grants RR00166 (to E.A.C.) and DK21723 (to J.A.B.).

² Current address: Department of Molecular Biotechnology, Box 357650, University of Washington, Seattle, WA 98195.

³ Current address: Division of Reproductive Biology, Stanford University School of Medicine, Stanford, CA 94305-5317.

⁴ Address correspondence and reprint requests to Dr. Edward A. Clark, Regional Primate Research Center, Box 357330, University of Washington, Seattle, WA 98195.

⁵ Abbreviations used in this paper: PDE, phosphodiesterase; IL-2R, IL-2 receptor complex; LTR, long terminal repeat; PIC, preintegration complexes; PKA, protein kinase A; Rp, rabbit peptide.

⁶ L. Li, Y. Sun, N. A. Glavas, F. Lau, E. A. Clark, and J. A. Beavo, 1999. Proliferative responses of memory CD4⁺ T cells dependent on constitutive expression of phosphodiesterase-7. Submitted for publication.

Received for publication December 1, 1999. Accepted for publication May 23, 2000.

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