Tat Protein Induces Human Immunodeficiency Virus Type 1 (HIV-1) Coreceptors and Promotes Infection with both Macrophage-Tropic and T-Lymphotropic HIV-1 Strains

Copyright © 1998, American Society for Microbiology. All Rights Reserved.

Tat Protein Induces Human Immunodeficiency Virus Type 1

(HIV-1) Coreceptors and Promotes Infection with both

Macrophage-Tropic and T-Lymphotropic HIV-1 Strains

LILI HUANG,1,2_{* IRENE BOSCH,}1,2_{WOLFGANG HOFMANN,}3,4_{JOSEPH SODROSKI,}3,4

ANDARTHUR B. PARDEE1,2

Divisions of Cancer Biology1_{and Human Retrovirology,}3_{Dana-Farber Cancer Institute, and Departments of Biological}

Chemistry and Molecular Pharmacology2_{and Pathology,}4_{Harvard Medical School, Boston, Massachusetts 02115}

Received 27 May 1998/Accepted 6 August 1998

Chemokine receptors CCR5 and CXCR4 are the primary fusion coreceptors utilized for CD4-mediated entry by macrophage (M)- and T-cell line (T)-tropic human immunodeficiency virus type 1 (HIV-1) strains, respec-tively. Here we demonstrate that HIV-1 Tat protein, a potent viral transactivator shown to be released as a soluble protein by infected cells, differentially induced CXCR4 and CCR5 expression in peripheral blood mononuclear cells. CCR3, a less frequently used coreceptor for certain M-tropic strains, was also induced. CXCR4 was induced on both lymphocytes and monocytes/macrophages, whereas CCR5 and CCR3 were induced on monocytes/macrophages but not on lymphocytes. The pattern of chemokine receptor induction by Tat was distinct from that by phytohemagglutinin. Moreover, Tat-induced CXCR4 and CCR5 expression was dose dependent. Monocytes/macrophages were more susceptible to Tat-mediated induction of CXCR4 and CCR5 than lymphocytes, and CCR5 was more readily induced than CXCR4. The concentrations of Tat effective in inducing CXCR4 and CCR5 expression were within the picomolar range and close to the range of extra-cellular Tat observed in sera from HIV-1-infected individuals. The induction of CCR5 and CXCR4 expression correlated with Tat-enhanced infectivity of M- and T-tropic viruses, respectively. Taken together, our results define a novel role for Tat in HIV-1 pathogenesis that promotes the infectivity of both M- and T-tropic HIV-1 strains in primary human leukocytes, notably in monocytes/macrophages.

Human immunodeficiency virus type 1 (HIV-1) Tat protein, a potent viral transactivator, is implicated in HIV-1 pathogen-esis not only by its indispensability for virus replication (23) but also by its capacity to prime quiescent T cells for productive HIV-1 infection (31) and induce apoptosis in uninfected T cells (30, 56, 61). The mechanism(s) underlying Tat-induced permissivity for productive HIV-1 infection by T-cell line (T)-tropic strains (31) has yet to be revealed. The inefficiency of HIV-1 infection in quiescent T cells may be due to decreased entry of HIV-1, premature termination of reverse transcrip-tion, or inefficient integration of HIV-1 provirus into chromo-somes (19, 38, 55, 59). Tat could directly or indirectly affect multiple steps in the virus life cycle to facilitate HIV-1 infec-tion, considering its pleiotropic biological properties, such as regulation of both viral and cellular gene expression (7, 23, 32, 51, 57, 60) and modulation of growth of various cell types (26–28, 31, 45), as well as its release from infected cells and its acting on bystander uninfected cells in a paracrine fashion (9, 16, 30, 36, 56, 61, 62).

In an attempt to elucidate the mechanism(s) underlying Tat-enhanced HIV-1 infection, we tested the possibility that Tat facilitates virus entry into target cells by regulating HIV-1 entry receptors. HIV-1 entry requires CD4 as well as other corecep-tors, which primarily include chemokine receptors CXCR4 and CCR5 (1, 3, 10, 12, 14, 17). Macrophage (M)-tropic HIV-1 strains, which infect primary macrophages and lymphocytes, mainly utilize CCR5 as a coreceptor (1, 10, 12, 14), while T-tropic HIV-1 strains, which infect lymphocytes and T-cell

lines, utilize CXCR4 as a coreceptor (3, 17). Moreover, dual-tropic HIV-1 strains, which infect macrophages, lymphocytes, and T-cell lines, utilize both CCR5 and CXCR4 (13). In addi-tion to CCR5 and CXCR4, other chemokine receptors, such as CCR3, CCR2b, and ChemR1, are also utilized by a restricted subset of M-tropic and dualtropic virus strains (10, 13, 49). The importance of the expression levels and patterns of these che-mokine receptors for HIV-1 infectivity is underscored by the recent findings from in vivo studies that individuals homozy-gous for defective CCR5 alleles are resistant to primary M-tropic HIV-1 infection and individuals heterozygous for the mutated CCR5 alleles have slower disease progression than those homozygous for normal CCR5 alleles (11, 22, 33, 39, 41, 50). It has also been shown by in vitro studies that increased cell surface expression of coreceptors correlates with increased infectivity of HIV-1 (1, 3, 8, 10, 12, 14, 17, 25, 44, 47, 58). Therefore, we examined the effect of Tat on chemokine recep-tor expression and also tested the possibility that Tat promotes HIV-1 entry. We found that Tat differentially induced CXCR4, CCR5, and CCR3 expression in peripheral blood mononuclear cells (PBMCs) and that the induction of CCR5 and CXCR4 expression correlated with Tat-enhanced infectivity of M- and T-tropic viruses, respectively.

MATERIALS AND METHODS

Cell cultures. PBMCs were isolated from healthy donors by Ficoll-Paque (Pharmacia, Uppsala, Sweden) density gradient centrifugation and were cultured in RPMI 1640 supplemented with 10% heat-inactivated fetal bovine serum, 2

mML-glutamine, 100 U of penicillin/ml, and 100mg of streptomycin/ml. In most

assays (for immunofluorescence staining or single-round infection), cells were

seeded into 6-well plates at a density of 106_{per ml and were cultured for the}

indicated number of days at 37°C in the presence of Tat protein or phytohemag-glutinin (PHA; Sigma Chemical, St. Louis, Mo.). Control cells received the same amount of Tat-dissolving buffer. For antibody blocking experiments, prior to addition to PBMC cultures, Tat protein (100 ng/ml) was incubated at room

* Corresponding author. Mailing address: Division of Cancer Biol-ogy, Dana-Farber Cancer Institute, 44 Binney St., Boston, MA 02115. Phone: (617) 632-4688. Fax: (617) 632-4680. E-mail: lhuang@mbcrr .harvard.edu.

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isothiocyanate-labeled anti-CD14 MAb (Pharmingen), followed by indirect staining for CXCR4, CCR5, or CCR3. Stained cells were then washed, fixed in 3.7% formaldehyde in PBS, and analyzed on a FACScan flow cytometer (Becton Dickinson, San Jose, Calif.).

Reverse transcriptase PCR (RT-PCR).Two micrograms of total RNA isolated from PBMCs by using TRIzol reagent (Gibco BRL, Grand Island, N.Y.) was

primed with oligo(dT) and reverse transcribed into cDNA in a 30-ml reaction

mixture containing Moloney murine leukemia virus reverse transcriptase (RT;

Gibco BRL). A series of increasing amounts of cDNA, 0.5, 1, and 2ml, from the

cDNA reaction mixture were subjected to PCR amplification by using AmpliTaq DNA polymerase (Perkin-Elmer Cetus, Norwalk, Conn.) in a TRIO-Thermo-block (Biometra) for 30 cycles of denaturing at 94°C for 30 s, annealing at 60°C for 40 s, and extension at 72°C for 1.5 min, followed by a final extension at 72°C

for 5 min. The following primers were used: for CXCR4, 59-GTTACCATGGA

GGGGATCAG-39and 59-CAGATGAATGTCCACCTCGC-39; for CCR5, 59

-GGTGGAACAAGATGGATTAT-39 and 59-CATGTGCACAACTCTGACT

G-39; for CCR3, 59-TGTGGCTATCCTTCTCTCTTCC-39and 59-AGGCAATT

TTCTGCATCTGACC-39; and forb-actin, 59-CATCCTCACCCTGAAGTAC

C-39and 59-GGTGAGGATCTTCATGAGGT-39.

Single-round infection assays.Reporter recombinant HIV-1 viruses with

dif-ferent envelope (Env) proteins were generated by cotransfection of 23106

HeLa cells by the calcium phosphate method with 20mg of an HIV-1 reporter

plasmid and 2mg of pSVIIIenv, encoding Env proteins from T-tropic (HXBc2)

or M-tropic (ADA or YU2) isolates. We used two kinds of HIV-1 reporter

plasmids, a CAT reporter plasmid, pHXBDBglCAT, and a green fluorescent

protein (GFP) reporter plasmid, pHIVec2GFP, each of which contains an HIV-1 provirus with a deletion in the env gene and a replacement of the nef gene with a CAT or GFP gene. For transfections with the GFP reporter plasmid, pCMVPack, encoding the packaging signals, was also added. The transfected HeLa supernatant containing the recombinant viruses was collected and assayed for RT activity. Equal numbers of RT units of the recombinant viruses were used to infect PBMCs which had been preincubated with a series of increasing con-centrations of Tat for 4 days and then washed. Cells were lysed at 48 h postin-fection for measurement of CAT activity by thin-layer chromotography, or they were fixed with 3.7% formaldehyde in PBS at day 3, 5, 7 or 9 postinfection for visualization of GFP by fluorescence microscopy or FACScan analysis.

RESULTS

Tat protein induces CXCR4, CCR5, and CCR3 expression in PBMCs.The effects of extracellular Tat on chemokine re-ceptor expression in PBMCs isolated from healthy donors were first examined by RT-PCR. Treatment of primary cultures of PBMCs with Tat (100 ng/ml) for 3 days induced high levels of mRNA expression for CXCR4, CCR5, and CCR3, as demon-strated by semiquantitative RT-PCR (Fig. 1). Further quanti-tative analysis by densitometry revealed approximately fivefold induction of CXCR4 and CCR5 mRNA expression and ap-proximately twofold induction of CCR3 mRNA expression. These results indicate that Tat induced chemokine receptor expression at the mRNA level.

To further determine if Tat induces the surface protein expression of these chemokine receptors, we used indirect immunofluorescence and flow cytometric analysis. Consistent with the RT-PCR results, treatment of PBMCs with Tat (100 ng/ml) for up to 4 days led to four- to fivefold induction of

CXCR4 (Fig. 2A) and CCR5 protein expression (Fig. 2B), as well as approximately 1.5-fold induction of CCR3 protein ex-pression (Fig. 2C), in PBMCs. Antibody blocking experiments demonstrated that both an anti-Tat polyclonal antibody and an anti-Tat MAb inhibited the Tat-induced up-regulation of CXCR4 (Fig. 2D) and CCR5 (Fig. 2E) expression, indicating that the observed induction of chemokine receptor expression is indeed due to Tat protein. The overall patterns of induction from all donors tested were similar to those represented in Fig. 2, although there was some individual variability in the abso-lute values. Time course studies showed that the induction of CXCR4 and CCR5 expression by Tat at 100 ng/ml was de-tected at day 2, further increased at day 4, sustained until day 8, and decreased to initial pretreatment levels by day 12 (data not shown).

Tat protein differentially induces CXCR4, CCR5, and CCR3 expression on monocytes/macrophages and lymphocytes. In uninfected PBMCs, CXCR4 and CCR5 are expressed mainly on lymphocytes and monocytes (5, 35, 58). These cell types are major reservoirs for HIV-1 infection, especially monocyte-de-rived macrophages, which are the primary targets for M-tropic strains isolated at early stages of HIV-1 infection. Therefore, we wanted to determine if Tat differentially induced the che-mokine receptors on different leukocyte subsets. Lymphocytes were gated according to their forward and side scatter, and monocytes/macrophages were gated by using CD14 (a marker for monocytes/macrophages) surface labeling. Interestingly, CXCR4 was induced on both lymphocytes and monocytes/ macrophages (Fig. 2A), whereas CCR5 (Fig. 2B) and CCR3 (Fig. 2C) were induced on monocytes/macrophages but not on lymphocytes. Furthermore, the induction of CXCR4, CCR5, and CCR3 on monocytes/macrophages was dramatic. After Tat treatment, the proportion of monocytes/macrophages ex-pressing CXCR4 and CCR5 increased from 16.1 to 89.4% and from 19.4 to 81.7%, respectively (Fig. 2A and B). The propor-tion of monocytes/macrophages expressing CCR3 upon Tat treatment increased from 49.9 to 86.3% (Fig. 2C). The in-creased surface labeling of CXCR4, CCR5, and CCR3 on monocytes/macrophages was not due to an increased number of monocytes/macrophages in Tat-treated PBMC cultures, since the number of monocytes/macrophages gated in each sample was the same and Tat treatment did not affect the absolute number of monocytes/macrophages in PBMCs (data not shown).

amplification. RT-PCR ofb-actin mRNA was included and served as a loading

control. The amplified PCR products (1,023 bp for CXCR4, 1,115 bp for CCR5,

321 bp for CCR3, and 394 bp forb-actin) are shown.

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FIG. 2. Tat induces CXCR4, CCR5, and CCR3 surface protein expression in PBMCs. (A through C) Indirect immunofluorescence staining and FACScan analysis of CXCR4 (A), CCR5 (B), and CCR3 (C) expression in PBMCs cultured in the absence (control) or presence of Tat (100 ng/ml) or PHA (1mg/ml) for 4 days were carried out as described in Materials and Methods. Lymphocytes were gated according to forward and side scatter, and monocytes/macrophages were gated by using CD14 surface labeling. Percentages of positive cells were set based on negative controls (not shown) and are indicated. (D and E) Inhibition of Tat-induced up-regulation of CXCR4 (D) and CCR5 (E) expression in PBMCs by anti-Tat rabbit antiserum and anti-Tat MAb. Tat was incubated with Tat antiserum or MAb before addition to PBMC cultures. Tat pretreated with control (Con.) rabbit serum and Tat pretreated with control hybridoma supernatant were also included as controls for pretreatment with Tat antiserum or MAb. Fold induction of CXCR4 and CCR5 expression in Tat-treated PBMCs was obtained by comparison with expression in untreated cells, which was set to 1. Data are mean values of duplicates. Results shown are representative of three independent experiments.

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The pattern of chemokine receptor induction by Tat protein is distinct from that by PHA.Activation of PBMCs by certain cytokines and mitogens leads to up-regulation of chemokine receptors, including CXCR4 and CCR5 (5, 34, 53). In order to determine if Tat-induced chemokine receptor expression is similar to other activation-induced expression, we treated PBMCs in parallel with the mitogen PHA and examined che-mokine receptor expression. PHA (1mg/ml) induced CXCR4, CCR5, and CCR3 expression in PBMCs, but to a lower extent than Tat (Fig. 2A through C). The effects of PHA on chemo-kine receptor expression on lymphocytes were similar to those of Tat in that both PHA and Tat significantly induced CXCR4, but not CCR5 and CCR3, expression on lymphocytes. How-ever, in contrast to Tat, which dramatically induced the expres-sion of CXCR4, CCR5, and CCR3 on monocytes/macro-phages, PHA treatment had no significant effects on CXCR4, CCR5, and CCR3 expression on monocytes/macrophages. The expression of CCR5 and CCR3 was induced by PHA on PBMCs but not on lymphocytes or monocytes/macrophages, suggesting that PHA may induce CCR5 and CCR3 expression on other leukocyte subsets. Taken together, these results indi-cate that Tat differentially induced the expression of the T-tropic coreceptor (CXCR4) and the M-T-tropic coreceptors (CCR5 and CCR3), probably through a mechanism distinct from that of PHA.

Tat protein-induced CXCR4 and CCR5 expression is dose dependent.We further determined if Tat-induced chemokine receptor expression was dose dependent. Since CXCR4 and CCR5 are the primary coreceptors for T- and M-tropic HIV-1 strains, respectively, and the overall expression pattern of CCR3, a minor coreceptor for certain M-tropic and dualtropic strains, was similar to that of CCR5, we further examined the expression of CXCR4 and CCR5. Freshly isolated PBMCs were treated with a series of increasing concentrations of Tat (1, 10, 50, 100, 500, and 1,000 ng/ml) for 4 days and then were analyzed for CXCR4 and CCR5 expression. Tat exhibited a dose-response effect of induction of CXCR4 and CCR5 ex-pression in PBMCs, reaching plateaus at around 500 ng/ml (Fig. 3). When PBMCs were gated on monocytes/macrophages and lymphocytes, respectively, different patterns of CXCR4 and CCR5 induction appeared. For CXCR4, Tat-induced ex-pression was observed on both monocytes/macrophages and lymphocytes, and the induction on monocytes/macrophages

was much more dramatic than that on lymphocytes (Fig. 3A), consistent with the results shown in Fig. 2A. Moreover, on monocytes/macrophages, Tat was able to give rise to approxi-mately 3.5-fold induction at 10 ng/ml and to the highest level of induction (around sevenfold) at 50 ng/ml, whereas on lympho-cytes the induction of CXCR4 expression required Tat at higher concentrations (.10 ng/ml) and reached the highest levels (around fourfold) at 500 ng/ml (Fig. 3A). For CCR5, Tat-induced expression was observed on monocytes/macro-phages but not on lymphocytes (Fig. 3B), again in agreement with the data shown in Fig. 2B. Induction of CCR5 required lower concentrations of Tat than induction of CXCR4, since Tat at 1 ng/ml caused approximately threefold induction of CCR5 expression on monocytes/macrophages (Fig. 3B), whereas no induction of CXCR4 expression occurred at this concentration (Fig. 3A). Overall, monocytes/macrophages were more suscep-tible to Tat-mediated induction of CXCR4 and CCR5 expres-sion than lymphocytes, and CCR5 was more readily induced than CXCR4.

FIG. 2—Continued.

FIG. 3. Tat-induced CXCR4 and CCR5 expression is dose dependent. CXCR4 (A) and CCR5 (B) expression was determined by indirect immunoflu-orescence staining of PBMCs cultured in the presence of a series of increasing concentrations of Tat for 4 days. Lymphocytes and monocytes/macrophages were gated as described in the Fig. 2 legend. The fold induction of CXCR4 and CCR5 expression in Tat-treated cells was obtained by comparison to expression in untreated cells. Data shown are representative of three independent experi-ments.

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Tat protein-induced CXCR4 and CCR5 expression corre-lates with Tat-enhanced HIV-1 infectivity.Increased cell sur-face expression of coreceptors correlates with increased infec-tivity of HIV-1 (1, 3, 8, 10, 12, 14, 17, 25, 44, 47, 58). Thus, significant induction of CXCR4 and CCR5 expression by Tat suggests that Tat is able to promote the infectivity of both T-and M-tropic HIV-1 strains. To confirm this, we used a single-round infection assay to evaluate the entry and early-phase infectivity of HIV-1 viruses containing different Env proteins (10, 20). In the assay, recombinant HIV-1 viruses were gener-ated by cotransfection of HeLa cells with a CAT reporter HIV-1 vector containing an HIV-1 provirus with a deletion in the env gene and a replacement of the nef gene with the CAT gene, as well as with a plasmid expressing Env protein derived from a laboratory-adapted T-tropic isolate (HXBc2) or an M-tropic primary HIV-1 isolate (ADA or YU2). The recom-binant viruses were then used at equal numbers of RT units to infect PBMCs which had been cultured with a series of increas-ing concentrations of Tat (0, 10, 50, 100, 500, and 1,000 ng/ml) for 4 days. At 48 h postinfection, CAT activity in whole-cell lysates was measured to assess the efficiency of the single-round infection. In PBMCs infected with the CAT reporter HIV-1 recombinant viruses containing the T-tropic HXBc2 Env proteins, CAT activity increased in a dose-dependent manner (Fig. 4A and 5A). Treatment with Tat at lower con-centrations (,100 ng/ml) resulted in a significant increase in CAT activity. Such an increase was unlikely due to a direct transactivation of HIV-1 LTR by Tat, since direct transactiva-tion of HIV-1 LTR required much higher concentratransactiva-tions ($500 ng/ml) of extracellular Tat (references 16 and 31 and data not shown). At 500 ng/ml or higher, Tat could directly transactivate HIV-1 LTR, thus probably contributing to the higher CAT activity observed. Overall, Tat-induced expression of the T-tropic coreceptor CXCR4 correlated with Tat-en-hanced infectivity of the T-tropic HIV-1 strain (Fig. 5A). Sim-ilar results were obtained when the CAT reporter HIV-1 re-combinant viruses containing the M-tropic (ADA or YU2) Env proteins were used (Fig. 4B and C and 5B). The single-round infection assays with Env proteins from either of the two M-tropic strains gave very similar results. In both assays, a roughly twofold increase in CAT activity was achieved at 10 ng of

Tat/ml, and a plateau was reached at 100 ng of Tat/ml. The expression of CCR5 was fully induced at 10 ng of Tat/ml, but the CAT activity, reflecting early-phase infectivity, reached a maximum at 100 ng of Tat/ml (Fig. 5B), suggesting that Tat also increased HIV-1 infectivity by other mechanisms besides the induction of chemokine receptors.

We further carried out single-round infection assays with GFP reporter recombinant HIV-1 viruses in order to directly examine the proportion of infected cells affected by Tat. Start-ing from day 3 after infection with GFP reporter viruses con-taining either the T-tropic (HXBc2) or the M-tropic (YU2) Env proteins, we detected a significant increase in the pro-portion of GFP-positive cells among Tat-treated PBMCs compared to untreated cells, by visualization of GFP with a

FIG. 4. Tat promotes the infectivity of CAT reporter HIV-1 recombinant viruses. PBMCs were cultured in the presence of increasing concentrations of Tat for 4 days, infected with equal numbers of RT units of CAT reporter recombinant viruses containing HXBc2 (A), ADA (B), or YU2 (C) Env proteins, and analyzed for CAT activity at 48 h postinfection. The experiments were repeated three times, and comparable results were obtained.

FIG. 5. Tat-induced CXCR4 and CCR5 expression correlates with Tat-en-hanced infectivity of both CAT and GFP reporter HIV-1 recombinant viruses. CXCR4 (A) and CCR5 (B) expression was analyzed by indirect immunofluores-cence staining of PBMCs cultured in the presence of a series of increasing concentrations of Tat protein for 4 days. The fold increase in the percentage of CXCR4- or CCR5-positive cells in Tat-treated PBMCs was obtained by com-parison to untreated cells. The CAT assay results with CAT reporter HIV-1 viruses containing either the T-tropic (HXBc2) (A) or the M-tropic (ADA or YU2) (B) Env proteins were obtained from the autoradiograms shown in Fig. 4, which were scanned with a densitometer. The CAT activity was expressed as the percent chloramphenicol conversion. The fold increase in CAT activity in Tat-treated PBMCs was calculated by comparison to unTat-treated cells. The GFP results were obtained by infecting PBMCs with GFP reporter HIV-1 viruses containing either HXBc2 (A) or YU2 (B) Env proteins for 9 days and analyzing the GFP fluorescence of intact cells with a flow cytometer. The fold increase in the percentage of GFP-positive cells among Tat-treated PBMCs was calculated by comparison to untreated cells.

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which correlated with its differential effects on CCR5 and CXCR4 expression in monocytes/macrophages and lympho-cytes.

Tat promotes both M- and T-tropic HIV-1 infection in monocytes/macrophages. Tat-mediated enhancement of HIV-1 infection in PBMCs and the dramatic induction by Tat of CXCR4, CCR5, and CCR3 expression on monocytes/macro-phages implicate Tat in the promotion of HIV-1 infection in monocytes/macrophages. As demonstrated by single-round in-fection assays using GFP reporter HIV-1 recombinant viruses, Tat greatly facilitated the infectivity of both M-tropic (Fig. 6A through D) and T-tropic (Fig. 6E through H) viruses in adher-ent monocytes/macrophages. Subsequadher-ent FACScan analysis of the monocytes/macrophages showed that Tat induced a four-to fivefold increase in the percentage of GFP-positive cells (Fig. 7). These results indicate an important role of Tat in HIV-1 infection in monocytes/macrophages.

DISCUSSION

In this study, we demonstrated that extracellular Tat protein differentially induced the expression of chemokine receptors CXCR4, CCR5, and CCR3 in primary cultures of PBMCs. CXCR4, a C-X-C chemokine receptor, was induced on both lymphocytes and monocytes/macrophages, whereas CCR5 and CCR3, the C-C chemokine receptors, were induced on cytes/macrophages but not on lymphocytes. Moreover, mono-cytes/macrophages were more susceptible to Tat-mediated in-duction of CXCR4 and CCR5 expression than lymphocytes, and CCR5 was more readily induced than CXCR4. The great susceptibility of monocytes/macrophages to Tat-mediated in-duction of chemokine receptors and the selective inin-duction of the M-tropic HIV-1 coreceptors CCR5 and CCR3 on mono-cytes/macrophages are of particular interest, since monocytes/ macrophages are the major target for HIV-1 infection in vivo, especially at early stages of virus infection, and are a primary reservoir for persistent infection (43).

The induction of CXCR4 and CCR5 expression by Tat was dose dependent. The extracellular Tat used here induced high levels of CXCR4 and CCR5 expression at concentrations well below those required for a direct transactivation as determined by us (unpublished data) and others (16, 31), suggesting that Tat mediates CXCR4 and CCR5 expression via an indirect pathway. Two different pathways have been described to me-diate cellular and viral effects of extracellular Tat (16, 31, 63). One is through signal transduction and requires lower Tat concentrations; the other is through the direct transactivating effect of Tat and requires higher Tat concentrations. For the signaling pathway, integrin receptors are implicated in medi-ating extracellular Tat-induced effects (2, 6, 16, 31), and Tat

distinct from that by PHA. PHA stimulation induced the ex-pression of CXCR4, but not CCR5 and CCR3, on lympho-cytes, which is similar to the effect of Tat on lymphocytes and is also consistent with results obtained by others (5). However, in contrast to Tat, which dramatically induced CXCR4, CCR5, and CCR3 expression on monocytes/macrophages, PHA, an activator of monocytes/macrophages (4), had no significant effect on chemokine receptor expression on this cell type. These results suggest that Tat probably induces chemokine receptor expression via a mechanism distinct from that of PHA.

To our best knowledge, Tat is the first virus-encoded protein shown to induce the expression of HIV-1 coreceptors. Other viral proteins, such as envelope glycoprotein gp120, Nef, and Vpu, have been shown to down-regulate CD4 expression in primary monocytes/macrophages and lymphocytes (24, 29, 37). Tat, in contrast, did not have significant effects on CD4 expres-sion in monocytes/macrophages and lymphocytes (unpublished data). Recent studies demonstrate that the CD4 and CCR5 cell surface concentrations required for efficient infection of M-tropic HIV-1 are interdependent and that the requirement for one is increased when the other is present in a limiting amount (47). In monocytes/macrophages, CD4 expression is low (21, 42), and it is further down-regulated in HIV-1-in-fected individuals (48). Under those circumstances, changes in coreceptor expression could readily affect the susceptibility of macrophages to HIV-1 infection. Therefore, Tat-mediated in-duction of coreceptors CXCR4, CCR5, and CCR3 in PBMCs, especially in monocytes/macrophages, could greatly enhance the susceptibility of these cells to HIV-1 infection. This is confirmed by our single-round infection assays using both CAT and GFP reporter HIV-1 recombinant viruses pseudotyped with T- or M-tropic Env proteins. Our results showed that Tat enhanced the early-phase infectivity of T- and M-tropic HIV-1 strains in PBMCs and that such enhancement in T- and M-tropic HIV-1 infectivity correlated with Tat-mediated induc-tion of the expression of CXCR4 and CCR5, respectively. Notably, Tat greatly enhanced the susceptibility of monocyte-derived macrophages to infection by both M- and T-tropic HIV-1 strains. In our system, the T-tropic HIV-1 virus, which is thought to be unable to infect primary monocytes/macro-phages, can infect these cells, in agreement with results from others (54). Overall, our results suggest that Tat facilitates HIV-1 infection by inducing coreceptors and thus promoting virus entry.

Besides the induction of chemokine receptors, other mech-anisms for the Tat-mediated increase in HIV-1 infectivity were also implied by our data. Tat may modulate other cellular activities and therefore facilitate postentry events of HIV-1 infection. In this regard, Tat has been shown to modulate

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FIG. 6. Tat promoted the infectivity of the GFP reporter HIV-1 recombinant viruses in monocytes/macrophages. PBMCs were cultured in the absence (A, B, E, and F) or presence (C, D, G, and H) of Tat (100 ng/ml) for 4 days and were infected with GFP reporter HIV-1 viruses containing the M-tropic (YU2) (A through D) or T-tropic (HXBc2) (E through H) Env proteins for 9 days. After removal of the medium, the adherent cells, largely containing monocytes and monocyte-derived macrophages, were washed off nonadherent cells, fixed, and visualized by fluorescence microscopy to detect GFP fluorescence. Shown here are representative pictures depicting phase-contrast (A, C, E, and G) and fluorescence (B, D, F, and H) imaging. Results obtained from cells infected for 5 to 7 days were comparable to these.

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cytokine expression (7, 51, 57) and increase the activation states of T cells and monocytes (26–28, 31, 45).

Our findings are relevant to in vivo infection in that the concentrations of Tat effective in inducing chemokine receptor expression in our experiments are within the picomolar range and close to the range of extracellular Tat observed in sera from HIV-1-infected individuals (56). In fact, Tat is likely to reach even higher levels in lymphoid tissues of HIV-1-infected individuals due to active viral replication (15, 46, 56). Whether these chemokine receptors are up-regulated in HIV-1-infected individuals has not been well documented. The in vivo situa-tion would be expected to be more complicated than the in vitro situation, in that chemokine receptor expression is regu-lated by multiple factors, including negative and positive reg-ulators.

In summary, Tat protein induced the expression of HIV-1 primary coreceptors CXCR4 and CCR5 in PBMCs. Such in-duction in CXCR4 and CCR5 expression correlated with Tat-enhanced infectivity of M- and T-tropic HIV-1 strains, respec-tively. Moreover, Tat-mediated increases in the infectivity of both M- and T-tropic strains in monocytes/macrophages are prominent and of pathological significance, since macrophages are major reservoirs for HIV-1 infection and primary trans-mission sites to T cells (43). Our results define a novel role for Tat in HIV-1 pathogenesis. By up-regulating HIV-1 corecep-tors and increasing the activation states of T cells and mono-cytes (26–28, 31, 45), as well as stimulating viral replication (23), Tat greatly promotes HIV-1 infection. This, together with Tat-induced apoptosis in uninfected T lymphocytes (30, 56, 61), contributes to CD41_{T-cell loss in AIDS patients. Thus,} our findings should contribute to our understanding of the molecular mechanisms of HIV-1 infection and to better design of prophylactic and therapeutic strategies. Anti-Tat agents and vaccines might potentially be effective in interfering with HIV-1 infection.

ACKNOWLEDGMENTS

We thank Hyeryun Choe for kindly providing the Env protein-expressing plasmids; the AIDS Research and Reference Reagent Pro-gram, Division of AIDS, NIAID, NIH, for Tat antiserum (contributed by B. Cullen), anti-Tat MAb (contributed by K. Krohn and V. Ovod), anti-CXCR4 MAb 12G5 (contributed by J. Hoxie), and anti-CCR3 MAb 7B11 (contributed by LeukoSite, Inc.); Isaac Rondon and

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