By
From the * Regional Primate Research Center and the Department of Microbiology, University of
Washington, Seattle, Washington 98195
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Abstract |
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We previously found that activation of primary CD4+ T cells via both the T cell antigen receptor (TCR) and CD28 is required for HIV-1 DNA to be translocated from the cytoplasm to the nucleus. Here we report that expression of c-Myc protein in CD4+ T cells is induced only after such costimulation. In addition, cyclosporin A not only inhibits nuclear import of HIV-1 DNA but also inhibits expression of c-Myc protein. Because of these correlations, we tested whether c-Myc is necessary for nuclear import of HIV-1 DNA. Specific c-myc antisense, but not sense or non-sense, phosphorothioate oligodeoxynucleotides selectively induced the accumulation of two NH2-terminally truncated c-Myc proteins and abolished HIV-1 genome entry into host nuclei. Consequently, both virus replication and HIV-1-induced apoptotic cell death were inhibited. Synthesis of viral full-length DNA was not affected. Specific c-myc antisense oligonucleotide inhibited HIV-1 infection under conditions that did not affect cell cycle entry or proliferation. Thus, c-Myc appears to regulate HIV-1 DNA nuclear import via a mechanism distinct from those controlling entry into the cell cycle.
Key words: c-Myc; HIV-1 DNA; T cell; nuclear import; apoptosis ![]() |
Introduction |
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The life cycle of human immunodeficiency virus 1 (HIV-1) in infected cells can be divided into pre- and postintegrated stages. After HIV-1 binds to the surface of T cells through interaction of the envelope protein gp120 with CD4 and a seven-span transmembrane chemokine receptor, the virus fuses with the host cell, enters the cytoplasm, and disassociates from the cell membrane (1). Viral reverse transcription is initiated, then linear double-stranded viral DNA is synthesized, followed by formation of virus preintegration complexes (PIC)1 (5). Double-stranded viral DNA within these complexes migrates to the host nucleus and integrates into the host cell genome, or forms circular molecules without the capacity to integrate (6, 7). After a latency period, proviruses can be induced by a variety of stimuli to replicate and this in turn can lead to depletion of CD4+ T cells by a process of programmed cell death (8, 9).
Full-length HIV-1 DNA synthesis and translocation to the nucleus are dependent upon activation of T cells (10- 16). Two T cell activation signals are required for the synthesis and nuclear translocation of simian immunodeficiency virus (SIV) or HIV-1 DNA (14, 17): one signal through the TCR, which normally regulates the G0 to G1 transition, induces full-length viral DNA synthesis; the second signal, through CD28 or the IL-2 receptor complex (IL-2R), which regulates the G1 to S transition, controls viral DNA entry into the nucleus. Furthermore, cyclosporin A (CSA), a T cell activation inhibitor, and mimosine, a late G1 phase inhibitor, abrogate nuclear import of SIV or HIV-1 genomes (14, 17, 18). However, the cellular factors involved in regulation of this process are not well understood.
One candidate molecule regulated by lymphocyte activation is c-Myc, a transcription factor that has been implicated in regulation of cell activation, differentiation, cell cycle progress, transformation, and apoptosis (19). The c-myc proto-oncogene is an immediate-early gene rapidly induced during the G0 to G1 transition in activated T cells (20). An IL-2R-dependent signaling pathway is required for induction of c-myc expression (26) and CSA suppresses c-myc gene transcription (31). These observations suggested that c-Myc might play a key role in the regulation of HIV-1 DNA nuclear import.
Here we present evidence that expression of c-Myc occurs as a consequence of T cell costimulation. In addition, blocking c-Myc by CSA correlates with this drug's inhibitory effect on translocation of HIV-1 genome to the nucleus. Furthermore, specific c-myc antisense, but not corresponding sense, non-sense, or scrambled phosphorothioate oligodeoxynucleotides (PS-ODNs), selectively abolished HIV-1 DNA entry into host nuclei and induced 46- and 50-kD truncated c-Myc proteins whose NH2-terminal transactivation domains are deleted. As a result, both replication and the cytopathic effects of HIV-1 were inhibited. Specific c-myc antisense PS-ODNs inhibited HIV-1 infection without affecting cell cycle entry or proliferation, suggesting that c-Myc regulates HIV-1 DNA nuclear import via a mechanism distinct from those controlling entry into the cell cycle.
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Materials and Methods |
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Reagents.
PS-ODNs used in this study were synthesized by Oligo Etc. Sequences used were as previously described (23): c-myc antisense, AACGTTGAGGGGCAT, located in exon 2 of initiation site of translation; sense c-myc, ATGCCCCTCAACGTT; non-sense, AGTGGCGGAGACTCT; and scrambled, AAGCATACGGGGTGT containing a GGGG motif (32). The oligonucleotides were dissolved in 30 mM Hepes (pH 7.0). Purified mAbs to human CD8 (G10-1, IgG2a), CD16 (FC-2, IgG2b), CD20 (1F5, IgG2a), and HLA-DR (HB10a, IgG2a) were produced in our lab and used to purify human primary CD4+ T cells as previously described (14). Goat anti-mouse IgG conjugated to magnetic microbeads was purchased from Miltenyi Biotec. mAbs to human CD3 (64.1, IgG2a) and CD28 (9.3, IgG2a) were used to activate CD4+ T cells as previously described (14). Phospho-c-Myc (Thr58/Ser62) polyclonal antibody was purchased from New England Biolabs. Anti-human c-Myc mAb (9E10, IgG1), rabbit polyclonal antibody specific to NH2-terminal region 1-262 amino acids of c-Myc (N-262), and rabbit polyclonal anti-ERK1 (c-16) antiserum were obtained from Santa Cruz Biotechnology. PE-conjugated anti-HIV-1 p24 protein mAb was purchased from Coulter Corp. TUNEL (TdT-mediated dUTP nick-end labeling) detection kits were obtained from Boehringer Mannheim.CD4+ T Cell Isolation.
Enriched preparations of human CD4+ T cells were isolated from peripheral blood samples from healthy, HIV-seronegative donors as follows: PBLs were obtained by centrifugation over Ficoll-Hypaque, and then E-rosette-positive (Er+) cells were isolated as previously described (33). CD4+ T cells were obtained by negative selection of Er+ cells depleting CD8+, CD16+, CD20+, and HLA-DR+ cells with mAb-coated beads. The purity of isolated CD4+ cells was >97% as monitored by flow cytometry. Cells were cultured in RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovine serum, 2 mM glutamine, 10 U/ml penicillin, 10 mg/ml streptomycin, 1 mM pyruvate, and nonessential amino acids.HIV-1 Infection.
HIV-1 strain Lai was prepared as previously described (14). Cells were infected with HIV-1 at a multiplicity of infection of 0.01 per cell.PCR to Monitor Initiation and Elongation of HIV-1 DNA Synthesis and Viral DNA Nuclear Import.
DNA was extracted from HIV-1- and heat-inactivated HIV-1-infected cells as previously described (14). PCR was performed as described (14) with some modifications including: 50 ng of DNA/sample for amplification ofWestern Blot Analysis.
After various treatments, 5 × 106 primary CD4+ T cells were lysed in 500 µl lysis buffer (2% NP-40, 0.5% sodium deoxycholate, 0.2% SDS, 25 mM Tris-HCl, 50 mM NaCl, 1 mM PMSF, 1 mM Na3VO4, 10 µM E-64 [trans-epoxysuccinylt-L-leucylamido (4-guanidino)-butane], 1 µg/ml pepstatin, 10 µg/ml leupeptin, and 0.1% aprotinin). After incubation on ice for 30 min the cells were sonicated. The cell lysates (equivalent to 106 primary CD4+ T cells) were mixed with 2× SDS loading buffer (125 mM Tris-HCl [pH 6.8], 4% SDS, 20% glycerol, 83 mM dithiothreitol, and 0.02% bromophenol blue), incubated at 100°C for 5 min, electrophoresed by 8% SDS-polyacrylamide gel, and then transferred to nitrocellulose membranes (Schreicher & Schuell). The membranes were blocked with 5% nonfat milk-TBST (50 mM Tris-HCl [pH 7.4], 150 mM NaCl, 0.1% Tween 20) at 4°C overnight, followed by incubation with primary antibodies (in 5% BSA-TBST) at 4°C overnight. After washing, membranes were incubated with horseradish peroxidase-conjugated second antibodies at room temperature for 1 h. Bands on the blotted membranes were detected by incubation with enhanced chemiluminescence reagent (ECL) (Amersham) for 1 min and exposure to Kodak X-Omat film (Eastman-Kodak Co.).Flow Cytometry.
Apoptotic cell death followed by HIV-1 infection was detected by TUNEL according to the manufacturer's protocol (Boehringer Mannheim). After TUNEL staining, cells were resuspended in 100 µl PBS containing 1% BSA; PE-conjugated anti-HIV-1 p24 mAb was added and incubated at 4°C for 20 min. The cells were washed with cold PBS, suspended in 1% paraformaldehyde (Sigma Chemical Co.), and kept at 4°C in the dark until flow cytometry analysis by means of FACScan® (Becton Dickinson). Cell cycle stages were determined by measuring DNA content with propidium iodide as previously described (34).Cell Proliferation.
Cell proliferation was estimated by [3H]thymidine incorporation: 105 primary CD4+ T cells in the presence or absence of oligodeoxynucleotides were stimulated with CD3 (10 µg/ml) and CD28 (20 µg/ml) mAbs in triplicate in 96-well plates. The cells were incubated at 37°C in a 5% CO2 incubator for 3 d. Each well was pulsed for 16 h with 0.5 µCi [3H]thymidine, and then the incorporation of [3H]thymidine radioactivity was monitored by a beta counter. ![]() |
Results and Discussion |
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In our previous study we found that HIV-1 nuclear import required a CSA-sensitive pathway, and that both TCR and CD28 ligation are essential for this process (14). Similarly, the expression of c-Myc in primary CD4+ T cells required costimulation with CD3 and CD28 mAbs (Fig. 1 A); neither CD3 nor CD28 ligation alone induced c-Myc expression. Time course experiments showed that c-Myc expression increased by 4 h, peaked at 24 h after costimulation, and was sustained for 48 h. Moreover, CSA inhibited c-Myc expression (Fig. 1 A, bottom). Because of this correlation, we tested whether c-Myc might be a key regulator of HIV-1 DNA nuclear import in primary T cells. Since no c-Myc-specific inhibitor is yet available, we used a c-myc antisense PS-ODN to inhibit c-Myc function. By competitively inhibiting HIV-1 reverse transcriptase binding to the virus genome-cellular primer complex, PS-ODNs have an inhibitory effect on the initiation of HIV-1 reverse transcription in a sequence-independent manner (35, 36). However, sequence-independent PS-ODNs do not exhibit any anti-HIV-1 activity once initiation of virus reverse transcription has begun (35). To avoid nonspecific anti-HIV activity of sequence-independent PS-ODNs, we first infected activated CD4+ T cells with HIV-1 for 24 h and then administrated graded doses of c-myc antisense, sense, or non-sense PS-ODNs to the infected cells. As shown in Fig. 1 B, initiation of reverse transcription (LTR/LTR product) and full-length viral DNA synthesis (LTR/gag product) were not affected by the c-myc antisense PS-ODN.
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However, nuclear import of HIV-1 DNA (LTR circles) was blocked by c-myc antisense PS-ODN even at doses as low as 1 µM. Doses below to 0.2 µM were less efficient at inhibiting LTR circle formation (data not shown). Neither c-myc sense nor non-sense PS-ODN had any effect on viral DNA nucleus translocation up to 8 µM (Fig. 1 B). Consequently, HIV-1-infected cells treated with c-myc antisense PS-ODN did not produce p24 gag protein or undergo apoptosis (Fig. 1 C). Under conditions in which HIV-1 had already entered the nucleus (e.g., at 48 h), c-myc antisense PS-ODN did block viral p24 expression (data not shown). Lack of an effect by c-myc antisense PS-ODN on full-length viral DNA synthesis was not simply because the oligonucleotides were added too late to the cultures (after 24 h infection), as full-length viral DNA was not detectable until at least 40 h after HIV infection in activated CD4+ T cells (reference 14 and data not shown). Thus, c-myc antisense PS-ODN apparently selectively acts on the stage of HIV-1 DNA nuclear import.
We next studied whether c-myc antisense PS-ODN specifically inhibited full-length c-Myc protein expression. Using mAb 9E10 specific to the COOH-terminal end of c-Myc (39), we consistently observed that in the presence of c-myc antisense, sense, or non-sense PS-ODNs, the two major forms of c-Myc proteins, p64 and p67, remained relatively unchanged (Fig. 2). However, c-myc antisense PS-ODN selectively induced the accumulation of 46- and 50-kD proteins, whose expression levels were higher than that of the full-length c-Myc. Neither the c-myc sense nor non-sense PS-ODN induced accumulation of these two proteins (Fig. 2, top). These data are consistent with previous studies showing that expression of c-Myc short (c-MycS) proteins in some tumor cell lines arised from two translational initiation sites downstream of the full-length c-Myc start codon (40). These downstream-initiated c-MycS proteins lack most of the NH2-terminal transactivation domain; they are produced through a leaky scanning mechanism, since optimization of the traditional initiation codon for full-length c-Myc results in less synthesis of the c-MycS proteins (45). Because the c-myc antisense oligonucleotide we used corresponds to the initiation site of full-length c-Myc mRNA, and the two smaller proteins we detected are about the same size as c-MycS isoforms, it seemed likely that the 46- and 50-kD proteins are produced through the same mechanism leading to deletion of the NH2-terminal region. This possibility was substantiated by the fact that antibodies specific to either NH2-terminal phosphorylated Thr58/Ser62 or the whole NH2 terminus region of c-Myc failed to recognize 46- and 50-kD proteins (Fig. 2, middle and bottom). However, both antibodies were able to recognize p64 and p67 full-length c-Myc, which did not change expression in cells treated with different PS-ODNs (Fig. 2). The same result was obtained in a CD4+ lymphoid cell line, CEM (data not shown). Thus, the c-myc antisense oligonucleotides, but not control PS-ODNs, selectively induce NH2-terminally truncated c-Myc proteins that are known to act as dominant negative inhibitors by competitively suppressing full-length c-Myc functions (45). Blockage of HIV-1 DNA nuclear import by c-myc antisense PS-ODN most likely is mediated by these NH2-terminally truncated c-Myc proteins.
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Finally, we tested whether c-myc antisense PS-ODN could inhibit the entry of cell cycle and proliferation induced in primary CD4+ T cells after TCR and CD28 ligation. Treating CD4+ T cells with 6 µM of c-myc antisense oligonucleotide, which efficiently blocked HIV-1 LTR circle formation, could not inhibit cell cycle progression (Fig. 3 A). Similarly, c-myc antisense, sense, and non-sense PS-ODN had no effects on CD4+ T cell proliferation induced by CD3 plus CD28 mAbs (Fig. 3 B). These data are consistent with previous findings that NH2-terminally truncated c-Myc proteins do not interfere with cell growth (45, 49).
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The study presented here reveals a novel function of
c-Myc for regulation of HIV-1 nuclear import. Blocking of
HIV-1 DNA nuclear import by c-myc antisense PS-ODN
appeared to be mediated through the presence of 46- and
50-kD NH2-terminally truncated c-Myc proteins, which
do not affect cell cycle progression or cell proliferation (Fig.
3, A and B). Our data imply that the mechanism by which c-Myc controls HIV-1 DNA nuclear import is distinct
from those controlling cell cycle progression. However,
precisely where and how c-Myc is required for HIV-1
DNA nuclear import in proliferating CD4+ T cells remains
to be discovered. NH2-terminal-defective c-Myc proteins
are able to heterodimerize with Max, translocate to nucleus, repress gene expression, stimulate cellular proliferation, and induce cell apoptosis (49). However, c-MycS
proteins are not able to activate gene transcription (49). It is
probable that c-Myc regulates HIV-1 DNA nuclear import
through its transactivation activity by regulation downstream gene expression. The ability of HIV-1 to infect
nondividing cells, such as monocytes, terminally differentiated macrophages, mucosal dendritic cells, or -irradiated cells, is believed to be a unique feature since oncoretroviruses only can establish infection when the cells undergo
mitosis (50). The ability of HIV-1 to infect nondividing
cells is presumably related to the fact that its PIC can be
recognized by the cell nuclear import machinery (58)
and actively transported through nucleopores (62). Moreover, a cellular serine/threonine protein kinase, mitogen-activated protein kinase (MAPK), can associate with HIV-1
PIC to facilitate nuclear targeting of viral DNA (63). It
is unclear whether HIV-1 DNA nuclear import in proliferating CD4 T cells is regulated through the identical pathway seen in nondividing cells. A reasonable possibility is
that c-Myc affects the expression of genes encoding cellular
proteins involved in nuclear transport. Further elucidation
of the role of c-Myc in regulation of expression of cellular
nuclear importing molecules might help us to understand
how c-Myc regulates HIV-1 DNA nuclear import.
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Footnotes |
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Address correspondence to Edward A. Clark, Regional Primate Research Center, Box 357330, University of Washington, Seattle, WA 98195. Phone: 206-543-8706; Fax: 206-685-0305; E-mail: eclark{at}bart.rprc.washington.edu
Received for publication 21 October 1998 and in revised form 12 February 1999.
We thank Ms. M. Domenowske for preparation of figures; Dr. Aaron Marshall and Ms. Kate Elias for editorial assistance; Drs. James Mullins and Michael Katze for critical review of the manuscript; Drs. Andrew Craxton, Raymond T. Doty, and Aaron Marshall and Mr. Aimin Jing for helpful discussion; and members of the Clark laboratory for technical assistance.
This work was supported by National Institutes of Health grant RR00166.
Abbreviations used in this paper c-MycS, c-Myc short; CSA, cyclosporin A; PS-ODN, phosphorothioate oligodeoxynucleotide; PIC, pre-integration complexes; TUNEL, TdT-mediated dUTP nick-end labeling.
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