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The Journal of Immunology, 1999, 163: 906-912.
Copyright © 1999 by The American Association of Immunologists

Processing, Secretion, and Anti-HIV-1 Activity of IL-16 With or Without a Signal Peptide in CD4+ T Cells

Paul Zhou1, Krishnakumar Devadas, Deepanker Tewari, Aleksandra Jegorow and Abner Louis Notkins

Experimental Medicine Section, Oral Infection and Immunity Branch, National Institute of Dental Research, National Institutes of Health, Bethesda, MD 20892


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
CD4+ T cells transfected with the C-terminal 130 aa of human IL-16 are rendered resistant to HIV infection. Whether the constitutively expressed IL-16 acts intracellularly, extracellularly, or both is not clear. To address this question and to further study the processing of IL-16, new constructs containing either the C-terminal 130 aa or the C-terminal 100 aa (PDZ-like motif) were constructed with and without a signal peptide. Pulse-chase experiments and treatment of cells with brefeldin A and/or tunicamycin showed that IL-16 is secreted despite the absence of a signal peptide, but with a signal peptide IL-16 is processed through the endoplasmic reticulum-golgi pathway and is glycosylated. Cells expressing IL-16 linked to a signal peptide secrete considerably more IL-16 into the supernatant than cells expressing IL-16 without a signal peptide and are considerably more resistant to HIV replication. Resistance extends to almost 25 days for cells expressing IL-16 with signal peptide as compared with only 15 days for cells without signal peptide. Cells expressing the C-terminal 100 aa not linked to a signal peptide are poor secretors of IL-16 and show little if any resistance to HIV. In contrast, cells expressing the C-terminal 100 aa linked to a signal peptide secrete IL-16 and are resistant to HIV replication. It is concluded that the secretion of IL-16 is required for HIV inhibition.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Interleukin-16 (IL-16) is a CD8+ T lymphocyte-derived cytokine that is chemotatic for CD4+ T lymphocytes, monocytes, and eosinophils at nanomolar concentrations (1, 2, 3, 4). In addition to its chemotatic activity, IL-16 induces expression of IL-2R and MHC class II, increases intracellular Ca+ and inositol-1,4,5-trisphosphate in CD4+ T lymphocytes (5), and suppresses both the MLR and the activation of CD4+ T lymphocytes via the TCR/CD3 complex (6). Human IL-16 cDNA has been cloned (7, 8) and shown to encode a 631-aa precursor that lacks a signal peptide in its N terminus (8). In primary CD8+ T lymphocytes, precursor proteins are constitutively synthesized. Upon activation, these proteins are processed and released from cells (9, 10). The processing of the precursor into the mature form may involve a member of caspase family, caspase-3 (11), but the mechanism for IL-16 release is still unknown.

Recently, Baier et al. showed that the recombinant C-terminal 130 aa of human and simian IL-16, produced in bacteria, were capable of suppressing HIV-1 replication in CD8+-depleted PBMCs (12). To explore the potential of human IL-16 for gene therapy, we generated stable human CD4+ T cell transfectants expressing the C-terminal 130 aa of human IL-16. The constitutive expression of this portion of the molecule rendered the CD4+ transfectants resistant to HIV-1. Further studies showed that the inhibition of HIV-1 by IL-16 was not at viral entry or reverse transcription, but at viral mRNA expression (13). Maciaszek et al. drew similar conclusions from their work in a virus-free system (14). However, it is not clear whether the inhibition of HIV-1 replication requires intracellular IL-16 or extracellular IL-16 or both. Moreover, it is not known whether the entire C-terminal 130 aa of IL-16 or a smaller segment containing the 90-aa PDZ-like motif (15) is capable of inhibiting HIV. The PDZ motif has been found in over 50 proteins that direct specific protein-protein interactions mainly in the cytoplasmic membrane (16, 17).

To further investigate the requirement for intracellular vs extracellular IL-16 and the potential anti-HIV-1 activity of the PDZ motif, human CD4+ Jurkat cells were transfected with the C-terminal 130 aa and the C-terminal 100 aa (PDZ-like motif) of human IL-16, with and without an Ig signal peptide. Here, we report comparative studies on the processing, secretion, and anti-HIV-1 activity of the C terminus of IL-16.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Gene constructs and stable transfectants

The oligonucleotide primers for generating signal(Ig)-IL-16 (C-terminal 130 aa of human IL-16), PDZ (C-terminal 100 aa of human IL-16), and signal(Ig)-PDZ were as follows: I, 5'-tatcatatgccaagtacgcccccta-3'; II, 5'-tcgggcatggacaggacccatctgggag-3'; III, 5'-tcctgtccatgcccgacctcaactcctc-3'; IV, 5'-caatctagactaggagtctccagcagctgtgg-3'; V, 5'-accaagcttaccatgtgcacggtgacactggagaag-3'; VI, 5'-accgtgcaggacaggacccatctgggag-3'; and VII, 5'-tcctgtcctgcacggtgacactggagaagatg-3'.

Primers I, II, III, and IV were used to generate signal(Ig)-IL-16. At the 5' ends of primers I and IV the NdeI and XbaI restriction sites (italicized) were introduced, respectively. At the 5' ends of primers II and III, a short 16 overlapping nucleotides between them was introduced. By using pRC/CMV/TCR enhancer (TCRenh)-single chain Fv (18) as template, part of the pRC/CMV promoter and signal peptide (Ig) sequence was synthesized with primers I and II. By using pRC/CMV/TCRenh-IL-16 (13) as template, the sequence containing the C-terminal 130 aa of human IL-16 was synthesized with primers III and IV. The amplified sequences were put together by overlapping PCR (18, 19). The overlapping PCR-amplified product was ligated into a TA vector system for sequence analysis (Invitrogen, San Diego, CA). Conditions for regular PCR and overlapping PCR were as described (18, 20). The insert containing the right sequence was recloned into the NdeI and XbaI doubly digested mammalian expression vector pRC/CMV/TCRenh (13).

Primers I, VI, VII, and IV were used to generate signal(Ig)-PDZ. By using pRC/CMV/TCRenh-single chain Fv (18) as template, part of the pRC/CMV promoter and signal peptide (Ig) sequence was synthesized with primers I and VI, and by using pRC/CMV/TCRenh-IL-16 (13) as template, the PDZ sequence was generated with primers VII and IV. Overlapping PCR, ligation, sequencing, and recloning into mammalian expression vector were the same as described above.

Primers V and IV were used to generate PDZ with pRC/CMV/TCRenh-IL-16 (13) as template. The insert containing the right sequence was recloned into a HindIII and XbaI doubly digested mammalian expression vector.

To generate stable transfectants, the resulting plasmids, designated pRC/CMV/TCRenh/signal(Ig)-IL-16, pRC/CMV/TCRenh/PDZ, pRC/CMV/TCRenh/signal(Ig)-PDZ, and the previously generated pRC/CMV/TCRenh/IL-16 and pRC/CMV/TCRenh vector (13) were linearized at an XmnI site. Twenty micrograms of linearized DNA per construct were mixed with 106 Jurkat cells in 0.8 ml RPMI 1640 (Life Technologies, Rockville, MD). Electroporation was performed at 960 µF and 300 V/0.4 cm. Stable transfectants were generated by G418 selection (1.5 mg/ml; Life Technologies) for 2–3 wk and then by limiting dilution.

Immunoprecipitation

To study expression and stability of IL-16 in transfectants, pulse-chase experiments were performed in Jurkat transfectants containing vector alone (designated J-V),2 pRC/CMV/TCRenh-IL-16 (designated JIL-16), pRC/CMV/TCRenh-signal(Ig)-IL-16 (designated JIL-16-signal), pRC/CMV/TCRenh/PDZ (designated J-PDZ), pRC/CMV/TCRenh/signal(Ig)-PDZ (designated J-PDZ-signal). Briefly, 1.0 x 106 cells were incubated with 2 ml of methionine-free medium for 30 min and then metabolically labeled with [35S]methionine (150 µCi/ml; DuPont Pharmaceuticals, Wilmington, DE) for 2 h. After labeling, the cells were pelleted and supernatants were collected. The pellets were washed three times with HBSS and cultured in 4 ml of DMEM medium supplemented with 10% FBS, 2 mM L-glutamine, 50 U/ml of penicillin and streptomycin, and 1 mM sodium pyruvate for various times. At each time point, 1.0 ml of cell suspension was harvested. The cells were pelleted and supernatants were collected. The cell pellets then were solubilized with lysis buffer (10 mM Tris-HCl, pH 7.4, 1% Nonidet P-40 (w/v), 150 mM NaCl, 1 mM EDTA, 1 mg/ml BSA, and 1 mM PMSF). The radioactivity of labeled proteins in cell lysates was determined by TCA precipitation. IL-16 was immunoprecipitated with a rabbit anti-IL-16 serum previously described (13). The precipitated samples then were electrophoresed on 15% SDS/PAGE gels. The gels were fixed and treated with EN3HANCE (DuPont/NEN, Boston, MA) and dried before autoradiography.

To further study the IL-16 processing, pulse-chase experiments were performed in J-V, JIL-16, and JIL-16-signal cell lines in the presence of 10 µg/ml of brefeldin A (BFA) (Sigma, St. Louis, MO) and/or 5 µg/ml of tunicamycin (TM) (Sigma). TCA precipitation, immunoprecipitation, SDS/PAGE, and autoradiography were performed as described above.

Expression of CD4

Surface expression of CD4 molecules on J-V, JIL-16, JIL-16-signal, J-PDZ, and J-PDZ-signal lines was analyzed by FACS as described (13).

ELISA

To quantify the amount of IL-16 inside and outside cells, J-V, JIL-16, JIL-16-signal, J-PDZ, and J-PDZ-signal cell lines (2 x 106 cells) were cultured in 2 ml of high glucose DMEM medium supplemented with 10% FBS, 2 mM L-glutamine, 1 mM sodium pyruvate, 50 U/ml of penicillin, and 50 U/ml of streptomycin for 24 h. The cells then were pelleted and supernatants were collected. The cell pellets were incubated in 400 µl of lysis buffer for 30 min at 4°C. After incubation, lysate debris was pelleted and supernatants were collected. The amount of IL-16 in the culture supernatants and cell lysates was determined by using an IL-16 ELISA kit (BioSource International, Camarillo, CA) according to the manufacturer’s instructions.

HIV-1 infection and p24 assay

Individual or pooled J-V, JIL-16, JIL-16-signal, J-PDZ, and J-PDZ-signal cell lines (1 x 106 cells) were incubated at 37°C for 2 h with HIV-1 strain IIIB (60,000 cpm reverse transcriptase activity) in a final volume of 0.5 ml. Cells then were washed two times with HBSS and resuspended in 6 ml of DMEM medium supplemented with 10% FBS, 2 mM L-glutamine, 1 mM sodium pyruvate, 50 U/ml of penicillin, 50 U/ml of streptomycin and incubated at 37°C for 17–25 days. Every 3 days, 4 ml of cell suspension was harvested and replaced with fresh medium. The harvested cell suspension was pelleted, and 1 ml of supernatant was collected. HIV-1 p24 gag protein was measured by an ELISA kit (DuPont) according to the manufacturer’s instructions. The percent inhibition was calculated as follows: levels of p24 in vector control minus level of p24 in JIL-16, JIL-16-signal, J-PDZ, or J-PDZ-signal divided by level of p24 in vector control.

To test for IL-16 and anti-HIV-1 activity in supernatants, 50 x 106 cells of pooled J-V, JIL-16, and JIL-16-signal lines (at 1 x 106 cells/ml) were cultured in the fresh medium for 4 days. Supernatants were then collected, concentrated 8-fold using Centriprep-10 (Amicon, Beverly, MA), and sterilized through a 0.2-µm Acrodisc filter (Gelman Sciences, Ann Arbor, MI). The amounts of IL-16 in the supernatants were determined by ELISA as described above. The anti-HIV-1 activity in the supernatants was determined as follows: various amounts of concentrated supernatants were cultured with 1.0 x 106 CEM cells (American Type Culture Collection, Manassas, VA) for 2 days in 1.0 ml of fresh RPMI 1640 medium supplemented with 10% FBS, 2 mM L-glutamine, 1 mM sodium pyruvate, 50 U/ml of penicillin, and 50 U/ml of streptomycin. Cells then were infected with HIV-1 strain IIIB (60,000 cpm reverse transcriptase activity) for 2 h, washed two times with HBSS, then suspended (5 x 105 cells) in 2 ml of fresh RPMI 1640 medium together with various concentrated supernatants and incubated at 37 °C for 12 days. Every 2 days, 1.0 ml of cell suspension was harvested and replaced with fresh medium and various concentrated supernatants. The harvested cells were pelleted, and supernatants were collected. The amount HIV-1 p24 gag protein in the supernatants was determined as described above.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Expression of IL-16

Fig. 1Go depicts the four constructs used in the present experiments (IL-16, IL-16-signal, PDZ, and PDZ-signal). The sequence encoding the C-terminal 130 aa of IL-16 and the C-terminal 100 aa of IL-16 (PDZ) were first fused with the sequence encoding the 19 aa of the signal peptide of the human Ig VH-IV gene family (21) and then cloned into the pRC/CMV/TRCenh vector (13). This signal peptide was previously used by us to target anti-HIV-1 gp41 single-chain Fv into secretory pathway compartments (18).



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  		FIGURE 1. Schematic diagram of IL-16 and IL-16-PDZ constructs with and without a signal peptide. The signal peptide comes from the consensus sequence of the human Ig VH-IV gene family (21 ). The TCRenh and the CMV promoter were used to drive the construct (13 ). A putative N-linked glycosylation site (B) is underlined.

 
To study the expression and processing of the C-terminal 130 aa of human IL-16, pulse-chase experiments were performed with stable transfectants. Figs. 2Go, A and B show the processing of IL-16 in the presence or absence of BFA in the cell lines designated JIL-16 and JIL-16-signal. In the JIL-16 line, an 18-kDa species corresponding to the expected size of the C-terminal 130 aa of IL-16 was stably expressed within the cells. At the end of a 24-h chase, some of the 18-kDa species existed as a smaller 15-kDa breakdown intermediate. In contrast, in the JIL-16-signal cell line, a 23-kDa species was found in addition to the 18-kDa species. The half-life of both molecules was <30 min, and the 15-kDa breakdown intermediate was not seen. Examination of the supernatants of the JIL-16 line revealed the presence of the 18-kDa species beginning at 3 h and the 15-kDa intermediate at 24 h. The supernatants of the JIL-16-signal cell line showed diffuse 18-kDa and 23-kDa bands at 0 h, which persisted for 24 h (Fig. 2GoB). At 24 h, 40- to 60-kDa bands were seen in the supernatants of both the JIL-16 and JIL-16-signal cell lines and also in the supernatants of the J-V lines (data not shown). These bands, most likely, represent incompletely processed endogenous IL-16.



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  		FIGURE 2. Pulse-chase analysis of IL-16 proteins in cell lysates (A) and supernatants (B) of stably transfected Jurkat cells with vector alone (J-V), IL-16 (JIL-16), or IL-16-signal (JIL-16-signal) with or without BFA treatment. Cells were labeled with [35S]methionine for 2 h and then chased for 0, 3, 6, and 24 h. Exposure time: A, 2 days; B, 9 days.

 
To further evaluate the processing of the C-terminal 130 aa, JIL-16 and JIL-16-signal cell lines were treated with BFA, which is known to block protein export from the endoplasmic reticulum (ER) to the golgi network (22). As shown in Fig. 2Go, A and B, the treatment with BFA did not significantly alter the processing or release of the 18-kDa species or its 15-kDa breakdown intermediate in the JIL-16 line. However, BFA dramatically altered the processing of IL-16 in the JIL-16-signal cell line. The half-life of both the 18-kDa and 23-kDa species inside the cells was significantly increased (Fig. 2GoA), but little was secreted into the supernatant (Fig. 2GoB). Taken together, these experiments show that despite the absence of a signal peptide, IL-16 is released from the JIL-16 cell line, arguing that IL-16 does not traffic from the ER to the golgi.

Glycosylation of IL-16

Because the C-terminal 130 aa of human IL-16 contains a putative N-linked glycosylation site at position 5–7 (Fig. 1GoB), we suspected that the 23-kDa species observed in the JIL-16-signal cell line might be the glycosylated form of the 18-kDa species. To study this possibility, we performed pulse-chase experiments in the presence of BFA and TM, an inhibitor of N-linked glycosylation. Fig. 3Go shows that in the JIL-16-signal cell line, TM treatment completely eliminated the 23-kDa species resulting in a single 18-kDa species, whereas in the JIL-16 cell line the 23-kDa species did not appear and TM treatment did not result in any band shifts. The increase in size from 18 kDa to 23 kDa suggests that the glycosylation is due to a typical core oligosaccharide (23, 24). This finding further argues that the C-terminal 130 aa of IL-16 in the JIL-16 cell line does not enter into ER, whereas in the JIL-16-signal cell line it does.



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  		FIGURE 3. Pulse-chase analysis of IL-16 proteins in cell lysates of Jurkat cells stably transfected with IL-16 (JIL-16) or IL-16-signal (JIL-16-signal) with or without BFA and/or TM treatment. Cells were labeled with [35S]methionine for 60 min and then chased for 0 and 90 min. Exposure time: 3 days.

 
Quantitation of intracellular and extracellular IL-16

To quantitate the amount of IL-16 in J-V, JIL-16, and JIL-16-signal cell lines, ELISA assays were performed. As shown in Fig. 4GoA, the amount of IL-16 released into supernatants by the JIL-16-signal cell lines in 24 h was 5- to 6-fold greater than that released by the JIL-16 cell lines. Inside the JIL-16-signal cell lines, the amount of IL-16 found at 24 h was one-sixth to one-ninth that found in the JIL-16 cells (Fig. 4GoB). Thus, these studies show that by adding a heterologous signal peptide to the C-terminal 130 aa of human IL-16, the processing pathway of IL-16 is completely altered.



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  		FIGURE 4. Quantitative ELISA analyses of amount of IL-16 synthesized by 1 x 106 cells from six individual J-V, JIL-16, and JIL-16-signal lines in 24 h. A, Supernatants. B, Cell lysates. The mean and SD were calculated from triplicate wells.

 
Inhibition of HIV-1 replication in CD4+ cells

Because CD4 is the receptor for HIV-1 gp120 (25) and this same molecule may be involved in the biological functions of IL-16 (7), we compared the surface expression of CD4 on JV, JIL-16, and JIL-16-signal cell lines. CD4 expression as measured by FACS was essentially the same on all three cell lines (data not shown). To compare the level of resistance of the JIL-16 and JIL-16-signal cell lines to HIV-1 infection, cells were infected with HIV-1 IIIB and p24 levels were measured over 25 days. As seen in Table IGo, HIV-1 replication was inhibited by 90–99% for the first 12 days after infection as compared with J-V controls. For 15 days postinfection, but not thereafter, HIV replication was inhibited in the JIL-16 cells. In contrast, HIV replication was inhibited for up to 25 days in the cells expressing IL-16 with the signal peptide. These experiments were repeated five time with similar results.


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  		Table I. Concentration of p24 in HIV-1-infected J-V, JIL-16, or JIL-16-signal vector

 
In other experiments supernatant from uninfected JIL-16 and JIL-16-signal cells containing up to 3000 pg/ml of IL-16, as well as supernatant of vector control cells, were added in a single dose to fresh CEM cells, which then were infected with HIV. Compared with HIV replication in CEM cells treated with supernatant of vector control cells, HIV replication in CEM cells treated with supernatants from JIL-16 or JIL-16-signal cells was inhibited by up to 80% at 10 and 12 days postinfection.

Expression of the C-terminal 100 aa of IL-16

To study the expression and stability of the C-terminal 100 aa (PDZ) of human IL-16, pulse-chase experiments were performed. As shown in Fig. 5GoA, PDZ was constitutively expressed in both J-PDZ and J-PDZ-signal cell lines. The amount of PDZ within the cells was comparable in the two lines with a half-life of <30 min. PDZ was found in the supernatant of the J-PDZ-signal cell line, but not in the supernatants of J-PDZ cell line. The short half-life of PDZ in both the cell lysates and supernatants argue that the C-terminal 100 aa is much less stable than the C-terminal 130 aa of IL-16 (Fig. 2GoA).



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  		FIGURE 5. A, Pulse-chase analysis of IL-16-PDZ in cell lysates and supernatants of J-PDZ and J-PDZ-signal cell lines. Cells were first labeled with [35S]methionine for 2 h and then chased for 0, 3, and 6 h. Exposure time: 7 days. Quantitative ELISA analyses of amount of PDZ synthesized by 1 x 106 cells from six individual J-V, J-PDZ, and J-PDZ-signal lines in 24 h. B, Supernatants. C, Cell lysates. The mean and SD were calculated from triplicate wells.

 
ELISA was used to quantitate the amount of PDZ inside the cells and supernatants. As seen in Fig. 5Go, nearly equal amounts of PDZ were detected within the J-PDZ and J-PDZ-signal cell lines (i.e., lysates) (Fig. 5GoC), whereas in the supernatants, PDZ was detected in the J-PDZ-signal cell lines, but not in J-PDZ cell lines (Fig. 5GoB).

HIV replication in cells expressing the C-terminal 100 aa of IL-16

To determine whether the C-terminal 100 aa of IL-16 retains anti-HIV-1 activity, the individual J-PDZ and J-PDZ-signal cell lines along with the vector control (J-V) were infected with HIV-1 IIIB. As seen in Table IIGo, HIV-1 replication in the PDZ cell lines was comparable to or even greater than in the cell lines with the vector control. Because very little PDZ is secreted extracellularly (Fig. 5GoB), these findings argue that the expression of the PDZ domain of IL-16 inside cells does not inhibit HIV-1 replication. In contrast, HIV-1 replication in the J-PDZ-signal cell lines was inhibited by as much as 98% at day 8, 11, and 14 postinfection. At day 17 postinfection, inhibition was still at the 90% level. This experiment was repeated twice with similar results. Taken together with the data from Fig. 5Go, these findings argue that the PDZ domain of IL-16 retains anti-HIV-1 activity and that the inhibition of HIV-1 is mediated primarily by extracellularly secreted IL-16.


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  		Table II. Concentration of p24 in HIV-1-infected J-V, J-PDZ, or J-PDZ-signal

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Our studies show that although IL-16 is constitutively expressed both in cells with (IL-16-signal) and without (IL-16) the signal peptide, the processing pathways are totally different (Table IIIGo). With the signal peptide, IL-16 enters the conventional secretory pathway (i.e., ER to golgi), is N-link glycosylated, and quickly released into the culture supernatants. In contrast, without the signal peptide, IL-16 does not enter the conventional secretory pathway, is not glycosylated, and is partially processed into a 15-kDa breakdown intermediate, which together with the 18-kDa precursor is released slowly into the culture supernatants. Adding the signal peptide results in a 5- to 6-fold increase in the amount of IL-16 secreted into the supernatants and a marked reduction in the amount of IL-16 within the cells. In terms of anti-HIV-1 activity, HIV-1 was inhibited in both lines by as much as 99%, but the inhibition in the JIL-16-signal cells was prolonged to 25 days. Thus, by altering the processing pathway and releasing more IL-16 into supernatants, the anti-HIV-1 activity of IL-16 is prolonged, arguing that the anti-HIV-1 activity of IL-16 is mediated by extracellular, not intracellular, IL-16. This argument is further strengthened by the fact that cells that fail to secrete IL-16 (i.e., J-PDZ) show little if any resistance to HIV replication (Fig. 5Go and Table IIGo). Treatment of cells with IL-16 supernatants (added every other day throughout the experiment) rendered the cells partially resistant to HIV-1 (i.e., 80%), but the resistance fell far short of that seen in cells actually secreting IL-16 (i.e., 99% resistance). This is thought to be due to the continuous presence of IL-16 in the supernatants of cells constitutively expressing and secreting the molecule.


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  		Table III. Comparison of protein processing and anti-HIV-1 activity of the C-terminal 130 aa of IL-16 with or without a signal peptide in CD4+ T cells

 
The experiment with BFA and TM showed that without a signal peptide IL-16 did not traffic through the conventional secretory pathway. The processing of the C-terminal 130 aa of IL-16 in our study (Figs. 2Go and 3Go) resembles the processing of the IL-16 precursor in activated CD8+ T cells. In primary CD8+ T cells, the IL-16 precursor, which lacks a signal peptide at the N terminus, is constitutively synthesized in the cytosolic compartment (8, 9). Upon activation by Ags, mitogens, histamine, or serotonin, the precursor of IL-16 is processed and released from cells (9, 10). Recently, it was shown that caspase-3 is involved in processing of the IL-16 precursor (11). The 15-kDa breakdown intermediate observed in our study, most likely, is the mature form of IL-16 (2, 8). Based upon the molecular size of the breakdown intermediate, the cleavage site is probably at aspartate residue 9 in the C-terminal 130 aa of IL-16 or at aspartate residue 510 of the IL-16 precursor (7, 8, 11). If this turns out to be the case, the 15-kDa breakdown intermediate is the C-terminal 121 aa of IL-16. The release of processed IL-16 appears to be independent of the activity of caspase-3 because the C-terminal 130 aa of IL-16 is released before it is processed into the C-terminal 121 aa (Fig. 2GoB).

We also have compared the protein processing and anti-HIV-1 activity of the C-terminal 100 aa (PDZ-like motif) of human IL-16 with or without a signal peptide in human CD4+ cells. Although comparable amounts of PDZ were constitutively expressed inside both J-PDZ and J-PDZ-signal cells, PDZ was detected only in the supernatants of the J-PDZ-signal cells and not in the supernatants of the J-PDZ cells (Fig. 5Go). When PDZ was expressed only inside cells (J-PDZ), it did not have any inhibitory effect on HIV-1 replication (Table IIGo). In contrast, when PDZ was expressed and released into the supernatants (J-PDZ-signal), it profoundly inhibited HIV-1 replication. These results show that the C-terminal 100 aa (PDZ motif) of IL-16 retains anti-HIV-1 activity and that the activity is mediated by extracellular IL-16.

The PDZ-like motif has been found in over 50 proteins that direct intracellular protein-protein interactions. Our finding that the PDZ-like motif of IL-16 released into the supernatant renders cells resistant to HIV-1 suggests that, in addition to its role in signaling, adhesion, and ion channel clustering (16), PDZ-like motif-containing proteins also may be involved in extracellular ligand-receptor interaction. Recently, two PDZ-like domains (hDlg-3 and PSD-95-3) have been crystallized (26, 27). Structural analyses showed that both domains consist of a five-stranded anti-parallel ß-barrel flanked by three {alpha}-helices with a hydrophobic pocket on the surface. It is known that distinct PDZ-like motifs recognize unique C-terminal amino acid residues (28). Insight into IL-16 structure-function relationship might be obtained by modeling the three-dimensional structure of the C-terminal 100 aa of IL-16 based on known structural PDZ-like domains. In fact, just recently, using an nuclear magnetic resonance approach, Muhlhahn et al. showed that IL-16 is similar to hDlg-3 in terms of protein folding at the {alpha}-helices and anti-parallel ß-barrels, but because of an occluded peptide-binding site, IL-16 did not exhibit the expected peptide-binding properties of PDZ-like domains (29).

In addition to IL-16, there are a number of other proteins that act extracellularly and yet are synthesized intracellularly without a signal peptide. Among them are IL-1{alpha} and ß (30, 31, 32), IL-18 (33, 34), fibroblast growth factors (acidic and basic) (35, 36), yeast a-factor (37), and bacterial hemolysins (38). While the mechanisms for the secretion of the yeast a-factor and the bacterial hemolysins have been characterized (39, 40), the mechanism for the secretion of mammalian proteins without signal peptides remains unresolved. Of particular interest, all these proteins are very potent agents and exert their biological activity at pico- and nanomolar concentrations. Because of their potent activity, release from cells must require tight control. It is tempting to speculate that there might be a common mechanism controlling their release, and future studies directed at elucidating the export apparatus should prove rewarding.


   Acknowledgments
 
We thank Nikki Sumwalt for excellent editorial assistance.


   Footnotes
 
1 Address correspondence and reprint requests to Dr. Paul Zhou at the current address: Southwest Foundation for Biomedical Research, Department of Virology and Immunology, P.O. Box 760549, San Antonio, TX 78245-0549. Back

2 Abbreviations used in this paper: J-V, Jurkat transfectant containing vector alone; TCRenh, TCR enhancer; JIL-16, Jurkat transfectant containing pRC/CMV/TCRenh-IL-16; JIL-16-signal, Jurkat transfectant containing pRC/CMV/TCRenh-signal(Ig)-IL-16; J-PDZ, Jurkat transfectant containing pRC/CMV/TCRenh-PDZ; J-PDZ-signal, Jurkat transfectant containing pRC/CMV/TCRenh-signal(Ig)-PDZ; BFA, brefeldin A; TM, tunicamycin; ER, endoplasmic reticulum. Back

Received for publication January 26, 1999. Accepted for publication May 6, 1999.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Center, D. M., W. Cruikshank. 1982. Modulation of lymphocyte migration by human lymphokines. I. Identification and characterization of chemoattractant activity for lymphocytes. J. Immunol. 128:2563.[Medline]
  2. Cruikshank, W., D. M. Center. 1982. Modulation of lymphocyte migration by human lymphokines. II. Purification of a lymphotatic factor (LCF). J. Immunol. 128:2569.[Medline]
  3. Cruikshank, W. W., J. S. Berman, A. C. Theodore, J. Bernardo, D. M. Center. 1987. Lymphocyte activation of T4+ T lymphocytes and monocytes. J. Immunol. 138:3817.[Abstract]
  4. Rand, T., W. W. Cruikshank, D. M. Center, P. F. Weller. 1991. CD4-mediated stimulation of human eosinophils: lymphocyte chemoattractant factor and other CD4-binding ligands elicit eosinophil migration. J. Exp. Med. 173:1521.[Abstract/Free Full Text]
  5. Cruikshank, W. W., J. L. Greenstein, A. C. Theodore, D. M. Center. 1991. Lymphocyte chemoattractant factor induces CD4-dependent intracytoplasmic signaling in lymphocytes. J. Immunol. 146:2928.[Abstract]
  6. Center, D. M., H. Kornfeld, W. W. Cruikshank. 1996. Interleukin 16 and its function as a CD4 ligand. Immunol. Today 17:476.[Medline]
  7. Cruitshank, W. W., D. M. Center, N. Nisar, M. J. Wu, B. Natke, A. C. Theodore, H. Kornfeld. 1994. Molecular and functional analysis of a lymphocyte chemoattractant factor: association of biological function with CD4 expression. Proc. Natl. Acad. Sci. USA 91:5109.[Abstract/Free Full Text]
  8. Baier, M., N. Bannert, A. Werner, K. Lang, R. Kurth. 1997. Molecular cloning, sequence, expression, and processing of the interleukin 16 precursor. Proc. Natl. Acad. Sci. USA 94:5273.[Abstract/Free Full Text]
  9. Laberge, S., W. W. Cruikshank, H. Kornfeld, D. M. Center. 1995. Histamine-induced secretion of lymphocyte chemoattractant factor from CD8+ T cells is independent of transcription and translation: evidence for constitutive protein synthesis and storage. J. Immunol. 155:2902.[Abstract]
  10. Laberge, S., W. W. Cruikshank, D. J. Beer, D. M. Center. 1996. Secretion of IL-16 (lymphocyte chemoattractant factor) from serotonin-stimulated CD8+ cells in vitro. J. Immunol. 156:310.[Abstract]
  11. Zhang, Y., D. M. Center, D. M. H. Wu, W. W. Cruikshank, Y. Yuan, D. W. Andrew, H. Kornfeld. 1998. Processing and activation of pro-interleukin-16 by caspase-3. J. Biol. Chem. 273:1144.[Abstract/Free Full Text]
  12. Baier, M., A. Wermer, N. Bannert, K. Metzner, R. Kurth. 1995. HIV supression by interleukin-16. Nature 378:563.[Medline]
  13. Zhou, P., S. Goldstein, K. Devadas, D. Tewari, A. L. Notkins. 1997. Human CD4+ cells transfected with IL-16 cDNA are resistant to HIV-1 infection: inhibition of mRNA expression. Nat. Med. 3:659.[Medline]
  14. Maciaszek, J. W., N. A. Parada, W. W. Cruikshank, D. M. Center, H. Kornfeld, G. A. Viglianti. 1997. IL-16 represses HIV-1 promoter activity. J. Immunol. 158:5.[Abstract]
  15. Bazan, J. F., T. J. Schall. 1996. Interleukin-16 or not?. Nature 381:29.[Medline]
  16. Craven, S. E., D. S. Bredt. 1998. PDZ proteins organize synaptic signaling pathway. Cell 93:495.[Medline]
  17. Ponting, C. P., C. Phillip. 1995. DHR domains in syntrophins, neuronal NO synthases and other intracellular proteins. Trends Biochem. Sci. 20:102.[Medline]
  18. Zhou, P., S. Goldstein, K. Devadas, D. Tewari, A. L. Notkins. 1998. Cells transfected with a non-neutralizing antibody gene are resistant to HIV infection: targeting the endoplasmic reticulum and transgolgi network. J. Immunol. 160:1489.[Abstract/Free Full Text]
  19. Zhou, P., H. Cao, M. Smart, C. S. David. 1993. Molecular basis of genetic polymorphism in major histocompatibility complex-linked proteasome gene (LMP-2). Proc. Natl. Acad. Sci. USA 90:2681.[Abstract/Free Full Text]
  20. Clackson, T., H. R. Hoggenboom, A. D. Griffiths, G. Winter. 1991. Making antibody fragments using phage display libraries. Nature 352:624.[Medline]
  21. Lee, K. H., F. Matsuda, T. Kinashi, M. Kodaira, T. Honjo. 1987. A novel family of variable region genes of the human immunoglobulin heavy chain. J. Mol. Biol. 195:761.[Medline]
  22. Lippincott-Schwartz, J., L. C. Yuan, J. S. Bonifacino, R. D. Klausner. 1989. Rapid redistribution of golgi proteins into the ER in cells treated with brefeldin A: evidence for membrane cycling from golgi to ER. Cell 56:801.[Medline]
  23. Tanner, W., L. Lehle. 1987. Protein glycosylation in yeast. Biochem. Biophys. Acta 906:81.[Medline]
  24. Kukuruzinska, M. A., M. L. E. Bergh, B. J. Jackson. 1987. Protein glycosylation in yeast. Annu. Rev. Biochem. 56:915.[Medline]
  25. Dalgleish, A. G., P. C. Beverley, P. R. Clapham, D. H. Crawford, M. F. Greaves, R. A. Weiss. 1984. The CD4 (T4) antigen is an essential component of the receptor for the AIDS retrovirus. Nature 312:763.[Medline]
  26. Doyle, D. A., A. Lee, J. Lewis, E. Kim, M. Sheng, R. Mackinnon. 1996. Crystal structures of a complexed and peptide-free membrane protein-binding domain: molecular basis of peptide recognition by PDZ. Cell 85:1067.[Medline]
  27. Cabral, J. H., C. Petosa, M. J. Sutcliffe, S. Raza, O. Byron, F. Poy, S. M. Marfatia, A. H. Chishti, R. C. Liddington. 1996. Crystal structure of a PDZ domain. Nature 382:649.[Medline]
  28. Songyang, Z., A. S. Fanning, C. Fu, S. M. Marfatia, A. H. Chishti, A. Crompton, A. C. Chan, J. M. Anderson, L. C. Cantley. 1997. Recognition of unique carboxyl-terminal motifs by distinct PDZ domains. Science 275:73.[Abstract/Free Full Text]
  29. Muhlhahn, P., M. Zweckstetter, J. Georgescu, C. Ciosto, C. Renner, M. Lanzendorfer, K. Lang, D. Ambroius, M. Baier, R. Kurth, T. A. Holak. 1998. Structure of interleukin 16 resembles a PDZ domain with an occluded peptide binding site. Nat. Struct. Biol. 5:682.[Medline]
  30. Auron, P. E., A. C. Webb, L. J. Rosenwasser, S. F. Mucci, A. Rich, S. M. Wolff, C. A. Dinarello. 1984. Nucleotide sequence of human monocyte interleukin 1 precursor cDNA. Proc. Natl. Acad. Sci. USA 81:7907.[Abstract/Free Full Text]
  31. March, C. J., B. Mosley, A. Larsen, D. P. Cerretti, G. Braedt, V. Price, S. Gillis, C. S. Henney, S. R. Kronheim, K. Grabstein, P. J. Conlon, T. P. Hopp, D. Cosman. 1985. Cloning, sequence and expression of two distinct human interleukin-1 complementary DNAs. Nature 315:641.[Medline]
  32. Lomedico, P. T., U. Gubler, C. P. Hellmann, M. Dukovich, J. G. Giri, Y.-C. E. Pan, K. Collier, R. Seminnonow, A. O. Chua, S. B. Mizel. 1984. Cloning and expression of murine interleukin-1 cDNA in Escherichia coli. Nature 312:458.[Medline]
  33. Nakamura, K., H. Okamura, K. Nagata, T. Komatsu, T. Tamura. 1993. Purification of a factor which provides a costimulatory signal for {gamma} interferon production. Infect. Immun. 61:64.[Abstract/Free Full Text]
  34. Okamura, H., T. Komatsu, M. Yatsudo, A. Hakara, T. Tanimoto, K. Torigoe, T. Okura, Y. Nukada, K. Hattori, K. Akita, M. Namba, F. Tanabe, K. Konishi, S. Fukuda, M. Kurimoto. 1995. Cloning of a new cytokine that induces IFN-{gamma} production by T cells. Nature 378:88.[Medline]
  35. Abraham, J. A., A. Mergia, J. L. Whang, A. Tumolo, J. Friedman, K. A. Hjerrild, D. Gospodarwicz, J. C. Fiddes. 1986. Nucleotide sequence of a bovine clone encoding the angiogenic protein, basic fibroblast growth factor. Science 233:545.[Abstract/Free Full Text]
  36. Jaye, M., R. Howk, W. Burgess, G. A. Ricca, I.-M. Chiu, M. W. Ravera, S. J. O’Brien, W. S. Modi, T. Maciag, W. N. Drohan. 1986. Human endothelial cell growth factor: cloning, nucleotide sequence, and chromosome location. Science 233:541.[Abstract/Free Full Text]
  37. Schafer, W. R., R. Kim, R. Sterne, J. Thorner, S.-H. Kim, J. Rie. 1989. Genetic and pharmacological suppression of oncogenic mutations in RAS genes of yeast and humans. Science 245:379.[Abstract/Free Full Text]
  38. Felmlee, T., S. Pellett, E.-Y. Lee, R. A. Welch. 1985. Escherichia coli hemolysin is released extracellularly without cleavage of a signal peptide. J. Bacteriol. 163:88.[Abstract/Free Full Text]
  39. Koronakis, V., E. Koronakis, C. Hughes. 1989. Isolation and analysis of the C-terminal signal directing export of Escherichia coli hemolysin protein across bacterial membrane. EMBO J. 8:595.[Medline]
  40. McGrath, J. P., A. Varshavsky. 1989. The yeast STE6 gene encodes a hemologue of the mammalian multidrug resistance P-glycoprotein. Nature 340:400.[Medline]



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