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Suppression of human immunodeficiency virus type 1 replication by arginine deiminase of Mycoplasma arginini

¹ Department of Immunotherapeutics, Graduate School, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8519, Japan
² Japanese Foundation for AIDS Prevention, Tokyo 105-0001, Japan

Correspondence
Mari Kannagi
kann.impt{at}tmd.ac.jp

	ABSTRACT

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It was found previously that human immunodeficiency virus type 1 (HIV-1)-irrelevant CD8⁺ cytotoxic T lymphocytes (CTLs) from uninfected donors suppressed HIV-1 replication in a cell-contact-dependent manner. However, one of these CTL lines (CTL-3) also significantly suppressed HIV-1 replication through its supernatant. Here, the suppressive fraction from CTL-3 supernatant was purified and analysed by mass spectrometry. A protein band specific for the suppressive fraction was identified as arginine deiminase from Mycoplasma arginini, which catalyses the hydrolysis of arginine to citrulline. Addition of L-arginine or the use of antibiotics against mycoplasma restored supernatant-mediated but not cell-contact-dependent suppression of HIV-1 replication by CTL-3, clearly indicating that arginine deiminase of M. arginini in the supernatants suppressed HIV-1 replication, which is independent of CD8⁺ T-cell-mediated HIV-1 suppression via cell contact. Arginine deiminase is known to be a chemotherapeutic agent against arginine-requiring tumours and these results suggest that it also has potential application in antiviral therapy.

	MAIN TEXT

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CD8⁺ cells including human immunodeficiency virus type 1 (HIV-1)-specific cytotoxic T lymphocytes (CTLs) play important roles in controlling HIV-1 infection (Borrow et al., 1994; McMichael & Rowland-Jones, 2001). It has been shown that CD8⁺ cells of asymptomatic carriers produce an unknown CD8⁺ T-cell antiviral factor that can suppress HIV-1 replication at a transcriptional level without causing cell death (Mackewicz et al., 1995).

We have demonstrated previously that anti-HIV-1 activities of CD8⁺ cells of asymptomatic carriers exhibit both major histocompatibility complex (MHC) I-restricted and -unrestricted suppression (Kannagi et al., 1990; Ohashi et al., 1999), and that HIV-1-irrelevant CD8⁺ CTLs derived from uninfected donors also inhibit X4 and R5 HIV-1 replication (Liu et al., 2003). Therefore, we hypothesized that MHC I-unrestricted suppression of HIV-1 replication might be a common property of CD8⁺ CTLs, regardless of HIV-1 infection in the host. However, in our system, such CD8⁺ cell-mediated suppression required direct contact between CD8⁺ cells and infected cells.

Whilst investigating CD8⁺ cell-mediated HIV-1 suppression, we established four allo-specific CD8⁺ CTL lines, CTL-1 (Liu et al., 2003), CTL-2, CTL-3 and CTL-4, from four uninfected healthy donors by stimulating peripheral blood mononuclear cells with mitomycin C (MMC)-treated Raji (Pulvertaft, 1964) cells in a long-term culture in the presence of recombinant human interleukin-2 (rhIL-2) as described previously (Liu et al., 2003). These CTLs were not cytotoxic to autologous CD4⁺ cells, but significantly suppressed HIV-1 replication in HIV-1-infected autologous CD4⁺ cells when directly co-cultured.

Among these CTL lines, we found that culture supernatants of CTL-3 suppressed HIV-1 replication in addition to cell-mediated suppression. In the present study, we purified and identified the suppressive factor in the supernatant of CTL-3 by serial high-performance liquid chromatography (HPLC) and mass spectrometry.

Fig. 1 shows representative data of HIV-1 suppression by the established allo-specific CD8⁺ CTL lines. CTLs from both the CTL-2 and CTL-3 lines markedly suppressed HIV-1 replication when directly co-cultured with HIV-1-infected autologous CD4⁺ cells, whereas culture supernatants from CTL-3 but not CTL-2 suppressed HIV-1 replication (Fig. 1a). Culture supernatants of CTL-3 suppressed replication of both X4 HIV-1 strain NL4-3 (Adachi et al., 1986) and R5 HIV-1 strain JR-CSF (Koyanagi et al., 1987) (Fig. 1b). CTL-2 and CTL-3 culture supernatants did not alter the viability of CD4⁺ T cells during 4 days of culture.

View larger version (10K): [in this window] [in a new window]   Fig. 1. Cell- and soluble factor-mediated suppression of HIV-1 replication by CD8+ CTLs. (a) Cells (3x105 cells per well) (filled bar) or culture supernatants (50 % vol.) (open bar) of two allo-specific CD8+ CTL lines (CTL-2 and -3) established from two uninfected donors were co-cultured with phytohaemagglutinin-stimulated autologous CD4+ cells (105 cells per well) infected with HIV-1 NL4-3 for 2 h in vitro. After culturing for 4 days, the amount of HIV-1 p24 in the supernatants was measured by ELISA (Cellular Products). The results indicate the amounts of HIV-1 p24 (%) compared with infected CD4+ cells alone. The CTL culture supernatants used were prepared as fetal bovine serum (FBS) free and supplemented with 10 % FBS for use. To prepare FBS-free supernatants, CTLs were washed 24 h after stimulation with MMC-treated Raji cells and the supernatants were harvested following another 48 h of culture at a concentration of 5x106 cells ml–1 in FBS-free RPMI 1640 in the presence of 50 U rhIL-2 ml–1. The supernatants were passed through 0.22 µm filters before use. (b) Similarly prepared culture supernatants of CTL-2 and -3 were added at 50 % concentrations into acutely HIV-1 NL4-3 (filled bar)- or JR-CSF (open bar)-infected CD4+ cell cultures for 4 days and the amount of HIV-1 p24 in the supernatants was measured by ELISA. The results indicate the mean±SD of duplicate wells. Similar results were obtained when CTL supernatants prepared in the presence of FBS were used. The viability of CD4+ T cells after 4 days of culture with medium or with CTL-2 and CTL-3 culture supernatants was 95.37±0.13, 97.84±0.15 and 98.64±0.08 %, respectively, as determined by flow cytometry following staining with fluorescein isothiocyanate–annexin V and 7-aminoactinomycin D.

View larger version (44K): [in this window] [in a new window]   Fig. 2. Purification and identification of an HIV-1-suppressive factor in CTL-3 culture supernatants by HPLC. (a) FBS-free culture supernatants of CTL-3 prepared as described in Fig. 1 legend were concentrated with Amicon Ultra-4 (Millipore), applied to an anion-exchange column (UNO-Q1; Bio-Rad) and eluted with a linear gradient (0–1.0 M NaCl) in 10 mM potassium phosphate buffer at a flow rate of 1 ml min–1 using an HPLC system (BioCAD; Applied Biosystems). Protein peaks, conductivity and fraction numbers are indicated. (b) Each fraction was dialysed against PBS, cleared through a 0.22 µm filter, supplemented with 10 % FBS and added to an HIV-1 NL4-3-infected CD4+ cell culture at a concentration of 50 %. The same volume of PBS with 10 % FBS was used as a control (open bar). After 4 days of incubation, the amount of HIV-1 p24 in the culture supernatants was measured by ELISA. The results indicate the mean±SD of duplicate wells. (c) Fraction #26 separated by anion-exchange HPLC was further subjected to gel filtration on a TSK gel 2000 column (Tosoh) using HPLC at a flow rate of 0.5 ml min–1 in PBS. Protein peaks, conductivity and fraction numbers are indicated. (d) Each gel-filtrated HPLC fraction was evaluated for HIV-1-suppressive activity as described in (b). (e) Fractions #26-13, -14 and -15, separated as described in (c) from two independently prepared samples, were subjected to 7.5 % SDS-PAGE at a constant current of 24 mA per gel for 5 h. The proteins were visualized by silver staining (ProteoSilver Plus Silver Stain kit; Sigma-Aldrich). Similarly purified fractions from culture supernatants containing MMC-treated Raji cells alone served as controls. The arrow at 45 kDa indicates the bands specific for the fractions with HIV-1-suppressive activity. (f) The protein band at 45 kDa was excised from the SDS-polyacrylamide gel, digested with lysyl endopeptidase and subjected to MALDI-TOF-MS using an Ultraflex TOF/TOF mass spectrometer (Bruker Daltonics) as described previously (Yamagata et al., 2002). The MS spectrum of this protein as shown was analysed by MASCOT software (Matrix Science) with the NCBInr database and identified as arginine deiminase from M. arginini.

Next, HIV-1-suppressive fractions #26-14 and -15, serially purified by anion-exchange and gel-filtrated HPLC from CTL-3 supernatants, were resolved by 7.5 % SDS-PAGE. As a control, similarly purified fractions from culture supernatants of MMC-treated Raji cells were used. As shown in Fig. 2(e), silver staining showed protein bands at 45 kDa that were observed only in #26-14 and -15, and not in the control fractions. The 45 kDa protein bands were cut from a separately prepared negatively stained gel (Wako) and subjected to matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS).

Fig. 2(f) demonstrates the MS spectrum of peptides extracted from the 45 kDa band after digestion with lysyl endopeptidase, obtained using an Ultraflex TOF/TOF mass spectrometer (Kristensen et al., 2000; Yamagata et al., 2002). Several peaks were analysed further by MS/MS (data not shown). Computer analysis of the MS and MS/MS spectra using MASCOT software (Matrix Science) with the NCBInr database identified the protein as arginine deiminase from Mycoplasma arginini. The results strongly indicated that the CTL-3 cells had been contaminated by M. arginini, although growth of the CTL-3 cells was not affected.

Finally, we examined whether the suppressive effects of CTL-3 supernatants on HIV-1 replication were attributed to arginine deiminase. Arginine deiminase is a mycoplasma enzyme that catalyses the imine hydrolysis of arginine to citrulline and ammonia. When L-arginine (10 mM) was added to an HIV-1-infected CD4⁺ cell culture together with CTL-3 culture supernatant, the suppression of HIV-1 replication was almost completely restored (Fig. 3a), clearly indicating that the suppressive effects were mediated mostly by arginine deiminase. L-Glysine (10 mM) as a control showed no effect. In addition, treatment of the CTL-3 culture with antibiotic MC210 against mycoplasma for approximately 2 weeks abolished the HIV-1-suppressive activity of the culture supernatants (Fig. 3a).

View larger version (16K): [in this window] [in a new window]   Fig. 3. L-Arginine and MC210 restore the supernatant- but not cell-mediated suppression of HIV-1 replication by CTL-3. (a) Culture supernatants from CTL-3 cells and CTL-3 cells treated with an antibiotic against mycoplasma (MC210; Dainippon Pharmaceutical) for 2 weeks (CTL-3/MC210) were added to an acutely HIV-1 NL4-3-infected CD4+ cell culture in the absence (filled bar) or presence of 10 mM L-arginine (shaded bar) or L-glycine (open bar). HIV-1 p24 concentration in the supernatants was measured by ELISA 4 days after infection. (b) CTL-3 or CTL-3/MC210 cells were directly co-cultured with phytohaemagglutinin-stimulated autologous CD4+ cells (105 cells per well) acutely infected with HIV-1 NL4-3 at a CTL : CD4+ cell ratio of 3 in the absence (filled bar) or presence (shaded bar) of 10 mM L-arginine for 4 days. The amount of HIV-1 p24 in the supernatants was measured by ELISA. (c) Culture supernatants or cells from another mycoplasma-free CTL line (CTL-4) were co-cultured with HIV-1 NL4-3-infected autologous CD4+ cells in the absence (filled bar) or presence of 10 mM L-arginine (shaded bar) or L-glycine (open bar) for 4 days and HIV-1 p24 concentration in the supernatants was measured. The results indicate the mean±SD of duplicate wells.

Our results clearly indicate that arginine deiminase from M. arginini suppresses HIV-1 replication in CD4⁺ cells in vitro. Although arginine is a non-essential amino acid for humans and mice, some cancers have an elevated requirement for arginine. Arginine deiminase inhibits the growth of arginine-requiring tumours such as human melanomas and hepatocellular carcinoma in vitro and in vivo, suggesting its potential use as a chemotherapeutic reagent (Curley et al., 2003; Ensor et al., 2002).

Mycoplasma contamination of HIV-1-infected culture causes various effects in vitro, such as enhancement of the cytopathic effects associated with HIV-1 replication (Lo et al., 1991), inhibition of CD4 expression and gp120 binding (O'Toole & Lowdell, 1990) and apparent reduction of reverse transcriptase activity, probably due to nuclease activity (el-Farrash et al., 1994; Shang et al., 1995; Vasudevachari et al., 1990). Mycoplasma penetrans isolated from HIV-1-infected individuals potentially activates T lymphocytes and HIV-1 replication, suggesting its contribution to disease progression (Sasaki et al., 1995). This variety of effects might partly be due to the variety of Mycoplasma species.

In the present study, we identified arginine deiminase as a suppressive factor of HIV-1 replication. Although we found it in a CD8⁺ CTL line possessing HIV-1-suppressive activity, only supernatant-mediated and not cell-contact-dependent suppression of HIV-1 was attributed to arginine deiminase. The precise mechanisms of arginine deiminase-mediated inhibition of the HIV-1 replication cycle remain to be clarified.

	ACKNOWLEDGEMENTS

 
We thank ProPhoenix Co. Ltd (Higashi-Hiroshima, Japan) for analysis of protein bands by MALDI-TOF-MS. This work was supported by grants from the Ministry of Health, Welfare and Labor, Japan.

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Received 21 September 2005; accepted 31 January 2006.

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