Laboratory of Virology1, Laboratory of HaematologyOncology2 and Laboratory of Clinical Biochemistry3, Istituto Superiore di Sanità, Viale Regina Elena 299, 0061 Roma, Italy
Author for correspondence: Maurizio Federico. Fax +39 06 49903002. e-mail federico{at}iss.it
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Abstract |
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Introduction |
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The nef gene is expressed very early and large amounts of Nef protein are synthesized during the HIV replication cycle. Expression of nef is critical for in vitro virus replication in resting peripheral blood lymphocytes (PBLs) (Aiken & Trono, 1995 ; Chowers et al., 1994
; Miller et al., 1994
; Spina et al., 1994
) as well as for in vivo pathogenesis. A role for Nef therein has been deduced from results obtained in animal models, such as monkeys infected with nef-deleted SIV strains (Kestler et al., 1991
) and nef-transgenic mice (Hanna et al., 1998
). In addition, molecular studies on HIV isolates from seropositive cohorts indicated the possibility that detection of deleted/mutated nef genes represents a marker for non-progression (Brambilla et al., 1999
; Deacon et al., 1995
; Kirchoff et al., 1995
), although results inconsistent with this hypothesis have been published also (Huang et al., 1995
; Michael et al., 1995
). Nef typically induces down-regulation of both CD4 (Aiken et al., 1994
; Bandres et al., 1995
) and class I major histocompatibility complex (MHC) molecules (Schwartz et al., 1996
) through mechanisms that have been described in great detail (Greenberg et al., 1998a
; Piguet et al., 1999
). Data regarding Nef functions have been obtained mostly by analysing the effects of the endogenously expressed protein. Conversely, relatively few data on the effects of extracellular Nef have been published so far. Indeed, Nef is a cytoplasmic protein that associates with membranes through N-terminal myristoylation (Allan et al., 1985
): thus shedding of Nef from infected cells is not an obvious possibility. However, both antibodies (Abs) and cytotoxic T-lymphocytes against Nef have been found in a large proportion of infected individuals (Ameisen et al., 1989
; Bahraoui et al., 1990
). This suggests that in vivo Nef is processed and presented by APC, as the result of uptake of extracellular Nef possibly released by infected apoptotic cells. In this paper, we report that recombinant Nef protein (rNef) from the T-cell (T)-tropic NL4-3 HIV strain is able to enter human MDM and induce CD4 down-regulation. This correlates with a strong inhibition of HIV replication. We also show that rNef treatment of lymphocyte (T)- and macrophage (M)-tropic HIV doubly infected CD4+ lymphocyteMDM co-cultures led to preferential replication of the T-tropic HIV strain. These observations support the hypothesis that the HIV replication block induced by extracellular Nef in MDM could contribute to the switching from M- to T-tropic HIV strains frequently observed during AIDS progression.
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Methods |
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Monocytopoietic unilineage liquid cultures were obtained from CD34+ human progenitor cells (HPC) purified by negative selection from PBMC of healthy donors as described (Chelucci et al., 1999 ). HPC were seeded at 105 cells/ml and induced to monocytic differentiation in 20% FCS medium containing 1 ng/ml interleukin-6, 500 U/ml monocyte-colony stimulation factor and 100 ng/ml FLT3-ligand. Morphology analyses were performed by cytocentrifugation onto glass slides and staining with MayGrunwaldGiemsa (Sigma).
PBLs were obtained from PBMC adherence supernatants. Purified CD4+ lymphocytes were obtained by immunodepletion of PBMC with anti-CD8, -CD14 and -CD19 MAbs (Dako) followed by treatment with anti-mouse IgG Dynabeads. Lymphocyte expansion was performed by activating cells with 0·5 µg/ml phytohaemagglutinin (Sigma) and cultivating them in the presence of 100 U/ml interleukin-2 (Roche).
HeLaCD4 and 293 cell lines were grown in Dulbeccos modified minimum essential medium supplemented with 10% heat-inactivated FCS. HSB-2 (Ablashi et al., 1987 ), C8166 and CEMss cells were grown in RPMI 1640 supplemented with 10% heat-inactivated FCS.
The lymphocyte activation assay was performed by cultivating CD4+ lymphocytes for 2 days in supernatants from rNef-treated MDM. Then, cells were washed, seeded at 3x106/ml in 100 µl RPMI20% FCS in the presence of 2 µCi [methyl-3H]thymidine (2 Ci/mmol; Amersham) and incubated for 16 h at 37 °C. Finally, cells were washed, lysed in 1% SDS buffer and amounts of TCA-insoluble 3H-labelled macromolecules were determined by liquid scintillation counting.
Virus preparations, infections and detection.
Both ADA and BaL virus preparations were obtained as supernatants of peripheral blood (PB) MDM infected 7 days after purification and plating. Both NL4-3 and 89.6 virus preparations were obtained as supernatants of 293 cells 48 h after transfection of respective molecular clones by the calcium phosphate method (Wigler et al., 1979 ). The T-tropic NL4-3 strain was titred by scoring the syncytium number on C8166 cells 5 days after infection (Federico et al., 1993
). Infectivity titres of supernatants containing M- or dual-tropic HIV strains were evaluated by the limiting dilution method 10 days after infection of day 7 MDM. HIV p24 contents in supernatants were measured by quantitative ELISA (Abbott). Supernatants obtained 48 h after co-transfection of 293 cells with vectors expressing ADA HIV-1 and vesicular stomatitis virus envelope glycoprotein (VSV-G) (molar ratio 1:3) served as source of pseudotyped ADA virions. One ng/105 cells of either parental or pseudotyped ADA HIV-1 was used to infect day 7 MDM. Virus entry was evaluated 48 h thereafter by scoring the number of cells expressing HIV Gag-related products in cytofluorimetric analyses.
rNef preparation and FITC labelling.
rNef was obtained as a 6x His tagged fusion protein. The nef gene from HIV strain NL4-3 (Adachi et al., 1986 ) was amplified by PCR and cloned in-frame with the 6x His tag into the 5' BamHI/3' SalI sites of pQE 30 vector (Qiagen). Sequences of oligoprimers used in PCR are 5' GTTGGATCCCATAAGATGGGTGGCAAGTGG 3' for the 5' end (to obtain rNef mutated in the myristoylation acceptor the underlined codon was changed to GCT, leading to a Gly
Ala substitution), and 5' CTCGTCGACTCAGCAGTTCTTGAAGTACTC 3' for the 3' end. The correct reading frame of the inserted fragment was checked by sequencing (Sequenase II kit; US Biochemical). rNef was purified from bacterial lysates by using NiNTA resin (Qiagen) following the manufacturers recommendations. rNef was eluted stepwise with 100, 250 and 500 mM imidazole, and the fractions were collected and analysed by 12% SDSPAGE. rNef-containing fractions were pooled and dialysed extensively against 1x PBS to remove the urea completely. Finally, 10 µg aliquots of rNef were analysed by SDSPAGE and found to be devoid of non-specific bands on Coomassie brilliant blue staining. Recombinant protein preparations were scored as negative for the presence of bacterial endotoxins by using the Limulus amoebocyte lysate assay. Preparation of 6x His Uvp1 protein (a 21·5 kDa plasmidic DNARNA invertaseresolvase) was as previously described (Tosini et al., 1998
).
Crystallized BSA and rNef were labelled by reacting each protein with dissolved FITC (Pierce) following the manufacturers recommendations. Gel filtration chromatography was then performed on the reaction products and recovery of FITC-conjugated protein evaluated by quantitative Western blot analysis as described (dAloja et al., 1998 ).
rNef immunodepletion.
To ensure a total and specific depletion of rNef, complete medium supplemented with 100 ng/ml rNef was incubated for 8 h at 4 °C with a 1:50 dilution of a cocktail containing six different mono- and polyclonal anti-Nef Abs (all obtained from the NIH AIDS Research and Reference Program). As a control, rNef-complemented medium was incubated with irrelevant isotype- and species-matched Abs. Then, immunocomplexes were reacted with detergent-free protein ASepharose beads (Sigma) overnight at 4 °C. Afterwards, immunocomplexes bound to protein ASepharose were discarded after centrifugation, and supernatants were filtered (0·22 µm pore diameter) and added to MDM cultures. The efficiency of rNef immunodepletion was checked by a quantitative Western blot analysis on the protein ASepharose fraction (not shown).
FACS analyses and chemokine detection.
Direct and indirect FACS analyses were performed as previously described (Baiocchi et al., 1997 ) by using a Becton Dickinson cytofluorimeter. Anti-CD4PE, PE- or TRITC-conjugated anti-CD14, CD45FITC, and class II MHC (HLA-DR)FITC MAbs were obtained form Becton Dickinson. Anti-CXCR4 (clone 12G5) MAb was obtained from the NIH AIDS reagent program, and anti-CCR5 (clone 2D7)PE MAb was from R&D Systems. All FACS analyses on monocytemacrophage cells were performed after an Fc-blocking step, carried out by incubating cells for 15 min at room temperature with 10 µg/ml human IgG (Dako).
Percentages of cells expressing intracytoplasmic HIV-1 Gag-related products were evaluated by FACS analyses after treatment with Permeafix (Ortho Diagnostic) for 30 min at room temperature and labelling with a 1:50 dilution of KC57-RD1 PE-conjugated anti-HIV-1 Gag MAb (Coulter).
For confocal microscope analyses, >95% pure monocytes were seeded in complete medium on glass coverslips. After 7 days, medium was replaced and 50 µg/ml BSAFITC in BSA-free medium or 100 ng/ml of either wild-type or mutated rNefFITC in complete medium was added. Sixteen hours after treatment, cells were labelled for 1 h at 4 °C with anti CD14TRITC MAb. Then, cells were fixed in 2% (v/v) formaldehyde buffer, observed, and images were analysed by using a CS4D confocal microscope (Leika).
ELISA kits for the detection of MIP-1/-1
chemokines in supernatants were obtained from R&D Systems.
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Results |
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rNef is internalized by MDM
We asked how MDM interact with rNef, i.e. through binding of a specific receptor and/or cell internalization. MDM were incubated for 1 h at 4 °C with 100 ng/ml of FITC-labelled rNef and analysed by FACS. Fig. 2 shows that there was no rNef binding on MDM cell membrane, indicating the absence of a highly represented specific receptor recognizable by rNef. However, cells scored positive in FACS analysis as early as 6 h after the pulse at 37 °C. Levels of rNef internalization peaked 16 h after treatment (Fig. 2
) and progressively declined thereafter (not shown). We reproduced these results on both 7-day-old (Fig. 2A
) and 14-day-old (Fig. 2B
) MDM cultures. In general, we observed a more efficient rNef uptake in the less-aged MDM cultures. Very similar results were obtained by using day 14 HPC-derived monocytopoietic cultures (not shown). Conversely, neither binding nor internalization of rNef were observed in CD4+ lymphocytes, either quiescent or activated by supernatants from rNef-treated MDM (not shown).
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rNef treatment of MDM induces CD4-dependent inhibition of HIV entry
We observed that rNef treatment of MDM induces a CD4 down-regulation correlating with a strong impairment of HIV replication. Thus, a block at the virus entry step could be envisaged. In order to determine more specifically the point of action of the rNef-induced HIV inhibition, we utilized a CD4-independent HIV infection model. Day 7 MDM, treated with 100 ng/ml of rNef or left untreated, were infected with VSV-G-pseudotyped ADA HIV-1 and, as control, with the parental virus strain. Pseudotyped virions do not require HIV-specific receptors to enter MDM. Thus, we were able to determine the role of HIV receptors in the rNef-induced HIV inhibition. Virus entry was evaluated by scoring the number of cells producing HIV Gag-related antigens, as detectable by FACS analysis 48 h after infection. As shown in Fig. 5, there was a decrease in the number of Gag-positive cells as a consequence of rNef treatment of cells infected with ADA HIV-1. Conversely, the number of Gag-expressing cells after infection with pseudotyped ADA HIV-1 was not influenced by rNef treatment.
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Preferential replication of T-tropic HIV correlates with the presence of MDM
We established that replication of T-tropic over M-tropic HIV strains is favoured in CD4+ lymphocyteMDM co-cultures in the presence of rNef. This phenomenon may be the consequence of a block of M-tropic HIV replication in rNef-treated MDM or, alternatively, of the effect(s) of soluble factors (e.g. chemokines; see Table 1) released by rNef-stimulated MDM. Of note, enhancement of T-tropic HIV replication induced in lymphocytes by chemokine treatment has been described previously (Dolei et al., 1998
; Kinter et al., 1998
). In order to define better the mechanism of T-tropic preference in rNef-treated co-cultures, CD4+ lymphocytes were doubly infected as described above and cultivated in the absence of MDM but in the presence of medium conditioned by autologous MDM previously treated or not with rNef. Seven days after NL4-3 challenge, supernatants were collected and titres of M- and T-tropic viruses were determined. Neither reproducible variations in M-tropic HIV nor increases in T-tropic HIV amounts were detected (data not shown). This result supports the idea that preferential replication of T-over M-tropic HIV in rNef-treated CD4+ lymphocyteMDM co-cultures is the consequence of a block in HIV replication induced in MDM.
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Discussion |
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It has been shown that rNef treatment of CD4+ replicating T-cells induces inhibition of cell growth and, upon cross-linking through anti-Nef Abs, apoptosis (Fujii et al., 1996 ; Okada et al., 1997
). We did not detect anticellular effects with a large range of rNef doses (from 0·05 to 5 µg/ml) on both lymphocyte and monocyte cell populations. As our findings were obtained mostly in non-replicating cell populations, it may be inferred that anticellular effects of Nef are addressed mainly to events involved in cell duplication.
It has been reported that a Nef peptide (aa 123160) binds class II DR antigen on Raji cell membrane (Torres & Johnson, 1994 ). However, in our experiments, no binding with membrane of either monocytemacrophages or lymphocytes was observed. This appears consistent with data reported by Okada et al. (1997)
who demonstrated rNef binding in lectin-stimulated lymphocytes only. It is conceivable that, in our conditions, cells did not expose levels of class II DR and/or possible accessory molecules sufficient to allow rNef binding. However, we cannot formally exclude possible low-level expression of a potential cell-surface Nef receptor that could not be revealed by FACS analysis on rNefFITC-treated cells.
The evidence that rNef apparently does not bind MDM at 4 °C suggests that rNef crosses the MDM cell membrane by exploiting the constitutive endocytic MDM machinery. Quantitative differences in rNef uptake between 7- and 14-day-old MDM cultures could be related to decreased pinocytic/phagocytic activity in older cell cultures.
We observed that CD4 exposure is down-regulated by rNef treatment. This result reproduced a typical effect widely observed in CD4+ cells endogenously expressing Nef (Aiken et al., 1994 ; Anderson et al., 1994
; Bandres et al., 1995
; Garcia & Miller, 1991
; Greenway et al., 1994
; Rhee & Marsh, 1994
). Importantly, immunodepletion of rNef prevents the negative effect on MDM CD4 expression and, at the same time, induces virus rescue in infected MDM. However, we cannot formally exclude the possibility that as yet unidentified soluble factor(s) induced by rNef treatment may cooperate with rNef action in down-regulating MDM CD4. Infection with pseudotyped HIV allowed us to identify virus entry as the critical step involved in rNef-induced virus restriction.
rNef was obtained by bacterial recombinant technology, and thus it was not N-terminally myristoylated. However, the typical intracytoplasmic punctate pattern and the location of rNefFITC at the cell margin, clearly detectable in confocal analyses of MDM, are reminiscent of observations for endogenously expressed GFPNef (Greenberg et al., 1998b ), and could be suggestive of intracellular myristoylation of rNef. The hypothesis that rNef reaches the cell membrane after myristoylation was supported by the fact that internalized rNefFITC mutated in the myristoylation signal failed to locate to the cell margin.
By use of doubly infected CD4+ lymphocyteMDM co-cultures, we tried to reproduce events occurring in lymph nodes of seropositive patients when CD4+ lymphocytes prevalently replicating M-tropic HIV quasispecies interact with both infected and uninfected infiltrating monocytemacrophages. We reasoned that the HIV replication block induced in MDM by extracellular Nef could limit strongly spread of M-tropic virus, normally sustained by the ability to target both lymphocytes and MDM. Conversely, no inhibitory effects should be observed in the replication of HIV T-tropic strains which are unable to enter MDM. Our data appear in line with such an hypothesis. Increased amounts of a T-tropic HIV strain in rNef-treated co-cultures could be the consequence of enhanced availability of target cells in conditions where M-tropic replication is severely impaired in MDM. Of note, we evaluated the replication efficiency of T-tropic strains in activated CD4+ lymphocytes as being approximately 20-fold greater than that of M-tropic strains (unpublished observations).
The detection of anti-Nef Abs in sera from AIDS patients (Ameisen et al., 1989 ; Bahraoui et al., 1990
) was considered to be an indication that Nef is present in vivo in an extracellular form. This hypothesis was confirmed by Fujii et al. (1996)
who detected free Nef antigen by ELISA in a majority of HIV patients. Even if here we describe effects mediated by doses of rNef (10100 ng/ml) somewhat exceeding those detected in patients sera (110 ng/ml) (Fujii et al., 1996
), significantly higher Nef concentrations would conceivably be found in those microenvironments (e.g. lymph nodes) where macrophages and infected lymphocytes tightly interact. However, whether there is a specific secretion pathway for unmyristoylated Nef or the presence of extracellular Nef in vivo is the consequence of lysis of dead infected cells remains a matter of question. Also, effects on MDM of already myristoylated Nef possibly released by HIV-infected cells should be investigated.
It is conceivable that factors released in vivo by MDM as a result of an inflammatory stimulus (either dependent or not on HIV infection) attract and activate both infected and uninfected CD4+ lymphocytes that can be induced to productive HIV replication and cell death. Nef released by dead cells may be internalized by uninfected macrophages that, as a consequence, are induced to produce additional amounts of HIV stimulating factors (included chemokines) becoming, at the same time, resistant to HIV. This mechanism may be at least in part the basis of the switch from M- to T-tropic HIV strains (Cheng-Mayer et al., 1988 ; Fauci, 1996
; Schellekens et al., 1992
; Schuitemaker et al., 1992
; Tersmette et al., 1989
) frequently observed in seropositive patients progressing to AIDS.
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Acknowledgments |
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This work was supported by grants from AIDS project of the Ministry of Health, Rome, Italy.
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Received 22 May 2000;
accepted 24 August 2000.