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(The FASEB Journal. 1999;13:2265-2276.)
© 1999 FASEB

Effects of the human CD38 glycoprotein on the early stages of the HIV-1 replication cycle

ANDREA SAVARINO*, FLAVIA BOTTAREL{dagger},1, LILIANA CALOSSO*, MARIA JOSÈ FEITO{dagger}, THEA BENSI{dagger}, MANUELA BRAGARDO{dagger}, JOSÈ MARIA ROJO§, AGOSTINO PUGLIESE*, ISABELLA ABBATE{ddagger}, MARIA R. CAPOBIANCHI{ddagger}, FERDINANDO DIANZANI{ddagger}, FABIO MALAVASI|| and UMBERTO DIANZANI{dagger}2

* Department of Medical and Surgical Sciences, University of Turin, Italy;
{dagger} Department of Medical Science, University ‘A. Avogadro’ of Eastern Piedmont, Novara, Italy;
{ddagger} Department of Experimental Medicine and Pathology, University ‘La Sapienza’ of Rome, Italy;
§ Department of Immunology, Centro de Investigaciones Biologicas, CSIC, Madrid, Spain; and
|| Institute of Biology and Genetics, University of Ancona, Italy

2Correspondence: Department of Medical Science, University ‘A. Avogadro’ of Eastern Piedmont, via Solaroli 17, I-28100 Novara, Italy.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
CD38 displays lateral association with the HIV-1 receptor CD4. This association is potentiated by the HIV-1 envelope glycoprotein gp120. The aim of this work was to evaluate the CD38 role in T cell susceptibility to HIV-1 infection. Using laboratory X4 HIV-1 strains and X4 and X4/R5 primary isolates, we found that CD38 expression was negatively correlated to cell susceptibility to infection, evaluated as percentage of infected cells, release of HIV p24 in the supernatants, and cytopathogenicity. This correlation was at first suggested by results obtained in a panel of human CD4+ T cell lines expressing different CD38 levels (MT-4, MT-2, C8166, CEMx174, Supt-1, and H9) and then demonstrated using CD38 transfectants of MT-4 cells (the line with the lowest CD38 expression). To address whether CD38 affected viral binding, we used mouse T cells that are non-permissive for productive infection. Gene transfection in mouse SR.D10.CD4-.F1 T cells produced four lines expressing human CD4 and/or CD38. Ability of CD4+CD38+cells to bind HIV-1 or purified recombinant gp120 was significantly lower than that of CD4+CD38- cells. These data suggest that CD38 expression inhibits lymphocyte susceptibility to HIV infection, probably by inhibiting gp120/CD4-dependent viral binding to target cells.—Savarino, A., Bottarel, F., Calosso, L., Feito, M. J., Bensi, T., Bragardo, M., Rojo, J. M., Pugliese, A., Abbate, I., Capobianchi, M. R., Dianzani, F., Malavasi, F., and Dianzani, U. Effects of the human CD38 glycoprotein on the early stages of theHIV-1 replication cycle.


Key Words: susceptibility to HIV-1 • gp120/CD4-dependent virus binding


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
CELL SUSCEPTIBILITY TO infection by human immunodeficiency virus type-1 (HIV-1) depends on a complex system of virus/host interactions, in which cell-surface molecules play a pivotal role (1 2 3) . Viral attachment involves interaction of the HIV-1 envelope glycoprotein gp120 with CD4 acting as the main viral receptor, and several members of the chemokine receptor family acting as co-receptors (1) . Three co-receptors play a crucial role in infection of different types of human cells. CXCR4 (also known as fusin or LESTR) is the receptor for the stromal-derived factor (SDF-1) and is mainly used by the ‘lymphocyte-tropic’ X4 HIV-1 strains, which generally develop in the advanced stages of infection (3 , 4) . CCR5 is a receptor for the ß-chemokines macrophage inflammatory protein-1{alpha} (MIP-1{alpha}), MIP-1ß, and RANTES, and is mainly used by the ‘macrophage-tropic’ R5 HIV strains, which predominate in the early stages and can infect many cell types (3 , 4) . CCR3 is the receptor for eotaxin and is considered to be an important HIV-1 co-receptor on microglia (5) .

Other cell-surface proteins modulate susceptibility either by direct interference with virus attachment or through their signaling pathways. For instance, expression of CD26 or CD44 promotes and triggering of CD28 either increases or decreases susceptibility to several viral strains (6 7 8 9 10) . Moreover, during viral budding from infected cells, several cell-surface molecules are incorporated into viral envelopes. Some are adhesion molecules and contribute to viral attachment by interacting with their physiological ligands of the target cells (1 , 11) .

We have previously shown that CD4 binding by gp120 induces lateral associations with several leukocyte surface molecules (12 , 13) . Because HIV-1 infection depends on multiple intermolecular interactions on the cell surface and precise steric interactions, we suggested that some of these associations positively or negatively influence cellular infection (12) : CD38 was an attractive candidate, its expression being negatively correlated with HIV-1 infection in a panel of CD4+ T cell lines (12 13 14) .

Human CD38, a single-chain transmembrane type II glycoprotein, is surface-expressed by early hematopoietic cells, lost by mature cells, and re-expressed on cell activation (15) . It is detectable at high levels on mature thymocytes and activated T cells and at low levels on resting naive cells (CD45RA+R0- cells), whereas resting memory cells (CD45RA-R0+ cells) are CD38- (15 , 16) . CD38 is thought to exercise the following three functions on T cells: 1) as an ectoenzyme, it leads to the formation of cyclic ADP-ribose, a crucial compound in regulation of intracellular Ca2+ (15 , 17) ; 2) as an adhesion molecule, it mediates the interactions between leukocytes and vascular endothelial cells through CD31 (15 , 16 , 18) ; 3) as a molecule involved in transmembrane signaling, its engagement results in Ca2+ mobilization, costimulates cell activation, and modulates cytokine production (15) . CD38 signaling exploits other molecules specialized in signaling, such as CD3 in T cells (19) , BCR in B lymphocytes, (20) , and CD16 in natural killer (NK) cells (R. Mallone, A. Funaro, and F. Malavasi, unpublished). By contrast, the relationship between its signaling and ectoenzyme functions is not known, and CD38-induced Ca2+ mobilization seems to be independent from its enzyme activity (21) .

In HIV-1 infection, CD38 has so far been studied mainly from a prognostic perspective. Its expression is high on peripheral blood lymphocytes in primary infection, decreases during transition to the asymptomatic phase, and then increases during progression to AIDS (22) . High expression on peripheral blood CD8+ T cells is a negative prognostic factor and is decreased by successful HAART (highly active antiretroviral therapy) in both adults and children (22 23 24 25) . By contrast, high expression on CD4+ T cells is positively correlated with survival in children (26 , 27) . These findings have been attributed to the ability of CD38 to mark activation of the immune response (23) . However, the fact that CD38 association with CD4 is increased by gp120 (12 , 13) and its expression is correlated with HIV-1 infection, suggests that its role is direct (14 , 28) . The aim of the present work was to investigate this possibility by using human and mouse cell lines transfected with the human CD38 cDNA and assessing the effects of CD38 expression on various steps of HIV-1 replication.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Monoclonal antibodies
Fluorescein isothiocyanate (FITC)-labeled mouse monoclonal antibodies (mAbs) to human CD4 were purchased from Coulter, Hialeah, FL; R-phycoerythrin (R-PE)-conjugated mAbs to human CXCR-4 (12G5) were from PharMingen, San Diego, CA; FITC-conjugated mouse anti-HIV-1 p24 mAbs were from Virostat, Portland, ME; unconjugated anti-CD26 mAbs were a generous gift of Dr. E. C. Bosmans, Eurogenetics, Belgium; FITC-conjugated anti-CD38 mAbs (IB4) were obtained as described previously (29) ; FITC-conjugated rabbit anti-murine immunoglobulins were from Pierce, Rockford, IL. Appropriate isotype-matched mAbs were used as negative controls.

Cells
The following human T cell lines were used: H9, Supt-1, C 8166, MT-2, MT-4, and CEMx174 (14 , 30 31 32 33) . They were grown in RPMI-1640 medium (Life Technologies, Inc., Gaithersburg, MD), supplemented with 10% (v/v) fetal calf serum (FCS; Techno-Genetics, Milan, Italy), 200 µg/ml glutamine (Merck, Darmstadt, Germany), and 40 µg/ml gentamicin (Schering-Plow, Milan, Italy).

SR.D10-CD4-.F1 was a CD4- mutant cell line cloned from the mouse CD4+ TH2 cell line D10.G4.1 (34) . It was grown in Click medium with 10% FCS, 9% (v/v) ß-mercaptoethanol, 5 U/ml interleukin-2 (IL-2); 10 U/ml IL-4, and 25 pg/ml IL-1{alpha}.

Expression vectors
The SR.hCD4 line expressing human CD4 was produced by transfecting the human CD4 cDNA inserted in the pNeoSR{alpha} plasmid in SR.D10.CD4-.F1 cells as previously reported (35) .

CD38 transfectants were produced by transfecting the human CD38 cDNA in SR.D10.CD4-F1, SR.hCD4, and MT4 cells. Full-length human CD38 cDNA (generous gift of Dr. D. Jackson, Oxford, UK) was excised from the pCDM8 plasmid with XbaI and cloned into the corresponding site of a pcDNA3.1/zeo expression vector carrying ampicillin/zeocin resistance, or pcDNA3/neo carrying ampicillin/neomycin resistance. Sequencing of the CD38 cDNA after cloning ruled out the presence of mutations in the sequence and confirmed the correct position of the cDNA insertion in the plasmid. Plasmids were linearized with PvuI and transfected at 10 µg/ml in the appropriate cell line [5x106 cells in 0.8 ml of phosphate-buffered saline (PBS)] by electroporation at 960 µF and 260 V using a Gene Pulser (Bio-Rad, Hercules, CA). Transfectants carrying pcDNA3/neo were grown in culture medium containing 0.8 mg/ml G-418 (selection medium), whereas transfectants carrying pcDNA3.1/zeo were grown in medium containing 0.8 mg/ml zeocin.

Flow cytometry
To determine the expression of cellular and viral antigens, cells were pooled and washed in PBS with NaN3 (0.02%) and bovine serum albumin (2%; PBS A/A), and subsequently treated as follows.

To detect surface antigen expression, cells were suspended in PBS A/A (2.5x106 cells/ml) and incubated with saturating concentrations of the appropriate mAb. The negative control samples were incubated with isotype-matched mAbs. When unconjugated mAbs were used, cells were washed in PBS A/A and subsequently incubated with FITC-labeled goat anti-mouse Ig antibodies under the same conditions. Finally, the cells were washed three times and immediately analyzed by flow cytometry (FACScan, Becton-Dickinson, Mountain View, CA).

To determine the percentage of infected cells, pellets were fixed, permeabilized, and stained with mAbs to HIV-1 p24 using a commercially available kit (Caltag, Burlingame, CA), as described previously (32 , 36) . The fixed samples were analyzed by flow cytometry.

Fluorescence data were collected on a 4-decade log scale and the relative fluorescence intensity was stated as the median channel number (MeFI). Log values were mathematically converted to linear fluorescence intensity and the control antibody values for each experiment were subtracted. Fluorescence was also evaluated in terms of percentage of fluorescent cells beyond the threshold value established using cells stained with the isotype control reagents.

Infection with HIV-1
Stocks of HIV-1IIIB (a laboratory X4 strain) were obtained from the supernatant of H9 IIIB cells, a persistently HIV-1-infected H9 cell line, as described elsewhere (30) . Stocks of HIV-1P1 (a laboratory-adapted X4 strain) were obtained from acutely-infected C 8166 cells, as described previously (33) . Stocks of HIV-1BAL (a laboratory-adapted R5 strain) (37) were obtained from Dr. C. Balotta, ‘Sacco’ Hospital, Milan, Italy. Stocks of the X4 and R5/X4 primary isolates were obtained from Dr. F. Piro (‘Amedeo di Savoia’ Hospital, Turin, Italy) and Dr. G. Poli (DIBIT, Milan, Italy), respectively. Both primary isolates were grown in phytohemagglutinin (PHA)-activated peripheral blood mononuclear cells (PBMC). Stocks of pRRL.sin.hPGK.GFP, a lentiviral vector pseudotyped with the vesicular stomatitis virus (VSV)-G envelope glycoprotein (38) , were a generous gift of Dr. L. Naldini (IRCC Candiolo, Italy).

Viral stocks were titrated immunoenzymatically using p24 antigen enzyme-linked immunosorbent assay (ELISA) kits and biologically by the 50% end point dilution method, using MT-2 cells (laboratory strains) or PHA-activated PBMC (primary isolates). The infectious titer was expressed as cell culture infecting doses (CCID50)/ml (39) .

Cells to be infected were suspended at 5 x 105 cells/ml in fresh culture medium 24 h before the infection. Cell pellets were infected with the HIV-1 stock suspensions at a titer of 5–10 x 105 CCID50/ml; the number of cells was adjusted so as to have a multiplicity of infection (MOI) of ~1. The cells were then incubated at 37°C for 2 h, washed three times with PBS, resuspended at 2.5 x 105 cells/ml in fresh culture medium, transferred to 24-well plastic microtiter trays (Nunc, Kamstrup, Denmark), and incubated at 37°C in a 5% CO2 humidified atmosphere. At different times after infection, cell viability was assessed microscopically by the trypan blue-dye exclusion method. In the case of MT-4 cells, which display a typical clustered pattern, clusters were dissociated by pipetting, and reclustering was examined microscopically after a 3-h incubation at 37°C (14 , 30) .

Proviral DNA was detected by PCR 2 days after infection. Cells were lysed in 500 µl of 500 mM Tris-HCl pH 9, 2 mM EDTA, 10 mM NaCl, 1% SDS plus 80 ng of proteinase K/ml at 55°C for 24 h. Cellular DNA was prepared by phenol extraction, followed by ethanol precipitation. DNA content was determined spectrophotometrically at 260 nm and equal amounts of DNA for each experimental condition underwent serial dilutions that were amplified by PCR using primers for a conserved gag region sequence (sense: 5'-ATAATCCACCTATCCCAGTAGGAGAAAT-3'; anti-sense: 5'-TTTGGTCCTTGTCTTATGTCC-AGAATGC-3'), detecting full-length, double-stranded viral DNA in both the integrated and unintegrated form. The following amplification conditions were employed: denaturation (5 min at 94°C) was followed by 35 cycles of denaturation (1 min at 94°C), annealing (1 min at 55°C), extension (1 min at 72°C), and final incubation (10 min at 72°C). The amplification products of 114 bp obtained were electrophoresed on 3% agarose gels and visualized by ethidium bromide staining (37) . As a control, the human gene bax was amplified by PCR in the same DNA preparations (sense: 5'-TCTCCTGCAGGATGATTGC-3'; anti-sense: 5'-TCCCCAGGTCCTCACAGAT-3'). A standard curve for the quantification of proviral DNA copies was prepared using serial 10-fold dilutions of the DNA extracted from a suspension of 8E5 cells, containing one proviral copy per cell (40) , and amplified under the same conditions. Amplified products of the correct size were quantified using Gel Doc (Bio-Rad, Hercules, CA). The intensity units x mm2 (INTxmm2) of the serially diluted standard samples were used to construct the standard curve, using the least squares method. The proviral copy numbers of the experimental samples were calculated by reporting the values of the last positive sample dilution on the standard curve.

HIV-1 binding to cells
HIV-1 binding to cells was evaluated as previously reported (41) . Briefly, 106 cells were incubated with 1 ml of the viral suspensions (titer: 10 ng of p24/ml) for 2 h at 37 or 4°C. Cells were then washed twice in PBS, lysed in 500 µl PBS with 0.5% Triton X-100 (v/v), and p24 was quantitated immunoenzymatically in cell lysates using an ELISA kit (NENTM Life Science Product, Inc., Boston, MA).

Cell staining with purified HIV-1 gp120 was evaluated by incubating cells (2.5x106 cells/ml) with saturating concentrations (10 µg/ml) of FITC-conjugated gp120 (Intracell Corp, London, UK) in PBS A/A for 30 min at 4°C. Cells were then washed and analyzed by flow cytometry. The negative control samples were incubated with PBS A/A alone.

FRET assay
The OKT4 mAb (ATCC) was conjugated to Cy3 dye with the FluoroLink-Ab Cy3 Labeling Kit (Amersham, Little Chalfont, Buckinghamshire, UK). Conjugations were judged to be successful by spectrophotometric and spectrofluorometric measurements. Cells were washed with ice-cold PBS + 5% FCS, 0.1% NaN3, incubated on ice for 1 h simultaneously with the FITC-conjugated mAb (the donor fluorophore) and the Cy3-conjugated OKT4 (the accepting fluorophore), then washed, resuspended in PBS + 0.1% NaN3, and analyzed immediately. A FACScan flow cytometer was used to determine energy transfer between FITC and Cy3-labeled proteins on the cell surface. Fluorescence resonance energy transfer (FRET) to Cy-3 was detected by using standard methods (42) . FITC was excited at 488 nm and Cy3 emissions were collected at >600 nm. Data from 10,000 cells/test were stored in ‘list mode’ and analyzed with LYSYS II software (Becton Dickinson). The median linear channel of fluorescence was calculated and used as the indicator for the presence (a positive shift over background) or absence (no shift or negative shift) of energy transfer.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Cell phenotype and susceptibility to HIV-1 infection
The first step was to compare expression of CD38 and infection by the X4 HIVIIIB strain in a panel of human CD4+ T cell lines selected on account of their permissivity to HIV-1 infection and homogeneous expression of the main receptors for X4 strains (i.e., >=90% of CD4+ and CXCR-4+ cells). HIV-1 infection, evaluated as proportion of infected cells scored 4 days after infection by flow cytometry (IF%), negatively correlated to the CD38 expression level (Table 1 ), evaluated both as MeFI and proportion of positive cells (r = -0.92 and -0.87, respectively). A similar correlation was obtained between CD38 expression and the proportion of dead cells at 5 days after infection (r=-0.95). These results were in line with our previous observations with different approaches (14) . By contrast, IF% and cytopathic effects did not correlate with the expression levels of CD4 and CXCR4, nor of activation markers other than CD38 (CD26, CD71, HLA Class II).


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  		Table 1. Correlation between baseline expression levels of surface antigens and proportion of infected cells (IF%) after HIV-1 inoculation in a panel of human CD4+ T cell lines

HIV-1 replication in human transfectants expressing CD38
These data suggested that CD38 is a negative regulator of HIV-1 infection, even though differences in susceptibility due to other unknown features of the cell lines, such as differences in growth kinetics or differential expression of molecules other than CD38, could not be ruled out. To address this issue, we transfected the human CD38 cDNA (inserted in the pcDNA3.1neo plasmid) into MT-4 cells and evaluated HIV-1 infection of transfected clones expressing different levels of CD38. MT-4 cells were chosen because they had the lowest CD38 expression and the highest susceptibility to infection. Three transfectants expressing low (MT-4.38L), intermediate (MT-4.38I), and high (MT-4.38H) levels of CD38, respectively, were selected. A control mock transfectant (MT-4.M) was produced by transfecting MT-4 cells with the empty pcDNA3.1neo plasmid (Fig. 1 ). In all transfectants, expression of CD4 and CXCR-4, as assessed by immunofluorescence and flow cytometry, was similar to that displayed by parental MT-4 cells (data not shown). Transfectants and parental cells were incubated with HIV-1IIIB, washed, and cultured at 37°C. Infection was evaluated in terms of p24 release in the supernatants. Results indicate that HIV-1 replication was negatively correlated to CD38 expression (Fig. 2 ). At day 2 and day 5 after infection, parental MT-4 cells and mock MT-4-M cells displayed the highest levels of p24, followed in order by MT-4.38L, MT-4.38I, and MT-4.38H. In MT-4M cells, maximal levels of p24 were reached at day 5 after infection, and in MT-4.38 cells at day 7. At the latter time, differences were no longer significant, suggesting that CD38 affects early events of infection. In MT-4.38I and MT-4.38H, the low levels of virus replication were strictly paralleled by reduced cytopathic effects, as shown by the cell viability curves (Fig. 2) . Similar results were obtained when cytopathic effects were assessed as loss of ability to form cell clusters in infection driven by HIV-1IIIB (data not shown). In MT-4 cells, in fact, HIV-1IIIB acts as a slow/low syncytium-inducing strain whose cytopathic effect consists of loss of cell ability to cluster and is strictly correlated to the level of viral replication (14 , 30) . When infected with HIV-1IIIB, MT-4.38I and MT-4.38H cells maintained a partial ability to form clusters, whereas MT-4.38L, MT-4.M, and parental cells lost this ability. Similar impairment of HIV-1 replication in CD38-expressing cells was also observed by using another laboratory-adapted X4 strain (HIV-1P1; data not shown).



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  		Figure 1. Staining for surface CD38 in MT-4 cell transfectants: A) MT-4. M, B) MT-4.38L, C) MT-4.38I, D) MT-4.38H. Fluorescence using control isotype-matched mAbs (black) and fluorescence of transfectants carrying the CD38 cDNA (gray). The bold number shows the MeFI, i.e., the median fluorescence intensity from each histogram mathematically converted to linear fluorescence intensity and subtracted by the control antibody values.



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  		Figure 2. Inhibition of HIV-1IIIB replication in MT-4 cells transfected with the CD38 cDNA. A) p24 levels in supernatants at 2 and 5 days post-infection; B) correlation between baseline expression of CD38 and relative production of p24 in transfectants at day 5 post-infection; C) cell survival after HIV-1 infection. Cells were infected with HIV-1IIIB and grown in selection medium; p24 was measured in supernatants and expressed as means ± SD from three experiments. In A, asterisks indicate values significantly different than those displayed by MT-4. M cells (significance threshold: P<0.05, repeated-measures ANOVA+Student-Neuman-Keuls test). In B, mean p24 values at day 5 were plotted against CD38 MeFIs of clones (average from three independent experiments) and the line that best fitted the points was estimated by the least squares method (solid line: r=-0.97, P<0.05, t test for correlation). The dotted lines mark the 95% confidence limits of the regression line. In C, the results of one representative experiment are reported.

To investigate whether the inhibitory effect of CD38 expression on HIV replication was restricted to laboratory-adapted HIV-1 strains, we used two primary isolates, an X4 rapid/high syncytium-inducing and an X4/R5 slow/low syncytium-inducing. The results showed that both primary isolates had lower replication levels in CD38-expressing cells than in parental CD38-negative MT4 cells (Fig. 3 ).



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  		Figure 3. Inhibition of primary HIV-1 isolate replication in MT-4 cells transfected with the CD38 cDNA. A) p24 levels in supernatants of cultures infected with a X4 primary isolate; B) cell survival after infection with the X4 primary isolate. C) p24 levels in supernatants of cultures infected with a R5/X4 primary isolate; D) cell survival after infection with the R5/X4 primary isolate. Cells were infected with the clinical isolates (MOI=1) and grown in selection medium; p24 and counts of viable cells were measured at different times after infection. In panels C and D, cells were split 1:2 every other day from day 4 after infection. One experiment representative of two.

To assess whether CD38 affected the proviral DNA formation, we evaluated HIV-1 proviral genome in cellular DNA extracted from HIV-1IIIB-infected MT-4.38H and MT-4.M cells by semi-quantitative PCR on day 2. The amount of proviral DNA was lower in MT-4.38H cells than in MT-4.M cells (Fig. 4 ).



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  		Figure 4. Semi-quantitative PCR analysis of HIV-1 gag in DNA extracted from MT-4.38H and MT-4.M cells on day 2 after infection. A) PCR products obtained with primers for HIV-1 gag and the human gene bax from serial dilutions of DNA extracted from MT-4.38H and MT-4. M cells. The amount of gag DNA was lower in MT-4.38H than in MT-4. M cells. By contrast, no difference was found when the exon 4 of the human gene bax was amplified. B) Quantitative analysis of the PCR signal. Two thousand nanograms of DNA were loaded in lanes A, 666 ng in lanes B, 222 ng in lanes C, and 74 ng in lanes D. Estimation of proviral copies, performed by comparison with a standard curve, indicated that MT-4.38H cells contained 1.5 copies of HIV-DNA per cell, whereas MT-4.M cells contained 5.2 copies per cell.

HIV-1 binding to mouse T cell expressing the human CD4 and/or CD38
These data suggested that CD38 expression could affect some event(s) preceding proviral DNA completion. One possibility was that the lateral association of CD38 with CD4 interferes with viral binding. To investigate this possibility, we used mouse T cells that can be rendered susceptible to HIV binding by transfection with human CD4, although they remain non-permissive for productive infection. Therefore, we transfected human CD4 (inserted in the pNeoSRa plasmid) and/or CD38 (inserted in the pcDNA3.1zeo plasmid) in different combinations into the mouse SR.D10.CD4-.F1 T cell clone, a CD4- variant of the D10 TH2 T cell clone. Three clones expressing human CD4 (SR.hCD4), human CD38 (SR.hCD38), or both molecules (SR.hCD4.38), respectively, were selected (Fig. 5 ). Because CD4 and CD38 display a basal level of lateral association in human T cells (12) , we used FRET to evaluate whether human CD4 and CD38 maintained lateral associations in the murine environment. These experiments showed that energy transfer was obtained when SR.hCD4.38 cells stained with the Cy3-conjugated mAb to CD4 were co-stained with the FITC-conjugated mAb to CD38. In contrast, no transfer was detected when they were co-stained with FITC-conjugated control mAbs to mouse CD2 or CD3 (Fig. 6 ).



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  		Figure 5. Staining for human CD4 and human CD38 of mouse SR. D10. CD4-. F1-derived transfectants: control SR. D10. CD4-.F1, SR.hCD4 (human CD4+), SR.hCD38 (human CD38+), SR. hCD4.38 (human CD4+CD38+). Black curves show the background fluorescence obtained by staining cells with the control isotype-matched mAbs. Gray curves show the fluorescence of cells stained with the mAbs to human CD4 and CD38, as indicated.



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  		Figure 6. FRET between human CD4 and CD38 in SR.hCD4.38 cells. CD4 displays FRET with CD38, but not with mouse CD2 or CD3. Cells were stained with Cy3-conjugated CD4 mAb and the indicated FITC-conjugated mAb. FITC was excited at 488 nm and Cy3 emissions were collected at >600 nm. The FACS profiles show one representative experiment. Each quadrant shows Cy3 emissions at >600 nm in the absence and in the presence of the indicated FITC-conjugated mAb. A right shift of the curve indicates FRET. The bar graph shows the mean ± SD of the median fluorescence intensities, expressed as median fluorescent channels, from three experiments.

The murine clones were used to evaluate the effects of CD38 expression on CD4-dependent virus/cell binding in the absence of other human molecules. Moreover, they did not express endogenous murine CD38 and CD4 (35) , thus avoiding interference of the human molecules with their mouse homologs. Transfectants and parental cells were incubated with HIV-1IIIB for 2 h at 37°C, washed, and lysed. Cell-associated p24 was evaluated by ELISA in cell lysates (Fig. 7A ). SR.hCD4 cells displayed higher levels of cell-associated p24 than parental and SR.hCD38 cells. The double-transfectant SR.hCD4.38 displayed significantly lower levels than those displayed by SR.hCD4 cells and similar to those displayed by CD4- clones. The difference cannot be attributed to discrepancies in expression levels of human CD4, which were similar in SR.hCD4 and SR.hCD4.38 cells. Similar results were obtained using HIV-1P1, and performing the experiments at 4°C (data not shown). Because at 4°C the entry process is significantly reduced, these results support the hypothesis that CD38 carries out its inhibitory effects at the step of HIV-1 binding to target cells. The generality and specificity of this effect was further investigated by incubating cells with the R5 HIV-1BAL strain and using an HIV-1 vector pseudotyped with the VSV-G envelope glycoprotein as a positive control. Figure 7B shows that SR.hCD4.38 displayed higher levels of cell-associated p24 than those displayed by SR.hCD4 cells where HIV-1BAL was used, whereas no differences were found where the pseudotyped vector was used.



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  		Figure 7. Levels of virus/cell association in mouse SR. D10 cell lines expressing human CD4 and/or human CD38 incubated for 2 h with HIV-1. A) HIV-1IIIB displays higher association with SR.hCD4 cells (CD4) than with SR.hCD4.38 (CD4 CD38), SR.hCD38 (CD38) or parental SR. D10 cells (control). B) Comparison between HIV-1BAL and pRRL.sin.hPGK. GFP (VSV-env) association with SR.hCD4 and SR.hCD4.38 cells. Cells were incubated with the viral suspensions (titer: 10 ng of p24/ml), washed and immediately lysed. pRRL.sin.hPGK. GFP is an HIV-1 vector pseudotyped with the VSV-G envelope glycoprotein. Levels of virus/cell association are expressed as picograms of p24 in 2 x 106 cells (means±SD from three experiments). Asterisks indicate values significantly different than those displayed by SR.hCD4 cells (significance threshold: P<0.05, repeated-measures ANOVA+Student-Neuman-Keuls test).

To further confirm the possibility that CD38 inhibits HIV-1 binding to target cells, cells were incubated with FITC-labeled HIV-1IIIB gp120 for 30 min at 4°C, washed, and analyzed by flow cytometry. We found that gp120 staining was significantly lower in CD4+CD38+ cells than in their CD38- counterparts (Table 2 ).


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  		Table 2. Staining of mouse SR.D10 cells transfected with human CD4 and/or human CD38 with FITC-conjugated HIV-1 gp120 or mAb to human CD4


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
This work shows that CD38 expression negatively modulates CD4+ T cell susceptibility to infection by different HIV-1 strains. This effect was initially suggested by evaluating the correlation between CD38 expression levels and susceptibility to infection in several human CD4+ T cell lines. The results were then confirmed by transfecting the CD38 gene in a human CD4+CD38- T cell line and in a murine T cell clone expressing human CD4. The results showed that productive infection was significantly delayed in cells expressing high levels of CD38. This effect was not restricted to laboratory-adapted X4 virus strains, since it was observed also with X4 and X4/R5 primary isolates.

The effect seemed to be mediated by early events in the replication cycle of HIV because 1) reduction of virus yield was maximal at early times of infection and 2) at 48 h after infection, cells expressing high CD38 levels displayed lower levels of full-length proviral DNA than cells expressing low CD38 levels. It is likely that the effect is mainly exerted at pre-entry stages because it was detectable in binding experiments with whole virus or purified gp120 and was detectable in both human (data not shown) and murine cells.

Inhibition of HIV-1 infection by the lateral association between CD38 and CD4 potentiated by gp120 was postulated in earlier studies (12 13 14) . The ability of gp120 to recruit CD38 seems to be highly conserved, since it was detected with glycoproteins derived from HIV-1IIIB, HIV-1SF2, HIV-1MN, and HIV-1451 (13) . Moreover, it did not presumably involve chemokine receptors because no lateral association was detected between CD38 and CXCR4 or CCR5 in the presence or absence of gp120 (U. Dianzani, personal observation). It could be that CD38 docking to CD4 affects the gp120 interaction with CD4 and/or viral co-receptors by steric interference. However, we cannot rule out the possibility that steps following gp120/CD4 interaction may also be affected by CD38.

The results with CD38+ transfectants minimize the possibility that the negative correlation between CD38 expression and HIV-1 infection is due to factors independently controlling both variables. This possibility was raised by the observation that retinoic acid-responsive elements (RARE) control the CD38 gene as well as the expression of HIV-1: endogenous RARE positively regulate the CD38 gene, whereas viral RARE in the long-terminal repeats (LTR) negatively regulate HIV-1 genes (28 , 43) . CD38 expression, however, was not under the control of RARE in transfectants.

Inhibition could also be linked to the enzymatic or co-stimulatory functions of CD38. CD38 produces nicotinamide (NAm), a compound reported to inhibit HIV-1 replication and cytopathogenicity (14 , 44) probably through inhibition of poly(ADP-ribose) polymerase (45) . This possibility seems to be unlikely because the concentrations of NAm required to inhibit HIV-1 replication are much greater than those produced by CD38 in cell culture (P. Deterre, personal communication). Alternatively, co-stimulation might enable CD38 to inhibit in the same manner as CD28, whose triggering can decrease cell susceptibility to macrophage-tropic HIV-1 strains by inhibiting expression of CCR5 (46) . However, this possibility appears equally unlikely because CD38-mediated inhibition was detectable in the mouse model in the absence of viral co-receptors and productive infection. It was also detectable in short-term binding experiments at 4°C, which minimized the effect of putative signals triggered by gp120 through CD38.

The inhibition of HIV-1 replication by CD38 expression may account for the lower susceptibility of resting/naive (CD45RA+) than memory (CD45R0+) CD4+ T cells (47 48 49) , since resting naive cells constitutively express CD38, whereas resting memory cells are CD38- (16) . Moreover, it may play a role in the favorable prognostic value of high levels of CD38 on CD4+ cells in HIV+ children (26) and in the relative resistance to HIV-1 infection shown by children born to HIV+ mothers (50) . CD38 expression, in fact, is basically higher in children than in adults, whereas the highest levels are found in newborns, who have high counts of recent thymic emigrants and naive T cells (51 52 53) . By contrast, our observation is in apparent contrast with the notion that PHA-activated T cells, which express high levels of CD38 (CD38bright), are more susceptible to HIV infection than resting T cells. A reasonable speculation is that CD38-mediated protection may be highly effective in resting CD38bright cells, such as recent thymic emigrants and naive cells, but not in activated CD38bright cells, which express a different pattern of molecules involved in viral binding and entry (such as chemokine receptors, CD26, or CD44) and are much more efficient in supporting viral replication at post-entry levels, which may overwhelm inhibition by CD38 (41 , 51 , 54) . Therefore, CD38-mediated protection may be highly effective in children, whose CD38bright cells are predominantly resting cells, but not in adults, where they are predominantly activated (53) .

In conclusion, our data suggest that CD38 expression negatively modulates lymphocyte susceptibility to CD4-dependent infection by HIV-1, probably by inhibiting the gp120/CD4-dependent virus/cell binding. It is noteworthy that CD38 may also be expressed by cells of the monocyte/macrophage lineage, another crucial HIV-1 target (28) . In this regard, we are currently developing a model to test whether the CD38-related inhibitory effects found in this study may be extended to macrophage infection by R5 HIV-1 strains.

To our knowledge, this is the first report of a naturally occurring surface molecule capable of directly inhibiting susceptibility to HIV-1 infection. Elucidation of the inhibitory mechanism(s) of CD38 may help to clarify those of HIV-1 infection and provide ways of preventing it. Current antiretroviral drugs inhibit after HIV-1 attachment/entry. Strategies aimed at blocking such entry are being extensively sought in the effort to inhibit HIV-1 replication at several steps (55) .


   ACKNOWLEDGMENTS
 
This work was supported by the AIDS project (Istituto Superiore di Sanità, Rome), the Italian National Association against AIDS (ANLAIDS), Piedmont Section, Turin; Associazione Italiana Ricerca sul Cancro (AIRC, Milan), and grant FIS 98/0037 (Madrid). M. Bragardo was supported by ANLAIDS, Rome, M. J. Feito by the AIDS project. We thank Dr. G. Poli, Dr. C. Balotta, Milan, Italy, Dr. L. Naldini, Dr. A. Follenzi, and Dr. F. Piro, Turin, Italy, for providing materials and suggestions. A. Savarino is personally grateful to Dr. P. Gioannini (Turin, Italy) for enlightening and encouraging discussion.


   FOOTNOTES
 
1 The first two authors contributed equally to this work.

Received for publication February 22, 1999. Revised for publication August 9, 1999.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Wyatt, R., Sodroski, J. (1998) The HIV-1 envelope glycoproteins: fusogens, antigens, and immunogens. Science 280,1884-1888[Abstract/Free Full Text]
  2. Platt, E., Madani, N., Kozak, S. L., Kabat, D. (1997) Infectious properties of human immunodeficiency type 1 mutants with distinct affinities for the CD4 receptor. J. Virol. 71,883-890[Abstract]
  3. Dittmar, M. T., McKnight, A., Simmons, G., Clapham, P. R., Weiss, R. A., Simmonds, P. (1997) HIV-1 tropism and co-receptor use. Nature (London) 385,495-496[Medline]
  4. Cohen, J. (1997) Exploiting the HIV-chemokine nexus. Science 275,1261-1264[Free Full Text]
  5. He, J., Chen, Y., Farzan, M., Choe, H., Ohagen, A., Gartner, S., Busciglio, J., Yang, X., Hofmann, W., Newman, W., Mackay, C. R., Sodroski, J., Gabuzda, D. (1997) CCR3 and CCR5 are co-receptors for HIV-1 infection of microglia. Nature (London) 385,645-649[Medline]
  6. Callebaut, C., Krust, B., Jacotot, E., Hovanessian, A. G. (1993) T cell activation antigen, CD26, as a cofactor for entry of HIV in CD4+ cells. Science 262,2045-2050[Abstract/Free Full Text]
  7. Oravecz, T., Roderiquez, G., Koffi, J., Wang, J., Ditto, M., Bou-Habib, D. C., Lusso, P., Norcross, M. A. (1995) CD26 expression correlates with entry, replication and cytopathicity of monocytotropic HIV-1 strains in a T-cell line. Nature Med 1,919-926[Medline]
  8. Dukes, C. S., Yu, Y., Rivadeneira, E. D., Sauls, D. L., Liao, H. X., Haynes, B. F., Weinberg, J. B. (1995) Cellular CD44S as a determinant of human immunodeficiency virus type 1 infection and cellular tropism. J. Virol. 69,4000-4005[Abstract]
  9. Levine, B. L., Mosca, J. D., Riley, J. L., Carroll, R. G., Vahey, M. T., Jagodzinski, L. L., Wagner, K. F., Mayers, D. L., Burke, D. S., Weislow, O. S., St. Louis, D. C., June, C. H. (1996) Antiviral effect and ex vivo CD4+ T cell proliferation in HIV-positive patients as a result of CD28 costimulation. Science 272,1939-1943[Abstract]
  10. Kitchen, S. G., Korin, Y. D., Roth, M. D., Landay, A., Zack, J. A. (1998) Costimulation of naive CD8(+) lymphocytes induces CD4 expression and allows human immunodeficiency virus type 1 infection. J. Virol. 72,9054-9060[Abstract/Free Full Text]
  11. Frank, I., Stoiber, H., Godar, S., Stockinger, H., Steindl, F., Katinger, H. W., Dierich, M. P. (1996) Acquisition of host cell-surface-derived molecules by HIV-1. AIDS 10,1611-1620[Medline]
  12. Dianzani, U., Bragardo, M., Buonfiglio, D., Redoglia, V., Funaro, A., Portoles, P., Rojo, J., Malavasi, F., Pileri, A. (1995) Modulation of CD4 lateral interaction with lymphocyte surface molecules induced by HIV-1 gp120. Eur. J. Immunol. 25,1306-1311[Medline]
  13. Feito, M. J., Bragardo, M., Bonissoni, S., Bottarel, F., Malavasi, F., Dianzani, U. (1997) gp 120s derived from four different syncytium-inducing strains induce different patterns of CD4 association with lymphocyte surface molecules. Int. Immunol. 9,1141-1147[Abstract/Free Full Text]
  14. Savarino, A., Pugliese, A., Martini, C., Pich, P. G., Pescarmona, G. P., Malavasi, F. (1996) Investigation of the potential role of membrane CD38 in protection against cell death induced by HIV-1. J. Biol. Reg. Homeost. Agents 10,13-18
  15. Mehta, K., Shahid, U., Malavasi, F. (1996) Human CD38, a cell surface molecule with multiple functions. FASEB J 10,1408-1417[Abstract]
  16. Dianzani, U., Funaro, A., Di Franco, D., Garbarino, G., Bragardo, M., Redoglia, V., Buonfiglio, D., De Monte, L. B., Pileri, A., Malavasi, F. (1994) Interaction between endothelium and CD4+CD45RA+ lymphocytes: role of the human CD38 molecule. J. Immunol. 153,952-959[Abstract]
  17. Howard, M., Grimaldi, J. C., Bazan, J. F., Lund, F. E., Santos-Argumedo, L., Parkhouse, R. M. E., Walseth, T. F., Lee, H. C. (1993) Formation and hydrolysis of cyclic ADP-ribose catalyzed by lymphocyte antigen CD38. Science 262,1056-1059[Abstract/Free Full Text]
  18. Deaglio, S., Morra, M., Mallone, R., Ausiello, C. M., Prager, E., Gambarino, G., Dianzani, U., Stockinger, H., Malavasi, F. (1998) Human CD38 (ADP-ribosyl cyclase) is a counter-receptor of CD31, an Ig superfamily member. J. Immunol. 160,1106-1115[Abstract/Free Full Text]
  19. Morra, M., Zubiaur, M., Terhost, J., Sancho, J., Malavasi, F. (1998) CD38 is functionally dependent of TCR/CD3 complex in human T cells. FASEB J 330,1129-1135
  20. Lund, F. E., Yu, N., Kim, K-M., Reth, M., Howard, M. (1996) Signaling through CD38 augments B cell antigen receptor (BCR) responses and is dependent on BCR expression. J. Immunol. 159,1455-1467
  21. da Silva, C. P., Schweitzer, K., Heyer, P., Malavasi, F., Mayr, G. W., Guse, A. H. (1998) Ectocellular CD38-catalyzed synthesis and intracellular Ca2+-signalling activity of cyclic ADP-ribose in T-lymphocytes are not functionally related. Immunol. Rev. 161,79-93[Medline]
  22. Liu, Z., Hultin, L. E., Cumberland, W. G., Hultin, P., Schmidt, I., Matud, J. L., Detels, R., Giorgi, J. V. (1996) Elevated relative fluorescence intensity of CD38 antigen expression on CD8+ cells is a marker of poor prognosis in HIV infection: results of 6 years of follow-up. Cytometry 26,1-7[Medline]
  23. Liu, Z., Cumberland, W. G., Hultin, L. E., Kaplan, A. H., Detels, R., Giorgi, J. V. (1998) CD8+ T lymphocyte activation in HIV-1 disease reflects an aspect of pathogenesis distinct from viral burden and immunodeficiency. J. Acquir. Immune Defic. Syndr. Hum. Retroviruses 18,332-340[Medline]
  24. Autran, B., Carcelain, C., Li, T. S., Blanc, C., Mathez, D., Tubiana, R., Katlama, C., Debré, P., Leibowitch, J. (1997) Positive effects of combined antiretroviral therapy on CD4+ T cell homeostasis and function in advanced HIV disease. Science 277,112-116[Abstract/Free Full Text]
  25. Viganò, A., Saresella, M., Rusconi, S., Ferrante, P., Clerici, M. (1998) Expression of CD38 on CD8 T cells predicts maintenance of high viraemia in HAART-treated HIV-1-infected children. Lancet 352,1905-1906[Medline]
  26. De Martino, M., Rossi, M. E., Azzari, C., Gelli, M. G., Galli, L., Vierucci, A. (1998) Different meaning of CD38 molecule expression on CD4+ and CD8+ cells of children perinatally infected with human immunodeficiency virus type-1 infection surviving more than five years. Pediatr. Res. 43,752-758[Medline]
  27. Sirera, R., Bayona, A., Carbonell, F., Perez-Tamarit, D., Otero, M. C., Asensi, F., Gonzalez-Molina, A. (1998) The expression of CD38 and DR are markers of immune activation and disease progression in HIV+ children. 12th World AIDS Conference: Geneva, Switzerland, June 28-July 3 1998 ,528 Casparie Meerhugowaard The Netherlands. Abstract 213*/31166
  28. Ferrero, E., Malavasi, F. (1999) The metamorphosis of a molecule: from soluble enzyme to the leukocyte receptor CD38. J. Leukoc. Biol. 65,151-161[Abstract]
  29. Malavasi, F., Calligaris-Cappio, F., Milanese, C., Dellabona, P., Richiardi, P., Carbonara, A. (1984) Characterization of a murine monoclonal antibody specific for human early lymphohemopoietic cells. Hum. Immunol. 9,9-20[Medline]
  30. Pauwels, R., De Clerq, E., Desmyter, J., Balzarini, J., Goubau, P., Herdewijn, P., Vanderhaeghe, H., Vandeputte, M. (1987) Sensitive and rapid assay on MT-4 cells for detection of antiviral compounds against the AIDS virus. J. Virol. Meth. 16,171-185[Medline]
  31. Strizki, J. M., Turner, J. D., Collman, R. G., Hoxie, J., Gonzàlez-Scarano, F. (1997) A monoclonal antibody (12G5) directed against CXCR-4 inhibits infection with the dual-tropic human immunodeficiency virus type 1 isolate HIV-1(89.6) but not the T-tropic isolate HIV-1(HxB). J. Virol. 71,5678-5683[Abstract]
  32. Savarino, A., Calosso, L., Piragino, A., Martini, C., Gennero, L., Pescarmona, G. P., Pugliese, A. (1999) Modulation of surface transferrin receptors in lymphoid cells de novo infected with human immunodeficiency virus type 1. Cell Biochem. Funct. 17,47-55[Medline]
  33. Scheglovitova, O., Capobianchi, M. R., Antonelli, G., Guanmu, D., Dianzani, F. (1993) CD4-positive lymphoid cells rescue HIV-1 replication from abortively infected human primary endothelial cells. Arch. Virol. 132,267-280[Medline]
  34. Diez-Orejas, R., Ballester, S., Feito, M. J., Ronda, M., Ojeda, G., Criado, G., Portoles, P., Rojo, J. M. (1994) Genetic and immunological evidence for CD4-dependent association of p56lck with the {alpha}ß T-cell receptor (TCR): regulation of TCR-induced activation. EMBO J 13,90-99[Medline]
  35. Bragardo, M., Buonfiglio, D., Feito, M. J., Bonissoni, S., Redoglia, V., Rojo, J. M., Ballester, S., Portoles, P., Garbarino, G., Malavasi, F., Dianzani, U. (1997) Modulation of lymphocyte interaction with endothelium and homing by HIV-1 gp120. J. Immunol. 159,1619-1627[Abstract]
  36. North, M. E., Ivory, K., Funachi, M., Webster, A. D. B., Lane, A. C., Farrant, J. (1996) Intracellular cytokine production by human CD4+ and CD8+ T cells from normal and immunodeficient donors using directly conjugated anti-cytokine antibodies and three-colour flow cytometry. Clin. Exp. Immunol. 105,517-522[Medline]
  37. Capobianchi, M. R., Abbate, I., Antonelli, G., Turriziani, O, Dolei, A., Dianzani, F. (1998) Inhibition of HIV type 1 BaL replication by MIP-1{alpha}, MIP-1 223, and RANTES in macrophages. AIDS Res. Hum. Retroviruses 14,233-240[Medline]
  38. Dull, T., Zufferey, R., Kelly, M., Mandel, R. J., Nguyen, M., Trono, D., Naldini, L. (1998) A third-generation lentivirus vector with a conditional packaging system. J. Virol. 72,8463-8471[Abstract/Free Full Text]
  39. Nakashima, H., Pauwels, R., Baba, M., Schols, D., Desmyter, J., De Clercq, E. (1989) Tetrazolium-based plaque assay for HIV-1 and HIV-2, and its use in the evaluation of antiviral compounds. J. Virol. Meth. 26,319-329[Medline]
  40. Greenough, T. C., Brettler, D. B., Somasundaran, M., Panicali, D. L., Sullivan, J. L. (1997) Human immunodeficiency virus type 1-specific cytotoxic T lymphocytes (CTL), virus load, and CD4 load, and CD4 T cell loss: evidence supporting a protective role for CTL in vivo. J. Infect. Dis. 176,118-125[Medline]
  41. Dolei, A., Biolchini, A., Serra, C., Curreli, S., Gomes, E., Dianzani, F. (1998) Increased replication of T-cell-tropic HIV strains and CXC-chemokine receptor-4 induction in T cells treated with macrophage inflammatory protein (MIP)-1alpha, MIP-1beta and RANTES beta-chemokines. AIDS 12,183-190[Medline]
  42. Szollosi, J., Tron, L., Damjanovich, S., Helliewell, S. H., Arndt, D. J., Jovin, T. M. (1994) Fluorescence energy transfer measurements on cell surfaces: a critical comparison of steady-state fluorimetric and flow cytometric methods. Cytometry 5,210-216
  43. Maciaszek, J. W., Coniglio, S. J., Talmage, D. A., Viglianti, G. A. (1998) Retinoid-induced repression of human immunodeficiency virus type-1 core promoter activity inhibits virus replication. J. Virol. 72,3394-3400[Abstract/Free Full Text]
  44. Murray, M. F., Srinivasan, A. (1995) Nicotinamide inhibits HIV-1 in both acute and chronic in vitro infection. Biochem. Biophys. Res. Commun. 210,954-959[Medline]
  45. Furlini, G., Re, M. C., La Placa, M. (1991) Increased poly(ADP-ribose)polymerase activity in cells infected by human immunodeficiency virus type 1. Microbiologica 14,141-148[Medline]
  46. Carroll, R. G., Riley, J. L., Levine, B. L., Feng, Y., Kaushal, S., Ritchey, D. W., Bernstein, W., Weislow, O. S., Brown, C. R., Berger, E. A., June, C. H., St. Louis, D. C. (1997) Differential regulation of HIV-1 fusion cofactor expression by CD28 costimulation of CD4+ T cells. Science 276,273-276[Abstract/Free Full Text]
  47. Schnittman, S. M., Lane, H. C., Greenhouse, J., Justement, J. S., Baseler, M., Fauci, A. (1990) Preferential infection of CD4+ memory T cells by human immunodeficiency virus type 1: evidence for a role in the selective T-cell functional defects observed in infected individuals. Proc. Natl. Acad. Sci. USA 87,6058-6062[Abstract/Free Full Text]
  48. Cayota, A., Vuillier, F., Scott-Algara, D., Feuillie, V., Dighiero, G. (1993) Differential requirements for HIV-1 replication in naive and memory CD4 T cells from asymptomatic HIV-1 seropositive carriers and AIDS patients. Clin. Exp. Immunol. 91,241-248[Medline]
  49. Ostrowski, M. A., Chun, T. W., Justement, S. J., Motola, I., Spinelli, M. A., Adelsberger, J., Ehler, L. A., Mizell, S. B., Hallahan, C. W., Fauci, A. S. (1999) Both memory and CD45RA+/CD62L+ naive CD4(+) T cells are infected in human immunodeficiency virus type 1-infected individuals. J. Virol. 73,6430-6435[Abstract/Free Full Text]
  50. Matheson, P. B., Weedon, J., Cappelli, M., Abrams, E. J., Shaffer, N., Bamji, M., Krasinski, K., Lambert, G., Kaul, A., Grimm, K., et al (1995) Comparison of methods of estimating the mother-to-child transmission rate of human immunodeficiency virus type 1 (HIV-1). New York City Perinatal HIV Transmission Collaborative Study Group. Am. J. Epidemiol. 142,714-718[Abstract/Free Full Text]
  51. Bofill, M., Akbar, A. N., Salmon, M., Robinson, M., Burford, G., Janossy, G. (1994) Immature CD45RA(low)RO(low) T cells in the human cord blood. I. Antecedents of CD45RA+ unprimed T cells. J. Immunol. 152,5613-5623[Abstract]
  52. Amlot, P. L., Tahami, F., Chinn, D., Rawlings, E. (1996) Activation antigen expression on human T cells. I. Analysis by two-colour flow cytometry of umbilical cord blood, adult blood and lymphoid tissue. Clin. Exp. Immunol. 105,176-182[Medline]
  53. McCloskey, T. W., Cavaliere, T., Bakshi, S., Harper, R., Fagin, J., Kohn, N., Pahwa, S. (1997) Immunophenotyping of T lymphocytes by three-color flow cytometry in healthy newborns, children, and adults. Clin. Immunol. Immunopathol. 84,46-55[Medline]
  54. Mo, H., Monard, S., Pollack, H., Ip, J., Rochford, G., Wu, L., Hoxie, J., Borkowsky, W., Ho, D. D., Moore, J. P. (1998) Expression patterns of the HIV type 1 coreceptors CCR5 and CXCR4 on CD4+ T cells and monocytes from cord and adult blood. AIDS Res. Hum. Retroviruses 14,607-617[Medline]
  55. Chan, D. C., Kim, P. S. (1998) HIV entry and its inhibition. Cell 93,681-684[Medline]



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