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Originally published In Press as doi:10.1074/jbc.M312453200 on March 19, 2004

J. Biol. Chem., Vol. 279, Issue 22, 22996-23006, May 28, 2004
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HIV-1 Nef Enhances Both Membrane Expression and Virion Incorporation of Env Products

A MODEL FOR THE NEF-DEPENDENT INCREASE OF HIV-1 INFECTIVITY*

Ilaria Schiavoni{ddagger}, Susanna Trapp§, Anna Claudia Santarcangelo{ddagger}, Valentina Piacentini{ddagger}, Katherina Pugliese{ddagger}, Andreas Baur§, and Maurizio Federico{ddagger}

From the {ddagger}Laboratory of Virology, Istituto Superiore di Sanità, Rome, 00161 Italy and the §Department of Dermatology, University of Erlangen, Erlangen, 91052 Germany

Received for publication, November 13, 2003 , and in revised form, February 27, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The expression of human immunodeficiency virus Nef increases the viral infectivity through mechanisms still not fully elucidated. Here we report that wild-type (wt) human immunodeficiency virus, type 1 (HIV-1), particles were neutralized by higher concentrations of either anti-Env glycoprotein (gp) 41 antibodies or recombinant soluble human CD4 compared with {Delta}nef HIV-1. This appeared to be the result of a Nef-induced increase of virion incorporation of both gp41 (transmembrane (TM)) and surface gp120 Env products likely originating from enhanced steady-state levels of cell membrane-associated Env products. This, in turn, seemed to be the consequence of a reduced retention of the Env precursor. Most interesting, we found that both the Nef-directed increase of Env membrane expression and the Nef-induced enhancement of HIV-1 infectivity relied on the presence of the intracytoplasmic domain of TM, supporting the hypothesis of a functional correlation between these effects. Mutagenesis studies allowed us to establish that the two leucine residues at the TM C terminus, which are part of a sorting motif involved in the control of Env membrane expression, and the 181–210-residue Nef C-terminal region were critically involved in the Nef/Env functional interaction. In conclusion, we propose that Nef increases the infectivity of HIV-1 at least in part by enhancing the amounts of Env products incorporated into virus particles.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Nef is a non-enzymatic, myristoylated protein of 27–34 kDa, expressed exclusively by human immunodeficiency virus types 1 and 2 (HIV-1/2)1 and simian immunodeficiency virus (SIV) lentiviruses (for reviews see Refs. 1 and 2). Despite its original denomination (i.e. Negative factor), Nef acts as an enhancer of the viral infectivity through mechanisms still not fully clarified. Although previous observations suggested that the expression of Nef in the producer cells increases the retrotranscription activity in the target cells (3, 4), more recent papers suggested that Nef acts by increasing the viral infectivity through the stimulation of the intravirion retrotranscription activity (5) or by increasing the cytoplasmic delivery of virions through a mechanism acting at the level of viral entry (6). Finally, the possibility that Nef influences the functions of the viral envelope has been proposed (7).

HIV-1 Env products originate from the translation of single spliced viral RNA species into a highly glycosylated gp160 precursor. Most of the Env precursor is retained in the endoplasmic reticulum or in the cis-Golgi compartment (8–10). Only a small part of the Env precursor is transported to the trans-Golgi network and then to the cell membrane, where it is inserted upon cleavage by a cell protease. The outcome is the expositions of trimers of the SU gp120 subunit that are retained at the cell membrane through non-covalent interactions with trimers of the transmembrane gp41 subunit (TM) (11). This is characterized by a long (i.e. near 150 amino acids) intracytoplasmic domain (ICD) carrying sequences involved in the intracellular trafficking. These include a tyrosine-based, membrane-proximal 712YXX{Phi}715 motif (where {Phi} indicates an amino acid with a bulky aliphatic side chain) that induces TM endocytosis by means of the interaction with the adaptor complex (AP)-2 molecules (12, 13), whereas two additional distal elements have been reported to induce retention at the Golgi apparatus of internalized TM (14). Moreover, the well conserved C-terminal dileucine motif of the ICD-TM acts as a retention signal by binding the AP-1 clathrin adaptor, ultimately controlling the levels of Env transported to the cell surface (15).

Increasing bodies of evidence indicate that the release of infectious lentivirus particles relies on a coordinated interaction among different viral proteins. As an example, it was reported that the interaction between HIV-1 matrix protein and ICD-TM is critical for the correct assembling of infectious HIV-1 particles (16, 17). By investigating the mechanisms involved in the Nef-induced increase of HIV-1 infectivity, we observed that both Env products are more efficiently incorporated in wt than in {Delta}nef HIV-1 particles, possibly as the consequence of a Nef-dependent enhanced membrane expression of both Env products. In addition, we established that both the C-terminal dileucine motif of the ICD-TM and the structured Nef C-terminal region were critically involved in such a Nef/Env functional interaction. In sum, our data are consistent with the idea that Nef increases the infectivity of HIV-1 at least in part by enhancing the Env incorporation into the infectious virus particles.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Molecular Constructs—All HIV-1 molecular clones were derivatives of the NL4-3 strain (18). The {Delta}nef HIV-1 (19), the HIV-1 expressing a TM deprived of the whole ICD (pl94/NL4-3) (20), and that deleted of the CD4 binding domain (pl227/NL4-3) have been described preciously (20). The {Delta}nef derivative of pl94 was obtained by means of an XhoI-generated frameshift at nucleotide 103. NL4-3 nef and mutants thereof, as well as the VSV-G receptor protein, were expressed under the control of the immediate early cytomegalovirus promoter. The CD8/and {Delta}NGFr (the low affinity receptor for the human nerve growth factor truncated in its ICD, 21)/ICD-TM constructs were obtained by means of overlapping PCR following the standard procedures. In detail, the sequences coding either the CD8, the {Delta}NGFr, and the ICD-TM from the NL4-3 strain were amplified from pUc19{Delta}NGFr (21), pEF-BOS/CD8 (22), and pNL4-3 plasmids, respectively. The tyrosine to cysteine or isoleucine amino acid substitutions were created by mutating the first two nucleotides of the tyrosine codon of the 712YXX{Phi}715 TM domain from TA to TG or AT, respectively. Purified PCR products were then appropriately mixed in a single PCR amplification (100 ng each in 50 µl of final volume of reaction) by using exclusively the forward primer of CD8 or {Delta}NGFr (carrying the HindIII site) and the reverse primer specific for the ICD-TM (carrying the XbaI site). The final PCR product was purified, HindIII- and XbaI-digested, and inserted in the homologous sites of the pcDNA3 vector. The dileucine ICD-TM deletion mutant was recovered using a TM reverse primer lacking the two C-terminal codons. The enhanced green fluorescence protein (GFP) and the NL4-3 Nef-GFP fusion protein were expressed by immediate early cytomegalovirus-promoted pCsg25GFP and pCNefsg25GFP vectors, respectively. Vectors expressing the Nef mutants fused with GFP were obtained by amplifying each Nef sequence through oligoprimers carrying the SacII and NheI restriction sites at the 5' and 3' ends, respectively, and afterward inserting the PCR products in the homologous restriction sites of the pCsg25GFP vector. The construction of the CD8-fused Nef deletion mutants was described previously (22, 23). All PCR-generated molecular constructs were fully sequenced by the dideoxy chain termination method in order to exclude the presence of undesired mutations.

Cell Cultures—293T and HeLaCD4 cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% decomplemented fetal calf serum (dFCS). CD4 highly positive CEMss and CEM-GFP (24) cells were cultivated in RPMI supplemented with 10% dFCS. CD4+ human peripheral blood lymphocytes were recovered as described previously (25) from the buffy coat obtained from 20–40-year-old healthy male blood donors, activated with 2 µg/ml phytohemagglutinin (Sigma), and cultivated in RPMI containing 20% dFCS in the presence of 100 units/ml interleukin-2 (Roche Applied Science).

HIV-1 Production and Purification, Infections, and Detection—Supernatants from both 293T-transfected cells and CEMss-infected cells were the source of virus preparations. 293T cells were transfected by the calcium phosphate method with the respective pNL4-3 molecular clones and, for recovering pseudotyped HIV-1, the VSV-G expressing vector in a 5:1 molar ratio. Supernatants were harvested 48 h thereafter, clarified, and for the most part of purposes, concentrated by ultracentrifugation as described (26). In some instances, the concentrated virus preparations were purified through an additional ultracentrifugation step (SW 60 rotor, 30,000 x g, 4 h at 4 °C) by loading the viral pellet on a 20% sucrose cushion. Virus preparations were also recovered upon the infection of CEMss cells with VSV-G pseudotypes performed by adsorbing the viral inoculum for 1 h at 37 °C and, after washing, seeding the cells at the concentration of 5 x 105/ml and recovering supernatants 48 h after the challenge. For HIV-1 purification, viral pellets recovered upon ultracentrifugation on a 20% sucrose cushion were resuspended in 200 µl of PBS and ultracentrifuged in a 20–60% sucrose gradient for 14 h at 100,000 x g in an SW 60 rotor. Twenty fractions of 200 µl were then collected, diluted to 1.5 ml in PBS, centrifuged for 20 min at 60,000 x g in a TL100 (Beckman Coulter, Palo Alto, CA) ultracentrifuge, and resuspended in 100 µl of PBS. Fractions included in the peak of reverse transcriptase activity were pooled, pelleted as described above, and analyzed by Western blot. Virus preparations were titrated by both anti-p24 Gag quantitative enzyme-linked immunosorbent assay (Abbott), and reverse transcriptase assay (27). The infectivity of different HIV-1 strains was evaluated by infecting CEM-GFP cells with serial dilutions of the viral preparations and scoring the number of green fluorescent cells 48 h after the challenge by FACS.

Virus Neutralization Assay—Ten µl of complete medium containing 2 ng of HIV-1 were incubated in 96-well plates for 1 h at 4 °C with 10 µl of complete medium containing dilutions of either the anti-TM mAb 2F5 or the recombinant soluble human (rsh) CD4. Afterward, 2 x 104 CEM-GFP cells were added in a volume of 20 µl and incubated for an additional 4 h at 37 °C. Sixty µl of complete medium was added, and the cultures were carried out for an additional 2 days. Finally, cells were washed twice in PBS, fixed, and the percentages of fluorescent cells scored by FACS. In the case of experiments carried out with untreated HIV-1 supernatants, the cells were scored 4 days after the challenge due to the lower virus input used (i.e. 0.2 ng).

HIV-1 Binding Assay—35S-Labeled HIV-1 preparations purified as described above served for the binding assay. These were recovered by transfecting 293T cells with wt or {Delta}nef HIV-1-expressing vectors, labeling cells 24 h later with 1.85 MBq of both 35S-methionine and -cysteine in methionine/cysteine-free medium in the presence of 5% dialyzed dFCS, and recovering supernatants after an additional 24 h. CEMss cells (2.5 x 105) were first incubated at 4 °C with an excess (i.e. 200 ng) of unlabeled HIV-1 strain defective for the CD4-binding site (CD4– HIV-1), in order to saturate the nonspecific binding sites on the cell membrane. After two washings, cells were incubated with increasing amounts (i.e. from 6 to 45 ng, corresponding to 36,000–270,000 cpm) of 35S-labeled wt, {Delta}nef, or CD4– HIV-1 preparations. After an additional hour of incubation at 4 °C, cells were extensively washed, and the cell-bound radioactivity was evaluated upon trichloroacetic acid precipitation.

Immunofluorescence Analyses—For the detection of TM on the cell membrane, 3 x 105 HIV-1-infected CEMs cells were incubated with a 1:30 dilution of the MD-1 human anti-TM mAb for 1 h at 4 °C, and, thereafter, incubated with fluorescein isothiocyanate (FITC)-conjugated anti-human IgG. The membrane expression of SU was scored similarly, except cells were labeled with a 1:30 dilution of the 4G10 mouse anti-SU mAb. Regardless of the viral receptor detected, cells were then treated with Permeafix (Ortho Diagnostic, Raritan, NJ) for 30 min at room temperature and labeled for 1 h at room temperature with a 1:50 dilution of KC57-RD1 phycoerythrin (PE)-conjugated anti-Gag mAb (Coulter Corp., Hialeah, FL). The levels of membrane expression of the CD8/or {Delta}NGFr/ICD-TM products were evaluated either by direct immunofluorescence analyses using 1:50 dilutions of PE-conjugated anti-CD8 mAb (BD Biosciences) or by indirect FACS analysis by labeling the cells with anti-NGFr mAb (21) and, in the second step, with FITC-conjugated anti-mouse IgG.

Protein Detection—Both cells and purified viral preparations cells were lysed in PBS, 1% Triton X-100 in the presence of anti-proteolytic agents. For the preparation of cytoplasmic extracts, whole cell lysates were centrifuged at 6,000 x g for 10 min at 4 °C, and the supernatants were frozen at –80 °C. Aliquots of 30 µg of total cell proteins were used for the Western blot assays. The following mono- or polyclonal Abs served for the revelation of both virus and cell-associated HIV-1 products: HT3 goat anti-Env gp160 antiserum, Chessie 8 anti-TM mAb, AG3.0 anti-Gag mAb, ARP 444 sheep anti-Nef antiserum, and a pool of human anti-HIV-1 antisera. The pulse-chase labeling assay was performed by infecting CEMss cells with wt or {Delta}nef VSV-G HIV-1 (m.o.i. 0.5) and, 48 h thereafter, labeling the cells either for 1 h with [35S]methionine and -cysteine after 1 h of starvation or for 8 h, in both cases in the presence of 10% dialyzed dFCS. Infected cell cultures were then extensively washed and re-seeded in complete medium. Cells were sampled at different times, and 100 µg of cell lysates was immunoprecipitated overnight with pooled human anti-HIV-1 antisera in the presence of protein A-G-agarose beads (Pierce). Immunoprecipitated proteins were finally resolved in a 10% SDS-PAGE and revealed by autoradiography. Specific signals were quantified by means of an Instant Imager software (Packard Instrument Co.).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
The Expression of Nef in Virus-producing Cells Correlates with Decreased Sensitivity of the Released Virus Particles to both Anti-TM and Anti-SU Neutralization Factors—By attempting to determine whether the widely reproduced drop in the infectivity of {Delta}nef HIV-1 virions could be related to an altered molecular composition, we sought to evaluate the relative amounts of Env products in the virus particles. This was first approached through a virus neutralization assay carried out by incubating equivalent amounts of wt or {Delta}nef HIV-1 particles with increasing amounts of either anti-TM mAb or rshCD4. After 1 h of incubation at 4 °C, CEM-GFP cells (i.e. a CEM cell clone expressing the GFP under the control of HIV-1 long terminal repeat in a Tat-dependent manner) (24) were added, and the percentages of infected cells were scored after an additional 48 h by FACS analysis. The number of infected cells was expressed as percent of untreated infected cells. Of note, we found that the wt HIV-1 particles were neutralized with about 10-fold higher doses of anti-TM mAb than {Delta}nef HIV-1 preparations. In particular, the concentrations of anti-TM mAb neutralizing the 50% of wt HIV-1 particles ranged between 27 and 81 ng/ml for the wt HIV-1 and between 3 and 9 ng/ml for the {Delta}nef counterpart.

These results were obtained by infecting CEM-GFP cells with equal amounts of wt or {Delta}nef HIV-1 preparations. However, it is well known that the lack of nef expression leads to a reduction in the virus infectivity from 2- to 20-fold, depending on the target cells. This was also true for CEM-GFP cells that in our hands replicate {Delta}nef HIV-1 5-fold less efficiently than wt HIV-1 (not shown). To exclude the possibility that the striking differences we noticed in terms of anti-TM virus neutralization could be influenced by the differences in the infectivity between wt and {Delta}nef HIV-1, we repeated the neutralization assay by increasing the viral input of {Delta}nef HIV-1 by 5-fold. Also, the amounts of anti-TM mAb needed for neutralizing 50% of wt particles exceeded about 10 times those required for {Delta}nef HIV-1 (Fig. 1B).



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  		FIG. 1.
Wild-type HIV-1 particles are neutralized by higher concentrations of either anti-TM mAb or recombinant soluble human CD4 compared with {Delta}nef HIV-1. A, dose-response neutralization curves performed on CEM-GFP cells infected with preparations of wt or {Delta}nef HIV-1 pretreated with different doses of the 2F5 anti-TM mAb. Two nanograms of HIV-1 Gag p24 of either wt or {Delta}nef HIV-1 were treated with 1 ng/ml to 100 µg/ml of the 2F5 anti-TM mAb and then added to 2 x 104 CEM-GFP. After 48 h of incubation, cells were harvested and scored by FACS analysis for the GFP expression. In these conditions, the absolute percentages of infected cells ranged from 23 to 65%, and from 5 to 12% upon infection with wt or {Delta}nef HIV-1, respectively. Data were calculated on the basis of six independent experiments. B, dose-response neutralization curves performed as in A, except the viral input of {Delta}nef HIV-1 was 5-fold increased, and the 2F5 mAb concentrations used were from 1 to 720 ng/ml. In these conditions, the absolute percentages of infected cells were 61 and 56% upon infection with wt or {Delta}nef HIV-1, respectively. Values represent the mean percentages from two independent experiments. C, dose-response neutralization curves performed on CEM-GFP cells infected with wt or {Delta}nef HIV-1 pretreated with different doses of rshCD4. The experiments were carried out as described in the A, except concentrations from 0.15 ng/ml to 1 µg/ml of rshCD4 were used as the neutralization factor. In these conditions, the absolute percentages of infected cells ranged from 25 to 51% and from 6 to 19% upon infection with wt or {Delta}nef HIV-1, respectively. Data were calculated on the basis of the results from three independent experiments. D, anti-TM dose-response neutralization curves using unprocessed HIV-1 supernatants. The assay was performed as in A, except the viral input was 10-fold lower, and the cells were analyzed 4 days after the infection. In these conditions, the absolute percentages of infected cells were 16 and 4% for wt and {Delta}nef HIV-1, respectively. Values represent the mean percentages from two independent experiments. E, anti-SU dose-response neutralization curves performed using unprocessed HIV-1 supernatants. The experiments were carried out as described in D, except than different concentrations of rshCD4 were used as the neutralization factor. In these conditions, the absolute percentages of GFP-positive cells were 14 and 5% for wt or {Delta}nef HIV-1-infected cells, respectively. Data were calculated on the basis of the results from two independent experiments. In all panels, the proportions of infected cells are expressed as percent of cells not treated with the respective neutralization factor.

 
TM and SU products originated from a common gp160 Env precursor. Hence, monitoring the possible effects of Nef on Env SU might be helpful in clarifying which level Nef acts on the Env products. Thus, we repeated the neutralization assay by using different concentrations of rshCD4. Similarly to that already observed for anti-TM mAb, significantly higher doses (i.e. about 10-fold) of rshCD4 were required in order to neutralize 50% of the infectivity of wt compared with {Delta}nef HIV-1 particles (Fig. 1C). Coincident results were obtained using concentrated HIV-1 preparations upon purification on a sucrose cushion (not shown). Finally, we demonstrated that the virus concentration procedures did not influence the outcomes from both the anti-TM and anti-SU neutralization assays. In fact, we reproduced the results obtained previously by also neutralizing unprocessed HIV-1 supernatants (Fig. 1, D and E).

The Expression of Nef in the Producer Cells Leads to Increased Incorporation of Env Products in Virus Particles—The lower amounts of anti-Env neutralizing factors required for the neutralization of {Delta}nef compared with wt HIV-1 could imply a defect in the incorporation of Env products in the absence of Nef expression. To test such an hypothesis, we sought to evaluate the amounts of Env proteins in both wt and {Delta}nef virions. Viral particles emerging from CEMss cells infected with VSV-G HIV-1 were purified through a 20–60% sucrose gradient as described under "Experimental Procedures." Two-fold serial dilutions of viral lysates (from 50 to 6.25 ng of Gag p24 for the TM detection and, in view of its fainter signal, from 400 to 100 ng for SU; Fig. 2A) as well as the lysates from the respective producer cells (Fig. 2B) were analyzed by Western blot. Such analyses showed that the amounts of both SU and TM found in wt virions exceeded those detectable in {Delta}nef HIV-1 at 2–4-fold, in the absence of significant differences in the steady-state levels of such viral products in the producer cells (Fig. 2B). Of note, some reductions of both p55 and p32 Gag premature products were also detectable in {Delta}nef HIV-1. Consistent results were obtained by analyzing virus particles 48 h after the transfection of 293T cells with the respective HIV-1 molecular clones (not shown). In summary, we found that the decreased susceptibility to neutralization factors of HIV-1 produced in the presence of Nef coupled with increased virion incorporation of Env products.



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  		FIG. 2.
Wild-type virus particles incorporate higher amounts of Env products compared with {Delta}nef HIV-1. A, Western blot analysis on serial dilutions of purified wt or {Delta}nef HIV-1 virions. CEMss cells were infected with VSV-G wt or {Delta}nef HIV-1 (m.o.i. of 1), and 24 h thereafter, cell cultures were extensively washed. After an additional 24 h, supernatants were harvested, and viral particles were concentrated by ultracentrifugation and purified as described under"Experimental Procedures."Equivalent amounts of each HIV-1 preparation were run in 10% SDS-PAGE, and for the TM detection (on the left), signals were revealed by using 1:500 dilutions of both Chessie 8 anti-TM mAb and AG3.0 anti-Gag mAb. For the SU detection (on the right), signals were revealed by using a 1:1000 dilution of HT3 anti-gp160 antiserum and a 1:2000 dilution of AG3.0 anti-Gag mAb. As controls, 50 (in the TM assay) or 400 ng (in the SU assay) of {Delta}env HIV-1 as well as supernatants from mock-infected cells similarly processed were included. B, Western blot analyses of lysates of cells producing the HIV-1 particles analyzed in A. Replicated filters were incubated with a 1:1000 dilution of pooled human anti-HIV-1 antisera (upper panel) or a 1:1000 dilution of the sheep anti-Nef polyclonal Ab ARP 444 (lower panel). For all panels, the migration of major viral products is indicated on the left side, whereas the molecular marker sizes are reported on the right.

 
The Expression of Nef Induces Increased Expression of both TM and SU HIV-1 Proteins at the Cell Membrane—Data reported here indicate that the expression of Nef leads to an increased inclusion of both HIV-1 Env subunits in the virus particles. To inspect the underlying mechanism, we first were interested in evaluating the influence of Nef on the levels of cell membrane-associated Env products. For this purpose, CEMss cells were infected with an m.o.i. of 1 for VSV-G wt or {Delta}nef HIV-1. The use of pseudotypes was necessary for both increasing the overall infection efficiencies and alleviating the strong differences in the infectivity between the two HIV-1 strains. Infected cells were harvested at different times after the challenge, labeled with either anti-TM (Fig. 3A) or anti-SU (Fig. 3B) mAbs, and after cell permeabilization, stained with a mAb detecting HIV-1 Gag-related products. The FACS analyses revealed that the expression of Nef correlated with higher steady-state levels of both Env products at the cell membrane starting 24 h post-infection, as revealed by the increased percentages of double positive cells and/or by the enhanced mean fluorescence intensity values. Of note, such increases did not appear to be related to the extent of the viral genome expression, as indicated by the Gag-related fluorescence intensities. More prolonged observations were hindered by the rapid decline of the cell viability. In conclusion, we established a direct correlation among the Nef expressions in the producer cells, the amount of cell membrane-associated Env products, and the extent of their virion incorporation.



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  		FIG. 3.
The expression of Nef leads to increased cell membrane steady-state levels of both TM and SU Env products. Double parameter FACS analyses for the membrane expression of Env proteins and the cytoplasmic accumulation of Gag-related products in cells infected by wt or {Delta}nef HIV-1 are shown. CEMss cells were infected with an m.o.i. of 1 for VSV-G wt or {Delta}nef HIV-1, and the expression of the viral products was detected 16, 24, and 48 h thereafter. A, anti-TM/Gag FACS analysis. Cells harvested at the indicated times after the challenge were labeled with the MD-1 anti-TM human mAb and, in the second step, with FITC-conjugated anti-human IgG. Afterward, cells were treated with Permeafix and, as the final step, labeled with the PE-conjugated anti-Gag KC-57 mAb. B, anti-SU/Gag FACS analyses of the same cell populations analyzed in A. The cell membrane SU was detected using the 4G10 anti-SU mouse mAb, and, in a second incubation step, a FITC-conjugated anti-mouse IgG. For both panels, quadrants were set on the basis of cells harvested 48 h after the challenge and labeled with irrelevant, species specific, and isotype matched mAbs. Such a background fluorescence appeared identical to those detected by labeling uninfected cells with anti-Gag and anti-Env mAbs (not shown). The percentages of HIV-1 Gag-positive/TM or SU-negative, as well as of double positive cells are reported. The FITC-specific geometric mean fluorescence values (geoMFI) of HIV-1 Gag positive cells are also indicated.

 
The Nef Expression Couples with a Decreased Persistence of the gp160 Env Precursor—Backward in the Env life cycle, we next analyzed whether the presence of Nef could influence the fate of a newly synthesized Env precursor in HIV-1-infected cells. Cells infected with wt or {Delta}nef HIV-1 were labeled for 1 h and harvested at different time points until 8 h after the chase. Both Env gp160 and a product of its cleavage, i.e. SU gp120, were detected by means of immunoprecipitation (Fig. 4A). On the contrary, under these conditions the TM subunit was not reproducibly detectable. No major differences were noticed in such a first set of experiments, suggesting that both the stability and the maturation of the 35S-labeled gp160 Env precursor produced in 1 h of labeling were not affected by Nef. Since the 1 h of labeling allowed detecting Env products no longer than 8 h after the chase, we repeated the pulse-chase assay by prolonging the pulse to 8 h and the chase to 30 h (Fig. 4B). Clearly, under these conditions higher amounts of gp160 Env persisted in {Delta}nef HIV-1-infected cells, as was also detectable by scanning the specific signals (Fig. 4C). Consistently, the gp160/gp120 SU ratio appeared significantly higher in {Delta}nef HIV-1-infected cells both at the steady state and in all chase time points (Fig. 4D). This ratio decreased in the earlier chase time points, increasing between 4 and 24 h, which is indicative of an overall impaired production of Env mature products. Conversely, such a ratio constantly decreased in wt HIV-1-infected cells, which is suggestive of a gradual Env gp160 processing and membrane targeting of its cleavage products.



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  		FIG. 4.
The lack of Nef expression leads to persistence of the Env gp160 precursor. CEMss cells were infected with VSV-G wt, {Delta}nef, or {Delta}env HIV-1 (m.o.i. of 0.5) and, after 48 h, labeled in the presence of 10% dialyzed dFCS with 1.85 MBq/ml of both 35S-cysteine and -methionine either for 1 h after 1-h of starvation (A) or for 8 h (B). Thereafter, cell cultures were washed and re-seeded in complete medium. Cell were then harvested at the indicated times and processed by immunoprecipitation assay by using a pool of human anti-HIV-1 antisera. On the left side, the migration of gp160 and gp120 Env products is indicated, whereas the molecular marker sizes are reported on the right. C, the amounts of Env gp160 as calculated by scanning the specific signals shown in B by means of the Instant Imager apparatus and expressed as percentages relative to the time 0. D, Env gp160/gp120 ratios for each time point tested in B as calculated by scanning the specific signals. All results are representative of two independent experiments.

 
The fact that the expression of Nef correlated with a decreased persistence of the Env precursor is suggestive of its diminished retention at the cis-Golgi. This, in turn, could be the basis of the Nef-dependent increase in Env cell membrane expression we observed previously.

Wild-type HIV-1 Binds the Cell Membrane of CD4+ Cells More Efficiently than {Delta}nef HIV-1—Reported data strongly suggest that the presence of Nef in the producer cells increases the levels of Env products incorporated in HIV-1 virions. In order to test the biological relevance of such a finding, we compared the binding activity on CD4+ human cells of wt versus {Delta}nef HIV-1. After a pretreatment at 4 °C with an excess (i.e. 200 ng) of an HIV-1 strain defective for the CD4-binding site (CD4– HIV-1), CEMss cells were incubated with different amounts (i.e. from 6 to 45 ng) of purified, 35S-labeled wt, {Delta}nef, or, as control, CD4– HIV-1 preparations. After an additional hour of incubation at 4 °C, cells were extensively washed, and the cell-associated radioactivity was evaluated upon trichloroacetic acid precipitation. As shown in the Fig. 5, significantly higher amounts (i.e. 1.5–2-fold) of wt than {Delta}nef HIV-1 were found associated with CEMss cells, consistently with the observation that the expression of Nef in the producer cells leads to increased incorporation of Env products into virus particles.



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  		FIG. 5.
Wild-type HIV-1 binds more efficiently the membrane of CEMss cells than {Delta}nef HIV-1. 2.5 x 105 CEMss cells were incubated at 4 °C with an excess of HIV-1 deleted of the CD4 binding domain (CD4– HIV-1) and, in a second step, with different amounts of purified and 35S-labeled wt, {Delta}nef HIV-1, or, as negative control, CD4– HIV-1. The amounts of bound HIV-1 were evaluated by measuring the radioactivity associated with the cells considering that, in our hands, 1 ng of p24 HIV-1 corresponded to about 104 cpm. Values were calculated on the basis of duplicated conditions from two experiments.

 
The Nef-dependent Increase of Env Products at the Cell Membrane Relies on the Presence of the Intracytoplasmic Domain (ICD) of TM—In order to define the domain of Env responsive to the stimulatory effect of Nef, we measured the expression of both viral receptors on cells infected with VSV-G HIV-1 pseudotypes expressing HIV-1 Env-mutated/deleted products. Most interesting, we found that the presence of the ICD-TM was critical for the Nef-enhancing phenotype. Such a result was accomplished by infecting CEMss cells with VSV-G HIV-1 expressing a TM truncated in the whole ICD or, as a control, with the nef-defective counterpart. By monitoring the levels of both HIV-1 Gag and Env products by FACS 48 h after the challenge, we in fact observed that the Nef expression did not induce significant variations of the steady-state levels of both cell membrane-associated TM and SU (Fig. 6).



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  		FIG. 6.
The levels of Env products expressed by HIV-1 deleted of the ICD-TM are not influenced by the expression of Nef. Double FACS analysis for the membrane expression of Env proteins and cytoplasmic accumulation of Gag-related products in cells infected with wt or {Delta}nef HIV-1 strains both deleted of the ICD-TM. CEMs cells were infected with an m.o.i. 1 of VSV-G wt or {Delta}nef pl94 HIV-1 (i.e. a NL4-3 strain lacking the ICD-TM), and the expression of the viral products was detected 48 h thereafter. HIV-1 Gag-related products together with either TM (on the left side) or SU (on the right side) products were detected by labeling the transfected cells as described in the legend of Fig. 3. Quadrants were set on the basis of the fluorescence levels of infected cells harvested 48 h after the challenge and labeled with irrelevant, species-specific, and isotype-matched mAbs. The percentages of HIV-1 Gag-positive/TM- or SU-negative, as well as of double-positive cells are reported. The FITC-specific geoMFI values of HIV-1 Gag-positive cells are also indicated.

 
The Deletion of the ICD-TM Abrogates the Nef-induced Enhancement of Infectivity—Our results suggest that Nef induces increased Env virion incorporation by means of the interaction with the ICD-TM. To determine whether such a phenomenon is part of the effects of Nef on the HIV-1 infectivity, we expected that the lack of ICD-TM hinders at least in part the Nef-induced increase of infectivity. To test such an hypothesis, we assayed the extent of replication for a {Delta}ICD-TM HIV-1 mutant by challenging CD4+ quiescent human lymphocytes, i.e. cells that in the absence of Nef do not support HIV replication upon cell activation (28, 29).

Quiescent CD4+ lymphocytes (2.5 x 105 cells) from two healthy donors were separately infected with 5 ng (corresponding to an m.o.i. of 0.5 as measured on CEMss cells) of either wt, {Delta}nef, {Delta}ICD-TM, or {Delta}ICD-TM/{Delta}nef HIV-1 strains, and the release of virus particles was monitored until 15 days after the infection (Fig. 7). Either {Delta}nef, {Delta}ICD-TM, or {Delta}ICD-TM/{Delta}nef HIV-1 strains appeared to replicate inefficiently upon the activation of CD4+ lymphocytes. Most important, although the viability of cell cultures infected with wt HIV-1 dropped below 2% at day 10 post-infection, the cultures infected with each defective HIV-1 strain remained over 95% viable until day 15 after challenging (not shown). Of note, similar results were obtained even by increasing 4-fold the viral input (not shown). In sum, the presence of ICD-TM seems required for the Nef-induced enhancement of HIV-1 infectivity, supporting the idea that the Nef/ICD-TM interaction is important for the optimal infectivity of HIV-1.



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  		FIG. 7.
The Nef-induced enhancement of the viral infectivity is abrogated by the lack of ICD-TM. Quiescent CD4+ lymphocytes (2.5 x 105) from two healthy donors (A and B) were incubated with 5 ng of either wt, {Delta}nef, {Delta}ICD-TM, or {Delta}nef/{Delta}ICD-TM HIV-1. After 2 h of adsorption, cells were washed, cultivated for 3 days, and thereafter activated with PHA. Supernatants were harvested starting from 2 days after activation until 15 days post-infection, and the reverse transcriptase activities were measured as cpm/ml of supernatant normalized for 105 cells after background subtraction. Results are representative of two independent experiments.

 
The C-terminal Dileucine Motif in the ICD-TM Is Critical for the Nef-dependent Increase of TM at the Cell Membrane—The identification of the domains of the ICD-TM involved in the interaction with Nef was approached by constructing vectors expressing products of fusion between the ecto- and transmembrane domains of CD8 and the ICD-TM, as described previously (14, 15). Most important, such an experimental design successfully reproduced the Nef-induced enhancement of Env cell membrane expression, as we proved by co-transfecting 293T cells with vectors expressing the CD8/ICD-TM and Nef-GFP fusion proteins (Fig. 8A).



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  		FIG. 8.
The C-terminal TM dileucine motif is required for the Nef-dependent, TM-enhanced membrane expression. A, double FACS analyses for the presence of TM at the cell membrane in Nef-GFP expressing 293T. Cells were co-transfected with vectors expressing either GFP or Nef-GFP together with a construct expressing the CD8/ICD-TM fusion protein and, 40 h thereafter, analyzed by FACS after the staining with PE-conjugated anti-CD8 mAb. Quadrants were set on the basis of the fluorescence levels of mock-transfected cells labeled in a similar way. The percentages of GFP-positive/CD8-negative as well as of double-positive cells are indicated. The PE-specific geoMFI of GFP-positive cells are also reported. B, effects of the Nef expression on the levels of cell membrane expression of wt or 855–856 dileucine deleted ({Delta}LL) TM. 293T or HeLaCD4 cells were co-transfected with vectors expressing GFP or Nef-GFP, and the CD8 was fused with either the wt ICD-TM or its {Delta}LL-deleted form. After 40 h, the cells were harvested and labeled as described in A. Results are reported as the percentages of CD8-positive cells among the Nef-GFP-positive cell populations in percentage of cells expressing GFP alone. Percentage values are expressed as the means of six (for 293T cells) or two (for HeLaCD4) independent experiments. C, Western blot for the detection of CD8/ICD-TM or CD8/{Delta}LLICD-TM fusion proteins. Fractions of the cell populations utilized for the FACS analyses were lysed, and the cytoplasmic extracts were analyzed by Western blot for the expression of either the ICD-TM based fusion products (upper panel) or the actin protein (lower panel). On the left side, the arrows indicate the migration of the detected protein products, whereas the molecular marker sizes are reported on the right.

 
The HIV-1 ICD-TM contains internalization as well as retention signals. In particular, it was shown that the ICD-TM undergoes efficient cell internalization by means of a membrane-proximal tyrosine-based 712YXX{Phi}715 endocytosis signal (12). In this regard, we observed that the inactivation of such an internalization motif by replacing the tyrosine with either cysteine or isoleucine did not influence the Nef-induced enhancement of the TM membrane targeting (not shown), thus excluding that it is involved in the regulation of Env membrane expression.

The ICD-TM also carries a retention signal, i.e. a C-terminal dileucine motif appearing pretty well conserved among HIV and SIV isolates and that is involved in the control of the Env cell membrane targeting by means of association to the AP-1 clathrin adaptor (15). In order to test whether Nef interacts with such a motif, ICD-TM deleted of the two terminal dileucines (CD8/{Delta}LL ICD-TM) was expressed in 293T cells together with Nef-GFP or, as control, GFP-expressing vectors. Our results clearly show that the Nef-dependent increase of the steady-state membrane levels of CD8/ICD-TM was no more detectable in cells expressing the {Delta}LL ICD-TM, and overlapping results have been obtained by testing HeLaCD4 cells (Fig. 8B). Of note, Western blot analysis proved that both CD8/ICD-TM and CD8/{Delta}LL ICD-TM products appeared to be expressed at comparable levels (Fig. 8C). These results suggest that Nef counteracts the Env retention exerted by the host cell through the TM C-terminal dileucine motif.

The C-terminal Region of Nef Accounts for the Enhanced Expression of TM at the Cell Membrane—We next attempted to identify the Nef domains involved in the functional interaction with Env first by testing previously characterized Nef motifs. To this end, we set up a series of experiments by co-transfecting 293T cells with vectors expressing either mutants of Nef alone, in the form of GFP fusion proteins together with the CD8/ICD-TM, and/or by infecting CEMss cells with VSV-G HIV-1-expressing diverse Nef mutants and evaluating the levels of TM membrane expression 40 or 48 h thereafter. The Nef mutants we tested could be classified in terms of their defective functions as follows: cell membrane targeting (i.e. G2A) in view of the lack of the N-terminal myristoylation site (30); interaction with the SH3 domain of signaling cell proteins (i.e. P72A/X73X/X74X/P75A) (31); binding the cell thioesterase (i.e. D123G) (32); and interaction with the endocytotic machinery (i.e. E154Q/E155Q, L164A/L165A, and D174A/D175A), leading to a decreased internalization activity (33–35). Notably, all Nef mutants tested increased the Env membrane targeting similarly to the wt counterpart (not shown).

As an alternative approach, we transfected 293T cells with vectors expressing a series of CD8-based fusion proteins including different Nef deletion mutants as ICD. Indeed, such molecular constructs were already proven to be an effective tool in revealing the influence of Nef on the cell signaling (23). This set of experiments was performed by using a vector expressing the {Delta}NGFr/ICD-TM fusion protein, whose membrane expression, similar to that already observed in the case of CD8-based fusion products, is enhanced upon Nef co-expression (Fig. 9A). By means of double parameter FACS analyses carried out 40 h after the co-transfection, we found that the expression of a quite short C-terminal region of Nef, including the amino acids 181–210, was still able to increase the ICD-TM membrane exposition (Fig. 9B). Of note, the effects of SF2 Nef, from which all Nef deletion mutants have been recovered, appeared quite similar to those induced by NL4-3 Nef (Fig. 10B). Most important, all Nef-deleted constructs appeared to be efficiently expressed, as detected by Western blot analysis (Fig. 9C). These results indicate that the Nef effects on Env relies on the presence of a short C-terminal Nef region.



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  		FIG. 9.
Effects of the expression of Nef deletion mutants on the TM expression. A, double parameters FACS analyses for the membrane expression of CD8 and NGFr in 293T co-transfected with vectors expressing either CD8T or CD8-Nef and the NGFr/ICD-TM fusion protein. Forty h after transfection, the cells were analyzed by FACS upon staining with anti-NGFr mAb coupled, in a second step, with FITC-conjugated anti-mouse IgG, followed by the labeling with a PE-conjugated anti-CD8 mAb. Quadrants were set on the basis of the fluorescence levels of mock-transfected cells labeled in a similar way. The percentages of CD8-positive/NGFr-negative, as well as of double-positive cells are indicated. The FITC-specific geoMFI of CD8-positive cells are also reported. B, efficiency of ICD-TM membrane targeting upon co-expression of diverse Nef deletion mutants. 293T cells were co-transfected with vectors expressing the NGFr/ICD-TM fusion protein together with CD8-based chimeric proteins bearing, as the intracytoplasmic tail, different Nef deletion mutants. As controls, vector expressing CD8 fused with NL4-3 or SF2 Nef alleles (i.e. from two distinct wild-type HIV-1 strains) or expressing a CD8 truncated in its ICD (T) were used. After 40 h, cells were labeled for the membrane expression of both NGFr and CD8. Negative controls of the fluorescence levels were set up by labeling the cells with an irrelevant mouse mAb, together with a PE-conjugated, isotype-matched IgG. The number of NGFr-positive cells among the CD8-expressing cell populations are expressed in percent of the CD8T-transfected cells and were calculated as the means of three independent experiments with duplicate conditions. C, Western blot for the detection of CD8/Nef fusion proteins and deletion mutants thereof. Fractions of the cell populations utilized for the FACS analyses were lysed, and the cytoplasmic extracts were analyzed by Western blot analysis using the ARP444 anti-Nef sheep antiserum. The molecular marker sizes are reported on the right side.

 



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  		FIG. 10.
The expression of CD8/181–210 Nef rescues both the infectivity and the neutralization phenotypes of {Delta}nef HIV-1. 293T cells were co-transfected with the {Delta}nef HIV-1 molecular clone together with vectors expressing either the CD8T, CD8/wtNef, or CD8/181–210Nef. Forty eight h thereafter, cell supernatants were harvested, concentrated, and used to infect CEM-GFP cells in the absence (A) or in the presence of different concentrations of the anti-TM 2F5 mAb (B). After an additional 2 days, infected cell cultures were scored by FACS analysis for the GFP expression. The infectivity of each viral preparation was evaluated through the proportion of infected cells and expressed in A as percentage of {Delta}nef HIV-1-infected cells (Ctrl, whose absolute percent of infected cells averaged 8%). B, the proportions of infected cells are expressed as percentages of untreated infected cells. Data were calculated as the mean values from two independent experiments.

 
The Expression of the 181–210-Residue Region of Nef Rescues Both the Infectivity and the Neutralization Phenotypes of {Delta}nef HIV-1—The fact that the expression of the C-terminal region of Nef largely recapitulates the effects of Nef on the ICD-TM should imply that the expression of such a Nef deletion mutant would also be able to rescue the functional defects of {Delta}nef HIV-1. In this regard, we analyzed both the infectivity and neutralization phenotypes on HIV-1 particles recovered from the supernatants of 293T cells co-transfected with the {Delta}nef HIV-1 molecular clone together with CD8/wt Nef or its 181–210-residue deletion mutant. CEM-GFP cells were then infected either by the viral particles as such, to measure the respective efficiencies of infectivity, or by neutralization with different concentrations of anti-TM mAb, to evaluate the neutralization phenotype. Taken together, the results we obtained indicated that the expression of the 181–210-residue Nef peptide rescued both the neutralization and the infectivity phenotypes in amounts comparable with that induced by the full-length wt Nef (Fig. 10). Possibly as the consequence of a more rapid sorting toward the cell membrane due to the CD8 moiety, the effects induced by CD8/wtNef, however, appeared to be of a reduced potency compared with those induced by either untagged or virus-expressed Nef (data not shown). In any case, these data are consistent with our previous data pointing out the 181–210-residue Nef as a region important for the enhancement of Env expression.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
The evidence that the lack of the in cis Nef expression can almost be completely restored in terms of viral infectivity by expressing Nef in trans in the producer cells (36) suggests that Nef influences the composition of the emerging virus particles. By means of a GFP reporter-based neutralization assay, we demonstrated that the virus particles emerging from cells expressing {Delta}nef HIV-1 were about 10-fold more efficiently neutralized than wt HIV-1 by treatment with either anti-TM antibodies or rshCD4. On the other hand, the Western blot analysis on purified viral particles showed that wt HIV-1 incorporated at least 2-fold more Env products than the {Delta}nef counterpart. These quantitative discrepancies could be explained considering that the quantitative evaluation of Env products by Western blot could not discriminate between infectious and defective virus particles, as in the case of the neutralization assays. In this regard, it has been reported that the amounts of defective viral particles largely override the actually infective virions in virtually all viral preparations (37). Significantly enough, such a phenomenon increases with the efficiency of viral release from the producer cells. Thus, it is conceivable that the Western blot analysis does not fully account for the effects of Nef in the really infectious virions.

A previously reported Western blot assay on {Delta}nef HIV-1 virus particles emerging from H9 cells 13–17 days after the challenge failed to reveal significant variations in the Env contents compared with the wt virions (38). Possibly, the fact that the analysis we reported here has been carried out on virus particles harvested 48 h after the challenge strongly reduced the possibility of the outgrowth of HIV-1 particles mutated back to the wt phenotype, indeed a frequent occurrence in the presence of re-infection events. Conversely and consistently with our results, a more recent report (39) describes a strong Nef-dependent increase of Env incorporation in virions produced by CD4-expressing cells. This, however, was no longer observed in CD4-negative cells, where only a single Env product disappeared in the absence of Nef. It is conceivable that the discrepancy with data we obtained by producing HIV-1 upon 293T cell transfection depended, at least in part, on the analysis carried out on crude rather than purified virus preparations.

Additional inconsistencies concerning the consequences of Nef on the Env cell membrane expression can be found in the literature. In fact, although no effects of Nef on the cell membrane association of SU have been reported in a first instance (3), a more recent paper, on the contrary, reports that the expression of Nef correlated with increased amounts of SU on the cell membrane. Such an apparent contradiction might be explained in view of the different experimental systems used, i.e. the infection with Env-expressing vaccinia vectors of stable Nef-expressing cells in the first case, and the infection of CD4+ lymphocytes with either NL4-3 or a primary HIV-1 isolate in the latter case (40). In this regard, we describe here a nice correlation among the effects of Nef on the membrane expression of Env products, their incorporation into the virions, the susceptibility of the virus particles to either anti-TM or anti-SU neutralization factors, and the respective binding activity.

Notably, the results we obtained by transfecting ICD-TM-based molecular chimeras appeared consistent with the data from infection experiments, thus tentatively excluding a major role of additional viral proteins in the described Nef phenotype. Furthermore, the results from the infection of quiescent CD4+ lymphocytes with HIV-1 strains deleted of the ICD-TM represent a significant link between the influence of Nef on the Env incorporation into virus particles and its effects on virus infectivity.

It has been reported that the ICD-TM binds AP-1 clathrin adaptor molecules through its C-terminal dileucine motif, and that such an interaction increases the Env retention thus contributing to controlling the amounts of Env products targeted to the cell membrane (15). The data we obtained upon the expression of a ICD-TM deleted in the C-terminal dileucine motif suggest that Nef interferes with the retention control exerted by the AP-1/TM interaction on the Env cell membrane transport. This seems to agree with the results from our pulse-chase experiments suggesting that in the presence of Nef part of the gp160 Env precursor molecules escapes the retention at the endoplasmic reticulum/cis-Golgi compartment more efficiently, gaining access to the trans-Golgi, thus undergoing maturation, insertion in the cell membrane, and finally virion incorporation.

The analysis of Nef mutated in previously characterized functional domains offered additional suggestions about the mechanism underlying the Nef/Env interaction. In particular, the fact that the expression of the G2A Nef mutant, i.e. a Nef allele unable to target the cell membrane, reproduced the wt phenotype supports the idea that Nef interacts with Env during its anterograde pathway. Similarly, the evidence that Nef mutated in domains involved with the endocytotic cell machinery, showing a wt-like phenotype, is consistent with the hypothesis that events occurring after the Nef internalization are not involved in the effects on Env products. Consistently, none of the Nef domains we tested are included in the C-terminal 181–210-residue region we found involved in the TM-enhancing phenotype.

Taken together, our data could be of some help for the interpretation of previously described effects correlating with the expression of Nef. In particular, it is conceivable that pseudotyping HIV-1 with heterologous receptors like VSV-G overcomes the Nef-induced enhancement of the viral infectivity (41, 42) in that pseudotypes no longer utilize the HIV-1 Env viral receptors. Similarly, enhanced levels of Env products might account for the Nef-dependent increase of the HIV-1 cytoplasmic delivery (6).

Recent data obtained through a fluorescence-based fusion assay detecting the internalization of a heterologous enzyme (i.e. Escherichia coli {beta}-lactamase), fused with HIV-1 Vpr, have been interpreted as the inability of Nef in affecting the efficiency of HIV-1 fusion (43). However, conclusions based on the results from such types of assays should be made with caution, as they do not allow the discrimination of the fusion events underlying the entry of infectious viral particles from that of non-infectious ones, or even from the endocytosis-mediated viral entry (44, 45).

HIV/SIV lentiviruses possess an ICD-TM much longer than other retroviruses, i.e. about 150 versus 20–30 amino acids. Such a long tail contains redundant internalization/retention signals, leading to a very inefficient membrane expression of Env products. It might be considered the intriguing hypothesis that Nef evolved to counteract the strong activity of Env retention in the host cells. This effect appears synergic with that of the Gag matrix protein, which has been proven to inhibit the TM internalization and lysosome degradation (17). The overall effect is a strongly reduced anti-Env cellular activity, with an obvious advantage in terms of the infectivity of emerging virus particles.

Finally, the possibility that the potent Nef-induced CD4 down-regulation acts as a function complementary to the inhibition of Env retention should also be considered. In fact, enhanced membrane expression of SU would disappear in the presence of high levels of CD4, because the CD4-SU interaction at the cell membrane was proven to impair the infectivity of emerging virus particles (39, 46).

Rather than proposing a novel function for Nef, already showing an excess of activities, we sought to unravel the mechanism of a phenomenon already associated with the Nef expression. In addition, our data could open new avenues in studying the interactions among the HIV/SIV proteins required for the assembling of infectious virus particles and at the same time appearing to be of some value for the discovery of new antiviral drugs able to interfere with such a critical process.


   FOOTNOTES
 
* This work was supported by grants from the AIDS project of the Ministry of Health, Rome, Italy. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

To whom correspondence should be addressed: Laboratory of Virology, Istituto Superiore di Sanità, Viale Regina Elena, 299, 00161 Rome, Italy. Tel.: 39-6-49903248; Fax: 39-6-49903002; E-mail: federico{at}iss.it.

1 The abbreviations used are: HIV-1, human immunodeficiency virus, type 1; AP, adaptor complex; dFCS, decomplemented fetal calf serum; FITC, fluorescein isothiocyanate; GFP, green fluorescence protein; ICD, intracytoplasmic domain; mAb, monoclonal antibody; PE, phycoerythrin; SIV, simian immunodeficiency virus; SU, surface; TM, transmembrane; VSV-G, glycoprotein of the vesicular stomatitis virus; FACS, fluorescence-activated cell sorter; wt, wild type; m.o.i., multiplicity of infection; PBS, phosphate-buffered saline; rsh, recombinant soluble human; NGFr, nerve growth factor receptor; gp, glycoprotein; geoMFI, geometric mean fluorescence values. Back


   ACKNOWLEDGMENTS
 
The following reagents were obtained from the AIDS Research and Reference Program, Division of AIDS, NIAID, National Institutes of Health, Bethesda: 2F5 anti-TM mAb contributed by Dr. H. Katinger; rshCD4, MD-1 anti-TM mAb contributed by Dr. R. A. Myers; 4G10 anti-SU mAb contributed by Dr. A. Von Brunn; HT3 goat anti-gp160 Env antiserum; AG3.0 anti-Gag mAb contributed by Dr. J. Allan; and Chessie 8 anti-TM mAb contributed by Dr. G. K. Lewis. The ARP444 sheep anti-Nef antiserum was a kind gift of Dr. M. Harris, University of Leeds, UK. The {Delta}nef NL4-3 molecular clone was from Dr. J. C. Guatelli, University of California, San Diego. {Delta}ICD-TM and CD4– pNL4-3 molecular clones were the kind gifts from Dr. V. Bosch, University of Heidelberg, Germany. We are indebted to F. M. Regini for excellent editorial assistance.



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 DISCUSSION
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