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Journal of Virology, November 2006, p. 10663-10674, Vol. 80, No. 21
0022-538X/06/$08.00+0     doi:10.1128/JVI.02621-05
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Nef Alleles from Human Immunodeficiency Virus Type 1-Infected Long-Term-Nonprogressor Hemophiliacs with or without Late Disease Progression Are Defective in Enhancing Virus Replication and CD4 Down-Regulation{triangledown}

Andrea Crotti,1 Francesca Neri,2 Davide Corti,1 Silvia Ghezzi,2 Silvia Heltai,1 Andreas Baur,3 Guido Poli,1,4 Elena Santagostino,5 and Elisa Vicenzi2*

AIDS Immunopathogenesis Unit, San Raffaele Scientific Institute, Milan, Italy,1 Viral Pathogens and Biosafety Unit, San Raffaele Scientific Institute, Milan, Italy,2 University of Miami, School of Medicine, Department of Microbiology and Immunology, Miami, Florida,3 Vita-Salute, San Raffaele University, School of Medicine, Milan, Italy,4 A. Bianchi Bonomi Hemophilia and Thrombosis Center, IRCCS Maggiore Hospital, Mangiagalli and Regina Elena Foundation, Milan, Italy5

Received 16 December 2005/ Accepted 16 August 2006


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ABSTRACT
 
Infection with human immunodeficiency virus (HIV)-encoding defective nef variants may contribute to a relatively benign course of disease in a minority of long-term nonprogressors (LTNP). We have examined the functions of nef alleles from six individuals belonging to the same cohort of hemophiliacs infected with HIV-1 prior to 1985 and classified as LTNP in 1995. Three out of six individuals have progressed to HIV disease (late progressors [LP]), whereas the three remainders have maintained their LTNP status at least up to 2003. The nef alleles were obtained from both plasma virus and peripheral blood mononuclear cells of all six individuals in 1995 and 1998. The proportion of sequences containing mutations not yielding Nef expression significantly diminished in 1998 versus that in 1995. Several previously defined functional regions of intact nef alleles were highly conserved. However, the major variant obtained in 1998 from plasma RNA of five out of six individuals significantly reduced HIV infectivity/replication and impaired Nef-mediated CD4 but not major histocompatibility complex class I antigen down-modulation from the cell surface. Thus, functional alterations of the nef gene are present in both LP and LTNP, suggesting that Nef defectiveness in vitro is not necessarily associated with the long-term maintenance of LTNP status. Of interest is the fact that isolates from three out of three LP showed a dual CCR5/CXCR4 coreceptor use (R5X4), in contrast to those from LTNP, which were exclusively R5. Thus, in vivo evolution of gp120 Env to CXCR4 use appears to be associated with HIV disease progression in individuals infected with nef-defective viruses.


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INTRODUCTION
 
The natural history of human immunodeficiency virus (HIV) infection has highlighted that a small fraction (1 to 2%) of infected individuals remains disease free and with a high number (≥500 cells/µl) of peripheral CD4+ T cells in the absence of antiretroviral therapy for 7 or more years of infection. These subjects have been defined as long-term asymptomatics, long-term survivors, or long-term nonprogressors (LTNP). Most LTNP, however, progress towards AIDS (defined here as late progressors [LP]) whereas a small proportion has been shown to remain healthy for more than 15 years of infection (52).

The LTNP condition depends on a complex interplay between host and viral factors (1, 3, 19, 36, 47, 52). Infection with HIV-1 variants with attenuated replicative capacity may contribute to the beneficial course of disease in a minor group of LTNP (7). Among other viral genes, nef has been strongly associated with pathogenesis in vivo. Infection of rhesus macaques with viruses derived from infectious molecular clones of simian immunodeficiency virus (SIV) lacking the nef gene showed low viral load, normal circulating CD4+ T-cell counts, and no signs of disease progression (37). Furthermore, these animals were "vaccinated" against a challenge with wild-type (WT) virus (17). Subsequently, a few studies have identified human subjects infected with nef-defective HIV-1 with nonprogressing HIV infection (10, 12, 18, 22, 38, 46). Most of these individuals as well as baby and adult monkeys inoculated with SIV with nef deleted, however, have subsequently lost their LTNP condition and progressed to AIDS in spite of preservation of nef-defective SIV (6, 9).

Other reports have argued against the role of Nef in the establishment of an LTNP status, since most of the nef alleles isolated from LTNP were intact in sequence and function (33, 46). However, a higher proportion of disrupted over intact nef sequences can coexist within each individual (10). All but two of these studies have been carried out exclusively by cloning and sequencing nef from proviral DNA harbored in peripheral blood mononuclear cells (PBMC), whereas no information has been provided on whether nef deletions were present in plasma-associated virions (10, 54). In this regard, it is known that proviral DNA from circulating resting memory T lymphocytes can persist for their entire life span as an "archival" HIV DNA (56). In contrast, the majority of virions circulating in the bloodstream are the result of recent productive infection of cells residing mostly in the lymphoid tissues and organs (27, 31, 49, 60). The analysis of virion-associated RNA, therefore, provides reliable information on recently replicating virus (50).

In this study, we have characterized the nef variants obtained from plasma RNA and PBMC-associated DNA of six infected hemophiliacs that were selected as LTNP in 1995 out of a larger cohort (58). They naturally split into either LP or LTNP upon long-term follow-up. Their nef alleles from plasma were analyzed with regard to their capacity to alter the infectivity/replication and to mediate CD4 and major histocompatibility complex class I (MHC-I) antigen (Ag) down-regulation once inserted in the background of the full-length infectious molecular clone NL4-3 or in the subgenomic pcDNA3 expression vector under the control of the chicken beta-actin promoter. In addition, viral isolates were obtained from 1995 and 1998 PBMC and characterized for their coreceptor use along with the sequencing of the gp120 Env V3 region.


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MATERIALS AND METHODS
 
Blood samples from HIV-1-infected hemophiliacs. Peripheral whole venous blood was collected in EDTA-containing tubes; plasma was separated from blood and stored at –80°C, whereas PBMC were separated by Ficoll-Hypaque density gradient (Pharmacia Biotech AB, Uppsala, Sweden).

Analysis of nef alleles from patients. The nef alleles were PCR amplified by plasma HIV RNA as previously described (10). The nef alleles were cloned into pCRII vector (Topo-TA cloning; Invitrogen, Carlsbad, CA). The nef alleles were introduced into the infectious molecular clone NL4-3, in which the nef gene was removed and substituted with the cloning sites NotI and MluI. The coding sequence of each nef allele was amplified from pCRII nef-containing vector by the following primer pairs according to the allele sequences: NotP1, 5'-GCGGCCGCATGGGTGGCAAGTGGTC-3'; MluP1, 5'-ACGCGTTCAGCAGTTCTTAAAGTACTCCG-3'; NotP2, 5'-GCGGCCGCATGGGTGGTAAGTGGTC-3'; MluP2, 5'-ACGCGTTCAGCAGTCCTTGTAGTACTCCG-3'; NotP3, 5'-GCGGCCGCATGGGGGGCAAGTGGTC-3'; and MluP3, 5'-ACGCGTTCAGCAGTTCTTGTAGTACTCCG-3'. The nef alleles were cloned in the HindIII and XbaI sites of the pcDNA3 vector in which the cytomegalovirus promoter was substituted with that of the chicken beta-actin gene (kindly provided by Yves Collette, UMR599, Marseille, France).

Site-directed mutagenesis. Generation of single-mutated nef alleles has been performed with the use of a QuikChange multisite-directed mutagenesis kit (Stratagene, La Jolla, CA) following the manufacturer's instructions. Briefly, each sample reaction mixture was prepared with 40 ng of NL4-3 plasmid and 125 ng of the following primers: nl-{Delta} 5fw, 5'-GCTAGTACCAGTTGAGCCAGATAATAAAGGAGAGAACACC-3'; and nl-{Delta} 5rev, 5'-GGTGTTCTCTCCTTTATTATCTGGCTCAACTGGTACTAGC-3'.

Viral stock generation. NL4-3 infectious molecular clones carrying specific nef alleles were transfected into 293T kidney epithelial cells by Fugene 6 (Roche Diagnostics, Inc., Indianapolis, IN) according to the manufacturer's instructions. Culture supernatants were harvested after 48 h and assayed by standard Mg2+-dependent reverse transcriptase (RT) activity assay (59).

Immunoblot analysis of Nef expression. Immunoblot analysis of Nef protein expression was performed with aliquots of extracts prepared 48 h after transfection of 293T cells with the chimeric infectious molecular clones. Cells were lysed in buffer (20 mM HEPES, pH 7.9, 400 mM NaCl, 0.1 mM EDTA, 0.1 mM EGTA, 3 mM MgCl2, 10% glycerol, 0.5% NP-40) by five cycles of freezing and thawing and clarified by centrifugation. Samples containing 40 µg of proteins were denatured for 10 min at 95°C in reducing sample buffer, resolved on 12.5% polyacrylamide gels, and transferred to nylon membranes. Immunoblot analysis with a rabbit anti-Nef antiserum (AIDS Research and Reference Reagent Program, NIAID, NIH) or a serum from an AIDS patient recognizing all of the major HIV proteins was performed as described previously (10). The immunoblot was developed by an enhanced chemiluminescence detection system (Amersham, Little Chalfont, United Kingdom).

HIV infectivity assay. CEM-green fluorescent protein (GFP) cells (kindly provided by Xue Gongda, University of Zurich, Switzerland) were infected either with the CXCR4-dependent (X4) strain NL4-3 or with the NL4-3 nef variant. Viral supernatants containing 16 x 106 cpm of RT activity were added to 5 x 105/ml CEM-GFP cells in 24-well plastic plates (Falcon; Becton Dickinson Labware, Lincoln Park, NJ) in the presence of 2 µg/ml polybrene (Sigma-Aldrich, Milwaukee, WI). Fifty percent of the culture supernatant was replaced with fresh RPMI 1640 (Bio-Whittaker, Verviers, Belgium) at 72 h postinfection. After 5 days of infection, 5 x 105 cells were harvested, centrifuged at 1,500 rpm for 5 min, and stained with either phycoerythrin-Cy5 anti-human CD4 (BD Biosciences, San Jose, CA) or phycoerythrin-Cy5 anti-human HLA-ABC (BD Biosciences) monoclonal antibody for 20 min at 4°C. The cells were washed with phosphate-buffered saline (PBS) containing 2% fetal calf serum and 0.1% Na-azide, fixed in 2% formaldehyde-PBS, and analyzed by flow cytometry (FACScan; Becton Dickinson, Franklin Lakes, NJ). The percentage of infected cells in each sample was evaluated by analyzing the proportion of cells expressing GFP. During analysis by CellQuest software (Becton Dickinson), a lymphocyte gate in the forward scatter/side scatter diagram was used to include exclusively living cells.

Transient assays of receptor down-regulation by nef alleles. Jurkat E6 T cells (ATCC, Bethesda, MD) were washed in cold PBS and resuspended in electroporation buffer (Amaxa Biosystems, Cologne, GmbH, Germany). Five micrograms of plasmid pcDNA3 containing either NL4-3 Nef or the patient's allele was mixed with 0.1 ml of cell suspension containing 2 x 106 cells, transferred to a 2.0-mm electroporation cuvette, and nucleofected with an Amaxa nucleofector apparatus utilizing the S-18 program according to the manufacturer's directions. To evaluate the transfection efficiency, cells were cotransfected with the pmaxGFP plasmid (Amaxa Biosystems). The cells were seeded in 24-well plates at a concentration of 1 x 106 cells/ml. CD4 and MHC-I Ag expression on the cell surface and GFP fluorescence were analyzed by a FACScan (Becton Dickinson) at 24 h posttransfection, as described above.

HIV replication assay. PBMC from healthy HIV-seronegative donors were isolated by Ficoll-Paque Plus density gradient (Amersham Biosciences, Piscataway, NJ). Infected culture supernatants containing 2 x 105 cpm of RT activity were used to infect 1 x 106 resting PBMC. At 24 h postinfection, PBMC were stimulated by purified phytohemagglutinin (PHA, 5 µg/ml; Sigma Chemical Co., St. Louis, MS). After 72 h, the culture supernatant was replaced with fresh medium supplemented with 10 U/ml of recombinant interleukin-2 (Boehringer Mannheim, GmbH, Germany) and 10% fetal bovine serum (complete medium). Fifty percent of the culture medium was harvested and replaced with complete medium every 72 h. The kinetics of viral replication were determined by RT activity content in the culture supernatants stored at –80°C.

HIV isolation and chemokine coreceptor use. More than three million PBMC (3 x 106) from HIV-1-infected hemophiliacs were mixed with 6 x 106 allogeneic PHA-activated PBMC obtained from two uninfected healthy donors in the presence of 15 ml of complete medium. Virus production was measured in the coculture supernatant by RT activity every 72 h. The collected supernatants were stored at –80°C, and those corresponding to the peak of RT activity were pooled (primary isolates). The coreceptor use of these primary isolates was determined by infecting 105 U87 astrocytic cells stably expressing either CD4 or CD4 plus CCR5 or CXCR4 (30) with 100 µl of undiluted primary isolates in 1 ml of Dulbecco's modified Eagle's medium (Bio-Whittaker) supplemented with 10% fetal bovine serum. The kinetics of viral replication were determined by RT activity in the supernatants of the infected cultures.

Sequence analysis of primary isolates' gp120 Env V3 regions. Virion RNA was extracted from 140 µl of primary isolate stocks by the use of a viral RNA mini kit (QIAGEN GmbH, Hilden, Germany) according to the manufacturer's instructions. Nine microliters of viral RNA was retrotranscribed by poly(dN6) and Superscript II reverse transcriptase (Invitrogen). The following primer pair was used for amplification of the V3 region: sense, 5'-AAATGGCAGTCTAGCAGAAG-3'; and antisense, 5'-AATTTCTGGGTCCCCTCCTG-3'. Two independent PCR products from each sample were sequenced by fluorescence-labeled deoxynucleoside triphosphates in an automated sequencer (ABI Prism 3100; Applied Biosystems, Foster City, CA).


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RESULTS
 
Hemophilic LTNP cohort follow-up. Six individuals out of a cohort of 112 HIV-1-infected hemophiliacs, who received contaminated blood products prior to 1985 and attended on a regular schedule the A. Bianchi Bonomi Hemophilia and Thrombosis Center in Milan, fitted the definition of LTNP, i.e., documented infection lasting for at least 9 years, absence of clinical symptoms, healthy general condition, and CD4+ T-cell counts stably higher than 500/µl blood, without initiation of antiretroviral therapies at study entry in 1995 (58). This cohort of HIV-1-infected hemophilic LTNP consists of three men affected by severe hemophilia A (patients [pts] 2, 3, and 6), two men with hemophilia B (pts 1 and 5), and one woman (pt 4) who is a hemophilia B carrier. Extended follow-up of CD4+ T-cell counts in these individuals showed that subjects 4 and 5 maintained their CD4+ T-cell counts at ≥500 cells/µl up to the last evaluation in 2006 (Fig. 1A), whereas their viremia levels ranged from 2 to 4 log10 (Fig. 1B). All of these individuals are currently in stable, good, healthy condition. Subject 1 maintained CD4+ T-cell counts above 500 cells/µl until 2003, whereas he showed 473 cells/µl in 2005 and 317 cells/µl in 2006 (Fig. 1A). His viremia has also progressively increased with time (Fig. 1B). Since he has preserved his LTNP condition for at least 18 years (1985 to 2003), in this study he was still considered an LTNP. In contrast, subjects 2, 3, and 6 showed loss of CD4+ T-cell counts and increases in viremia levels between 1996 and 2000 when they started to assume conventional highly active antiretroviral therapy. These subjects have been redefined here as LP (LP-2, -3, and -6).


Figure 1
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  		FIG. 1. Follow-up of the hemophilic LTNP cohort. Peripheral blood CD4+ T-cell counts (A) and viremia levels (B) of HIV-1-infected hemophiliacs from study entry (1995) to 2006. Individuals 1, 4, and 5 (full symbols) have been maintaining their LTNP status from 1995 up to date (to 2003 in the case of subject 1), whereas individuals 2, 3, and 6 (open symbols) progressed to AIDS in 1996 (subject 6), 1998 (subject 2), and 2000 (subject 3) and have been considered LP. The initiation of highly active antiretroviral therapy in LP-2, -3, and -6 is indicated in panel B. The arrowheads indicate the sampling time.

Molecular analysis of nef alleles isolated in 1995 and 1998. The nef genes of all of these individuals were analyzed by cloning and sequencing from both plasma-associated virion RNA and PBMC-associated proviral DNA. A total of 186 sequences were obtained from blood samples collected in 1995 and 1998 (Table 1). The analysis of the sequences obtained in 1995 revealed that nef sequences with large deletions, frameshifts, and premature stop codons, defined here as grossly defective, coexisted with full-length nef sequences in all individuals but LP-6 (10). However, the proportion of grossly defective nef variants in 1998 (8 out of 109, 7%) was significantly smaller than that obtained in 1995 (29 out of 77, 38%; P = 0.02 by the paired t test after arcsine transformation) (Table 1). No differences were observed when the proportion of grossly defective sequences in LTNP was compared to that in LP either in 1995 or in 1998.


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  		TABLE 1. Frequency of grossly defective RNA and DNA nef sequences from hemophilic LTNP and LP

The sequence of nef allele shown to be the major species in each individual in the 1998 plasma samples was chosen and compared to the sequence shown to be the major plasma species in the 1995 analysis in the same individual. Their deduced amino acid translations were aligned using CLUSTALW (Fig. 2A). Several previously defined functional regions were highly conserved in all of the sequences obtained in 1995 and 1998. Among the domains involved in Nef structure and function, the motif MGXXXS(1-6) (where 1-6 represents amino acid positions 1 to 6), which is the signal for N-myristoylation of Nef (26), was not mutated. However, serine 9, which serves as a cyclic AMP-dependent kinase PKA phosphorylation site and is involved in Nef-mediated enhancement of viral replication in resting PBMC (41), was changed to a basic amino acid in LTNP-1 and -5 and in LP-2 (1998 only), whereas it was replaced with an isoleucine in 1995 and a methionine in 1998 in LP-6. An amino acid insertion that varied in size and sequence was present at the N termini of LP-3 and -6. The domain CAWLEA(68-73), which is the signal for specific cleavage by the viral protease of HIV-1 and contains a site for binding to the cytoplasmatic tail of CD4 (29), showed high conservation in all individuals. The acidic regions of four glutamic acids, EEEE(75-79), involved in down-modulation of MHC-I molecules (28) through the interaction with phosphofurin acid cluster sorting protein 1 (51), were characterized by a certain degree of heterogeneity in all subjects but LTNP-1 and LP-6. The PXXP proline-rich motif between residues 83 and 94, which mediates the interaction of Nef with tyrosine kinases of the Src family, such as Hck and Lck, (53) and molecules that participate with the T-cell receptor signaling, such as Vav (20), showed a high conservation in all individuals. The arginine in position 119, involved in the p21-activated kinase (PAK1/2) interaction together with arginine 120 (48), was substituted with a lysine. The leucine 126 and the FDP(135-138) motif, which bind to the cytoplasmatic tail of CD4 (29) and interact with human thioesterase II (14, 42), respectively, appeared to be highly conserved among all alleles from 1995 and 1998. In all nef alleles, the motif EXXXLL(174-179), involved in the interaction with adaptor protein (AP) complexes AP-1 and AP-2, was also present and conserved (11, 16, 28). The negatively charged region EE(168-169), symmetrically opposed in sequence from the other negatively charged region DD(188-189) (25), was conserved in all sequences except in those of LTNP-1, in which an in-frame deletion of five amino acids from the position 165 to the position 170 had occurred. All nef alleles contained a highly conserved DDPXXE(188-193) domain (the first position may be either an aspartic acid or an glutamic acid residue), which is the binding domain for c-Raf1 kinase that is involved in the mitogen-activated protein kinase pathway (32). The only exception was LTNP-1, in which the nef sequences showed the mutation P190T (Fig. 2A).


Figure 2
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  		FIG. 2. Evolution of nef alleles in LTNP and LP. (A) Alignment of the Nef protein sequences from LTNP and LP obtained in 1995 and 1998; the deduced amino acid sequences were aligned in the single-letter code. The reference NL4-3 Nef sequence is specified on top. Gaps (-) were introduced to maximize homology; dots indicate identical amino acid sequences. Each sequence is identified as follows: the first digit identifies the pt, the year of sample analysis is then reported, the following R indicates RNA-derived amplification, and the following digit identifies the clone number. Boxes indicate protein motifs, and their cellular binding partners are reported on top. (B) Immunoblot analysis of Nef proteins from chimeric viruses. Aliquots of protein extracts (40 µg) prepared from 293T cells transfected with NL4-3 expressing various nef alleles were immunoblotted with a rabbit anti-Nef antiserum.

nef alleles from LTNP and LP show reduced viral infectivity/early replication in CEM-GFP cells and lack CD4 down-modulation capacity. In order to analyze the impacts of different alleles on Nef function, the predominant nef variant out of 10 sequences amplified in 1998 from plasma-associated virus of each patient was inserted in place of Nef of the infectious molecular clone NL4-3. Six chimeric infectious clones, expressing the nef alleles from each individual (Fig. 2A), were transfected into 293T cells; virus production was measured in the supernatant by RT activity determination (data not shown). Cell pellets were harvested at 48 h posttransfection and subjected to Western blot analysis. Each chimeric infectious molecular clone carrying a specific nef allele expressed all of the major HIV-1 proteins in amounts comparable to those expressed by NL4-3 (data not shown). All but one of the recombinant viruses expressed a Nef protein of about 30 kDa (Fig. 2B) similar to that of WT virus. The chimeric clone from LTNP-1 (clone 1R5) expressed a Nef protein with a weight lower than that of NL4-3 Nef consistently with the presence of a five-amino-acid deletion in the flexible loop (Fig. 2A).

The infectivity/early-stage replication of chimeric infectious molecular clones was examined by the use of CEM-GFP cells that express GFP under the control of a subtype B HIV-1 long terminal repeat (23). Equal amounts of RT activity derived from 293T transfection with chimeric viruses were used to infect CEM-GFP cells. The extent of CD4 expression and GFP-positive (GFP+) infected cells was evaluated after staining with a CD4-specific monoclonal antibody by fluorescence-activated cell sorter analysis at 5 days postinfection. Since a mechanistic link has been proposed for CD4 down-regulation and increased HIV infectivity and replication (4), the correlation between these two functions of the viral protein was examined in six independent experiments. Indeed, CD4 surface expression was inversely correlated to the infectivity/early-stage replication in CEM-GFP cells (Spearman's r = –0.97, P < 0.0001) (Fig. 3A). In a representative experiment, the analysis of the infected cells revealed that four out of six nef alleles diminished the percentage of infected cells to an extent similar to that of {Delta}nef NL4-3. In particular, the infection with chimeric viruses expressing Nef of subjects 1 (1R5), 4 (4R4), 5 (5R8), and 6 (6R10) caused a minimal reduction of CD4 surface expression that was comparable to that of {Delta}nef NL4-3 (Fig. 3B). The nef alleles of subjects 1 (1R5), 4 (4R4), and 6 (6R10) significantly diminished the proportion of GFP+-infected cells compared to WT virus at 5 days postinfection (P < 0.001 by repeated-measure analysis of variance [ANOVA] computed with Bonferroni's multiple-comparison test) (Fig. 4A).Conversely, the percentage of CD4+ cells infected with the chimeric viruses 1R5, 4R4, and 6R10 was significantly higher than that of cells infected with NL4-3 (P < 0.01 by repeated-measure ANOVA computed with Bonferroni's multiple-comparison test) and was comparable to that of {Delta}nef NL4-3 (Fig. 4B). Since the infection of CEM-GFP cells with {Delta}nef NL4-3 virus resulted in a minimal reduction of the percentage of CD4+ cells, the data were reanalyzed by looking at the mean fluorescence intensities (MFI) after CD4 gating on the infected GFP+ cells. Upon infection with NL4-3, the CD4 MFI values of GFP+ cells were decreased 5-fold compared to those of mock-infected cells (Fig. 4C); in contrast, the MFI values of the chimeric viruses 1R5, 4R4, 5R8, and 6R10 were decreased only 1.5- to 2-fold (Fig. 4C).


Figure 3
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  		FIG. 3. Loss of infectivity and CD4 down-modulation capacity of nef alleles of LTNP and LP. (A) Correlation between percentage of GFP+ and CD4+ cells in CEM-GFP cells infected with NL4-3, {Delta}nef NL4-3, and chimeric viruses. A total of 54 samples obtained from six independent experiments were analyzed at 5 days postinfection according to Spearman's test (r = –0.97, P < 0.0001). (B) A representative experiment of Nef-induced CD4 down-modulation in HIV-1-infected CEM-GFP cells is shown. Infected cells are identified by the expression of GFP.


Figure 4
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  		FIG. 4. Loss of CD4 down-modulation capacity in nef alleles from LTNP and LP during infection of CEM-GFP cells. (A) The percentage of GFP+ cells infected with NL4-3 isogenic viruses differing in their nef alleles is reported as the means ± standard errors of the means (SEM) from eight independent experiments. (B) The percentage of CD4+ cells was evaluated by flow cytometry at 5 days postinfection, as reported for Fig. 3B. The means ± SEM from eight independent experiments are shown. The black bars indicate LTNP nef alleles, whereas the empty bars represent LP nef alleles. The grey, dark grey, and hatched bars represent the experimental controls. (C) The MFI of the GFP+ cells indicates that most alleles have lost their CD4 down-modulation ability, as analyzed in eight independent experiments (means ± SEM).

In order to properly isolate the effects of the nef alleles on CD4 down-regulation out of the context of virus replication, an expression vector in which the different nef alleles were expressed under the control of the chicken beta-actin promoter was introduced in the CD4+ Jurkat E6 T-cell line. As shown in Fig. 5A, all nef alleles but one (5R8) were less efficient than NL4-3 Nef in down-modulating CD4 Ag expression. Similar results were obtained by analyzing the MFI from three independent experiments, as shown in Fig. 5B.


Figure 5
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  		FIG. 5. Loss of CD4 down-modulation capacity in nef alleles from LTNP and LP after transient transfection of Jurkat E6 T cells. Cells were transfected with either an empty vector or vectors expressing NL4-3 Nef or the nef alleles, as described in Materials and Methods. (A) CD4 cell surface expression was analyzed by flow cytometry at 24 h posttransfection; the number in the upper right corner of each panel indicates the percentage of CD4-positive cells. (B) The MFI values from three independent experiments are reported as means ± standard deviations.

nef alleles from LTNP and LP maintain MHC-I Ag down-modulation capacity. We next examined the potential effects of these nef alleles in the down-modulation of MHC-I Ag as reported previously (40). In contrast to CD4 down-modulation, the infection with chimeric viruses expressing nef alleles caused a reduction of MHC-I Ag membrane expression comparable to that of NL4-3 (Fig. 6A). The MFI values for MHC-I of the GFP+ cells in three independent experiments confirmed that all six nef alleles down-regulated MHC-I Ag expression with a potency comparable to that of NL4-3 (Fig. 6B). Furthermore, the expression of the nef alleles in the pcDNA vector under the control of the chicken beta-actin promoter resulted in MHC-I Ag down-regulation comparable to that of NL4-3 Nef (Fig. 6C).


Figure 6
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  		FIG. 6. MHC-I Ag down-regulation in cells expressing WT nef and nef alleles. (A) The analysis of a representative experiment of MHC-I Ag down-modulation activity in HIV-1-infected CEM-GFP+ cells revealing down-regulating effects of Nef alleles is shown. (B) The MFI values of the MHC-I and GFP+ cells are reported as the means ± standard deviations from three independent experiments. (C) Jurkat E6 T cells were transfected with either an empty vector or vectors expressing NL4-3 Nef or the nef alleles, as described in Materials and Methods. After 24 h, cell surface expression of MHC-I Ag was analyzed by flow cytometry, as indicated by the MFI values from three independent experiments.

nef alleles of LTNP reduce the efficiency of virus replication in PBMC. Equal amounts of RT activity were loaded onto resting PBMC that were subsequently activated by PHA at 24 h postinfection. The levels of viral replication of each nef chimeric virus were compared to those of NL4-3 and {Delta}nef viruses at their peaks of replication, usually at 9 to 10 days postinfection. The replications of the chimeric viruses 1R5, 2R1, 5R8, and 6R10 were significantly impaired compared to that of NL4-3 (n = 4, P < 0.001 by repeated-measure ANOVA computed with Bonferroni's multiple-comparison test) (Fig. 7).


Figure 7
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  		FIG. 7. Defective replication of NL4-3-carrying nef alleles from LTNP or LP. Average RT activity levels released in the cell culture medium of PBMC collected from four donors and infected with NL4-3 variants over 9- to 10-day periods are shown. The whiskers indicate the range of virus replication, with the boxes extending from the 25th to the 75th percentile, whereas the horizontal lines show the median values. Statistical analysis was carried out according to the repeated-measure ANOVA computed with Bonferroni's multiple-comparison test (***, P < 0.001).

Since the 1R5 nef allele consistently diminished HIV replication in both CEM-GFP cells and PBMC and it was characterized by a deletion of five amino acids in the unsolved disordered loop (39), this deletion was introduced in the nef sequence of NL4-3 by site-directed mutagenesis. The kinetics of viral replication of the 1R5 NL4-3 chimeric virus and the {Delta}165-170 NL4-3 mutant compared to those of WT and {Delta}nef viruses are shown in Fig. 8. The 1R5 virus replicated less efficiently than WT virus; in contrast, the virus carrying only the deletion of five amino acids replicated as efficiently as WT virus. Thus, this single deletion mutation did not explain per se the defective phenotype associated with the allele isolated from LTNP-1.


Figure 8
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  		FIG. 8. Kinetics of replication of the chimeric 1R5 and mutant ({Delta}165-170 NL4-3) viruses compared to those of WT and {Delta}nef NL4-3 viruses, measured by RT activity in the supernatant of activated PBMC cultures collected every 3 to 4 days postinfection (PI). While the expression of the 1R5 nef allele impairs virus replication, the related five-amino-acid deletion alone does not. The means ± standard deviations from two independent experiments are shown.

Chemokine coreceptor use of primary HIV isolates from LTNP and LP and sequence analysis of the gp120 Env V3 region. Primary viral isolates were obtained from the cocultivation of PBMC from the infected hemophiliacs with allogeneic PHA blasts of seronegative donors. Only LTNP-4 PBMC did not yield virus isolation even after removal of CD8+ cells (data not shown). Virus isolation was positive for LTNP-1 only by PBMC collected in 1998. In contrast, virus isolates were obtained from PBMC of subjects 2, 3, 5, and 6 in both 1995 and 1998. Viral isolate coreceptor use is reported in Fig. 9. CCR5 use (R5) was detected in three out of four isolates obtained in 1995. However, two (from LP-3 and -6) out of these four isolates expanded their coreceptor use to CXCR4 (R5X4) in 1998. Since chemokine coreceptor usage is determined mostly by specific amino acid residues in the V3 loop of gp120 Env (21), we compared the V3 regions of the 1995 and 1998 isolates to analyze whether specific amino acid changes could be linked to this phenotypic change in our isolates. A basic residue was present in position 11 in the R5X4 dualtropic isolates of LP-2, whereas a nonpolar residue was found in either R5 or the remaining R5X4 viruses. Position 25 was characterized by the presence of either positively or no charged amino acids. Higher net charges were found in the R5X4 viral isolates (Fig. 9).


Figure 9
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  		FIG. 9. Chemokine coreceptor usage and predicted amino acid sequence alignment of the gp120 Env V3 region of sequential (1995 and 1998) primary viral isolates of HIV-infected hemophiliacs. No viral isolate was obtained from subject 4 at both time points and from subject 1 in 1995. The total amino acid charge of the V3 region was calculated by subtracting the number of negatively charged amino acids from the number of positively charged amino acids. ND, not determined.


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DISCUSSION
 
In this study, we have shown that the nef alleles obtained from HIV-1-infected hemophilic LTNP and LP are equally defective in HIV infection/replication and CD4 down-regulation capacities, though they maintain MHC-I Ag down-regulation activity. Evolution in gp120 Env coreceptor use from R5 to R5X4 viruses may explain disease progression in spite of the maintenance of nef defects.

A small proportion of HIV-infected individuals, LTNP, has been coping with HIV disease for several years, remaining disease free and in good, healthy condition without antiretroviral therapy. The reason(s) of resistance to disease progression is still unknown, and it is likely caused by multiple factors related to both the virus and the host. The continuous follow-up of LTNP is crucial for understanding the correlates of long-term virus control, since, thus far, no single infected individual has been reported to clear HIV infection. Indeed, LTNP are characterized by a diminished immunological damage associated with a lower degree of virus replication and T-cell activation compared to individuals with progressive disease (52). In our cohort of HIV-infected hemophiliacs, three out of the six individuals initially classified as LTNP in 1995 (58) have maintained their LTNP status throughout 2003, after 18 years or more of infection.

In spite of conflicting studies reporting either defective or normal functions of Nef in LTNP (10, 13, 18, 22, 38, 45, 46, 57), this viral gene is one of the best-characterized accessory genes in cohort studies. In our cohort, no single LTNP is characterized by mutations that lead to the lack of protein synthesis. Defective nef sequences coexisted with full-length nef open reading frames in all but one LTNP. However, the proportion of disrupted nef sequences within each individual has decreased over time, similar to our original observation for hemophilic progressors (10). These variations could be caused by a sampling bias, since only two independent PCR products were cloned, although several clones were sequenced. Alternatively, the decrease of defective clones in the 1998 sampling could have been the result of a selective loss of nef sequences that did not encode the full-length protein. In order to support this hypothesis, nef gene variants were obtained from plasma-derived virion RNA in addition to PBMC-associated DNA. The time-related evolution of the nef gene was also evident when a neighbor-joining analysis was performed on DNA and RNA sequences of single individuals. Most LTNP had DNA sequences that clustered apart from RNA sequences in 1995; however, the follow-up analysis revealed that the DNA sequences were intermingled with the RNA sequences in both LTNP and LP (data not shown), similar to what was observed with our progressor hemophiliacs (10).

In spite of nef gene evolution, full-length nef alleles obtained from the 1998 plasma sampling could impair infection/replication and CD4 cell surface expression. All but one out of six nef alleles showed an impaired capacity to fully support HIV replication in CEM-GFP cells and PBMC. We have previously reported that the 1995 nef alleles of subjects 1, 2, and 5 were defective in terms of enhancement of HIV infectivity/replication (10), suggesting that defective Nef proteins were likely contributing to keeping virus replication under partial control in these individuals. However, these effects were limited in time since functionally defective nef alleles were also found in individuals who have controlled disease progression up to at least 11 years of infection and have thereafter showed unequivocal signs of progression to HIV disease, defined here as LP. In this regard, a recent report showed that Nef is not required for efficient replication of viruses that use solely CCR5 for entry (44). In addition, evolution of either gp41 Env in macaques infected with SIV deleted of nef (2) or chemokine coreceptor use towards CXCR4 in individuals infected with nef-deleted HIV (34) has been reported. In this regard, we have obtained several viral isolates from the PBMC of our hemophiliacs and characterized their coreceptor usage. In 1995 and 1998, positive HIV isolation was obtained from PBMC in four out of six individuals (pts 2, 3, 5, and 6). At this second time point, HIV isolation was also positive in LTNP-1. Of interest is the fact that the three isolates of the LP were all R5X4 in 1998 (also in the case of LP-2 in 1995), whereas PBMC of LTNP either harbored R5 viruses (LTNP-1 and -5) or were negative for HIV isolation (LTNP-4). Thus, viral evolution of gp120 Env seems to characterize LTNP and LP in spite of nef functional defects.

Nef is expressed early in the HIV life cycle (61), and therefore, CD4 down-regulation could potentially favor the assembly and release of infectious particles coated with gp120 Env that otherwise could bind to CD4 intracellularly prior to incorporation in virions (55). Five nef alleles out of six conferred lower levels of infectivity to chimeric viruses than to NL4-3. However, in three out of these five alleles, the lower efficiency of infection/early replication was associated with CD4 cell surface expression levels higher than that of WT virus. These defective nef alleles were characterized by several point mutations located outside the highly conserved functional domains (8, 24). The 1R5 allele contained an in-frame deletion of amino acids 165 to 170 in the C-terminal flexible loop. The model representation of the flexible loop is characterized by an acidic stem made of eight adjacent aspartic and glutamic acid residues forming a strong negative-charged cluster at the N and C termini of the loop, while the dileucine-based motif (EXXXLL) is exposed at its center (25). The deletion of amino acids 165 to 170 does not disturb the integrity of the flexible loop, in which the dileucine motif was demonstrated to be essential for optimal viral infectivity (43). Indeed, this deletion alone did not influence infectivity/replication and CD4 expression when introduced in the NL4-3 Nef. The Nef flexible loop of NL4-3 (residues 162 to 194) differs from that of the 1R5 allele in a few positions (Fig. 2A). Therefore, other changes within the 1R5 allele must be responsible for the diminished infectivity and CD4 down-regulation. In this regard, a lysine was present instead of serine at position 9, and a recent report showed that the presence of serine at position 9 is critical for replication in quiescent primary cells and phosphorylation by cyclic AMP-dependent protein kinase (41). The enhancement of viral replication in resting PBMC was impaired in three additional nef alleles. These three alleles were characterized by a change of serine 9 into arginine (alleles 2.98.R1 and 5.98.R8) and methionine (6.98.R10) (Fig. 2A). Thus, our findings support the importance of the serine 9 PKA phosphorylation site in the enhancement of HIV replication by Nef (41).

An additional function ascribed to Nef, i.e., its ability to down-regulate MHC-I Ag expression from the surface of infected cells, can render them less susceptible to lysis by cytotoxic T lymphocytes (15). In this regard, it has been proposed that Nef-mediated down-regulation of MHC-I Ag is an important immune evasion mechanism exploited by HIV (35). In contrast to what was observed with infectivity and CD4 down-regulation capacity, our nef alleles obtained from either LTNP or LP down-regulate MHC-I Ag as efficiently as NL4-3 Nef. Although we cannot exclude that defects of this function could have been present in LTNP prior to our analysis, i.e., 1998, these findings suggest that the Nef-mediated down-modulation of MHC-I Ag is not necessarily associated with the maintenance of LTNP status.

In conclusion, selected functional alterations of the nef gene have been present in most LTNP and LP in our cohort of HIV-infected hemophiliacs. Three out of six individuals who were LTNP in 1995 progressed to HIV disease during the follow-up by the year 2000, and an additional one (LTNP-1) progressed to HIV disease by 2003. This clinical observation suggests that alterations in Nef functions are likely necessary but not sufficient to maintain LTNP status, as previously reported by independent investigators (5). Functional alterations in other accessory genes, such as Vif, Vpu, and Vpr, together with the evolution of the HIV-specific immune responses might therefore contribute to maintaining low levels of viral replication and delay disease progression. In addition, the detection of CXCR4 coreceptor use in primary isolates of LP, but not LTNP, suggests that the evolution of the Env gene towards higher virulence may occur and overcome the presence of nef-defective HIV. Thus, a constant follow-up of these individuals and their viruses will contribute to dissecting out both viral and host factors that could provide novel insights for designing better strategies for efficient long-term viral suppression.


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ACKNOWLEDGMENTS
 
This work was supported by a grant (40D.83; ELVIS Concerted Action for the Study of LTNP) from the V National Program of Research against AIDS of the Istituto Superiore di Sanità, Rome, Italy.


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FOOTNOTES
 
* Corresponding author. Mailing address: P2/P3 Laboratories, DIBIT, Via Olgettina 58, 20132 Milan, Italy. Phone: 39-02-2643-4908. Fax: 39-02-2643-4905. E-mail: vicenzi.elisa{at}hsr.it. Back

{triangledown} Published ahead of print on 30 August 2006. Back


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Journal of Virology, November 2006, p. 10663-10674, Vol. 80, No. 21
0022-538X/06/$08.00+0     doi:10.1128/JVI.02621-05
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



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