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The Journal of Immunology, 2008, 180, 4075 -4081
Copyright © 2008 by The American Association of Immunologists, Inc.
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*Gene*Protein

Functional Adaptation of Nef to the Immune Milieu of HIV-1 Infection In Vivo1

Martha J. Lewis2,*, Arumugam Balamurugan*, Ayako Ohno*, Stephanie Kilpatrick*, Hwee L. Ng* and Otto O. Yang*,{dagger}

* Division of Infectious Diseases, Department of Medicine, and AIDS Institute and {dagger} Department of Microbiology, Immunology, and Molecular Genetics, Geffen School of Medicine, University of California, Los Angeles, CA 90095


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Nef-mediated down-regulation of MHC class I (MHC-I) molecules on HIV-1-infected cells has been proposed to enhance viral persistence through evasion of host CTLs. This conclusion is based largely on demonstrations that Nef from laboratory HIV-1 strains reduces the susceptibility of infected cells to CTL killing in vitro. However, the function and role of Nef-mediated MHC-I down-regulation in vivo have not been well described. To approach this issue, nef quasispecies from chronically HIV-1-infected individuals were cloned into recombinant reporter viruses and tested for their ability to down-regulate MHC-I molecules from the surface of infected cells. The level of function varied widely between individuals, and although comparison to the immunologic parameters of blood CD4+ T lymphocyte count and breadth of the HIV-1-specific CTL response showed positive correlations, no significant correlation was found in comparison to plasma viremia. The ability of in vivo-derived Nef to down-regulate MHC-I predicted the resistance of HIV-1 to suppression by CTL. Taken together, these data demonstrate the functionality of Nef to down-regulate MHC-I in vivo during stable chronic infection, and suggest that this function is maintained by the need of HIV-1 to cope with the antiviral CTL response.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Nef is an accessory protein unique to HIVs and SIVs (1). Although Nef is not essential for viral replication, its reading frame is preferentially preserved in vivo, suggesting a functional role beyond enhancement of replication (2, 3, 4). Defective Nef appears to attenuate infection significantly (5). Experimental infection of rhesus macaques with SIV in which Nef has been deleted (SIV239{Delta}nef) results in low-to-undetectable levels of viremia, asymptomatic infection, and protection from subsequent challenge with wild type virus (6). In humans, several cases of individuals infected with Nef-defective HIV-1 have been reported, and many of these maintain low-to-undetectable levels of viremia with vigorous antiviral immunity and delayed disease progression (5, 7, 8, 9, 10).

Many effects of Nef have been proposed to contribute to its role in promoting viral pathogenicity. Chief among them are the abilities of Nef to modulate numerous cell-signaling pathways important for lymphocyte activation, and to down-regulate both CD4 and MHC class I (MHC-I)3 molecules from the surface of infected cells (11, 12, 13). Elimination of some or all of these functions leads to an attenuated infection. In particular, Nef-mediated down-regulation of MHC-I molecules resulting in evasion of host CTLs has been hypothesized to be a key function for increasing pathogenicity (14, 15).

Results of several in vitro studies have confirmed that this function does mediate reduced susceptibility of infected cells to CTL killing. Collins et al. (16) first demonstrated that Nef-mediated down-regulation of MHC-I did indeed decrease HIV-1-infected cell killing by CTL. Subsequently, Yang et al. and Tomiyama et al. (17, 18) performed functional assays that extended this finding to show that Nef renders HIV-1 relatively resistant to suppression of viral replication by CTL. Further in vitro modeling has shown that HIV-1 containing Nef with the ability to down-regulate MHC-I has a selective advantage compared with HIV-1 with Nef that cannot down-regulate MHC-I (19). Notably, all of these studies have been performed with essentially a single clone of Nef (HXB2 and NL4-3, both originating from LAV).

The role of Nef-mediated MHC-I down-regulation in pathogenesis has also been tested in the SIV-macaque model. Rhesus macaques infected with SIV containing Nef engineered to be specifically defective in MHC-I down-regulation function via hard-to-revert mutations showed trends for higher CTL levels and lower viremia in the first 14 wk of infection (20). Eventual failure to control SIV replication was accompanied by a striking pattern of Nef evolution to reconstitute the ability to down-regulate MHC-I via alternative motifs (distinct from the endogenous SIV sequences that had been experimentally deleted). These data strongly suggested the importance of this function in the pathogenesis of HIV-1 infection by decreasing CTL killing of virus-infected cells.

In vivo data in humans are few, but suggest that the ability of Nef to down-regulate MHC-I may vary according to stage of HIV-1 infection. Its MHC-I and CD4 down-regulatory functions are preserved in acute and early infection suggesting that these particular activities may impart a survival advantage for the virus during early stages of disease (21). However, it has been observed that the MHC-I down-regulatory function is absent in some groups such as certain pediatric patients and late stage AIDS patients, suggesting that there are disease states in which this function does not provide a functional benefit for the virus (22, 23, 24, 25).

Yet, for the vast majority of HIV-1-infected individuals, who are in the stable chronic stage of infection, the contribution of Nef-mediated MHC-I down-regulation to pathogenesis has not been described. Therefore, we tested primary isolate Nef sequences from chronically infected individuals for the ability to down-regulate MHC-I. This parameter was correlated to other parameters of HIV-1 infection in these individuals, and with the ability of Nef to mediate HIV-1 resistance to virus-specific CTL.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Subject population

Eleven chronically HIV-1-infected subjects with stable viremia and not on anti-retroviral therapy were recruited from the Los Angeles area. All subjects provided informed consent according to a protocol approved by the UCLA Institutional Review Board. Study subjects provided information on their most recent absolute CD4 count and viral load at the time of the study visit.

Mapping of HIV-1-specific CTL responses

PBMC were collected by standard Ficoll separation. Bulk CD8+ lymphocytes were expanded from 2 to 4 x 106 PBMC by the addition of a CD3/CD4 bi-specific Ab that allows for nonselective, polyclonal expansion of the CD8+ T lymphocyte subset (26). Cells were maintained in RPMI 1640 (Sigma-Aldrich) supplemented with 10% FCS and 50 U/ml IL-2 and were used 10–14 days after the addition of the Ab. Typically, the cells are >80% CD3+/CD8+ (27). Standard IFN-{gamma} ELISPOT assays (using anti-IFN-{gamma} Abs from Mabtech) screening a peptide library containing 15-mers representing the entire Clade B consensus HIV-1 proteome (National Institutes of Health AIDS Research and Reference Reagent Program, Rockville, MD) was used to map HIV-1 specific CTLs to the resolution of 15-mer peptides (28). Spot-forming cells (SFC) were counted using an automated ELISPOT reader (AID) and normalized to SFC/106 cells. A response was considered significant if it was >2 SDs above and double the triplicate negative control wells, and ≥50 SFC/106 cells.

HIV-1 nef quasispecies cloning from plasma

Viral RNA was isolated from 1 ml of plasma using the Ultrasens Viral Isolation kit (Qiagen) according to the manufacturer’s protocol. Viral RNA was used as a template for cDNA synthesis using Omniscript Reverse Transcriptase (Qiagen) and the following gene-specific primers: Nef 9589R, 5'-TAGTTAGCCAGAGAGCTCCCA; Nef 8670F, 5'-AATGCCACAGCCATAGCAGTG; Nef 8675F, 5'-GCAGTAGCTGAGGGGACAGATAGG; Nef 8687F, 5'-GTAGCTCAAGGGACAGATAGGGTTA; Nef 8736F, 5'-AGAGCTATTCGCCACATACC. RT products were then used for nested PCR using Master Taq kit (Eppendorf). The first PCR used the same primers used for reverse transcription. The nested PCR used the following primers: Nef 8787 XbaIF, 5'-GCTCTAGAATGGGTGGCAAGTGCTCAA and Nef 9495R, 5'-TTATATGCAGCATCTGAGGGC. Both PCR were conducted using the following conditions: 5 min at 95°C, 35 cycles of 95°C for 30 s, 54°C for 40 s, 72°C for 60 s, followed by a final extension at 72°C for 10 min. PCR products were gel-purified with Quick Spin Columns (Qiagen) and subsequently cloned in bulk by the TA method into pCR2.1-TOPO vector (Invitrogen). Ligation mixtures were grown in liquid culture with ampicillin and not subject to individual colony selection on solid medium to preserve the quasi-species mixture of cloned PCR products. Plasmid DNA was isolated and digested with XbaI and BspEI (New England Biolabs) and subsequently subcloned into the nef position of the half-genome construct p83-10 (29) (Fig. 1).


Figure 1
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  		FIGURE 1. Half-genome constructs and cloning strategy for making recombinant reporter viruses. Nef sequences were amplified from plasma viral RNA and cloned in bulk into p83-10, replacing the nef gene of the NL4-3-based parent vector. T1 lymphocytes were coelectroportated with EcoRI-digested p83-10 containing the various nef sequences and p83-2.1 containing mCD24/HSA as a reporter. Culture supernatant with infectious recombinant reporter virus was collected 11–13 days postelectroporation.

 
Sequencing and phylogenetic analysis

A minimum of 10 individual nef clones in the pCR2.1 TOPO vector were isolated per subject. Standard vector primers M13F and M13R were used for cycle sequencing with the Big Dye Terminator Reaction Kit 3.1 (Applied Biosystems). Sequencing products were purified using the CleanSeq reagent (Agencourt, Beckman Coulter) and run on an ABI3130 sequencer (Applied Biosystems). Electropherograms were edited using Sequence Analysis v5.2 software (Applied Biosystems). Duplicate sequences were excluded and the remaining sequences along with NL4-3 and Consensus B nef were aligned using CLUSTAL X and neighbor-joining trees were constructed using the DNADist and Neighbor programs of PHYLIP 3.64 (30). The tree was rooted with Consensus B and statistically evaluated with 1000 bootstrap replicates.

Production of nef recombinant reporter viruses

Recombinant reporter virus was constructed within half genome plasmids as previously described (29). The p83-2 plasmid contained the 3' end of HIV-1 in which murine CD24/HSA has been inserted in the Vpr reading frame. A parental p83-10 plasmid containing the HSA-HA reporter gene in the Nef reading frame was used as the vector for insertion of the above primary isolate nef quasispecies sequences. Viruses were produced with these constructs in parallel with p83-10 controls (Fig. 1) containing either wild-type NL4-3 nef or mutant M20A nef, by coelectroporation with p83-2 plasmid (19, 31). In brief, 10 µg of each half genome plasmid construct was digested with EcoRI. Both digested, gel-purified plasmids were mixed and electroporated into 10 million T1 cells using a GenePulser Electroporator (Bio-Rad). After a 20 min recovery on ice, cells were cultured in RPMI 1640 medium supplemented with 20% FCS. After day 7, culture supernatant was collected and tested for p24 by ELISA (PerkinElmer). Peak p24 production occurred 11–13 days postelectroporation. Genomic DNA from infected T1 cells was isolated and used as a template for nef PCR and sequencing as described above to confirm that no cross-culture contamination had occurred. Infected T1 cells were stained with Anti-HA-FITC (Roche) to confirm no contamination with the parent p83-10 vector (containing HSA-HA in nef) used for cloning. Infectious virus is produced only if the nef gene containing a large portion of the 3'LTR is present; therefore, all infectious recombinant reporter viruses contained nef inserts.

Flow cytometric measurement of HLA A*02 down-regulation by HIV-1-infected cells

The ability of Nef to down-regulate HLA A*02 on infected cells was assessed as previously reported (29). Briefly, two million T1 lymphocytes were infected at 37°C for 4 h with 1 ml of virus stock (containing 250–500 ng p24 Ag) of reporter virus carrying either wild-type NL4-3 nef, M20A nef, or nef quasispecies amplified from primary isolates described above. Cells were fed with 2 ml of RPMI 1640 supplemented with 10% FCS (Sigma-Aldrich). On day 5 postinfection, 2 x 105 cells were stained with 0.5 µl anti-murine CD24-PE and anti-human HLA A*02-FITC (BD Pharmingen), washed twice, and fixed with 1% paraformaldehyde. Uninfected T1 cells stained with isotype control Abs were used to set the negative quadrants. T1 cells stained with anti-human HLA A*02-FITC and WT NL4-3 Nef infected cells stained with anti-murine CD24-PE were used to establish appropriate compensation between FL1 and FL2 channels. At least 104 live cells were counted using a FACScan flow cytometer, and data were analyzed using CellQuest software (BD Biosciences). Maximum levels of HLA A*02 were determined using the M20A Nef mutant which is specifically deficient in MHC-I down-regulation (19, 31). Absolute percent HLA A*02 down-regulation (using M20A Nef-infected cells as maximum and isotype stained cells as minimum A*02) and percent relative to wild-type NL4-3 Nef were calculated based on results of three independent experiments.

Measurement of Nef impact on HIV-1 suppression by CTL

HIV-1 suppression assays were performed as previously reported (32, 33). In brief, 106 T1 cells were infected at an MOI of 0.1 for 4 h at 37°C. Cells were washed then cocultured in triplicate in 96-well plates with a CTL clone specific for the p17 (77–85) epitope SLYNTVATL at a ratio of 5 x 104 target cells with 1.25 x 104 CTL in RPMI 1640 medium supplemented with 10% FCS and 50 U/ml IL-2 (Sigma-Aldrich). Seven days postinfection, p24 levels were measured in triplicate by ELISA (PerkinElmer). Efficiency of suppression of viral replication was expressed (log10 p24 without CTL – log10 p24 with CTL)/log10 p24 without CTL.

Statistical analysis

Correlations and their statistical evaluation were performed using regression analysis as implemented by Microsoft Excel 2004 Analysis Tools.

Sequence accession numbers

The GenBank Accession no. were EU327403-EU327492.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Chronically HIV-1-infected subjects in varying stages of disease progression display a wide range of HIV-1-specific CTL responses

Eleven chronically infected subjects who were not on antiretroviral therapy had viremia ranging from 400 to >750,000 HIV-1 RNA copies/ml and peripheral blood CD4+ T lymphocyte counts ranging from 0 to 900 cells/mm3 (Table I). HIV-1-specific CTL responses were mapped using IFN-{gamma} ELISpot with a library of overlapping peptides spanning the entire Clade B consensus proteome sequence. These subjects displayed highly variable breadth and specificity in their CTL responses (Table I). Two subjects (00022 and 00037) in the terminal stages of AIDS had no detectable CTL responses, but the other subjects had ELISPOT responses detected against 3 to 14 peptides, with a mean and median of 6. In agreement with other studies (34, 35), neither the breadth nor magnitude of the measured CTL response correlated to viremia (data not shown). Overall, the study population of chronically infected subjects represented a broad range of clinical disease states, as determined by CD4+ T lymphocyte levels and viremia, with varying breadth of HIV-1 specific CTL responses.


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  		Table I. Study subject information

 
Plasma viral nef quasispecies are genetically distinct and display divergence between persons

To examine the evolutionary relationships of nef between these subjects, plasma viral RNA was sequenced. Bulk PCR-amplified nef was used throughout the cloning procedure to approximate the in vivo quasispecies. Multiple individual clones (4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19) from each subject were selected randomly for sequencing. Examination of sequences from 93 distinct clones revealed that 96% (89/93) encoded fully intact reading frames. Previously identified domains important for Nef-mediated MHC-I down-regulation were largely conserved: 98% (91/93) had intact myristoylation sites, 99% (92/93) had intact PxxP-PxxP domains, and 55% (51/93) had wild-type acidic (EEEE) domains. Subjects 00015, 00016, and 00030 had consistent substitutions at the second position of this acidic domain with either aspartic acid or asparagine (43% of all sequences); these substitutions occurs in some non-Clade B subtypes and SIV Nef (11). Subject 00037 had two sequences with lysine substitutions at the first or fourth positions (data not shown).

The nef genes were aligned with against NL4-3 and Clade B consensus sequences, and used to reconstruct a neighbor-joining phylogenetic tree (Fig. 2). The primary sequences were approximately equidistant from the consensus Clade B sequence, consistent with their being Clade B isolates. Sequences from 7 of 11 subjects formed statistically well-supported independent clusters indicating that these sequences were genetically distinct. However, in two cases – subjects 00015 and 00016, and subjects 00035 and 00039 – the sequences from two subjects clustered together with high statistical support indicating a common source for infection. Subjects 00015 and 00016 were a known transmission pair (44). The transmission sources of subjects 00035 and 00039 were unknown, although both were infected in the Los Angeles area within a span of about two years. With the exception of subject 00039, the sequences from each subject displayed typical quasispecies variation; sequences from subject 00039 fell into two statistically distinct groups, suggesting the possibility of dual infection. The results of sequencing and phylogenetic analysis generally confirmed that the cloned nef sequences were unique, typical Clade B isolates with a typical degree of in vivo sequence variation.


Figure 2
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  		FIGURE 2. Phylogenetic tree of nef quasispecies from primary isolates. A neighbor-joining tree was constructed using an alignment of 90 full-length nef coding sequences from the 11 study subjects along with NL4-3 and Consensus B nef sequences. The tree is unrooted and its topology was statistically evaluated with 1000 bootstrap replicates. The subject number is at the tips of the branches of each cluster. Each cluster was supported by a boostrap value ≥ 99%.

 
Nef quasispecies from different individuals display a range of MHC-I down-regulation function

The amplified nef quasispecies were cloned into the half-genome construct p83-10 and used to make recombinant NL4-3-based reporter viruses with the murine CD24/heat-stable Ag (mCD24/HSA) in the vpr reading frame (Fig. 1). T1 lymphocytes were infected with reporter virus carrying either wild-type NL4-3 nef, NL4-3 nef with the M20A mutation (specifically knocking out the ability to down-regulate MHC-I), or subject-derived nef quasispecies. Flow cytometric assessment of HLA A*0201 down-regulation on infected cells was performed 5 days after infection, directly comparing subject-derived Nef to the control NL4-3 wild-type and defective Nef (M20A mutant) proteins (Fig. 3A). Wild-type NL4-3 down-regulated HLA A*02 an average of 75% across multiple experiments, similar to previously published results (16, 33). Subject-derived Nef quasispecies displayed a broad range of overall HLA A*02 down-regulation efficiencies (Fig. 3B). Nef quasispecies from two subjects (00022 and 00037) were entirely inactive (similar to M20A Nef), while those of two others (00016 and 00021) were similar to wild-type NL4-3 Nef, and the remaining seven were intermediate. Interestingly, down-regulation appeared to be an all-or-none phenomenon. Subject-derived Nef with less than wild-type levels of function appeared to be mixtures of two distinct populations of virus - one that fully down-regulated and the other with no down-regulation (Fig. 3A, lower panels), with the relative proportions of the two populations determining the overall level of HLA A*02 down-regulation. In general, these data showed that the functional status of Nef varies widely in vivo.


Figure 3
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  		FIGURE 3. Measurement of Nef-mediated down-regulation of HLA A*02. T1 cells were infected with recombinant reporter viruses and tested on day 5 postinfection for both level of infection (detected by staining for mCD24) and level of HLA A*02. Cells were stained with anti-murine CD24-PE and anti-human HLA A*02-FITC. Approximately 20,000 live cells (as determined by forward and side scatter gating) were counted by flow cytometry. A, Representative histograms of A*02 levels on infected (mCD24/HSA positive) cells. Vertical lines represent maximum and minimum levels of A*02 as determined by mutant M20A (unable to down-regulate A*02) and isotype staining, respectively. B, Summarizes the absolute percentage of A*02 down-regulation for each isolate averaged from at least three separate infections. Error, SEM for the average of the three experiments.

 
The ability of Nef to down-regulate MHC-I predicts viral susceptibility to suppression by HIV-1-specific CTL

To determine the impact of varying levels of Nef-mediated MHC-I down-regulation on CTL antiviral function, these viruses were tested in a suppression assay with an HIV-1-specific CTL clone. The recombinant reporter viruses with primary isolate nef quasispecies, NL4-3 wild-type nef, and NL4-3 M20A nef were used to infect T1 lymphocytes and cultured in the absence or presence of a Gag-specific CTL clone (recognizing the A*0201-restricted epitope SLYNTVATL, p17 aa 77–85). In agreement with prior data, HIV-1 expressing Nef M20A was significantly more suppressed than Nef wild-type (Fig. 4, A and B). HIV-1 expressing Nef from the various subjects showed a range of susceptibility to suppression by CTL, ranging from less suppression than Nef wild-type to equivalent suppression to Nef M20A (Fig. 4, A and B). When this parameter was compared with the ability of the corresponding viruses to down-regulate MHC-I (Fig. 3), there was a clear relationship between susceptibility to inhibition by CTL vs MHC-I down-regulatory function (p = 0.0038, Fig. 4C). Thus, viral isolates that lost the ability to down-regulate HLA A*02 were more susceptible to CTL, and those that maintained this function were less susceptible. Overall, these findings indicated that this function of Nef has a direct impact on the susceptibility of HIV-1 to the CTL response.


Figure 4
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  		FIGURE 4. Impact of Nef-mediated MHC-I down-regulation on HIV-1 replication and CTL killing. T1 cells infected with recombinant reporter viruses were cocultured in the presence or absence of a CTL clone specific for HIV-1 p17 Gag. Coculture both with and without addition of the CTL clone was performed in triplicate. Levels of viral replication were assessed by p24 ELISA on day 7 postinfection. A, Examples of virus growth and inhibition are shown. B, The efficiency of suppression of replication each viral isolate was calculated by comparing p24 levels in the presence and absence of the CTL clone; means and SDs of the triplicates are plotted. C, The susceptibility to viral suppression by CTL was then correlated with the amount of Nef-mediated A*02 down-regulation for the corresponding virus constructs.

 
The MHC-I down-regulatory function of Nef correlates with breadth of the CTL response

To assess factors influencing this Nef function, the efficiency of Nef-mediated MHC-I down-regulation was compared with key virologic and immunologic parameters of the subjects. Comparison to level of viremia demonstrated perhaps a weak association of lower function with higher viremia (Fig. 5A). Comparison to peripheral blood CD4+ T lymphocyte counts (Fig. 5B) and breadth of the HIV-1-specific CTL response (Fig. 5C) showed significant trends. The ability of Nef to down-regulate MHC-I showed a positive correlation (p = 0.025) to CD4+ T lymphocyte counts (a global measurement of immunosuppressive state in HIV-1 infection) and breadth of virus-specific CTL responses (p = 0.033). As a whole, these data suggested that the ability of Nef to down-regulate MHC-I is greater when HIV-1 is under greater CTL-mediated immune pressure in vivo.


Figure 5
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  		FIGURE 5. Correlation of Nef function with clinical and immunological parameters. Amount of Nef-mediated class I down-regulation was assessed relative to total number of HIV-specific ELISpot responses (i.e., CTL breath) (A), absolute CD4 counts (B), and viral load (C). Univariate regression analysis was performed using levels of Nef function measured as percent wild-type NL4-3 Nef and the clinical and immunologic parameters given in Table I.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
A notable feature of HIV-1 is its high mutation rate and capacity to evolve in response to selective pressures. In the absence of drug treatment, the host CTL response is perhaps the greatest determinant of HIV-1 sequence evolution in vivo, due to viral mutation in recognized CTL epitopes (36, 37). Mutations usually incur a cost of reduced replicative capacity for any given organism; thus HIV-1 reaches equilibrium between escape from CTL and the cost of its escape mutations to maximize overall fitness in the face of the host CTL response. This phenomenon has been examined in detail for epitope mutations to directly avoid recognition via individual CTL epitope sequences (38, 39). However, Nef has been proposed to have an important role in HIV-1 evasion from CTL via MHC-I down-regulation on infected cells and is immunodominant (34, 35, 40), but the global evolution of Nef in response to CTL is not well understood. Data from SIV-infected macaques strongly suggests that Nef indeed adapts to reconstitute MHC-I down-regulatory function when it has been experimentally knocked out in vivo (20).

To our knowledge, this is the first comprehensive survey of this HIV-1 Nef function from primary isolates representing the in vivo quasispecies. Results from our experiments with a broad panel of primary nef quasispecies demonstrate that the ability of Nef to down-regulate MHC-I is generally present but rather variable in individuals in the quasi-stable chronic stage of HIV-1 infection. The degree of this function strongly influences the susceptibility of the virus to suppression by CTL, as predicted by earlier in vitro studies comparing CTL inhibition of HIV-1 containing functional and dysfunctional Nef (17, 33). This observation would suggest that maximizing MHC-I down-regulation would be beneficial for HIV-1 persistence in the setting of antiviral CTL in vivo, as suggested by evaluations in an in vitro model of HIV-1 selection (19).

Comparing the efficiencies of primary isolate Nef proteins in down-regulating MHC-I vs in vivo parameters reveals suggestive associations between this function and immune status in the infected individuals. When we compared with the level of viremia, this Nef function shows no apparent relationship, implying that the functional status of Nef does not directly influence the total level of viral replication (or vice-versa). However, there are associations of Nef-mediated MHC-I down-regulatory function with both higher blood CD4+ T lymphocyte counts and breadth of HIV-1-specific CTL responses. The former parameter is clinically highly predictive of global immune function in HIV-1 infection (e.g., risk of opportunistic infections), while the latter is a theoretical measurement of the responsiveness of cellular immunity against HIV-1. Notably, measurements of the overall magnitude and breadth of the CTL response have been found not to predict control of viremia, although recent data have shown a quantitative correlation of the Gag-specific CTL response with lower viremia when studied across a large cohort of infected persons (41, 42). Our findings are consistent with a scenario where Nef adapts in response to cellular immune pressure against HIV-1, while this immune pressure is only one factor influencing viral replication, and thus does not itself determine the final degree of viremia.

A question that arises is why Nef would not simply maximize MHC-I down-regulation constitutively. Nef is a small protein (~206 aa) that likely plays numerous additional roles in immunopathogenesis through its effects on infected cells, including down-regulation of CD4 and enhancement of cellular activation, which are proposed to increase viral replication and infectivity. Each of these effects occurs through distinct pathways and interactions with different molecules. It is, therefore, likely that this small protein cannot adapt to maximize all functions simultaneously, but must make functional trade-offs in response to the relative benefits of each effect. For example, in end-stage infection, when cellular immunity against the virus has collapsed and activated memory CD4+ T lymphocytes are severely depleted, it could be more important for Nef to serve the purpose of enhancing HIV-1 replication rather than down-regulating MHC-I. Indeed this has been suggested by Carl et al. (25) who tested several Nef functions from isolates obtained at different stages of disease. Some studies have suggested that the magnitude of the Nef-specific CTL response is positively correlated to viremia (34, 40, 43); the implication is unclear, but this could be consistent with increased Nef-targeting by CTL when Nef has evolved to enhance viral replication in the setting of an ineffective CTL response.

The causality of this association of Nef-mediated MHC-I down-regulation as an adaptation to immune pressure remains to be confirmed. An alternative explanation could be that immune function responds to the functional status of Nef. Indeed, clinical examples of the Sydney Cohort and experimental evidence from Nef-deleted SIV infection of rhesus macaques indicate that defective Nef allows more vigorous cellular immunity (6, 8). However, those situations involve virus that has irreparable deletions in Nef, while the protein is intact in the vast majority of natural infections. Given the tendency of HIV-1 to rapidly evolve in response to host pressures such as specific CTL responses and antiretroviral drug treatment, it is reasonable to presume that Nef adapts functionally to immune pressure in a similar manner. It is almost certain, however, that the interaction is bi-directional, and that the degree of viral escape from immunity leads to subsequent immune dysfunction.

An interesting side observation is that the ability of Nef to down-regulate MHC-I appears to be an all-or-none function for individual clones within the plasma quasispecies swarm in vivo. The significance is unclear, but raises the possibility that there is differential immune pressure in distinct compartments of HIV-1 replication. The relative proportions of differentially functional virus could reflect the relative rates of viral replication in separate compartments under differential CTL pressure. For example, virus replicating in a compartment (such as a particular anatomic location) that is inaccessible to CTL could maximize other functions(s) and lose MHC-I down-regulatory function. The virus-infected cells from this compartment that circulate to the peripheral blood compartment, where CTL are present, would then get preferentially cleared due to lack of MHC-I down-regulation, vs virus selected in the periphery in the presence of CTL (selected to down-regulate MHC-I). Further investigation will be required to explore this possibility.

Finally, a shortcoming of our study is its cross-sectional nature. Studying HIV-1-infected persons over time could provide more information about the pressures that affect Nef function in vivo. Because acute infection is a period of rapidly shifting immune pressure on the virus, in contrast to chronic infection, it would be interesting to examine the function of Nef in relationship to immune and virologic parameters longitudinally during early infection. It is possible that the rapid evolution of quasispecies diversity in acute infection in response to host selective pressures leads to the emergence of a particular Nef mutant or dominant quasispecies that may define disease progression. The longitudinal approach has been very useful for examining epitope escape mutations, and would also be helpful for evaluating the evolution of Nef in vivo.

In conclusion, our results demonstrate that Nef usually remains functional in chronic infection and influences susceptibility of HIV-1-infected cells to CTL killing. The MHC-I down-regulatory function of Nef appears to adapt to the immune milieu of the host, increasing in response to increased immune pressure. It, therefore, follows that blocking this function in vivo could be an effective strategy for maximizing CTL efficacy in chronic infection. Small molecule inhibitors or vaccine-directed CTL responses targeted at disrupting Nef function could be avenues could improve CTL activity against infected cells (reducing viral fitness and replication) and improve immune control.


   Acknowledgments
 
We are grateful to all the subjects who participated in this study.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
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.

1 This work was supported by Public Health Service Grants AI051970 (to O.O.Y.), AI043203 (to O.O.Y.), and AI068449 (to M.J.L.). Back

2 Address correspondence and reprint requests to Dr. Martha J. Lewis, Geffen School of Medicine, University of California, Department of Medicine, Division of Infectious Diseases, Center for Health Sciences 37-121, Los Angeles, CA 90095. E-mail address: malewis{at}mednet.ucla.edu Back

3 Abbreviations used in this paper: MHC-I, MHC class I; SFC, spot-forming cells. Back

Received for publication October 25, 2007. Accepted for publication December 31, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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