Xiaomei T. Kuang

Attenuation of Multiple Nef Functions in HIV-1 Elite Controllers

BackgroundImpaired HIV-1 Gag, Pol, and Env function has been described in elite controllers (EC) who spontaneously suppress plasma viremia to < 50 RNA copies/mL; however, activity of the accessory protein Nef remains incompletely characterized. We examined the ability of 91 Nef clones, isolated from plasma of 45 EC and 46 chronic progressors (CP), to down-regulate HLA class I and CD4, up-regulate HLA class II invariant chain (CD74), enhance viral infectivity, and stimulate viral replication in PBMC.ResultsIn general, EC Nef clones were functional; however, all five activities were significantly lower in EC compared to CP. Nef clones from HLA-B*57-expressing EC exhibited poorer CD4 down-regulation function compared to those from non-B*57 EC, and the number of EC-specific B*57-associated Nef polymorphisms correlated inversely with 4 of 5 Nef functions in these individuals.ConclusionResults indicate that decreased HIV-1 Nef function, due in part to host immune selection pressures, may be a hallmark of the EC phenotype.

Ability of HIV-1 Nef to downregulate CD4 and HLA class I differs among viral subtypes

BackgroundThe highly genetically diverse HIV-1 group M subtypes may differ in their biological properties. Nef is an important mediator of viral pathogenicity; however, to date, a comprehensive inter-subtype comparison of Nef in vitro function has not been undertaken. Here, we investigate two of Nef’s most well-characterized activities, CD4 and HLA class I downregulation, for clones obtained from 360 chronic patients infected with HIV-1 subtypes A, B, C or D.ResultsSingle HIV-1 plasma RNA Nef clones were obtained from N=360 antiretroviral-naïve, chronically infected patients from Africa and North America: 96 (subtype A), 93 (B), 85 (C), and 86 (D). Nef clones were expressed by transfection in an immortalized CD4+ T-cell line. CD4 and HLA class I surface levels were assessed by flow cytometry. Nef expression was verified by Western blot. Subset analyses and multivariable linear regression were used to adjust for differences in age, sex and clinical parameters between cohorts. Consensus HIV-1 subtype B and C Nef sequences were synthesized and functionally assessed. Exploratory sequence analyses were performed to identify potential genotypic correlates of Nef function. Subtype B Nef clones displayed marginally greater CD4 downregulation activity (p = 0.03) and markedly greater HLA class I downregulation activity (p < 0.0001) than clones from other subtypes. Subtype C Nefs displayed the lowest in vitro functionality. Inter-subtype differences in HLA class I downregulation remained statistically significant after controlling for differences in age, sex, and clinical parameters (p < 0.0001). The synthesized consensus subtype B Nef showed higher activities compared to consensus C Nef, which was most pronounced in cells expressing lower protein levels. Nef clones exhibited substantial inter-subtype diversity: cohort consensus residues differed at 25% of codons, while a similar proportion of codons exhibited substantial inter-subtype differences in major variant frequency. These amino acids, along with others identified in intra-subtype analyses, represent candidates for mediating inter-subtype differences in Nef function.ConclusionsResults support a functional hierarchy of subtype B > A/D > C for Nef-mediated CD4 and HLA class I downregulation. The mechanisms underlying these differences and their relevance to HIV-1 pathogenicity merit further investigation.

Genotypic and Functional Impact of HIV-1 Adaptation to Its Host Population during the North American Epidemic

HLA-restricted immune escape mutations that persist following HIV transmission could gradually spread through the viral population, thereby compromising host antiviral immunity as the epidemic progresses. To assess the extent and phenotypic impact of this phenomenon in an immunogenetically diverse population, we genotypically and functionally compared linked HLA and HIV (Gag/Nef) sequences from 358 historic (1979–1989) and 382 modern (2000–2011) specimens from four key cities in the North American epidemic (New York, Boston, San Francisco, Vancouver). Inferred HIV phylogenies were star-like, with approximately two-fold greater mean pairwise distances in modern versus historic sequences. The reconstructed epidemic ancestral (founder) HIV sequence was essentially identical to the North American subtype B consensus. Consistent with gradual diversification of a “consensus-like” founder virus, the median “background” frequencies of individual HLA-associated polymorphisms in HIV (in individuals lacking the restricting HLA[s]) were ∼2-fold higher in modern versus historic HIV sequences, though these remained notably low overall (e.g. in Gag, medians were 3.7% in the 2000s versus 2.0% in the 1980s). HIV polymorphisms exhibiting the greatest relative spread were those restricted by protective HLAs. Despite these increases, when HIV sequences were analyzed as a whole, their total average burden of polymorphisms that were “pre-adapted” to the average host HLA profile was only ∼2% greater in modern versus historic eras. Furthermore, HLA-associated polymorphisms identified in historic HIV sequences were consistent with those detectable today, with none identified that could explain the few HIV codons where the inferred epidemic ancestor differed from the modern consensus. Results are therefore consistent with slow HIV adaptation to HLA, but at a rate unlikely to yield imminent negative implications for cellular immunity, at least in North America. Intriguingly, temporal changes in protein activity of patient-derived Nef (though not Gag) sequences were observed, suggesting functional implications of population-level HIV evolution on certain viral proteins.

Dynamic range of Nef functions in chronic HIV-1 infection

HIV-1 Nef is required for efficient viral replication and pathogenesis. However, the extent to which Nefs functions are maintained in natural sequences during chronic infection, and their clinical relevance, remains incompletely characterized. Relative to a control Nef from HIV-1 strain SF2, HLA class I and CD4 down-regulation activities of 46 plasma RNA Nef sequences derived from unique chronic infected individuals were generally high and displayed narrow dynamic ranges, whereas Nef-mediated virion infectivity, PBMC replication and CD74 up-regulation exhibited broader dynamic ranges. 80% of patient-derived Nefs were active for at least three functions examined. Functional co-dependencies were identified, including positive correlations between CD4 down-regulation and virion infectivity, replication, and CD74 up-regulation, and between CD74 up-regulation and PBMC replication. Nef-mediated virion infectivity inversely correlated with patient CD4(±) T-cell count. Strong functional co-dependencies and the polyfunctional nature of patient-derived Nef sequences suggest a phenotypic requirement to maintain multiple Nef functions during chronic infection.

Impaired Nef Function Is Associated with Early Control of HIV-1 Viremia

ABSTRACT Host and viral factors influence the HIV-1 infection course. Reduced Nef function has been observed in HIV-1 controllers during the chronic phase, but the kinetics and mechanisms of Nef attenuation in such individuals remain unclear. We examined plasma RNA-derived Nef clones from 10 recently infected individuals who subsequently suppressed viremia to less than 2,000 RNA copies/ml within 1 year postinfection (acute controllers) and 50 recently infected individuals who did not control viremia (acute progressors). Nef clones from acute controllers displayed a lesser ability to downregulate CD4 and HLA class I from the cell surface and a reduced ability to enhance virion infectivity compared to those from acute progressors (all P < 0.01). HLA class I downregulation activity correlated inversely with days postinfection (Spearmans R = −0.85, P = 0.004) and positively with baseline plasma viral load (Spearmans R = 0.81, P = 0.007) in acute controllers but not in acute progressors. Nef polymorphisms associated with functional changes over time were identified in follow-up samples from six controllers. For one such individual, mutational analyses indicated that four polymorphisms selected by HLA-A*31 and B*37 acted in combination to reduce Nef steady-state protein levels and HLA class I downregulation activity. Our results demonstrate that relative control of initial HIV-1 viremia is associated with Nef clones that display reduced function, which in turn may influence the course of HIV-1 infection. Transmission of impaired Nef sequences likely contributed in part to this observation; however, accumulation of HLA-associated polymorphisms in Nef that impair function also suggests that CD8+ T-cell pressures play a role in this phenomenon. IMPORTANCE Rare individuals can spontaneously control HIV-1 viremia in the absence of antiretroviral treatment. Understanding the host and viral factors that contribute to the controller phenotype may identify new strategies to design effective vaccines or therapeutics. The HIV-1 Nef protein enhances viral pathogenesis through multiple mechanisms. We examined the function of plasma HIV-1 RNA-derived Nef clones isolated from 10 recently infected individuals who subsequently controlled HIV viremia compared to the function of those from 50 individuals who failed to control viremia. Our results demonstrate that early Nef clones from HIV controllers displayed lower HLA class I and CD4 downregulation activity, as well as a reduced ability to enhance virion infectivity. The accumulation of HLA-associated polymorphisms in Nef during the first year postinfection was associated with impaired protein function in some controllers. This report highlights the potential for host immune responses to modulate HIV pathogenicity and disease outcome by targeting cytotoxic T lymphocyte (CTL) epitopes in Nef.

No Evidence for Selection of HIV-1 with Enhanced Gag-Protease or Nef Function among Breakthrough Infections in the CAPRISA 004 Tenofovir Microbicide Trial

Background Use of antiretroviral-based microbicides for HIV-1 prophylaxis could introduce a transmission barrier that inadvertently facilitates the selection of fitter viral variants among incident infections. To investigate this, we assessed the in vitro function of gag-protease and nef sequences from participants who acquired HIV-1 during the CAPRISA 004 1% tenofovir microbicide gel trial. Methods and Results We isolated the earliest available gag-protease and nef gene sequences from 83 individuals and examined their in vitro function using recombinant viral replication capacity assays and surface protein downregulation assays, respectively. No major phylogenetic clustering and no significant differences in gag-protease or nef function were observed in participants who received tenofovir gel versus placebo gel prophylaxis. Conclusion Results indicate that the partial protective effects of 1% tenofovir gel use in the CAPRISA 004 trial were not offset by selection of transmitted/early HIV-1 variants with enhanced Gag-Protease or Nef fitness.

Differential Ability of Primary HIV-1 Nef Isolates To Downregulate HIV-1 Entry Receptors

ABSTRACT HIV-1 Nef downregulates the viral entry receptor CD4 as well as the coreceptors CCR5 and CXCR4 from the surface of HIV-infected cells, and this leads to promotion of viral replication through superinfection resistance and other mechanisms. Nef sequence motifs that modulate these functions have been identified via in vitro mutagenesis with laboratory HIV-1 strains. However, it remains unclear whether the same motifs contribute to Nef activity in patient-derived sequences and whether these motifs may differ in Nef sequences isolated at different infection stages and/or from patients with different disease phenotypes. Here, nef clones from 45 elite controllers (EC), 46 chronic progressors (CP), and 43 acute progressors (AP) were examined for their CD4, CCR5, and CXCR4 downregulation functions. Nef clones from EC exhibited statistically significantly impaired CD4 and CCR5 downregulation ability and modestly impaired CXCR4 downregulation activity compared to those from CP and AP. Nefs ability to downregulate CD4 and CCR5 correlated positively in all cohorts, suggesting that they are functionally linked in vivo. Moreover, impairments in Nefs receptor downregulation functions increased the susceptibility of Nef-expressing cells to HIV-1 infection. Mutagenesis studies on three functionally impaired EC Nef clones revealed that multiple residues, including those at novel sites, were involved in the alteration of Nef functions and steady-state protein levels. Specifically, polymorphisms at highly conserved tryptophan residues (e.g., Trp-57 and Trp-183) and immune escape-associated sites were responsible for reduced Nef functions in these clones. Our results suggest that the functional modulation of primary Nef sequences is mediated by complex polymorphism networks. IMPORTANCE HIV-1 Nef, a key factor for viral pathogenesis, downregulates functionally important molecules from the surface of infected cells, including the viral entry receptor CD4 and coreceptors CCR5 and CXCR4. This activity enhances viral replication by protecting infected cells from cytotoxicity associated with superinfection and may also serve as an immune evasion strategy. However, how these activities are maintained under selective pressure in vivo remains elusive. We addressed this question by analyzing functions of primary Nef clones isolated from patients at various infection stages and with different disease phenotypes, including elite controllers, who spontaneously control HIV-1 viremia to undetectable levels. The results indicated that downregulation of HIV-1 entry receptors, particularly CCR5, is impaired in Nef clones from elite controllers. These functional impairments were driven by rare Nef polymorphisms and adaptations associated with cellular immune responses, underscoring the complex molecular pathways responsible for maintaining and attenuating viral protein function in vivo.

Nef-mediated down-regulation of CD4 and HLA class I in HIV-1 subtype C infection: Association with disease progression and influence of immune pressure

Nef plays a major role in HIV-1 pathogenicity. We studied HIV-1 subtype C infected individuals in acute/early (n = 120) or chronic (n = 207) infection to investigate the relationship between Nef-mediated CD4/HLA-I down-regulation activities and disease progression, and the influence of immune-driven sequence variation on these Nef functions. A single Nef sequence per individual was cloned into an expression plasmid, followed by transfection of a T cell line and measurement of CD4 and HLA-I expression. In early infection, a trend of higher CD4 down-regulation ability correlating with higher viral load set point was observed (r = 0.19, p = 0.05), and higher HLA-I down-regulation activity was significantly associated with faster rate of CD4 decline (p = 0.02). HLA-I down-regulation function correlated inversely with the number HLA-associated polymorphisms previously associated with reversion in the absence of the selecting HLA allele (r = -0.21, p = 0.0002). These data support consideration of certain Nef regions in HIV-1 vaccine strategies designed to attenuate the infection course.

Consequences of HLA-B*13-Associated Escape Mutations on HIV-1 Replication and Nef Function.

ABSTRACT HLA-B*13 is associated with superior in vivo HIV-1 viremia control. Protection is thought to be mediated by sustained targeting of key cytotoxic T lymphocyte (CTL) epitopes and viral fitness costs of CTL escape in Gag although additional factors may contribute. We assessed the impact of 10 published B*13-associated polymorphisms in Gag, Pol, and Nef, in 23 biologically relevant combinations, on HIV-1 replication capacity and Nef-mediated reduction of cell surface CD4 and HLA class I expression. Mutations were engineered into HIV-1NL4.3, and replication capacity was measured using a green fluorescent protein (GFP) reporter T cell line. Nef-mediated CD4 and HLA-A*02 downregulation was assessed by flow cytometry, and T cell recognition of infected target cells was measured via coculture with an HIV-specific luciferase reporter cell line. When tested individually, only Gag-I147L and Gag-I437L incurred replicative costs (5% and 17%, respectively), consistent with prior reports. The Gag-I437L-mediated replication defect was rescued to wild-type levels by the adjacent K436R mutation. A novel B*13 epitope, comprising 8 residues and terminating at Gag147, was identified in p24Gag (GQMVHQAIGag140–147). No other single or combination Gag, Pol, or Nef mutant impaired viral replication. Single Nef mutations did not affect CD4 or HLA downregulation; however, the Nef double mutant E24Q-Q107R showed 40% impairment in HLA downregulation with no evidence of Nef stability defects. Moreover, target cells infected with HIV-1-NefE24Q-Q107R were recognized better by HIV-specific T cells than those infected with HIV-1NL4.3 or single Nef mutants. Our results indicate that CTL escape in Gag and Nef can be functionally costly and suggest that these effects may contribute to long-term HIV-1 control by HLA-B*13. IMPORTANCE Protective effects of HLA-B*13 on HIV-1 disease progression are mediated in part by fitness costs of CTL escape mutations in conserved Gag epitopes, but other mechanisms remain incompletely known. We extend our knowledge of the impact of B*13-driven escape on HIV-1 replication by identifying Gag-K436R as a compensatory mutation for the fitness-costly Gag-I437L. We also identify Gag-I147L, the most rapidly and commonly selected B*13-driven substitution in HIV-1, as a putative C-terminal anchor residue mutation in a novel B*13 epitope. Most notably, we identify a novel escape-driven fitness defect: B*13-driven substitutions E24Q and Q107R in Nef, when present together, substantially impair this proteins ability to downregulate HLA class I. This, in turn, increases the visibility of infected cells to HIV-specific T cells. Our results suggest that B*13-associated escape mutations impair HIV-1 replication by two distinct mechanisms, that is, by reducing Gag fitness and dampening Nef immune evasion function.

A robust and scalable TCR-based reporter cell assay to measure HIV-1 Nef-mediated T cell immune evasion

HIV-1 evades cytotoxic T cell responses through Nef-mediated downregulation of HLA class I molecules from the infected cell surface. Methods to quantify the impact of Nef on T cell recognition typically employ patient-derived T cell clones; however, these assays are limited by the cost and effort required to isolate and maintain primary cell lines. The variable activity of different T cell clones and the limited number of cells generated by re-stimulation can also hinder assay reproducibility and scalability. Here, we describe a heterologous T cell receptor reporter assay and use it to study immune evasion by Nef. Induction of NFAT-driven luciferase following co-culture with peptide-pulsed or virus-infected target cells serves as a rapid, quantitative and antigen-specific measure of T cell recognition of its cognate peptide/HLA complex. We demonstrate that Nef-mediated downregulation of HLA on target cells correlates inversely with T cell receptor-dependent luminescent signal generated by effector cells. This method provides a robust, flexible and scalable platform that is suitable for studies to measure Nef function in the context of different viral peptide/HLA antigens, to assess the function of patient-derived Nef alleles, or to screen small molecule libraries to identify novel Nef inhibitors.