Nef-mediated inhibition of NFAT following TCR stimulation differs between HIV-1 subtypes

Naidoo, Lisa; Mzobe, Zinhle; Jin, Steven; Rajkoomar, Erasha; Reddy, Tarylee; Brockman, Mark A.; Brumme, Zabrina L.; Ndung’u, Thumbi; Mann, Jaclyn K.

doi:10.1016/j.virol.2019.02.011

Cited by 9 publications

(17 citation statements)

References 77 publications

(142 reference statements)

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“…The dimerization-defective Nef mutant Nef-DD is included as a negative control. (B) Cell-surface MHC-I rescue by Nef inhibitor analogs based on median fluorescence intensity of MHC-I + cells and the following formula: [(Nef + MHC + /Nef – MHC-I + ) drug – (Nef + MHC + /Nef – MHC-I + ) no drug ] × 100, where Nef + cells are defined by GFP expression (analysis as per Naidoo et al). Bars represent mean values ± SE with 3 to 5 biological replicates in each case.…”

Section: Resultsmentioning

confidence: 99%

Tight-Binding Hydroxypyrazole HIV-1 Nef Inhibitors Suppress Viral Replication in Donor Mononuclear Cells and Reverse Nef-Mediated MHC-I Downregulation

et al. 2019

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The HIV-1 Nef accessory factor is critical to the viral life cycle in vivo and promotes immune escape of infected cells via downregulation of cell-surface MHC-I. Previously, we discovered small molecules that bind directly to Nef and block many of its functions, including enhancement of viral infectivity and replication in T cell lines. These compounds also restore cell-surface MHC-I expression in HIV-infected CD4 T cells from AIDS patients, enabling recognition and killing by autologous cytotoxic T lymphocytes (CTLs). In this study, we describe the synthesis and evaluation of a diverse set of analogs based on the original hydroxypyrazole Nef inhibitor core. All analogs were screened for the interaction with recombinant HIV-1 Nef by surface plasmon resonance (SPR) and for antiretroviral activity in TZM-bl reporter cells infected with HIV-1. Active analogs were ranked on the basis of an activity score that integrates three aspects of the SPR data (affinity, residence time, and extent of binding) with antiretroviral activity. The top scoring compounds bound tightly to Nef by SPR, with K D values in the low nM to pM range, and displayed very slow dissociation from their Nef target. These analogs also suppressed HIV-1 replication in donor peripheral blood mononuclear cells (PBMCs) with IC 50 values in the 1−10 nM range without cytotoxicity, inhibited Nef-mediated IL-2-inducible tyrosine kinase (Itk) and hematopoietic cell kinase (Hck) activation, and rescued MHC-I downregulation in a Nef-transfected T cell line. The development of Nef inhibitors based on the structure−activity relationships defined here has promise as a new approach to antiretroviral therapy that includes a path to eradication of HIV-infected cells via the adaptive immune response.

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Section: Resultsmentioning

confidence: 99%

Tight-Binding Hydroxypyrazole HIV-1 Nef Inhibitors Suppress Viral Replication in Donor Mononuclear Cells and Reverse Nef-Mediated MHC-I Downregulation

et al. 2019

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“…Furthermore, considering that some of the HLA-associated mutations we studied here were previously reported to significantly impact Nef function (71K and 88G) (Mann et al. 2015; Naidoo et al. 2019) but had very moderate effects here in the LANL consensus subtype B background, the fitness cost of these mutations may be dependent on sequence background.…”

Section: Resultsmentioning

confidence: 91%

Modelling and in vitro testing of the HIV-1 Nef fitness landscape

et al. 2019

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An effective vaccine is urgently required to curb the HIV-1 epidemic. We have previously described an approach to model the fitness landscape of several HIV-1 proteins, and have validated the results against experimental and clinical data. The fitness landscape may be used to identify mutation patterns harmful to virus viability, and consequently inform the design of immunogens that can target such regions for immunological control. Here we apply such an analysis and complementary experiments to HIV-1 Nef, a multifunctional protein which plays a key role in HIV-1 pathogenesis. We measured Nef-driven replication capacities as well as Nef-mediated CD4 and HLA-I down-modulation capacities of thirty-two different Nef mutants, and tested model predictions against these results. Furthermore, we evaluated the models using 448 patient-derived Nef sequences for which several Nef activities were previously measured. Model predictions correlated significantly with Nef-driven replication and CD4 down-modulation capacities, but not HLA-I down-modulation capacities, of the various Nef mutants. Similarly, in our analysis of patient-derived Nef sequences, CD4 down-modulation capacity correlated the most significantly with model predictions, suggesting that of the tested Nef functions, this is the most important in vivo. Overall, our results highlight how the fitness landscape inferred from patient-derived sequences captures, at least in part, the in vivo functional effects of mutations to Nef. However, the correlation between predictions of the fitness landscape and measured parameters of Nef function is not as accurate as the correlation observed in past studies for other proteins. This may be because of the additional complexity associated with inferring the cost of mutations on the diverse functions of Nef.

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“…Previously, other Nef functions were measured for the same Nef clones [ 15 , 16 ]. These included CD4 and HLA-I down-regulation, using flow cytometry-based methods similar to that for SERINC5 down-regulation [ 15 ].…”

Section: Resultsmentioning

confidence: 99%

“…Given the key role of Nef-mediated SERINC5 down-regulation in enhancing virion infectivity, we sought to determine whether this Nef activity contributes significantly to HIV-1 subtype C disease progression. The SERINC5 down-regulation activity of 106 Nef clones, isolated from patients in early infection and for which CD4 and HLA-I down-regulation activities [ 15 ] as well as alteration of TCR signalling activity [ 16 ] had previously been measured, was evaluated in a CEM-derived CD4 + T cell line using a flow cytometry-based assay and then related to subsequent rate of CD4 + T cell decline and viral load set point. In past work, computational models of the Nef fitness landscape that were based on correlated patterns of multiple mutations and generated using > 10 000 Nef sequences, were used to generate a predicted Nef fitness measure—an E value—for each of the same Nef clones [ 17 ].…”

Section: Introductionmentioning

confidence: 99%

HIV-1 subtype C Nef-mediated SERINC5 down-regulation significantly contributes to overall Nef activity

et al. 2023

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Background Nef performs multiple cellular activities that enhance HIV-1 pathogenesis. The role of Nef-mediated down-regulation of the host restriction factor SERINC5 in HIV-1 pathogenesis is not well-defined. We aimed to investigate if SERINC5 down-regulation activity contributes to HIV-1 subtype C disease progression, to assess the relative contribution of this activity to overall Nef function, and to identify amino acids required for optimal activity. We measured the SERINC5 down-regulation activity of 106 subtype C Nef clones, isolated from individuals in early infection, for which the Nef activities of CD4 and HLA-I down-regulation as well as alteration of TCR signalling were previously measured. The relationship between SERINC5 down-regulation and markers of disease progression, and the relative contribution of SERINC5 down-regulation to a Nef fitness model-derived E value (a proxy for overall Nef fitness in vivo), were assessed. Results No overall relationship was found between SERINC5 down-regulation and viral load set point (p = 0.28) or rate of CD4+ T cell decline (p = 0.45). CD4 down-regulation (p = 0.02) and SERINC5 down-regulation (p = 0.003) were significant determinants of E values in univariate analyses, with the greatest relative contribution for SERINC5 down-regulation, and only SERINC5 down-regulation remained significant in the multivariate analysis (p = 0.003). Using a codon-by-codon analysis, several amino acids were significantly associated with increased (10I, 11V, 38D, 51T, 65D, 101V, 188H and, 191H) or decreased (10K, 38E, 65E, 135F, 173T, 176T and, 191R) SERINC5 down-regulation activity. Site-directed mutagenesis experiments of selected mutants confirmed a substantial reduction in SERINC5 down-regulation activity associated with the mutation 173T, while mutations 10K, 135F, and 176T were associated with more modest reductions in activity that were not statistically significant. Conclusions These results suggest that SERINC5 down-regulation is a significant contributor to overall Nef function and identify potential genetic determinants of this Nef function that may have relevance for vaccines or therapeutics.

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Nef-mediated inhibition of NFAT following TCR stimulation differs between HIV-1 subtypes

Cited by 9 publications

References 77 publications

Tight-Binding Hydroxypyrazole HIV-1 Nef Inhibitors Suppress Viral Replication in Donor Mononuclear Cells and Reverse Nef-Mediated MHC-I Downregulation

Tight-Binding Hydroxypyrazole HIV-1 Nef Inhibitors Suppress Viral Replication in Donor Mononuclear Cells and Reverse Nef-Mediated MHC-I Downregulation

Modelling and in vitro testing of the HIV-1 Nef fitness landscape

HIV-1 subtype C Nef-mediated SERINC5 down-regulation significantly contributes to overall Nef activity

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