Modulation of TCR-dependent NFAT signaling is impaired in HIV-1 Nef isolates from elite controllers.

HIV-1 Nef modulates the activation state of CD4 T cells by altering signaling events elicited by the T cell receptor (TCR). Primary nef sequences exhibit extensive inter-individual diversity that influences their ability to downregulate CD4 and HLA class I; however, the impact of nef variation on modulation of T cell signaling is poorly characterized. Here, we measured TCR-mediated activation of NFAT transcription factor in the presence of nef alleles isolated from 45 elite controllers (EC) and 46 chronic progressors (CP).

Implications of HIV-1 Nef for "Shock and Kill" Strategies to Eliminate Latent Viral Reservoirs.

Finding a cure for HIV is challenging because the virus is able to integrate itself into the host cell genome and establish a silent state, called latency, allowing it to evade antiviral drugs and the immune system. Various "shock and kill" strategies are being explored in attempts to eliminate latent HIV reservoirs. The goal of these approaches is to reactivate latent viruses ("shock"), thereby exposing them to clearance by viral cytopathic effects or immune-mediated responses ("kill").

Inhibition of NF-κB-dependent HIV-1 replication by the marine natural product bengamide A.

HIV-1 inhibitors that act by mechanisms distinct from existing antiretrovirals can provide novel insights into viral replication and potentially inform development of new therapeutics. Using a multi-cycle HIV-1 replication assay, we screened 252 pure compounds derived from marine invertebrates and microorganisms and identified 6 (actinomycin Z, bastadin 6, bengamide A, haliclonacyclamine A + B, keramamine C, neopetrosiamide B) that inhibited HIV-1 with 50% effective concentrations (ECs) of 3.8 μM or less.

Relative Resistance of HLA-B to Downregulation by Naturally Occurring HIV-1 Nef Sequences.

Unlabelled: HIV-1 Nef binds to the cytoplasmic region of HLA-A and HLA-B and downregulates these molecules from the surface of virus-infected cells, thus evading immune detection by CD8(+) T cells. Polymorphic residues within the HLA cytoplasmic region may affect Nef's downregulation activity. However, the effects of HLA polymorphisms on recognition by primary Nef isolates remain elusive, as do the specific Nef regions responsible for downregulation of HLA-A versus HLA-B.

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

Unlabelled: 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.

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.

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

Unlabelled: 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.

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.

Impaired Nef function is associated with early control of HIV-1 viremia.

Unlabelled: 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).

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.

Effect of 3',5'-cyclic diguanylic acid in a broiler Clostridium perfringens infection model.

In an effort to explore strategies to control Clostridium perfringens, we investigated the synergistic effect of a ubiquitous bacterial second messenger 3',5'-cyclic diguanylic acid (c-di-GMP) with penicillin G in a broiler challenge model. All chicks were inoculated in the crop by gavage on d 14, 15, and 16 with a mixture of 4 C. perfringens strains.

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

Background: The 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.

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.

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 Nef's 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.

Attenuation of multiple Nef functions in HIV-1 elite controllers.

Background: Impaired 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.

Results: In general, EC Nef clones were functional; however, all five activities were significantly lower in EC compared to CP.