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. 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. Conclusion Results indicate that decreased HIV-1 Nef function, due in part to host immune selection pressures, may be a hallmark of the EC phenotype

Examining the function of primary HIV-1 Nef isolates in the setting of viral control

HIV is a global pandemic. While combination anti-retroviral therapy can block viral replication, HIV establishes latent reservoirs that persist for life. A rare group of HIV-infected individuals called “elite controllers” spontaneously suppress plasma viremia to clinically undetectable levels. Understanding how elite controllers contain HIV could assist in developing more effective interventions, such as vaccines or eradication strategies. The HIV Nef protein is critical for viral pathogenesis and progression to AIDS. Nef modulates cellular proteins to increase viral replication and to evade host immunity. Functional impairment of several HIV proteins has been seen in elite controllers, but prior to my thesis a systematic analysis of Nef had not been done. To explore this, I examined Nef clones from elite controllers and progressors. I observed that Nef clones from controllers displayed significantly lower activity for multiple functions. Furthermore, I identified natural polymorphisms in Nef that contributed to attenuated function and confirmed these by testing Nef mutants. In summary, I have shown that natural sequence variation in Nef results in substantial differences in protein function. Nef clones isolated from elite controllers displayed the poorest activity for multiple functions, indicating that attenuation of Nef may contribute to reduced pathogenesis in these cases. Therapeutic targeting of the Nef domains identified in my studies could contribute to HIV eradication strategies

Brockman, M.A., et al., Human Leukocyte Antigen (HLA) Class I Down-Regulation by Human Immunodeficiency Virus Type 1 Negative Factor (HIV-1 Nef): What Might We Learn From Natural Sequence Variants? Viruses 2012, 4, 1711-1730

In the original manuscript, the text in figure 1 is illegible. Furthermore, there is an unnecessary carriage return (page 1716, ~line 18) "crystallographic ... methods"

Effect of Basic Site Substituents on Concerted Proton–Electron Transfer in Hydrogen-Bonded Pyridyl–Phenols

Separated concerted proton–electron transfer (sCPET) reactions of two series of phenols with pendent substituted pyridyl moieties are described. The pyridine is either attached directly to the phenol (<b>HOAr-pyX</b>) or connected through a methylene linker (<b>HOArCH</b>_<b>2</b><b>pyX</b>) (X = 4-NO₂, 5-CF₃, 4-CH₃, and 4-NMe₂). Electron-donating and -withdrawing substituents have a substantial effect on the chemical environment of the transferring proton, as indicated by IR and ¹H NMR spectra, X-ray structures, and computational studies. One-electron oxidation of the phenols occurs concomitantly with proton transfer from the phenolic oxygen to the pyridyl nitrogen. The oxidation potentials vary linearly with the p<i>K</i>_a of the free pyridine (pyX), with slopes slightly below the Nerstian value of 59 mV/p<i>K</i>_a. For the <b>HOArCH</b>_<b>2</b><b>pyX</b> series, the rate constants <i>k</i>_sCPET for oxidation by NAr₃^•+ or [Fe­(diimine)₃]³⁺ vary primarily with the thermodynamic driving force (Δ<i>G</i>°_sCPET), whether Δ<i>G</i>° is changed by varying the potential of the oxidant or the substituent on the pyridine, indicating a constant intrinsic barrier λ. In contrast, the substituents in the <b>HOAr-pyX</b> series affect λ as well as Δ<i>G</i>°_sCPET, and compounds with electron-withdrawing substituents have significantly lower reactivity. The relationship between the structural and spectroscopic properties of the phenols and their CPET reactivity is discussed

Effect of Basic Site Substituents on Concerted Proton–Electron Transfer in Hydrogen-Bonded Pyridyl–Phenols

Separated concerted proton–electron transfer (sCPET) reactions of two series of phenols with pendent substituted pyridyl moieties are described. The pyridine is either attached directly to the phenol (<b>HOAr-pyX</b>) or connected through a methylene linker (<b>HOArCH</b>_<b>2</b><b>pyX</b>) (X = 4-NO₂, 5-CF₃, 4-CH₃, and 4-NMe₂). Electron-donating and -withdrawing substituents have a substantial effect on the chemical environment of the transferring proton, as indicated by IR and ¹H NMR spectra, X-ray structures, and computational studies. One-electron oxidation of the phenols occurs concomitantly with proton transfer from the phenolic oxygen to the pyridyl nitrogen. The oxidation potentials vary linearly with the p<i>K</i>_a of the free pyridine (pyX), with slopes slightly below the Nerstian value of 59 mV/p<i>K</i>_a. For the <b>HOArCH</b>_<b>2</b><b>pyX</b> series, the rate constants <i>k</i>_sCPET for oxidation by NAr₃^•+ or [Fe­(diimine)₃]³⁺ vary primarily with the thermodynamic driving force (Δ<i>G</i>°_sCPET), whether Δ<i>G</i>° is changed by varying the potential of the oxidant or the substituent on the pyridine, indicating a constant intrinsic barrier λ. In contrast, the substituents in the <b>HOAr-pyX</b> series affect λ as well as Δ<i>G</i>°_sCPET, and compounds with electron-withdrawing substituents have significantly lower reactivity. The relationship between the structural and spectroscopic properties of the phenols and their CPET reactivity is discussed

In vitro functional assessment of natural HIV-1 group M Vpu sequences using a universal priming approach

The HIV-1 accessory protein Vpu exhibits high inter- and intra- subtype genetic diversity that may influence Vpu function and possibly contribute to HIV-1 pathogenesis. However, scalable methods to evaluate genotype/phenotype relationships in natural Vpu sequences are limited, particularly those expressing the protein in CD4+ T-cells, the natural target of HIV-1 infection. A major impediment to assay scalability is the extensive genetic diversity within, and immediately upstream of, Vpu's initial 5' coding region, which has necessitated the design of oligonucleotide primers specific for each individual HIV-1 isolate (or subtype). To address this, we developed two universal forward primers, located in relatively conserved regions 38 and 90 bases upstream of Vpu, and a single universal reverse primer downstream of Vpu, which are predicted to cover the vast majority of global HIV-1 group M sequence diversity. We show that inclusion of up to 90 upstream bases of HIV-1 genomic sequence does not significantly influence in vitro Vpu expression or function when a Rev/Rev Response Element (RRE)-dependent expression system is used. We further assess the function of four diverse HIV-1 Vpu sequences, revealing reproducible and significant differences between them. Our approach represents a scalable option to measure the in vitro function of genetically diverse natural Vpu isolates in a CD4+ T-cell line