136 HIV-1 Nef regulates activity of endoplasmic reticulum chaperone calnexin

HIV-1 Nef promotes viral replication by downmodulating a number of cell surface transmembrane proteins, such as CD4, MHC-I and MHC-II, which are targeted by Nef to various degradation pathways. Nef is also responsible for downregulation of cellular cholesterol transporter ABCA1, and this effect contributes to development of atherosclerosis in HIV infected patients. Surprisingly, in contrast to CD4 and MHC I, to which Nef has to bind to exert downregulation, binding to ABCA1 turned out to be unnecessary for inactivation of ABCA1 by Nef. Here, we identified a novel mechanism by which Nef influences activity of host cell and viral proteins. We show that Nef interacts with an endoplasmic reticulum chaperone calnexin, which is essential for folding and maturation of glycosylated proteins. Nef disrupts calnexin interaction with ABCA1, thus impairing functionality of this protein, but increases affinity and enhances interaction of calnexin with gp160, promoting maturation and functionality of viral Env proteins. Knock-down of calnexin lead to reduced fusion activity of HIV-1 envelope and reduced virion infectivity, as well as to defective cholesterol efflux, which is mediated by ABCA1. However, gp160 and ABCA1 interacted with calnexin differently: while gp160 binding to calnexin was dependent on glycosylation, interaction of ABCA1 with calnexin was glycosylation-independent. Therefore, Nef binds to calnexin and stimulates interaction between calnexin and gp160 at the expense of ABCA1 and probably other ER proteins. These results provide a mechanistic explanation for previously unexplained effect of Nef on functionality of ABCA1, and suggest a mechanism for upregulation of HIV infectivity by Nef through stimulation of Env maturation

Lipid metabolism in patients infected with Nef-deficient HIV-1 strain

Background: HIV protein Nef plays a key role in impairing cholesterol metabolism in both HIV infected and bystander cells. The existence of a small cohort of patients infected with Nef-deficient strain of HIV presented a unique opportunity to test the effect of Nef on lipid metabolism in a clinical setting. Methods: Here we report the results of a study comparing six patients infected with Nef-deficient strain of HIV (Delta NefHIV) with six treatment-naive patients infected with wild-type HIV (WT HIV). Lipoprotein profile, size and functionality of high density lipoprotein (HDL) particles as well as lipidomic and microRNA profiles of patient plasma were analyzed. Results: We found that patients infected with Delta NefHIV had lower proportion of subjects with plasma HDL-C levels < 1 mmol/l compared to patients infected with WT HIV. Furthermore, compared to a reference group of HIV-negative subjects, there was higher abundance of smaller under-lipidated HDL particles in plasma of patients infected with WT HIV, but not in those infected with Delta NefHIV. Lipidomic analysis of plasma revealed differences in abundance of phosphatidylserine and sphingolipids between patients infected with Delta NefHIV and WT HIV. MicroRNA profiling revealed that plasma abundance of 24 miRNAs, many of those involved in regulation of lipid metabolism, was differentially regulated by WT HIV and Delta NefHIV. Conclusion: Our findings are consistent with HIV protein Nef playing a significant role in pathogenesis of lipid-related metabolic complications of HIV disease

Exosomes containing HIV protein Nef reorganize lipid rafts potentiating inflammatory response in bystander cells.

HIV infection has a profound effect on "bystander" cells causing metabolic co-morbidities. This may be mediated by exosomes secreted by HIV-infected cells and containing viral factors. Here we show that exosomes containing HIV-1 protein Nef (exNef) are rapidly taken up by macrophages releasing Nef into the cell interior. This caused down-regulation of ABCA1, reduction of cholesterol efflux and sharp elevation of the abundance of lipid rafts through reduced activation of small GTPase Cdc42 and decreased actin polymerization. Changes in rafts led to re-localization of TLR4 and TREM-1 to rafts, phosphorylation of ERK1/2, activation of NLRP3 inflammasome, and increased secretion of pro-inflammatory cytokines. The effects of exNef on lipid rafts and on inflammation were reversed by overexpression of a constitutively active mutant of Cdc42. Similar effects were observed in macrophages treated with exosomes produced by HIV-infected cells or isolated from plasma of HIV-infected subjects, but not with exosomes from cells and subjects infected with ΔNef-HIV or uninfected subjects. Mice injected with exNef exhibited monocytosis, reduced ABCA1 in macrophages, increased raft abundance in monocytes and augmented inflammation. Thus, Nef-containing exosomes potentiated pro-inflammatory response by inducing changes in cholesterol metabolism and reorganizing lipid rafts. These mechanisms may contribute to HIV-associated metabolic co-morbidities

Lipid metabolism in patients infected with Nef-deficient HIV-1 strain.

BACKGROUND: HIV protein Nef plays a key role in impairing cholesterol metabolism in both HIV infected and bystander cells. The existence of a small cohort of patients infected with Nef-deficient strain of HIV presented a unique opportunity to test the effect of Nef on lipid metabolism in a clinical setting. METHODS: Here we report the results of a study comparing six patients infected with Nef-deficient strain of HIV (ΔNefHIV) with six treatment-naïve patients infected with wild-type HIV (WT HIV). Lipoprotein profile, size and functionality of high density lipoprotein (HDL) particles as well as lipidomic and microRNA profiles of patient plasma were analyzed. RESULTS: We found that patients infected with ΔNefHIV had lower proportion of subjects with plasma HDL-C levels/l compared to patients infected with WT HIV. Furthermore, compared to a reference group of HIV-negative subjects, there was higher abundance of smaller under-lipidated HDL particles in plasma of patients infected with WT HIV, but not in those infected with ΔNefHIV. Lipidomic analysis of plasma revealed differences in abundance of phosphatidylserine and sphingolipids between patients infected with ΔNefHIV and WT HIV. MicroRNA profiling revealed that plasma abundance of 24 miRNAs, many of those involved in regulation of lipid metabolism, was differentially regulated by WT HIV and ΔNefHIV. CONCLUSION: Our findings are consistent with HIV protein Nef playing a significant role in pathogenesis of lipid-related metabolic complications of HIV disease

Prion infection impairs cholesterol metabolism in neuronal cells

Conversion of prion protein (PrPC) into a pathological isoform (PrPSc) during prion infection occurs in lipid rafts and is dependent on cholesterol. Here, we show that prion infection increases the abundance of cholesterol transporter, ATP-binding cassette transporter type A1 (ATP-binding cassette transporter type A1), but reduces cholesterol efflux from neuronal cells leading to the accumulation of cellular cholesterol. Increased abundance of ABCA1 in prion disease was confirmed in prion-infected mice. Mechanistically, conversion of PrPC to the pathological isoform led to PrPSc accumulation in rafts, displacement of ABCA1 from rafts and the cell surface, and enhanced internalization of ABCA1. These effects were abolished with reversal of prion infection or by loading cells with cholesterol. Stimulation of ABCA1 expression with liver X receptor agonist or overexpression of heterologous ABCA1 reduced the conversion of prion protein into the pathological form upon infection. These findings demonstrate a reciprocal connection between prion infection and cellular cholesterol metabolism, which plays an important role in the pathogenesis of prion infection in neuronal cells

A Mouse Model of Harlequin Ichthyosis Delineates a Key Role for Abca12 in Lipid Homeostasis

Harlequin Ichthyosis (HI) is a severe and often lethal hyperkeratotic skin disease caused by mutations in the ABCA12 transport protein. In keratinocytes, ABCA12 is thought to regulate the transfer of lipids into small intracellular trafficking vesicles known as lamellar bodies. However, the nature and scope of this regulation remains unclear. As part of an original recessive mouse ENU mutagenesis screen, we have identified and characterised an animal model of HI and showed that it displays many of the hallmarks of the disease including hyperkeratosis, loss of barrier function, and defects in lipid homeostasis. We have used this model to follow disease progression in utero and present evidence that loss of Abca12 function leads to premature differentiation of basal keratinocytes. A comprehensive analysis of lipid levels in mutant epidermis demonstrated profound defects in lipid homeostasis, illustrating for the first time the extent to which Abca12 plays a pivotal role in maintaining lipid balance in the skin. To further investigate the scope of Abca12's activity, we have utilised cells from the mutant mouse to ascribe direct transport functions to the protein and, in doing so, we demonstrate activities independent of its role in lamellar body function. These cells have severely impaired lipid efflux leading to intracellular accumulation of neutral lipids. Furthermore, we identify Abca12 as a mediator of Abca1-regulated cellular cholesterol efflux, a finding that may have significant implications for other diseases of lipid metabolism and homeostasis, including atherosclerosis

Lipids, biomarkers, and subclinical atherosclerosis in treatment-naive HIV patients starting or not starting antiretroviral therapy: Comparison with a healthy control group in a 2-year prospective study

Objective: To assess the effect of HIV infection and combined antiretroviral therapy (c-ART) on various proatherogenic biomarkers and lipids and to investigate their relationship with subclinical atherosclerosis in a cohort of treatment-naive HIV-infected patients. Methods: We performed a prospective, comparative, multicenter study of 2 groups of treatment-naive HIV-infected patients (group A, CD4>500 cells/mu L, not starting c-ART; and group B, CD4<500 cells/mu L, starting c-ART at baseline) and a healthy control group. Laboratory analyses and carotid ultrasound were performed at baseline and at months 12 and 24. The parameters measured were low-density lipoprotein (LDL) particle phenotype, lipoprotein-associated phospholipase A2 (Lp-PLA2), interleukin-6 (IL-6), high-sensitivity C-reactive protein (hs-CRP), sCD14, sCD163, monocyte chemoattractant protein-1(MCP-1), and asymmetric dimethylarginine (ADMA). A linear mixed model based on patient clusters was used to assess differences in biomarkers between the study groups and over time. Results: The study population comprised 62 HIV-infected patients (group A, n = 31; group B, n = 31) and 22 controls. Age was 37 (30-43) years, and 81% were men. At baseline, the HIV-infected patients had a worse LDL particle phenotype and higher plasma concentration of sCD14, sCD163, hs-CRP, and LDL-Lp-PLA2 than the controls. At month 12, there was an increase in total cholesterol (p = 0.002), HDL-c (p = 0.003), and Apo A-I (p = 0.049) and a decrease in sCD14 (p = <0.001) and sCD163 (p<0.001), although only in group B. LDL particle size increased in group B at month 24 (p = 0.038). No changes were observed in group A or in the healthy controls. Common carotid intima-media thickness increased in HIV-infected patients at month 24 (Group A p = 0.053; group B p = 0.048). Plasma levels of sCD14, sCD163, and hs-CRP correlated with lipid values. Conclusions: In treatment-naive HIV-infected patients, initiation of c-ART was associated with an improvement in LDL particle phenotype and inflammatory/immune biomarkers, reaching values similar to those of the controls. HIV infection was associated with progression of carotid intima-media thickness

Dose-Dependent Effect of Rosuvastatin on VLDL-Apolipoprotein C-III Kinetics in the Metabolic Syndrome

OBJECTIVE - Dysregulated apolipoprotein (apo)C-III metabolism may account for hypertriglyceridemia and increased cardiovascular risk in the metabolic syndrome. This study investigated the dose-dependent effect of rosuvastatin on VLDL apoC-III transport in men with the metabolic syndrome. RESEARCH DESIGN AND METHODS - Twelve men with the metabolic syndrome were studied in a randomized double-blind crossover trial of 5-week intervention periods with placebo, 10 mg rosuvastatin, or 40 mg rosuvastatin, with 2-week placebo washouts between each period. VLDL apoC-III kinetics were examined using a stable isotope method and compartmental modeling at the end of each intervention period. RESULTS - Compared with placebo, there was a significant dose-dependent reduction with rosuvastatin in plasma triglyceride and VLDL apoC-III concentrations. Rosuvastatin significantly (P P P P CONCLUSIONS - In this study, rosuvastatin decreased the production and increased the catabolism of VLDL apoC-III, a mechanism that accounted for the significant reduction in VLDL apoC-III and triglyceride concentrations. This has implications for the management of cardiometabolic risk in obese subjects with the metabolic syndrome