Role of HIV-1 Nef Lysine Residues 4 and 7 in the Interaction with Calnexin and Inhibition of ABCA1 Activity

Patients infected with HIV are at increased risk of developing atherosclerosis, a result due partly to the functional impairment of cellular cholesterol transporter, ATP-binding cassette transporter A1 (ABCA1). This transporter mediates efflux of cholesterol from cells to high density lipoprotein, thus allowing cells to maintain normal levels of cholesterol. When ABCA1 function is inhibited, cells, and in particular macrophages, accumulate cholesterol, resulting in formation of atherosclerotic plaques. The HIV-1 protein, Nef downregulates ABCA1 and inhibits the activity of this protein, thereby reducing cholesterol efflux and promoting atherosclerosis, but the molecular mechanism by which Nef inhibits ABCA1 is unknown. Previous studies demonstrated that Nef blocks interaction between ABCA1 and endoplasmic reticulum chaperone, calnexin. The calnexin-ABCA1 interaction is required for the functionality of ABCA1, as it was shown that cholesterol efflux was inhibited when calnexin expression was knocked down. It was shown that Nef binds to calnexin, and this binding is required for inhibition of ABCA1 activity. Bioinformatic analysis of Nef-calnexin interaction identified lysine residues at positions 4 and 7 of the Nef amino acid sequence as critical for interaction with calnexin. My project is to perform site-directed mutagenesis of Nef to introduce single and double mutations replacing lysines with alanines at positions 4 or 7. Experiments are ongoing to express these Nef mutants in 293T cells and analyze the contribution of each amino acid to Nef interaction with calnexin and down-regulation of ABCA1. These studies will contribute to the development of drugs targeting cholesterol deregulation caused by HIV-1 Nef

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

A Pathogenic Role for Splenic B1 Cells in SIV Disease Progression in Rhesus Macaques

B1 cells spontaneously produce protective natural antibodies which provide the first line of defense against a variety of pathogens. Although these natural antibodies share similar autoreactive features with several HIV-1 broadly neutralizing antibodies, the role of B1 cells in HIV/SIV disease progression is unknown. We report the presence of human-like B1 cells in rhesus macaques. During chronic SIV infection, we found that the frequency of splenic CD11b+ B1 cells positively correlated with plasma SIV viral load and exhausted T cells. Mechanistically, we discovered that splenic CD11b+ B1 cells express PD-L2 and IL-10, and were able to induce PD-1 upregulation on CD4+ T cells in vitro. These findings suggest that splenic CD11b+ B1 cells may contribute to the regulation of SIV plasma viral load by enhancing T cell exhaustion. Therefore, understanding the mechanisms that govern their function in rhesus macaques may lead to novel therapeutic strategies for impeding HIV/SIV disease progression

HIV-1 protein Nef inhibits activity of ATP-binding cassette transporter A1 by targeting endoplasmic reticulum chaperone calnexin.

HIV-infected patients are at increased risk of developing atherosclerosis, in part due to an altered high density lipoprotein profile exacerbated by down-modulation and impairment of ATP-binding cassette transporter A1 (ABCA1) activity by the HIV-1 protein Nef. However, the mechanisms of this Nef effect remain unknown. Here, we show that Nef interacts with an endoplasmic reticulum chaperone calnexin, which regulates folding and maturation of glycosylated proteins. Nef disrupted interaction between calnexin and ABCA1 but increased affinity and enhanced interaction of calnexin with HIV-1 gp160. The Nef mutant that did not bind to calnexin did not affect the calnexin-ABCA1 interaction. Interaction with calnexin was essential for functionality of ABCA1, as knockdown of calnexin blocked the ABCA1 exit from the endoplasmic reticulum, reduced ABCA1 abundance, and inhibited cholesterol efflux; the same effects were observed after Nef overexpression. However, the effects of calnexin knockdown and Nef on cholesterol efflux were not additive; in fact, the combined effect of these two factors together did not differ significantly from the effect of calnexin knockdown alone. Interestingly, gp160 and ABCA1 interacted with calnexin differently; although gp160 binding to calnexin was dependent on glycosylation, glycosylation was of little importance for the interaction between ABCA1 and calnexin. Thus, Nef regulates the activity of calnexin to stimulate its interaction with gp160 at the expense of ABCA1. This study identifies a mechanism for Nef-dependent inactivation of ABCA1 and dysregulation of cholesterol metabolism

Interaction Between HIV-1 Nef and Calnexin: From Modeling to Small Molecule Inhibitors Reversing HIV-Induced Lipid Accumulation.

OBJECTIVE: HIV-infected patients are at an increased risk of developing atherosclerosis, in part due to downmodulation and functional impairment of ATP-Binding Cassette A1 (ABCA1) cholesterol transporter by the HIV-1 protein Nef. The mechanism of this effect involves Nef interacting with an endoplasmic reticulum (ER) chaperone calnexin and disrupting calnexin binding to ABCA1, leading to ABCA1 retention in ER, its degradation and resulting suppression of cholesterol efflux. However, molecular details of Nef-calnexin interaction remained unknown, limiting translational impact of this finding. APPROACH AND RESULTS: Here, we used molecular modeling and mutagenesis to characterize Nef-calnexin interaction and to identify small molecule compounds that could block it. We demonstrated that interaction between Nef and calnexin is direct and can be reconstituted using recombinant proteins in vitro with a binding affinity of 89.1 nM measured by surface plasmon resonance. The cytoplasmic tail of calnexin is essential and sufficient for interaction with Nef, and binds Nef with affinity of 9.4 nM. Replacing lysine residues in positions 4 and 7 of Nef with alanines abrogates Nef-calnexin interaction, prevents ABCA1 downregulation by Nef, and preserves cholesterol efflux from HIV-infected cells. Through virtual screening of the NCI library of compounds, we identified a compound, 1[(7-Oxo-7H-benz[de]anthracene-3-yl)amino]anthraquinone, which blocked Nef-calnexin interaction, partially restored ABCA1 activity in HIV-infected cells, and reduced foam cell formation in a culture of HIV-infected macrophages. CONCLUSION: This study identifies potential targets that can be exploited to block the pathogenic effect of HIV infection on cholesterol metabolism and prevent atherosclerosis in HIV-infected subjects

Nuclear cysteine cathepsin variants in thyroid carcinoma cells

The cysteine peptidase cathepsin B is important in thyroid physiology by being involved in thyroid prohormone processing initiated in the follicular lumen and completed in endo-lysosomal compartments. However, cathepsin B has also been localized to the extrafollicular space and is therefore suggested to promote invasiveness and metastasis in thyroid carcinomas through, e.g., ECM degradation. In this study, immunofluorescence and biochemical data from subcellular fractionation revealed that cathepsin B, in its single- and two-chain forms, is localized to endo-lysosomes in the papillary thyroid carcinoma cell line KTC-1 and in the anaplastic thyroid carcinoma cell lines HTh7 and HTh74. This distribution is not affected by thyroid stimulating hormone (TSH) incubation of HTh74, the only cell line that expresses a functional TSH-receptor. Immunofluorescence data disclosed an additional nuclear localization of cathepsin B immunoreactivity. This was supported by biochemical data showing a proteolytically active variant slightly smaller than the cathepsin B proform in nuclear fractions. We also demonstrate that immunoreactions specific for cathepsin V, but not cathepsin L, are localized to the nucleus in HTh74 in peri-nucleolar patterns. As deduced from co-localization studies and in vitro degradation assays, we suggest that nuclear variants of cathepsins are involved in the development of thyroid malignancies through modification of DNA-associated proteins

Virus Control in Vaccinated Rhesus Macaques Is Associated with Neutralizing and Capturing Antibodies against the SHIV Challenge Virus but Not with V1V2 Vaccine–Induced Anti-V2 Antibodies Alone

The role of vaccine-induced anti-V2 Abs was tested in three protection experiments in rhesus macaques. In an experiment using immunogens similar to those in the RV144 vaccine trial (Anti-envelope [Env]), nine rhesus macaques were coimmunized with gp16092TH023 DNA and SIV gag and gp120A244 and gp120MN proteins. In two V2-focused experiments (Anti-V2 and Anti-V2 Mucosal), nine macaques in each group were immunized with V1V292TH023 DNA, V1V2A244 and V1V2CasaeA2 proteins, and cyclic V2CaseA2 peptide. DNA and protein immunogens, formulated in Adjuplex, were given at 0, 4, 12, and 20 weeks, followed by intrarectal SHIVBaL.P4 challenges. Peak plasma viral loads (PVL) of 106-107 copies/ml developed in all nine sham controls. Overall, PVL was undetectable in one third of immunized macaques, and two animals tightly controlled the virus with the Anti-V2 Mucosal vaccine strategy. In the Anti-Env study, Abs that captured or neutralized SHIVBaL.P4 inversely correlated with PVL. Conversely, no correlation with PVL was found in the Anti-V2 experiments with nonneutralizing plasma Abs that only captured virus weakly. Titers of Abs against eight V1V2 scaffolds and cyclic V2 peptides were comparable between controllers and noncontrollers as were Ab-dependent cellular cytotoxicity and Ab-dependent cell-mediated virus inhibition activities against SHIV-infected target cells and phagocytosis of gp120-coated beads. The Anti-Env experiment supports the role of vaccine-elicited neutralizing and nonneutralizing Abs in control of PVL. However, the two V2-focused experiments did not support a role for nonneutralizing V2 Abs alone in controlling PVL, as neither Ab-dependent cellular cytotoxicity, Ab-dependent cell-mediated virus inhibition, nor phagocytosis correlated inversely with heterologous SHIVBaL.P4 infection