Up-Regulation of the ATP-Binding Cassette Transporter A1 Inhibits Hepatitis C Virus Infection
Simone Bocchetta,# 1 , 2 Patrick Maillard,# 1 Mami Yamamoto,# 1 , 3 Claire Gondeau, 4 Florian Douam, 5 Stéphanie Lebreton, 6 Sylvie Lagaye, 7 Stanislas Pol, 7 , 8 François Helle, 9 Wanee Plengpanich, 10 , 11 Maryse Guérin, 10 Maryline Bourgine, 12 Marie Louise Michel, 12 Dimitri Lavillette, 5 Philippe Roingeard, 13 Wilfried le Goff, 10 and Agata Budkowska 1 , *
PLoS One. 2014; 9(3): e92140. Published online 2014 Mar 19. doi: 10.1371/journal.pone.0092140
Hepatitis C virus (HCV) establishes infection using host lipid metabolism pathways that are thus considered potential targets for indirect anti-HCV strategies. HCV enters the cell via clathrin-dependent endocytosis, interacting with several receptors, and virus-cell fusion, which depends on acidic pH and the integrity of cholesterol-rich domains of the hepatocyte membrane. The ATP-binding Cassette Transporter A1 (ABCA1) mediates cholesterol efflux from hepatocytes to extracellular Apolipoprotein A1 and moves cholesterol within cell membranes. Furthermore, it generates high-density lipoprotein (HDL) particles. HDL protects against arteriosclerosis and cardiovascular disease. We show that the up-regulation of ABCA1 gene expression and its cholesterol efflux function in Huh7.5 hepatoma cells, using the liver X receptor (LXR) agonist GW3965, impairs HCV infection and decreases levels of virus produced. ABCA1-stimulation inhibited HCV cell entry, acting on virus-host cell fusion, but had no impact on virus attachment, replication, or assembly/secretion. It did not affect infectivity or properties of virus particles produced. Silencing of the ABCA1 gene and reduction of the specific cholesterol efflux function counteracted the inhibitory effect of the GW3965 on HCV infection, providing evidence for a key role of ABCA1 in this process. Impaired virus-cell entry correlated with the reorganisation of cholesterol-rich membrane microdomains (lipid rafts). The inhibitory effect could be reversed by an exogenous cholesterol supply, indicating that restriction of HCV infection was induced by changes of cholesterol content/distribution in membrane regions essential for virus-cell fusion. Stimulation of ABCA1 expression by GW3965 inhibited HCV infection of both human primary hepatocytes and isolated human liver slices. This study reveals that pharmacological stimulation of the ABCA1-dependent cholesterol efflux pathway disrupts membrane cholesterol homeostasis, leading to the inhibition of virus–cell fusion and thus HCV cell entry. Therefore besides other beneficial roles, ABCA1 might represent a potential target for HCV therapy.
Association of ATP-Binding Cassette Transporter (ABC) Gene Polymorphisms with Viral Load in Patients with Genotype 1 Hepatitis C Virus Infection.
Chen L, Rao H, Zhang W, Liu F, Jiang D, Wei L
Clinical Laboratory, 01 Sep 2016, 62(9):1643-1649.
BACKGROUND:ATP-binding cassette transporters (ABC) gene polymorphisms are associated with various biological functions, including hepatitis C virus (HCV) infection. This study aims to explore the impact of ABC transporters polymorphisms on HCV viral load in chronic treatment-naïve hepatitis C patients. METHODS:We recruited 347 Chinese Han patients chronically infected with genotype 1 HCV in this study. Ten single nucleotide polymorphism (SNPs) in ABCA1, ABCB5, ABCB11, ABCG2, ABCG5, ABCG10 were analyzed by custom chip from Illumina. Allele frequency analysis and genotype frequency analysis were performed. RESULTS:Patients were categorized according to pretreatment HCV viral load (VL) with a cutoff level 600 000 IU/mL. No significant variations on gender and age were observed in the two groups. G allele of rs3890182 and C allele of rs1883025 in ABCA1 gene were significantly associated with lower HCV viral load (p = 0.013 and p = 0.006) in allele frequency analysis. GG genotype of rs3890182 and CC genotype of rs1883025 in ABCA1 gene were significantly associated with lower HCV viral load (p = 0.027 and p = 0.013) in genotype frequency analysis. Quantitative analysis showed significantly lower viral load in patients with CC genotype of rs1883025 (p = 0.012). Allele associated lower HCV viral load was reported to be associated with higher HDL cholesterol level. CONCLUSIONS:Our finding suggests that ABCA1 gene polymorphism in rs1883025 is significantly associated with HCV VL in patients infected with HCV genotype 1.
HIV hijacks cholesterol transporter ABCA1 to get access to cellular cholesterol
Michael Bukrinsky, Tatiana Pushkarsky, Angela Grant, Huanhuan L Cui & Dmitri Sviridov
Retrovirology volume 6, Article number: P17 (2009) Cite this article
Cholesterol plays an important role in the HIV life cycle, and cholesterol depletion impairs both production and infectivity of HIV virions. Such dependence on cholesterol suggests that HIV may have evolved mechanisms to ensure access to cellular cholesterol during viral assembly. Recently, we demonstrated that HIV-1, via its protein Nef, inhibits activity of the cellular cholesterol transporter ABCA1 and impairs reverse cholesterol transport (RCT) from infected cells. In this study, we examined interaction between Nef and ABCA1 and investigated the role of ABCA1 in cholesterol delivery to nascent HIV virions. ABCA1 is a 12-transmembrane domain protein, and co-immunoprecipitation analysis using Nef and various fragments of ABCA1 identified multiple sites of interaction. Importantly, Nef was found to interact with the 46-aminoacid carboxyl-terminal domain of ABCA1, which was previously implicated in RCT. Mutation of leucines to alanines in positions 2230, 2233 and 2235 impaired interaction of this C-terminal domain of ABCA1 with Nef. When these mutations were introduced in the full-length ABCA1, such mutant protein was able to support RCT but lost sensitivity to Nef. To further characterize the role of ABCA1 inhibition in HIV biology, we analyzed cholesterol content of lipid rafts and HIV virions produced in cells expressing or not ABCA1. This analysis demonstrated that ABCA1 expression significantly reduced lipid raft cholesterol content, resulting in a corresponding reduction of virus-associated cholesterol and viral infectivity. This result is consistent with ABCA1-mediated redirection of cholesterol from lipid rafts (sites of HIV assembly) to non-raft regions of the plasma membrane (physiological localization of ABCA1). Intriguingly, Nef expression induced re-localization of ABCA1 to lipid rafts, suggesting that HIV hijacks the ABCA1 pathway to deliver cholesterol to the sites of HIV assembly.
Conclusion.- Our results describe a novel interaction between HIV-1 Nef and ABCA1, the key cholesterol transporter of peripheral cells. We demonstrate that interaction of Nef with the C-terminal domain of ABCA1 is critical for Nef-mediated impairment of RCT. Intriguingly, Nef seems to hijack ABCA1 to deliver cholesterol to HIV assembly sites. It appears that in an infected cell there is a dynamic interaction between Nef and ABCA1: overexpression of Nef (a situation occurring in most HIV-infected cells) leads to inactivation of ABCA1 physiological activity (RCT), its re-localization to lipid rafts, delivery of cholesterol to assembling virions and production of highly infectious viral particles. In contrast, overexpression of ABCA1 (e.g., by drugs stimulating ABCA1 expression such as LXR agonists) stimulates RCT, reduces cholesterol in lipid rafts and virions, and inhibits HIV production and infectivity.
Ritonavir increases CD36, ABCA1 and CYP27 expression in THP-1 macrophages
Jordi Pou 1, Alba Rebollo, Núria Roglans, Rosa M Sánchez, Manuel Vázquez-Carrera, Juan C Laguna, Juan Pedro-Botet, Marta Alegret
Exp Biol Med (Maywood). 2008 Dec;233(12):1572-82. doi: 10.3181/0805-RM-144. Epub 2008 Oct 10.
Ritonavir, a protease inhibitor used in combination antiretroviral therapy for HIV-1 infection, is associated with an increased risk of premature atherosclerosis. The aim of the present study was to assess the effects of ritonavir, in the absence of added lipoproteins, on the expression of genes that control cholesterol trafficking in human monocytes/macrophages.
Design: THP-1 cells were used to study the effects of ritonavir on the expression of CD36, ATP binding cassette transporters A1 (ABCA1) and G1 (ABCG1), scavenger receptor B class I (SR-BI), caveolin-1 and sterol 27-hydroxylase (CYP27). Exposure to ritonavir (2.5 mug/ml) increased CD36 protein (28%, P < 0.05) and mRNA (38%, P < 0.05) in differentiated THP-1 macrophages, but not in undifferentiated monocytes. This effect was not related to the increase in PPARgamma expression (51%, P < 0.05) caused by ritonavir. Ritonavir also reduced SR-BI protein levels (46%, P < 0.05) and increased CYP27 (43%, P < 0.05) and ABCA1 (49%, P < 0.05) mRNA expression. Liver X receptor alpha (LXRalpha) mRNA, protein and binding activity were also increased by ritonavir treatment.
Conclusions: We propose that ritonavir induces ABCA1 expression in THP-1 macrophages through LXRalpha. The increase in ABCA1 and other cholesterol efflux mediators, such as CYP27, may compensate CD36 induction. Therefore, we suggest that the net effect of ritonavir on macrophages in the absence of lipoproteins is not clearly proatherogenic.
Analysis of ABCA1 and cholesterol efflux in HIV-infected cells
Nigora Mukhamedova,1 Beda Brichacek,2 Christina Darwish,2 Anastas Popratiloff,3 Dmitri Sviridov,1 and Michael Bukrinsky2
Methods Mol Biol. 2016; 1354: 281–292.
Cholesterol is an essential component of the cellular membranes and, by extension, of the HIV envelope membrane, which is derived from the host cell plasma membrane. Depletion of the cellular cholesterol has a inhibitory effect on HIV assembly, reduces infectivity of the produced virions, and makes the cell less susceptible to HIV infection. It is not surprising that the virus has evolved to gain access to cellular proteins regulating cholesterol metabolism. One of the key mechanisms used by HIV to maintain high levels of cholesterol in infected cells is Nef-mediated inhibition of cholesterol efflux and the cholesterol transporter responsible for this process, ABCA1. In this article, we describe methods to investigate these effects of HIV-1 infection.
Retinoic acid and liver X receptor agonist synergistically inhibit HIV infection in CD4+ T cells by up-regulating ABCA1-mediated cholesterol efflux
Hong Jiang 1, Yunden Badralmaa, Jun Yang, Richard Lempicki, Allison Hazen, Ven Natarajan
Lipids Health Dis. 2012 Jul 9;11:69. doi: 10.1186/1476-511X-11-69.
Background: Retinoic acids regulate the reverse cholesterol transport by inducing the ATP binding cassette transporter A1 (ABCA1) dependent cholesterol efflux in macrophages, neuronal as well as intestine cells. In the present study, we aim to test the effect of all trans retinoic acid (ATRA) on ABCA1 expression in human CD4+ T cells and the involvement of cholesterol in ATRA mediated anti-HIV effect.
Results: Treatment with ATRA dramatically up-regulated ABCA1 expression in CD4+ T cells in a time and dose dependent manner. The expression of ABCA1 paralleled with increased ABCA1-dependent cholesterol efflux. This induction was dependent on T cell receptor (TCR) signaling and ATRA failed to induce ABCA1 expression in resting T cells. Moreover, ATRA and liver X receptor (LXR) agonist-TO-901317 together had synergistic effect on ABCA1 expression as well as cholesterol efflux. Increased ABCA1 expression was associated with lower cellular cholesterol staining. Cells treated with either ATRA or TO-901317 were less vulnerable to HIV-1 infection. Combination of retinoic acid and TO-901317 further inhibited HIV-1 entry and their inhibitory effects could be reversed by cholesterol replenishment.
Methods: ABCA1 RNA and protein were determined by real-time PCR and immuno blot methods in cells treated with ATRA. Cholesterol efflux rate was measured in cells treated with ATRA and TO-901317.
Conclusions: ATRA up-regulates ABCA1 expression and cholesterol efflux in CD4+ T cells and combination of ATRA and liver X receptor (LXR) agonist further enhanced these effects. Increased cholesterol efflux contributed to reduced HIV-1 entry, suggesting that anti-HIV effect of ATRA is mediated through ABCA1.
HIV–Cholesterol Connection Suggests a New Antiretroviral Strategy
PLoS Biol. 2006 Nov; 4(11): e400. Published online 2006 Oct 31. doi: 10.1371/journal.pbio.0040400
Straddling the line between living and nonliving, a virus must commandeer the molecular machinery of the cell it infects to persist, and sometimes even alters the cellular environment toward that end. The HIV genome encodes nine proteins, some playing multiple roles to help the virus invade human immune cells and co-opt cellular proteins—and even normal cellular activities—to reproduce. For example, a recent study showed that an HIV protein called Nef, which is required for viral replication and infection, also binds cholesterol and delivers it to the cell membrane where new virus particles (called virions) are assembled. Once assembled, virions take part of the lipid-rich cell membrane with them as they bud off in search of new cells to infect.
Now, in a new study, Zahedi Mujawar, Michael Bukrinksy, Dmitri Sviridov, and colleagues show that the HIV Nef also disrupts cholesterol trafficking machinery in the macrophages infected by the virus. By preventing the normal efflux of cholesterol from macrophages, Nef ensures that nascent virions have access to a steady supply of cholesterol. Unfortunately, what’s good for the virus causes even more trouble for a person infected with HIV: reduced cholesterol efflux from macrophages may explain why patients with HIV face increased risk of atherosclerosis and coronary artery disease.
Normally, excess cholesterol is exported to molecules in the extracellular space. The authors found that cholesterol efflux to an extracellular molecule called apoA-I was impaired in HIV-infected macrophages. Lower levels of efflux correlated with higher levels of viral replication. To see whether Nef might affect efflux impairment, the authors infected macrophages with HIV strains carrying either a functional or mutated Nef gene (known to affect cholesterol binding and delivery). Cholesterol efflux to apoA-I was “substantially reduced” in cells infected with functional Nef. In contrast, cholesterol efflux in cells infected with Nef-deficient strains was similar to that seen in uninfected cells—allowing the authors to conclude that HIV needs Nef to block cholesterol efflux.
Cholesterol is removed from macrophages by proteins called ATP-binding cassette (ABC) transporters, which hand off their cargo to different molecules. Having shown that Nef hinders efflux to apoA-I, the authors reasoned that Nef does so by targeting apoA-I’s donor—ABCA1. This conclusion is supported by two lines of evidence: stimulating expression of ABCA1 in infected cells significantly reduced Nef-mediated impairment of efflux to ApoA-I, but efflux was unaffected when ABCA1 levels were very low. Nef mediates these effects, the authors show, by specifically down-regulating ABCA1 after transcription and limiting its abundance, and by keeping ABCA1 sequestered at the membrane. Whether these mechanisms work in tandem or operate independently is a question for future study.
HIV-infected macrophages that expressed Nef not only contained far more lipid-containing vacuoles than uninfected cells or cells infected with Nef-deficient HIV, they also synthesized molecules associated with cholesterol accumulation and cholesterol-laden “foam cells.” And when the authors analyzed atherosclerotic plaque sections taken from patients with HIV who had been treated with anti-retroviral therapy (HAART), they found HIV-infected, cholesterol-laden macrophages in the atherosclerotic plaque—suggesting that these cells contribute to arterial disease.
Altogether, the results show that HIV inhibits cholesterol efflux through Nef, leading to cholesterol accumulation and foam cell formation. They also suggest potential targets for controlling both HIV and atherosclerosis. When the authors increased ABCA1 levels to stimulate cholesterol efflux—having observed a link between reduced efflux and increased viral replication—the treatment restored efflux and reduced virion infectivity by reducing virion incorporation of cholesterol. This suggests that stimulating cholesterol efflux may not only reduce the risk of atherosclerosis but may also help control HIV replication in HIV-infected patients.
Cdc42 – A tryst between host cholesterol metabolism and infection
Dmitri Sviridov and Nigora Mukhamedova
Small GTPases. 2018; 9(3): 237–241. Published online 2016 Aug 31
Emerging evidence points to an important connection between pathogenesis of intracellular infections and host cholesterol metabolism. In our study we demonstrated that human cytomegalovirus exploits host small GTPase Cdc42 to hijack cellular cholesterol efflux pathway. It appears that the virus uses host machinery to stimulate cholesterol efflux by modifying lipid rafts and altering properties of plasma membrane, but the altered pathway is controlled by the viral protein US28 instead of the host ATP binding cassette transporter A1. We speculate that virus-controlled remodeling of plasma membrane facilitates immune evasion, exocytosis of viral proteins and cell-to-cell transmission of human cytomegalovirus. These mechanisms may be not unique for the cytomegalovirus and subverting reverse cholesterol transport pathway may be a generic mechanism used by pathogens to alter properties of host plasma membrane adapting it for their purposes—to hide and disseminate.
Lipid flippases promote antiviral silencing and the biogenesis of viral and host siRNAs in Arabidopsis
Zhongxin Guo 1, Jinfeng Lu 1, Xianbing Wang 1, Binhui Zhan 1, Wanxiang Li 1, Shou-Wei Ding 2
Proc Natl Acad Sci U S A. 2017 Feb 7;114(6):1377-1382. doi: 10.1073/pnas.1614204114. Epub 2017 Jan 25.
Dicer-mediated processing of virus-specific dsRNA into short interfering RNAs (siRNAs) in plants and animals initiates a specific antiviral defense by RNA interference (RNAi). In this study, we developed a forward genetic screen for the identification of host factors required for antiviral RNAi in Arabidopsis thaliana Using whole-genome sequencing and a computational pipeline, we identified aminophospholipid transporting ATPase 2 (ALA2) and the related ALA1 in the type IV subfamily of P-type ATPases as key components of antiviral RNAi. ALA1 and ALA2 are flippases, which are transmembrane lipid transporter proteins that transport phospholipids across cellular membranes. We found that the ala1/ala2 single- and double-mutant plants exhibited enhanced disease susceptibility to cucumber mosaic virus when the virus-encoded function to suppress RNAi was disrupted. Notably, the antiviral activity of both ALA1 and ALA2 was abolished by a single amino acid substitution known to inactivate the flippase activity. Genetic analysis revealed that ALA1 and ALA2 acted to enhance the amplification of the viral siRNAs by RNA-dependent RNA polymerase (RdRP) 1 (RDR1) and RDR6 and of the endogenous virus-activated siRNAs by RDR1. RNA virus replication by plant viral RdRPs occurs inside vesicle-like membrane invaginations induced by the recruitment of the viral RdRP and host factors to subcellular membrane microdomains enriched with specific phospholipids. Our results suggest that the phospholipid transporter activity of ALA1/ALA2 may be necessary for the formation of similar invaginations for the synthesis of dsRNA precursors of highly abundant viral and host siRNAs by the cellular RdRPs.