- Open Access
Caveolin-1 reduces HIV-1 infectivity by restoration of HIV Nef mediated impairment of cholesterol efflux by apoA-I
© Lin et al.; licensee BioMed Central Ltd. 2012
- Received: 18 June 2012
- Accepted: 26 September 2012
- Published: 15 October 2012
HIV infection results in inhibited cholesterol efflux by apolipoprotein A-I (apoA-I) in macrophages, and this impairment involves Nef mediated down-regulation and redistribution of ATP-binding cassette transporter A1 (ABCA-1). We investigated the effect of caveolin-1 (Cav-1) on the cholesterol efflux by apoA-I in HIV infected primary and THP-1 cell-differentiated macrophages as well as astrocyte derived glioblastoma U87 cells.
Our results reveal that Cav-1 restores the Nef -mediated impairment of cholesterol efflux by apoA-I in both cell types. Co-immunoprecipitation studies indicate a physical association of Cav-1 and Nef. The level of ABCA-1 expression remains the same whether Cav-1 is over-expressed or not. In addition, we examined the cholesterol composition of HIV particles released from Cav-1 treated cells and identified that the cholesterol content is dramatically reduced. The infectivity level of these virus particles is also significantly decreased.
These observations suggest that the interplay of Cav-1 with Nef and cholesterol subsequently counters Nef induced impairment of cholesterol efflux by apoA-l. The findings provide a cellular mechanism by which Cav-1 has an ability to restore HIV mediated impairment of cholesterol efflux in macrophages. This subsequently influences the cholesterol content incorporated into virus particles thereby inhibiting HIV infectivity and contributing to HIV’s persistent infection of macrophages.
- Cholesterol efflux
- Apolipoprotein A-I
Caveolin 1 (Cav-1), a 21~24-kDa scaffolding protein, is an important structural component of caveolae , small invaginations of the plasma membrane, which are enriched in cholesterol, phospholipids, and sphingolipids. This protein is highly expressed in terminally differentiated cells including endothelial cells, macrophages, dendritic cells and adipocytes [2, 3]. Functional studies have shown that Cav-1 is involved in a wide range of cellular processes, including cell cycle regulation, signal transduction, endocytosis, cholesterol trafficking and efflux [3–9]. Multiple lines of evidence indicate that Cav-1 acts as a scaffolding protein capable of directly interacting with and modulating the activity of caveolin-bound signaling molecules. The Cav-1 scaffolding domain (CSD), residues 82 to 101, is essential for both Cav-1 oligomerization and the interaction of caveolin with other proteins . Associations with other proteins through the CSD help provide coordinated and efficient signal transduction [11, 12]. The CSD serves as a receptor for binding proteins containing the sequence φXφXXXXφ, φXXXXφXXφ, or φXφXXXXφXXφ (φ representing any aromatic amino acid and X any other amino acid). HIV Env has been shown to interact with Cav-1 via a motif (WNNMTWMQW) localized within the ectodomain (the C-terminal heptad repeats) of HIV-1 gp41 [13–15]. Our group has shown the binding of Cav-1 with HIV Env in the lipid rafts which subsequently blocks cell fusion and innocent bystander killing mediated by HIV envelope . We have also demonstrated that HIV infection in primary human monocyte derived macrophages (MDMs) results in a dramatic up-regulation of Cav-1 expression mediated by HIV Tat . Furthermore, over-expression of Cav-1 causes significant reduction in HIV replication in macrophages. Cav-1 inhibits HIV replication through transcriptional repression of viral gene expression by modulating the NF-κB pathway . The up-regulation of Cav-1 by HIV infection and subsequent inhibition of HIV replication suggest a role for Cav-1 in macrophage persistent infection.
Cav-1 plays an important role in cellular cholesterol homeostasis, a process that controls intracellular lipid composition and prevents cholesterol accumulation. Cav-1 has been implicated in modulating the expression of lipoprotein receptors and interacts with many lipid transporter molecules [11, 19–21]. Furthermore, it is involved in the transport of newly synthesized cholesterol from the endoplasmic reticulum (ER) to the plasma membrane [11, 22, 23] and promotes cholesterol efflux in hepatic cells [9, 24]. HIV appears to manipulate cellular cholesterol metabolism to ensure that there is a sufficient supply of cholesterol and that it is located in the appropriate compartments such as lipid rafts for efficient virus release and subsequent infectivity [25–28]. Cholesterol is an important component that influences HIV production and efficient virus infectivity. Cholesterol depletion significantly reduces HIV-1 particle production [29–34]. Virus infectivity is also negatively affected when HIV is produced from cholesterol depleted cells [26, 35].
The HIV accessory protein Nef has an ability to exploit cholesterol metabolism. Proposed mechanisms for this strategy include binding to cholesterol and aiding the transport of newly synthesized cholesterol into lipid rafts and viral particles as well as enhancing cholesterol synthesis [36, 37]. Nef has also been shown to impair ATP binding cassette transporter protein 1 (ABCA-1)-dependent cholesterol efflux from human macrophages by down-regulation and redistribution of ABCA-1 . This suggests that Nef is involved in HIV mediated cholesterol accumulation. Since Cav-1 has a high affinity for cholesterol and aids in the transport of newly synthesized cholesterol from the ER to the plasma membrane and indirectly promoting the transfer to extracellular acceptors such as lipid free apolipoprotein A-I (apoA-I) we hypothesize it would influence the level of cholesterol accumulation as well as virus production and infectivity. Macrophages are major targets for HIV infection and also play an important role in its pathogenesis. The up-regulation of Cav-1 by HIV infection and the role of Cav-1 in cholesterol trafficking suggest a mechanism for a Cav-1/cholesterol mediated impact on HIV replication in macrophages. In this report, we establish evidence for a Cav-1/cholesterol mediated mechanism of inhibition of HIV replication for the first time providing a new angle in understanding HIV’s persistent infection of macrophages.
Cav-1 restores HIV Nef mediated impairment of cholesterol efflux by apoA-I in U87 cells and macrophages
In addition, to determine whether Cav-1 specifically restores Nef mediated impairment of cholesterol efflux to apoA-l, U87 cells were co-transfected with a Nef mutant (NefG2A) and Cav-1 expressing plasmids. The NefG2A is a Nef mutant that cannot undergo myristoylation . Its association with the plasma membrane is impaired [26, 35], and it lacks the ability to decrease apoA-l stimulated cholesterol efflux [36, 41]. As shown in Figure 3C, cells expressing Nef experienced 62% less cholesterol efflux to apoA-I compared to Mock. In contrast, NefG2A had no effect on apoA-l mediated cholesterol efflux in the presence of either endogenous or over-expressing Cav-1 cells. These studies, therefore, clearly establish that Cav-1 counters Nef mediated impairment of cholesterol efflux by apoA-l.
Cav-1 over-expression has no effect on ABCA-1 expression
Interaction of Nef and Cav-1
Cav-1 reduces HIV-1 infectivity by reducing the cholesterol content of virus particles
HIV has been indicated to manipulate host cholesterol metabolism, leading to excessive cholesterol accumulation in infected T cells or macrophages [38, 45], thereby supporting efficient viral replication. In the absence of proper esterification to fatty acid and efflux, cholesterol accumulates in the endoplasmic reticulum eventually leading to ER dysfunction and the activation of an ER stress associated apoptosis pathway [46–48]. Cav-1 is an important cellular cholesterol regulator, and its expression is dramatically enhanced in HIV infected macrophages , implicating a role for Cav-1 in HIV associated cholesterol alterations. Cav-1 is a structural component of Caveolae membrane microdomains, which have been suggested to play an important role in cholesterol trafficking and efflux. In this study, we investigate the effect of Cav-1 on the cholesterol efflux in HIV infected macrophages and human astrocytes-derived glioblastoma U87 cells. Our results show that Cav-1 restores the Nef induced impairment of cholesterol efflux by apoA-I. Furthermore, this restoration causes a reduction in the cholesterol composition of virus particles leading to decreased HIV infectivity. This suggests a role for Cav-1 in macrophage HIV persistent infection by enhancing cholesterol efflux.
Our results show neither Nef nor Cav-1 had significant effect on HDL mediated cholesterol efflux. HDL plays an important role in reverse cholesterol transport (RCT), in which HDL transports cholesterol from peripheral tissues to liver for excretion. RCT is a multifaceted, dynamic pathway which is involved with multiple molecules and effectors. The first step in RCT is ABCA-1 dependent efflux of cholesterol and phospholipids to apoA-I, the major component of HDL. ABCA-1 interacts with apoA-I and stimulates free cholesterol and phospholipids efflux responsible for nascent HDL formation . Wang et al. reported that ABCA-1 expression markedly increases apoA-I but not HDL mediated lipid efflux; the reason could be that compared with HDL, apoA-I is the preferred acceptor for ABCA1-promoted cholesterol and phospholipid efflux. We also found upon HIV infection Nef down regulates ABCA-1 expression, which dramatically inhibits apoA-I mediated cholesterol efflux, whereas HDL mediated cholesterol efflux was not affected by HIV infection. Over-expression of Cav-1 restores the impaired cholesterol efflux to apoA-I significantly, but not so much on intact HDL cholesterol efflux.
Promotion of cholesterol efflux by over-expression of Cav-1 is observed in hepatic cells . Cav-1 can enhance the transfer of cholesterol to cholesterol-rich domains in the plasma membrane, where it is accessible to efflux. Multiple mechanisms are proposed for Cav-1’s regulation of cholesterol homeostasis. These include the modulation of the expression of lipoprotein receptors and the activity of proteins involved in lipid metabolism as well as interactions with lipid transport or transport of cholesterol to the plasma membrane facilitating cholesterol efflux [6–8, 22, 43]. ABCA-1 expression is important in regulating cholesterol efflux to apoA-I and it has been implicated that ABCA-1 stimulates the reorganization of plasma membrane microdomains to facilitate cholesterol efflux to apoA-I [51, 52]. Cav-1 can regulate cholesterol homeostasis by modulating the expression of lipid regulators. Reduced levels of ABCA-1 have been observed in macrophages of Cav-1 knockout mice . Our results show that we observe no change in the level of ABCA-1 expression when Cav-1 is over-expressed suggesting that the endogenous Cav-1 expression is sufficient enough to maintain physiologically relevant levels of ABCA-1 and that additional amounts of Cav-1 does not have an impact on ABCA-1 expression. The reduced level of ABCA-1 observed in the knockout mice is in complete absence of Cav-1 expression. ABCA-1 dependent cholesterol efflux can be impaired by HIV Nef mediated down modulation and altering of the intracellular distribution of ABCA-1 [38, 42]. Similarly we observed a 69% decrease in ABCA-1 expression in the presence of Nef. Interestingly the decrease in ABCA-1 remains the same when additional amounts of Cav-1 are provided indicating that the reversal of Nef’s effect on cholesterol efflux by Cav-1 is not related to the level of ABCA-1 expression. Inhibition of ABCA-1 protein expression, as it pertains to Nef, in part depends upon the ER associated proteasomal degradation mechanism . An unknown additional pathway unrelated to proteasomal activity is also suggested to contribute to ABCA-1 degradation. Although ABCA-1 is shown to interact with Nef the physical association is not essential for Nef mediated down-regulation of ABCA-1 efflux activity [38, 42]. However, the influence of cellular distribution of ABCA-1 by Nef has been determined using confocal microscopy with Nef causing a prominent trapping of ABCA-1 in the ER . ABCA-1 expression has been implicated in influencing the redistribution of cholesterol and Cav-1 . Redistribution of Cav-1 from punctate caveolae-like structures to the general area of the plasma membrane is observed upon ABCA-1 expression. Our co-immunoprecipitation study reveals an interaction between Cav-1 and Nef. Furthermore, our observation that Cav-1 does not interact with the myristoylation defective Nef (NefG2A mutant) implicates an association of these proteins at the plasma membrane. These observations suggest that the interplay of Cav-1 with Nef and cholesterol subsequently counters Nef induced impairment of cholesterol efflux by apoA-l. In addition, since caveolae is a major source and platform for cholesterol efflux  over-expression of Cav-1 may induce the formation of more caveolae, which should subsequently enhance cholesterol efflux. The presence of Cav-1 in macrophages and its up-regulation upon HIV infection, therefore, can contribute to increased cholesterol efflux in these cells.
Cholesterol is an important structural component of HIV particles and their cholesterol content is tightly linked to HIV infectivity [25, 27, 44]. Cholesterol depletion significantly reduces HIV-1 particle production [29–34, 44]. There is also a marked decrease in infectivity of virions produced from such cells . The significant reduction correlates with the amount of virion-associated cholesterol . In the current study, we clearly established that Cav-1 significantly reduces infection with virions produced from Cav-1 treated cells when compared to that of the same number of virions obtained from untreated cells. We have previously shown that Cav-1 represses HIV gene expression by blocking the NF-κB pathway thus subsequently affecting virus production . The decrease in virus production is therefore in part due to transcriptional suppression of HIV gene expression. Here, we examined the cholesterol content of HIV particles produced from Cav-1 treated cells and clearly established a significant cholesterol decrease in virus particles. Furthermore, normalized amounts of virus in the infectivity assay of HIV released from Cav-1 treated cells shows that infectivity is markedly reduced. Normalized amounts of virus to assay for infectivity, rules out any concern regarding the level of virus release contributing to the reduction of infectivity. The major step that causes a decrease in virion infectivity related to cholesterol depletion is the fusion steps of infection . In support of this notion, we previously demonstrated that Cav-1 significantly suppressed Env-induced membrane hemifusion , indicating that the decrease in fusion partly involves a reduction in the cholesterol composition of the plasma membrane. Cav-1 can counter the influence of HIV on cholesterol metabolism by promoting cholesterol trafficking to the membrane subsequently enhancing cholesterol efflux, therefore, depriving the HIV virion of cholesterol. Since Cav-1 is involved in cholesterol metabolism the up-regulation of Cav-1 can have an impact on the level of cellular cholesterol thereby contributing to a reduction in virus production and infectivity, consequently contributing to a persistent infection of macrophages.
The HIV-1 proviral constructs pNL4-3 (T-tropic), pNL-AD8 (M-tropic), pWT/Bal (M-tropic), pNL4-3.Luc.R-E-, and pSG3Δenv were kindly provided by NIH AIDS Research and Reference Reagent Program [70–76]. The construct pNL4-3.Luc.R-E- is defective for env and nef where as pSG3Δenv has intact nef, but a deletion in env. An expression plasmid for vesicular stomatitis virus envelope G protein (pCI-VSV) was kindly provided by Jiing-Kuan Yee of City of Hope National Medical Center, Duarte, California. A Cav-1 expressing plasmid, pCZ-cav-1, was generated as described previously . pCZ-vector is the same as pCZ-cav-1 except it lacks the coding sequence of cav-1. The Nef expression plasmid pcDNA3.1SF2Nef was provided by NIH AIDS Research and Reference Reagent Program [77, 78]. A construct expressing Nef tagged with HA (pCI NL4-3 Nef-HA-WT) was purchased from Addgene Inc (Cambridge, MA). The NefG2A mutation plasmid was generated using a site-directed mutagenesis kit according to the manufacturer’s protocol (Strategene). Briefly, the mutation was generated by PCR amplification using pCI NL4-3 Nef-HA-WT as template and the following pair of primers: 5′-ggattttgctataagatggctggcaagtggtcaaaaagt-3′ and 5′-actttttgaccacttgccagccatcttatagcaaaatcc-3′. The PCR products were digested with the restriction enzyme DpnI to destroy template plasmids and were then transformed into DH5α competent cells. Introduction of the mutation (pCI NL4-3 NefG2A-HA) was confirmed by sequence analysis. Wild type AD8 and a replication competent nef defective AD8 derived HIV provirus DNA construct ADnefmut  were provided by Dr. Maureen Goodenow of the University of Florida. Adenovirus particles (Ad) for expressing Cav-1 (Ad-Cav-1) and GFP (Ad-GFP) were obtained from Vector Biolabs (Philadelphia, PA).
Human U87MG-CD4 cells stably transfected with CXCR4 (U87-CD4-CXCR4) or CCR5 (U87-CD4-CCR5), human acute monocytic leukemia (THP-1),and an indicator cell line for tittering HIV (TZM-bl) was kindly provided by the NIH AIDS Research and Reference Reagent Program. U87-CD4-CXCR4 were maintained in DMEM containing 15% FBS, penicillin-streptomycin (100 μg/mL), glutamine, puromycin (1μg/ml; Sigma Chemical), and neomycin (G418; 300μg/ml; Sigma). THP-1 cells were grown in RPMI-1640 containing 10% FBS, 1.0mM sodium pyruvate, and 0.05 mM 2-mercaptoethanol. For differentiation into macrophages, THP-1 cells were treated with 50 ng/ml of phorbol 12-myristate 13-acetate (PMA, Sigma Chemical) for 5 days until the cells adhered and exhibited macrophage-like morphology. TZM-bl and 293T cells were grown in DMEM medium supplemented with 10% FBS and penicillin-streptomycin (100μg/ml). All cultures were maintained at 37°C in a humidified atmosphere with 5% CO2.
Peripheral blood mononuclear cells (PBMCs) were isolated from buffy coats prepared from healthy donors by centrifugation through a Ficoll gradient (Sigma-Aldrich, St. Louis, MO). Monocytes were isolated by negative selection with a human monocyte enrichment kit according to the manufacturer’s instructions (EasySep® Human Monocyte Enrichment Kit, Stemcell Technologies). The monocyte preparations contained 97% CD14+ cells, as determined by flow cytometry. For differentiation of monocytes into macrophages (MDMs), monocytes were seeded into Biocoat poly-D-lysine plates (B.D. Bioscience), and cultured in DMEM, supplemented with 10% heat-inactivated human serum, gentamicin (50μg/ml), ciprofloxacin (10μg/ml), and M-CSF (1000U/ml) for 7 days. MDM culture medium was half-exchanged every 2 to 3 days.
Transfection of siRNA
Small interfering RNA (siRNA) targeting Cav-1 and control siRNA were purchased from Santa Cruz Biotechnology, Inc. Transfection of siRNA was performed using OligofectaminTM Reagent (Invitrogen Corp., Carlsbad, Calif.) according to the manufacturer’s protocol. Briefly, the day before transfection, U87cells were seeded into a 24 well plate and cultured with antibiotics free medium to 30% confluency. Cells were washed and resuspended in 200ul serum free medium. Transfection mixture was prepared by incubating 50pmol of siRNA duplexes with 3ul of Oligofectamin in a final volume of 50ul Opti-MEM I Medium. After a 5 hour incubation, 125ul of growth medium with 3 times the normal concentration of serum was added to cells. Transfection was repeated once the next day. For THP1 macrophages, cells were first transfected with siRNA followed by HIV infection, and cells were then transfected again with siRNA the day after infection. The efficiency of Cav-1 knock-down by the siRNA transfection was monitored using Western blot analysis.
Virus production and concentration
Infectious virus HIV-1 AD8, ADnefmut, Bal, and NL4-3 were generated by calcium phosphate transfection of monolayers of 293T cells in 75-cm2 flasks with 25μg provirus DNA. Supernatants containing virus were harvested 4 days after transfection and quantified using the TZM-bl indicator cells as well as by measuring reverse transcriptase and a p24 ELISA method as described previously . When required, virus was produced from U87-CD4-CXCR4 cells transfected with 18μg proviral HIV NL4-3 along with 9μg pCZ-Cav-1 or pCZ-vector. To generate pseudotyped HIV particles 20μg pSG3Δenv or pNL4-3.Luc.R-E- was co-transfected with 3μg pCI-VSV into monolayers of 293T cells in 75-cm2 culture flasks by the calcium phosphate method. Pseudotyped viral supernatants were harvested 4 days post-transfection and were clarified by centrifugation at 3,000 rpm for 20 min and then by filtering through a 0.45 μm-pore size filter. Virus particles were concentrated using virus precipitation reagent Retro-ConcentinTM (System Biosciences) according to the manufacturer’s protocol.
Oil red O staining
To determine the influence of Cav-1 on the level of lipid accumulation in HIV infected and uninfected cells oil red O staining was performed. THP-1 cells were differentiated into macrophages by treatment with 50 ng/ml PMA for 5 days then infected with HIV AD8 (moi, 0.01) or Bal (moi, 0.001). On day 10 post infection, cells were loaded with cholesterol by incubating with 50μg/ml Ac-LDL (Biomedical Technologies Inc., Stoughton, MA) for 48 h followed by 30μg/ml apoA-I stimulation for 18 hours. Differentiated THP-1 cell-differentiated macrophage cells were also infected with Ad-Cav-1 or Ad-GFP at an moi (multiplicity of infection) of 100. Twenty-four hours later they were infected with VSV pseudotyped HIV pSG3Δenv or pNL4-3.Luc.R-E- at an moi of 3 and incubated for 5 days. Oil red O staining was performed as previously described . Briefly, cells were rinsed with PBS, followed by fixation with 3.7% paraformaldehyde for 60 min. The cells were stained using freshly prepared Oil red O (Sigma) working solution at room temperature for 10min. Intensity of cell staining was observed using a light microscope.
Virus infectivity assay
To test Cav-1’s influence on HIV-1 infectivity, TZM-bl cells were infected with virus harvested from Cav-1 treated cells and the infectivity levels were measured by luciferase activity. MDMs were first infected with adenovirus expressing Cav-1 or GFP at an moi of 100 in serum free medium for 6 hours. The cells were then washed and incubated in serum-containing medium over-night, after which cells were infected with HIV AD8 at an moi of 0.1 for 6 hours, at which point they were washed and refreshed with new medium. On day 6 post infection, supernatants were subjected to RT assay or titered using the indicator TZM-bl cell line. Virus amounts were normalized with level of infectivity being assayed by measuring luciferase within TZM-bl cells . Normalized amounts of virus were used for subsequent infections.
Determination of cholesterol content and cholesterol replenishment assay
Equivalent amounts of virions were quantified by p24 assay and tested for cholesterol content using the Amplex Red cholesterol Assay Kit (Invitrogen, Carlsbad, CA) according to the manufacturer’s protocol. To replenish cholesterol virus amounts were also normalized by p24 assay and incubated in 0.5mM (2-Hydroxypropyl)-ß-Cyclodextrin solution (Sigma Aldrich) with 1.5mM cholesterol (Sigma Aldrich) at 37°C for 1 hour. These quantified and normalized amounts of virus were used to infect TZM-bl cells and monitored for luciferase activity.
U87 cells were transfected with pcDNA3.1 and pCZ-vector (mock), pcDNA3.1SF2Nef and pCZ-vector (Nef), pCZ-cav-1 and pcDNA3.1 (Cav-1), pcDNA3.1SF2Nef and pCZ-cav-1 (Nef plus Cav-1), pCI NL4-3 NefG2A-HA (NefG2A) and pCZ-vector, or pCI NL4-3 NefG2A-HA and pCZ-cav-1 (NefG2A plus Cav-1). Twenty-four hours after transfection cell culture medium was replaced with serum free medium containing 2 μCi/mL [3H] cholesterol and 1.5% BSA and incubated for 36 hours. Radioisotope-containing medium was then removed and cells were washed twice with PBS and cultured for an additional 18 hours in serum free medium in the presence or absence of 50 μg/ml ApoA-l (Biomedical Technologies Inc., Stoughton, MA). Cholesterol content was measured in the cell free media as well as within cells after lysing using 0.1N NaOH. ApoA-l specific cholesterol efflux was determined using the formula: apoA-l specific efflux = % cholesterol efflux with apoA-l - % cholesterol efflux without apoA-l (blank); cholesterol efflux= [cpm(supernatants)/cpm(supernatants+cells)] ×100%. HDL mediated cholesterol efflux is also examined by incubating cells for 18 hours in the presence or absence of 50 μg/ml HDL (Biomedical Technologies Inc., Stoughton, MA).
To determine cholesterol efflux from macrophages, MDMs were first infected with Ad-Cav-1 or Ad-GFP at an moi of 50 for 24 hours, which was followed by infection of pseudotyped HIV pSG3Δenv (psHIVwtNef) or pNL4-3.Luc.R-E- (psHIVΔNef). Five days post infection cells were then labeled with 1 μCi/mL [3H] cholesterol for 48 hours and apoA-l mediated cholesterol efflux was determined as described above. Similarly cholesterol efflux from THP-1 cell-differentiated macrophages was determined 21 days after infecting with HIV AD8 at an moi of 0.001. Cholesterol efflux was also determined 14 days after THP-1 cell-differentiated macrophages infected with an moi of 0.001, 0.01, or 0.1. In addition, primary macrophages (MDMs) were infected with AD8 or ADnefmut HIV with an moi 0.01 and then cultured cells were subjected to cholesterol efflux assay 15 days after infection. ABCA-1 expression was determined by Western blots in MDMs 14 days after co-infection with AD8 or ADnefmut HIV and with Ad-Cav-1 or Ad-GFP. Inhibition of HIV replication was performed by treating infected cells with 5 uM azidothymidine (AZT) (Sigma-Aldrich, St. Louis, MO).
Immunoprecipitation and Immunoblotting analyses
U87 cells were transfected with pCZ-Cav-1 and HA-tagged Nef (pCI NL4-3 Nef-HA-WT) or HA-tagged NefG2A (pCI NL4-3 NefG2A-HA), followed by incubation of medium containing cholesterol (30μg/ml) for 48 hours. Cells were then treated with apoA-I (20μg/ml) for 30 min. Cells were put on ice, washed twice with cold PBS and total cellular protein was extracted in lysis buffer (50 mM Tris pH 7.5,100 mM NaCl, 1 mM EDTA, 0.1% (v/v) Triton X-100, 10 mM NaF, 1 mM phenylmethyl sulfonyl fluoride, and 1 mmol/L vanadate) with a complete protease Inhibitor mixture (Roche Diagnostics, Indianapolis, IN). The concentration of extracted protein was determined and adjusted to 1 ug/ul. A total of 500 ul was used for each immunoprecipitation, to which 2μg of antibodies (anti-Cav-1 or anti-HA) or normal IgG were added. The mixtures were incubated at 4°C overnight. Following the overnight incubation, 25 μl of protein A/G-agarose beads (Santa Cruz Biotechnology, Santa Cruz, CA) were added and the mixtures were then rotated for 2 hours at 4°C. The beads were harvested by centrifugation and washed five times with lysis buffer. Loading buffer was added and boiled for 5 min. The samples were subjected to SDS-PAGE and analyzed by immunoblotting as described previously . The primary antibodies used for immunoblotting were rabbit polyclonal anti-Cav-1(Santa Cruz Biotechnology, Santa Cruz, CA), mouse monoclonal anti-Nef, rabbit Nef antiserum, and human monoclonal anti-Gag (NIH AIDS Research and Reference Reagent Program), goat polyclonal anti-HA (Genescript), mouse monoclonal anti-ABCA1 (abcam), and ß-actin protein antibody (Sigma, St. Louis, MO). The secondary antibodies were HRP-linked anti-rabbit, anti-mouse (Cell Signaling Technology, Inc., Danvers, MA), anti-human IgG (Sigma, St. Louis, MO) or anti-goat IgG (Santa Cruz Biotechnology, Santa Cruz, CA).
Student’s t test was applied to analyze the differences between sets of data. All analyses were performed with SPSS 12.0.1 for Windows, and were considered significant at p ≤ 0.05.
This research was supported by a grant from the National Institutes of Health (AI39126) to A.M.
- Rothberg KG, Heuser JE, Donzell WC, Ying YS, Glenney JR, Anderson RG: Caveolin, a protein component of caveolae membrane coats. Cell. 1992, 68: 673-682. 10.1016/0092-8674(92)90143-Z.View ArticlePubMedGoogle Scholar
- Harris J, Werling D, Hope JC, Taylor G, Howard CJ: Caveolae and caveolin in immune cells: distribution and functions. Trends Immunol. 2002, 23: 158-164. 10.1016/S1471-4906(01)02161-5.View ArticlePubMedGoogle Scholar
- Galbiati F, Volonte D, Liu J, Capozza F, Frank PG, Zhu L, Pestell RG, Lisanti MP: Caveolin-1 expression negatively regulates cell cycle progression by inducing G(0)/G(1) arrest via a p53/p21(WAF1/Cip1)-dependent mechanism. Mol Biol Cell. 2001, 12: 2229-2244.PubMed CentralView ArticlePubMedGoogle Scholar
- Fielding PE, Russel JS, Spencer TA, Hakamata H, Nagao K, Fielding CJ: Sterol efflux to apolipoprotein A-I originates from caveolin-rich microdomains and potentiates PDGF-dependent protein kinase activity. Biochemistry. 2002, 41: 4929-4937. 10.1021/bi012091y.View ArticlePubMedGoogle Scholar
- Gargalovic P, Dory L: Cellular apoptosis is associated with increased caveolin-1 expression in macrophages. J Lipid Res. 2003, 44: 1622-1632. 10.1194/jlr.M300140-JLR200.View ArticlePubMedGoogle Scholar
- Gargalovic P, Dory L: Caveolins and macrophage lipid metabolism. J Lipid Res. 2003, 44: 11-21. 10.1194/jlr.R200005-JLR200.View ArticlePubMedGoogle Scholar
- Le PU, Guay G, Altschuler Y, Nabi IR: Caveolin-1 is a negative regulator of caveolae-mediated endocytosis to the endoplasmic reticulum. J Biol Chem. 2002, 277: 3371-3379. 10.1074/jbc.M111240200.View ArticlePubMedGoogle Scholar
- Chao WT, Fan SS, Chen JK, Yang VC: Visualizing caveolin-1 and HDL in cholesterol-loaded aortic endothelial cells. J Lipid Res. 2003, 44: 1094-1099. 10.1194/jlr.M300033-JLR200.View ArticlePubMedGoogle Scholar
- Fu Y, Hoang A, Escher G, Parton RG, Krozowski Z, Sviridov D: Expression of caveolin-1 enhances cholesterol efflux in hepatic cells. J Biol Chem. 2004, 279: 14140-14146. 10.1074/jbc.M311061200.View ArticlePubMedGoogle Scholar
- Couet J, Li S, Okamoto T, Ikezu T, Lisanti MP: Identification of peptide and protein ligands for the caveolin-scaffolding domain Implications for the interaction of caveolin with caveolae-associated proteins. J Biol Chem. 1997, 272: 6525-6533. 10.1074/jbc.272.10.6525.View ArticlePubMedGoogle Scholar
- Quest AF, Leyton L, Parraga M: Caveolins, caveolae, and lipid rafts in cellular transport, signaling, and disease. Biochem Cell Biol. 2004, 82: 129-144. 10.1139/o03-071.View ArticlePubMedGoogle Scholar
- Williams TM, Lisanti MP: Caveolin-1 in oncogenic transformation, cancer, and metastasis. Am J Physiol Cell Physiol. 2005, 288: C494-C506.View ArticlePubMedGoogle Scholar
- Huang JH, Lu L, Lu H, Chen X, Jiang S, Chen YH: Identification of the HIV-1 gp41 core-binding motif in the scaffolding domain of caveolin-1. J Biol Chem. 2007, 282: 6143-6152.View ArticlePubMedGoogle Scholar
- Hovanessian AG, Briand JP, Said EA, Svab J, Ferris S, Dali H, Muller S, Desgranges C, Krust B: The caveolin-1 binding domain of HIV-1 glycoprotein gp41 is an efficient B cell epitope vaccine candidate against virus infection. Immunity. 2004, 21: 617-627. 10.1016/j.immuni.2004.08.015.View ArticlePubMedGoogle Scholar
- Benferhat R, Krust B, Rey-Cuille MA, Hovanessian AG: The caveolin-1 binding domain of HIV-1 glycoprotein gp41 (CBD1) contains several overlapping neutralizing epitopes. Vaccine. 2009, 27: 3620-3630. 10.1016/j.vaccine.2009.03.057.View ArticlePubMedGoogle Scholar
- Wang XM, Nadeau PE, Lo YT, Mergia A: Caveolin-1 modulates HIV-1 envelope-induced bystander apoptosis through gp41. J Virol. 2010, 84: 6515-6526. 10.1128/JVI.02722-09.PubMed CentralView ArticlePubMedGoogle Scholar
- Lin S, Wang XM, Nadeau PE, Mergia A: HIV infection upregulates caveolin 1 expression to restrict virus production. J Virol. 2010, 84: 9487-9496. 10.1128/JVI.00763-10.PubMed CentralView ArticlePubMedGoogle Scholar
- Wang XM, Nadeau PE, Lin S, Abbott JR, Mergia A: Caveolin 1 inhibits HIV replication by transcriptional repression mediated through NF-kappaB. J Virol. 2011, 85: 5483-5493. 10.1128/JVI.00254-11.PubMed CentralView ArticlePubMedGoogle Scholar
- Lin YC, Ma C, Hsu WC, Lo HF, Yang VC: Molecular interaction between caveolin-1 and ABCA1 on high-density lipoprotein-mediated cholesterol efflux in aortic endothelial cells. Cardiovasc Res. 2007, 75: 575-583. 10.1016/j.cardiores.2007.04.012.View ArticlePubMedGoogle Scholar
- Ikonen E, Parton RG: Caveolins and cellular cholesterol balance. Traffic. 2000, 1: 212-217. 10.1034/j.1600-0854.2000.010303.x.View ArticlePubMedGoogle Scholar
- Truong TQ, Brodeur MR, Falstrault L, Rhainds D, Brissette L: Expression of caveolin-1 in hepatic cells increases oxidized LDL uptake and preserves the expression of lipoprotein receptors. J Cell Biochem. 2009, 108: 906-915. 10.1002/jcb.22321.View ArticlePubMedGoogle Scholar
- Smart EJ, Ying Y, Donzell WC, Anderson RG: A role for caveolin in transport of cholesterol from endoplasmic reticulum to plasma membrane. J Biol Chem. 1996, 271: 29427-29435. 10.1074/jbc.271.46.29427.View ArticlePubMedGoogle Scholar
- Uittenbogaard A, Ying Y, Smart EJ: Characterization of a cytosolic heat-shock protein-caveolin chaperone complex Involvement in cholesterol trafficking. J Biol Chem. 1998, 273: 6525-6532. 10.1074/jbc.273.11.6525.View ArticlePubMedGoogle Scholar
- Truong TQ, Aubin D, Falstrault L, Brodeur MR, Brissette L: SR-BI, CD36, and caveolin-1 contribute positively to cholesterol efflux in hepatic cells. Cell Biochem Funct. 2010, 28: 480-489. 10.1002/cbf.1680.View ArticlePubMedGoogle Scholar
- Liao Z, Graham DR, Hildreth JE: Lipid rafts and HIV pathogenesis: virion-associated cholesterol is required for fusion and infection of susceptible cells. AIDS Res Hum Retroviruses. 2003, 19: 675-687. 10.1089/088922203322280900.View ArticlePubMedGoogle Scholar
- Zheng YH, Plemenitas A, Linnemann T, Fackler OT, Peterlin BM: Nef increases infectivity of HIV via lipid rafts. Curr Biol. 2001, 11: 875-879. 10.1016/S0960-9822(01)00237-8.View ArticlePubMedGoogle Scholar
- Maziere JC, Landureau JC, Giral P, Auclair M, Fall L, Lachgar A, Achour A, Zagury D: Lovastatin inhibits HIV-1 expression in H9 human T lymphocytes cultured in cholesterol-poor medium. Biomed Pharmacother. 1994, 48: 63-67. 10.1016/0753-3322(94)90077-9.View ArticlePubMedGoogle Scholar
- Carter GC, Bernstone L, Sangani D, Bee JW, Harder T, James W: HIV entry in macrophages is dependent on intact lipid rafts. Virology. 2009, 386: 192-202. 10.1016/j.virol.2008.12.031.View ArticlePubMedGoogle Scholar
- Aloia RC, Tian H, Jensen FC: Lipid composition and fluidity of the human immunodeficiency virus envelope and host cell plasma membranes. Proc Natl Acad Sci USA. 1993, 90: 5181-5185. 10.1073/pnas.90.11.5181.PubMed CentralView ArticlePubMedGoogle Scholar
- Bukrinsky M, Sviridov D: Human immunodeficiency virus infection and macrophage cholesterol metabolism. J Leukoc Biol. 2006, 80: 1044-1051. 10.1189/jlb.0206113.View ArticlePubMedGoogle Scholar
- Ding L, Derdowski A, Wang JJ, Spearman P: Independent segregation of human immunodeficiency virus type 1 Gag protein complexes and lipid rafts. J Virol. 2003, 77: 1916-1926. 10.1128/JVI.77.3.1916-1926.2003.PubMed CentralView ArticlePubMedGoogle Scholar
- Holm K, Weclewicz K, Hewson R, Suomalainen M: Human immunodeficiency virus type 1 assembly and lipid rafts: Pr55(gag) associates with membrane domains that are largely resistant to Brij98 but sensitive to Triton X-100. J Virol. 2003, 77: 4805-4817. 10.1128/JVI.77.8.4805-4817.2003.PubMed CentralView ArticlePubMedGoogle Scholar
- Nguyen DH, Hildreth JE: Evidence for budding of human immunodeficiency virus type 1 selectively from glycolipid-enriched membrane lipid rafts. J Virol. 2000, 74: 3264-3272. 10.1128/JVI.74.7.3264-3272.2000.PubMed CentralView ArticlePubMedGoogle Scholar
- Ono A, Freed EO: Plasma membrane rafts play a critical role in HIV-1 assembly and release. Proc Natl Acad Sci USA. 2001, 98: 13925-13930. 10.1073/pnas.241320298.PubMed CentralView ArticlePubMedGoogle Scholar
- Guyader M, Kiyokawa E, Abrami L, Turelli P, Trono D: Role for human immunodeficiency virus type 1 membrane cholesterol in viral internalization. J Virol. 2002, 76: 10356-10364. 10.1128/JVI.76.20.10356-10364.2002.PubMed CentralView ArticlePubMedGoogle Scholar
- Zheng YH, Plemenitas A, Fielding CJ, Peterlin BM: Nef increases the synthesis of and transports cholesterol to lipid rafts and HIV-1 progeny virions. Proc Natl Acad Sci USA. 2003, 100: 8460-8465. 10.1073/pnas.1437453100.PubMed CentralView ArticlePubMedGoogle Scholar
- Wout AB v ’t, Swain JV, Schindler M, Rao U, Pathmajeyan MS, Mullins JI, Kirchhoff F: Nef induces multiple genes involved in cholesterol synthesis and uptake in human immunodeficiency virus type 1-infected T cells. J Virol. 2005, 79: 10053-10058. 10.1128/JVI.79.15.10053-10058.2005.View ArticleGoogle Scholar
- Mujawar Z, Rose H, Morrow MP, Pushkarsky T, Dubrovsky L, Mukhamedova N, Fu Y, Dart A, Orenstein JM, Bobryshev YV, et al: Human immunodeficiency virus impairs reverse cholesterol transport from macrophages. PLoS Biol. 2006, 4: e365-10.1371/journal.pbio.0040365.PubMed CentralView ArticlePubMedGoogle Scholar
- Han X, Kitamoto S, Lian Q, Boisvert WA: Interleukin-10 facilitates both cholesterol uptake and efflux in macrophages. J Biol Chem. 2009, 284: 32950-32958. 10.1074/jbc.M109.040899.PubMed CentralView ArticlePubMedGoogle Scholar
- Welker R, Harris M, Cardel B, Krausslich HG: Virion incorporation of human immunodeficiency virus type 1 Nef is mediated by a bipartite membrane-targeting signal: analysis of its role in enhancement of viral infectivity. J Virol. 1998, 72: 8833-8840.PubMed CentralPubMedGoogle Scholar
- Djordjevic JT, Schibeci SD, Stewart GJ, Williamson P: HIV type 1 Nef increases the association of T cell receptor (TCR)-signaling molecules with T cell rafts and promotes activation-induced raft fusion. AIDS Res Hum Retroviruses. 2004, 20: 547-555. 10.1089/088922204323087804.View ArticlePubMedGoogle Scholar
- Mujawar Z, Tamehiro N, Grant A, Sviridov D, Bukrinsky M, Fitzgerald ML: Mutation of the ABCA1 C-terminus disrupts HIV-1 Nef binding but does not block the Nef enhancement of ABCA1 protein degradation. Biochemistry. 2010, 49: 8338-8349. 10.1021/bi100466q.PubMed CentralView ArticlePubMedGoogle Scholar
- Frank PG, Cheung MW, Pavlides S, Llaverias G, Park DS, Lisanti MP: Caveolin-1 and regulation of cellular cholesterol homeostasis. Am J Physiol Heart Circ Physiol. 2006, 291: H677-H686. 10.1152/ajpheart.01092.2005.View ArticlePubMedGoogle Scholar
- Hamard-Peron E, Muriaux D: Retroviral matrix and lipids, the intimate interaction. Retrovirology. 2011, 8: 15-10.1186/1742-4690-8-15.PubMed CentralView ArticlePubMedGoogle Scholar
- Morrow MP, Grant A, Mujawar Z, Dubrovsky L, Pushkarsky T, Kiselyeva Y, Jennelle L, Mukhamedova N, Remaley AT, Kashanchi F, et al: Stimulation of the liver X receptor pathway inhibits HIV-1 replication via induction of ATP-binding cassette transporter A1. Mol Pharmacol. 2010, 78: 215-225. 10.1124/mol.110.065029.PubMed CentralView ArticlePubMedGoogle Scholar
- Crowe SM, Westhorpe CL, Mukhamedova N, Jaworowski A, Sviridov D, Bukrinsky M: The macrophage: the intersection between HIV infection and atherosclerosis. J Leukoc Biol. 2010, 87: 589-598. 10.1189/jlb.0809580.PubMed CentralView ArticlePubMedGoogle Scholar
- Choudhury RP, Lee JM, Greaves DR: Mechanisms of disease: macrophage-derived foam cells emerging as therapeutic targets in atherosclerosis. Nat Clin Pract Cardiovasc Med. 2005, 2: 309-315. 10.1038/ncpcardio0195.View ArticlePubMedGoogle Scholar
- Feng B, Yao PM, Li Y, Devlin CM, Zhang D, Harding HP, Sweeney M, Rong JX, Kuriakose G, Fisher EA, et al: The endoplasmic reticulum is the site of cholesterol-induced cytotoxicity in macrophages. Nat Cell Biol. 2003, 5: 781-792. 10.1038/ncb1035.View ArticlePubMedGoogle Scholar
- Ohashi R, Mu H, Wang X, Yao Q, Chen C: Reverse cholesterol transport and cholesterol efflux in atherosclerosis. QJM. 2005, 98: 845-856. 10.1093/qjmed/hci136.View ArticlePubMedGoogle Scholar
- Wang N, Silver DL, Costet P, Tall AR: Specific binding of ApoA-I, enhanced cholesterol efflux, and altered plasma membrane morphology in cells expressing ABC1. J Biol Chem. 2000, 275: 33053-33058.View ArticlePubMedGoogle Scholar
- Argmann CA, Edwards JY, Sawyez CG, O'Neil CH, Hegele RA, Pickering JG, Huff MW: Regulation of macrophage cholesterol efflux through hydroxymethylglutaryl-CoA reductase inhibition: a role for RhoA in ABCA1-mediated cholesterol efflux. J Biol Chem. 2005, 280: 22212-22221. 10.1074/jbc.M502761200.View ArticlePubMedGoogle Scholar
- Landry YD, Denis M, Nandi S, Bell S, Vaughan AM, Zha X: ATP-binding cassette transporter A1 expression disrupts raft membrane microdomains through its ATPase-related functions. J Biol Chem. 2006, 281: 36091-36101. 10.1074/jbc.M602247200.View ArticlePubMedGoogle Scholar
- Bergamaschi A, Pancino G: Host hindrance to HIV-1 replication in monocytes and macrophages. Retrovirology. 2010, 7: 31-10.1186/1742-4690-7-31.PubMed CentralView ArticlePubMedGoogle Scholar
- Herbein G, Varin A: The macrophage in HIV-1 infection: from activation to deactivation?. Retrovirology. 2010, 7: 33-10.1186/1742-4690-7-33.PubMed CentralView ArticlePubMedGoogle Scholar
- Le Douce V, Herbein G, Rohr O, Schwartz C: Molecular mechanisms of HIV-1 persistence in the monocyte-macrophage lineage. Retrovirology. 2010, 7: 32-10.1186/1742-4690-7-32.PubMed CentralView ArticlePubMedGoogle Scholar
- Guillemard E, Jacquemot C, Aillet F, Schmitt N, Barre-Sinoussi F, Israel N: Human immunodeficiency virus 1 favors the persistence of infection by activating macrophages through TNF. Virology. 2004, 329: 371-380. 10.1016/j.virol.2004.08.030.View ArticlePubMedGoogle Scholar
- Salahuddin SZ, Rose RM, Groopman JE, Markham PD, Gallo RC: Human T lymphotropic virus type III infection of human alveolar macrophages. Blood. 1986, 68: 281-284.PubMedGoogle Scholar
- Gartner S, Markovits P, Markovitz DM, Kaplan MH, Gallo RC, Popovic M: The role of mononuclear phagocytes in HTLV-III/LAV infection. Science. 1986, 233: 215-219. 10.1126/science.3014648.View ArticlePubMedGoogle Scholar
- Sharova N, Swingler C, Sharkey M, Stevenson M: Macrophages archive HIV-1 virions for dissemination in trans. EMBO J. 2005, 24: 2481-2489. 10.1038/sj.emboj.7600707.PubMed CentralView ArticlePubMedGoogle Scholar
- Freed EO: HIV-1 and the host cell: an intimate association. Trends Microbiol. 2004, 12: 170-177. 10.1016/j.tim.2004.02.001.View ArticlePubMedGoogle Scholar
- Malim MH, Emerman M: HIV-1 Accessory Proteins. Ensuring Viral Survival in a Hostile Environment. Cell Host Microbe. 2008, 3: 388-398. 10.1016/j.chom.2008.04.008.View ArticlePubMedGoogle Scholar
- Liu L, Oliveira NM, Cheney KM, Pade C, Dreja H, Bergin AM, Borgdorff V, Beach DH, Bishop CL, Dittmar MT, McKnight A: A whole genome screen for HIV restriction factors. Retrovirology. 2011, 8: 94-10.1186/1742-4690-8-94.PubMed CentralView ArticlePubMedGoogle Scholar
- Giri MS, Nebozhyn M, Showe L, Montaner LJ: Microarray data on gene modulation by HIV-1 in immune cells: 2000–2006. J Leukoc Biol. 2006, 80: 1031-1043. 10.1189/jlb.0306157.View ArticlePubMedGoogle Scholar
- Giri MS, Nebozyhn M, Raymond A, Gekonge B, Hancock A, Creer S, Nicols C, Yousef M, Foulkes AS, Mounzer K, et al: Circulating Monocytes in HIV-1-Infected Viremic Subjects Exhibit an Antiapoptosis Gene Signature and Virus- and Host-Mediated Apoptosis Resistance1. J Immunol. 2009, 182: 4459-4470. 10.4049/jimmunol.0801450.PubMed CentralView ArticlePubMedGoogle Scholar
- Vazquez N, Greenwell-Wild T, Marinos NJ, Swaim WD, Nares S, Ott DE, Schubert U, Henklein P, Orenstein JM, Sporn MB, Wahl SM: HIV-1 induced macrophage gene expression includes p21, a target for viral regulation. J Virol. 2005, 79: 4479-4491. 10.1128/JVI.79.7.4479-4491.2005.PubMed CentralView ArticlePubMedGoogle Scholar
- Parton RG, Simons K: The multiple faces of caveolae. Nat Rev. 2007, 8: 185-194.View ArticleGoogle Scholar
- Fra AM, Williamson E, Simons K, Parton RG RG: De novo formation of caveolae in lymphocytes by expression of VIP21-caveolin. Proc Natl Acad Sci USA. 1995, 92: 8655-8659. 10.1073/pnas.92.19.8655.PubMed CentralView ArticlePubMedGoogle Scholar
- Hatanaka M, Maeda T, Ikemoto T, Mori H, Seya T, Shimizu A: Expression of Caveolin-1 in Human T Cell Leukemia Cell Lines. Bioch Bioph Res Comm. 1998, 253: 382-387. 10.1006/bbrc.1998.9744.View ArticleGoogle Scholar
- Vallejo J, Hardin CD: Expression of caveolin-1 in lymphocytes induces caveolae formation and recruitment of phosphofructokinase to the plasma membrane. FASEB J. 2005, 16: 586-587.Google Scholar
- Adachi A, Gendelman HE, Koenig S, Folks T, Willey R, Rabson A, Martin MA: Production of acquired immunodeficiency syndrome-associated retrovirus in human and nonhuman cells transfected with an infectious molecular clone. J Virol. 1986, 59: 284-291.PubMed CentralPubMedGoogle Scholar
- Freed EO, Englund G, Martin MA: Role of the basic domain of human immunodeficiency virus type 1 matrix in macrophage infection. J Virol. 1995, 69: 3949-3954.PubMed CentralPubMedGoogle Scholar
- Hwang SS, Boyle TJ, Lyerly HK, Cullen BR: Identification of the envelope V3 loop as the primary determinant of cell tropism in HIV-1. Science. 1991, 253: 71-74. 10.1126/science.1905842.View ArticlePubMedGoogle Scholar
- He J, Choe S, Walker R, Di Marzio P, Morgan DO, Landau NR: Human immunodeficiency virus type 1 viral protein R (Vpr) arrests cells in the G2 phase of the cell cycle by inhibiting p34cdc2 activity. J Virol. 1995, 69: 6705-6711.PubMed CentralPubMedGoogle Scholar
- Connor RI, Chen BK, Choe S, Landau NR: Vpr is required for efficient replication of human immunodeficiency virus type-1 in mononuclear phagocytes. Virology. 1995, 206: 935-944. 10.1006/viro.1995.1016.View ArticlePubMedGoogle Scholar
- Wei X, Decker JM, Liu H, Zhang Z, Arani RB, Kilby JM, Saag MS, Wu X, Shaw GM, Kappes JC: Emergence of resistant human immunodeficiency virus type 1 in patients receiving fusion inhibitor (T-20) monotherapy. Antimicrob Agents Chemother. 2002, 46: 1896-1905. 10.1128/AAC.46.6.1896-1905.2002.PubMed CentralView ArticlePubMedGoogle Scholar
- Wei X, Decker JM, Wang S, Hui H, Kappes JC, Wu X, Salazar-Gonzalez JF, Salazar MG, Kilby JM, Saag MS, et al: Antibody neutralization and escape by HIV-1. Nature. 2003, 422: 307-312. 10.1038/nature01470.View ArticlePubMedGoogle Scholar
- Raney A, Kuo LS, Baugh LL, Foster JL, Garcia JV: Reconstitution and molecular analysis of an active human immunodeficiency virus type 1 Nef/p21-activated kinase 2 complex. J Virol. 2005, 79: 12732-12741. 10.1128/JVI.79.20.12732-12741.2005.PubMed CentralView ArticlePubMedGoogle Scholar
- O'Neill E, Kuo LS, Krisko JF, Tomchick DR, Garcia JV, Foster JL: Dynamic evolution of the human immunodeficiency virus type 1 pathogenic factor, Nef. J Virol. 2006, 80: 1311-1320. 10.1128/JVI.80.3.1311-1320.2006.PubMed CentralView ArticlePubMedGoogle Scholar
- Theodore TS, Englund G, Buckler-White A, Buckler CE, Martin MA, Peden KW: Construction and characterization of a stable full-length macrophage-tropic HIV type 1 molecular clone that directs the production of high titers of progeny virions. AIDS Res Hum Retroviruses. 1996, 12: 191-194. 10.1089/aid.1996.12.191.View ArticlePubMedGoogle Scholar
- Howell KW, Meng X, Fullerton DA, Jin C, Reece TB, Cleveland JC: Toll-like Receptor 4 Mediates Oxidized LDL-Induced Macrophage Differentiation to Foam Cells. J Surg Res. 2011, 171: e27-e31. 10.1016/j.jss.2011.06.033.View ArticlePubMedGoogle Scholar
- Lin S, Wu M, Xu Y, Xiong W, Yi Z, Zhang X, Zhenghong Y: Inhibition of hepatitis B virus replication by MyD88 is mediated by nuclear factor-kappaB activation. Biochim Biophys Acta. 2007, 1772: 1150-1157. 10.1016/j.bbadis.2007.08.001.View ArticlePubMedGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.