HIV-1 Nef protein in the nucleus influences adipogenesis as well as viral transcription through the peroxisome proliferator-activated receptors
Otake, Kaoria; Omoto, Shinyab; Yamamoto, Takuyab; Okuyama, Harumib; Okada, Hidechikac; Okada, Norikoc; Kawai, Masahiroc; Saksena, Nitin Kd; Fujii, Yoichi Rb
From aNagoya University Hospital, Nagoya, the bMolecular Biology and Retrovirus Genetics Group, Division of Nutritional Sciences, Graduate School of Pharmaceutical Sciences and the cGraduate School of Medical Sciences, Nagoya City University, Nagoya Japan and the dRetroviral Genetics Laboratory, Center for Virus Research, Westmead Millennium Institute, Westmead Hospital, Sydney, Australia.
Correspondence to Y. R. Fujii, Molecular Biology and Retrovirus Genetics Group, Division of Nutritional Sciences, Graduate School of Pharmaceutical Sciences, Nagoya City University, Nagoya 467-8603, Japan.
Received: 6 April 2003; revised: 1 July 2003; accepted: 17 August 2003.
Background: Although the HIV-1 Nef protein (27 kDa) localizes primarily in cytoplasm, there is considerable evidence suggesting its occasional localization in the nucleus. Nef is known to play an important role in transcriptional events and viral replication, but the actual target of Nef in the nucleus remains to be identified.
Objective: To examine the functional roles of Nef in the nucleus and its possible interactions with other unknown factors in the nucleus.
Methods: High-density microarray analysis was used to screen directly the unique functions of Nef on host gene transcription. The nuclear localization of Nef and its effects on the expression of peroxisome proliferator-activated receptors (PPAR) was examined using PPAR promoter/reporter assay and immunoblotting. A long terminal repeat/reporter assay was used to investigated the effects of Nef and PPAR on viral transcription.
Results: Nef in the nucleus suppressed PPARγ expression and reduced fatty acid levels in human T and macrophage cell lines. Expression of Nef or PPAR suppressed viral replication; the effect of PPARγ or retinoid X receptor-α on viral replication were reduced by coexpression of Nef in MT(−)4 T cells.
Conclusion: Nef may be involved in both viral replication and the wasting syndrome associated with AIDS.
In HIV-1-infected humans and macaques experimentally infected with SIV, wasting is a common symptom defining AIDS . However, the underlying mechanisms for wasting disease, mainly seen at the onset of AIDS, remain unclear. From the clinical data, it is evident that survival time in AIDS is associated with hypertriglyceridaemia, which leads to a decrease in fat storage in adipose tissue . Further, some HIV-1-infected patients who have been treated with protease inhibitors also show dyslipidaemia and insulin resistance . However, the viral and host factors influencing fat distribution during HIV disease remain poorly defined . Protease inhibitors may accentuate the underlying peroxisome dysregulation. Dysregulation of peroxisomes is associated with a spectrum of biochemical changes seen in the lipodystrophies . Peroxisomes proliferate under the influence of peroxisome proliferators, which act as ligands for the peroxisome proliferator-activated receptors (PPAR) . PPAR are ligand-activated transcription factors belonging to the family of orphan nuclear factors (NR). PPARα is mainly expressed in fresh peripheral blood mononuclear cells and is found in many tissues such as liver, heart, kidney, lung, skeletal muscle and adipose tissue. PPARγ is expressed in abundance in adipose tissue, but also in skeletal muscle, liver, heart and bone marrow stromal cells. The predominant effect of PPARγ is on transcriptional regulation. Although there is little information on a functional role for PPAR in HIV, PPAR appears to have a considerable role in the regulation of lipid metabolism, glucose homeostasis and cellular differentiation .
How viral factors and/or viral proteins interact with PPARγ and modulate HIV pathogenesis remains unclear. We are interested in addressing the interaction of HIV Nef protein with PPARγ. As Nef protein is primarily localized in the cytoplasm, its occasional nuclear localization (especially with some strains of HIV) is intriguing. Findings by Hayes et al.  suggested that PPARγ reduces HIV-1 mRNA stability and so an interaction of Nef protein (synthesized early in viral life cycle) with the NR family could occur. Although, viral changes such as deletions in the nef accessory gene have been correlated with long-term non-progression in HIV-infected individuals , three of six members of the Sydney blood bank cohort harbouring nef-deleted viruses did progress to AIDS . In SIV, the Nef protein is required for induction of AIDS-like disease in monkeys  and the disease has slow progression in monkeys infected with nef-deleted SIV . This suggests that the presence of continued nef deletions cannot accentuate disease progression in HIV-infected humans and SIV-infected monkeys and that Nef is essential for efficient viral replication. This is supported by studies in vivo in severe combined immunodeficient mice engrafted with human immune systems (SCID-Hu mice) . Recently, microarray assays have been employed to show that Nef is involved in signal transduction as well as in the transcriptional programme [11,12]. Although there is substantial evidence suggesting a role for Nef in disease development and AIDS, the relation between Nef and the AIDS wasting disease, which might be induced by dysregulation of transcriptional factors such as PPAR, remains unclear. The present study examines how Nef could influence PPAR in order to obtain insight into how Nef affects the in vivo dominantly lentiviral pathogenesis and its possible relationship with wasting disease.
Cells, antibodies and viruses
HL60, U937, Gin-1, COS, HeLa and Molt-4 cell lines were maintained in RPMI-1640 supplemented with 10% heat-inactivated fetal bovine serum and antibiotics. Persistently HIV-1IIIB-infected MT-4 and U937 cells were cultured with RPMI-1640. Anti-Nef rabbit IgG and anti-Nef mouse monoclonal antibody F3 were prepared as described previously . Anti-human histone H1 monoclonal antibody was purchased from Biogenesis (Poole, UK). Anti-human transferrin receptor monoclonal antibody and alkaline phosphatase-conjugated goat anti-mouse IgG (H+L) antibody were purchased from Zymed (South San Francisco, California, USA). Anti-human actin monoclonal antibody was purchased from Progen (Heidelberg, Germany). The phycoerythrin-conjugated rat anti-mouse IgG was purchased from BD Biosciences (Franklin Lakes, California, USA). Anti-PPARγ rabbit serum was purchased from Sigma (St Louis, Missouri, USA). HIV-1SF2 and HIV-1SF162 viruses were gifted by Dr C. Chang-Mayer.
Construction of plasmids and generation of recombinant adenoviruses
To generate Tet-Off vectors, the pT2 Tet-Off plasmid derived from pTet-Splice  (gifted by Dr T. Takemori, National Institute of Infectious Disease of Japan) was digested with XhoI plus BamHI. The Tet-Off operator fragment was blunted with Klenow ploymerase. The pIRES-EGFP plasmid (Clontech, BD Biosciences) was digested with NurI plus EcoRV. The blunted Tet-Off operator fragment was inserted into the pIRES-EGFP plasmid. The pT2-IRES-EGFP plasmid was digested with EcoRI and then the polymerase chain reaction (PCR)-amplified HIV-1NL43 and HIV-1SF2 nef gene were inserted into the EcoRI site of pT2-IRES-EGFP (pT2-Nef-IRES-EGFP). The pTet-Off EF-TA plasmid (Clontech) was also used for the Tet-Off system. To prepare recombinant adenoviruses, the pT2-Nef-IRES-EGFP and pT2-IRES-EGFP were digested with HincII and pEF-TA plasmid was digested with ScaI plus NheI; the fragments from pT2-Nef-IRES-EGFP and pT2-IRES-EGFP were inserted into the SwaI site of the pAxCW cosmid vector (Takara Biochemicals, Kyoto, Japan). The fragment from pEF-TA was blunted by Klenow polymerase and inserted into the SwaI site of pAxCW. The recombinant adenoviruses and control vectors (AxT2-Nef-IRES-EGFP, AxT2-IRES-EGFP and AxEF-TA) were prepared according to the manufacturer's instructions (Takara Biochemicals). The recombinant cosmid vectors were transfected into 293 cells, which express E1A and E1B genes. After the transfection, the E1 gene-deleted adenviruses recombined with the wild-type virus were produced from 293 cells. In the Tet-Off system, in the absence of tetracycline (doxycycline, Dox), trans-activator expressed from AxEF-TA virus (a fusion protein consisting of the tetracycline repressor and the trans-activator domain of the VP6 of herpes simplex virus) binds to the promoter elements in AxT2-Nef-IRES-EGFP or AxT2-IRES-EGFP virus and activates expression of recombinant Nef and/or green fluorescent protein (GFP). Binding of trans-activator to the promoter element blocks subsequent gene activation in presence of Dox. Therefore, AxT2-Nef-IRES-EGFP or AxT2-IRES-EGFP virus was coinfected with AxEF-TA virus in the presence or absence of Dox. Cells were infected with a multiplicity of infection (MOI) of 2 to 10. For the detection of expressed Nef, cells were cultured in the Lab-Teck Chamber Slide System (Nalge Nunc International. Naperville, Illinois, USA). Cells were fixed with 3.7% paraformaldehyde in phosphate-buffered saline and were permeabilized with 0.5% Triton X-100 in phosphate-buffered saline. For the immunofluorescence assay, anti-Nef F3 monoclonal antibodies were added to the samples, which were left at 0°C for 4 h, then washed and incubated with phycoerythrin-conjugated anti-mouse IgG (Zymed) for 2 h at 0°C. All samples were examined using an MRC-1024 confocal microscope (Bio-Rad, Hercules, California, USA) and Facscalibur analyser (Becton Dickinson, San Jose, California, USA). For preparation of PPAR-expressing vectors, the human PPARα DNA fragment of pcDNA3-PPARα was digested with BamHI and ligated into the same enzyme site in pSVSPORT1 (GIBCO, Grand Island, New York, USA) (pSV-PPARα). The human PPARγ2 in pSVSPORT was also prepared. pCDNefNL43 was gifted by Dr N. L. Michael.
Expression and purification of recombinant Nef proteins
For the preparation of recombinant glutathione S-transferase (GST)-fused Nef protein, the entire nef DNA of NL43, SP-1-101, IP-1-1 and RP-1-3 HIV-1 strains was prepared by PCR and subcloned into pBluescript as described previously . The XhoI and HindIII fragment in pBluescript was blunted by Klenow polymerase, and the fragment was ligated in the pGEX-4T-1 (Pharmacia, Piscataway, New Jersey, USA) at a similarly blunted XhoI site. PPARγ2 gene in pSVSPORT was used as a template to amplify PPARγ DNA by PCR with primer A (5′-GTTATGGGTG AAACTCTGGGAGAT-3′) and primer B (5′-ATG TTCCTGAACATAATCGTC-3′). After TA cloning (Invitrogen, Carlsbad, California, USA), the PPARγ gene fragment was obtained from a SmaI–XhoI cut of pCR PPARγ plasmid. The fragment was introduced into SmaI–XhoI sites of pGEX-4T-1. The Nef–GST and PPAR–GST fusion proteins were expressed in Escherichia coli . The recombinant proteins were purified as described previously [13,15].
Internalization assay and subcellular fractionation
Cells (1 × 106) were incubated for 30 min at room temperature with various purified Nef proteins at a concentration of 0.1 μg/ml. After 24 h in culture, the plasma membrane, cytosolic and nuclear fractions of the cells were separated and each fraction was collected by sucrose density gradient centrifugation. These fractions were subjected to sodium dodecylsulphate polyacrylamide gel electrophoresis (SDS–PAGE) and analyzed by immunoblotting as previously reported .
RNA isolation and microarray analysis
Total RNA of the nef-transfected HeLa cells was prepared and purified with TRIZOL reagent (GIBCO) for the microarray assay. The microarray analysis was performed with AtlasTM Glass Human 1.0 Microarrays (Clontech) according to the manufacturer's instructions. Individual mRNA levels were scanned as intensities of Cy3 (532 nm) and Cy5 (635 nm) (Amersham Pharmacia Biotech, Little Chalfont, UK) fluorescence using a GenePix 4000 (Axon Instruments, Foster City, California, USA) and then scored and analyzed by GenePix Pro 3.0 (Axon Instruments). Relative intensity (RI) is the fluorescence intensity (Cy3) of mRNA expression in the control EGFP virus (AxT2-IRES-EGFP plus AxEF-TA)-infected cells divided by the fluorescent intensity (Cy5) in the Nef recombinant viral (AxT2-Nef-IRES-EGFP plus AxEF-TA) infected cells. HIV-1 mRNA was amplified by reverse transcriptase (RT)-PCR with TOYOBO RT-PCR Primer Sets (Toyobo Biochemicals, Tokyo, Japan) using HIV-1 gag primers, SK145 and SK431 (Perkin Elmer Cetus, Connecticut, USA).
Chloramphenicol acetyltransferase and luciferase assay
Cells (1 × 105) were transfected with 2.5 μg plasmids containing nef or PPAR DNA and the pRSVCAT119 plasmid mixed with 5 μg lipofectin (GIBCO). The cells were cultured for 48 h at 37°C in 0.5 ml RPMI-1640 medium containing 10% Nu-serum. After 30 h, the chloramphenicol acetyltransferase (CAT) activity was measured using the manufacturer's instructions (Clontech). The transfection efficiency was determined by the β-galactosidase control plasmid. The luciferase (Luc) assay is described as elsewhere .
Extraction and analysis of lipids
Lipids were extracted from cells at 40 h after transfection with pSV-PPARγ and pT2-Nef-IRES-EGFP plus pEF-TA plasmids, or infection with HIV-1SF2 or HIV-1SF162. The fatty acids were quantified by gas liquid chromatography using a GC-18A (Shimazu, Kyoto, Japan). To examine total lipids, cells were fixed with 10% formalin and stained with 0.25% Oil Red O (Sigma) in isopropanol. The number of stained particles in a dish or stained cells in three fields was counted under the microscope and the mean of three individual experiments was calculated.
Screening by microarray analysis
A microarray Atlas-Human 1.0 was used to monitor the change in Nef mRNA levels with time to screen for unique functional aspects of Nef through the transcription process. Expression of Nef was synchronized with EGFP gene expression. HeLa cells were coinfected with NefNL43-IRES-EGFP operator plus Tet-Off adenoviruses in the presence or absence of 1 μg/ml Dox and were screened at 12 h post-transfection. Expression of EGFP in HeLa cells cotransfected with NefNL43-IRES-EGFP plus EF-TA or a control EGFP (corresponding to control screening, addressing the influence of adenovirus infection) plus EF-TA viruses was increased > 98.5% compared with expression in uninfected cells (data not shown). Therefore, total RNA from the transduced cells was extracted at 18 h or 48 h after Dox treatment. Levels of gene expression (RI) in microarrays were restricted to < 10.0 and > 0.1. At 18 h after Dox treatment, expression of 19 of the 1081 genes screened decreased and only one gene increased expression. However, at 48 h after Dox, expression of 14 genes decreased and four increased compared with the control. Nef commonly suppressed approximately 100-fold the transcription of genes for NR in the control EGFP virus/the Nef recombinant viral infection at both 18 h (RI = 0.009) and 48 h (RI = 0.015). Other genes that had markedly changed expression levels at 18 and 48 h were of a very different nature. Nef strongly suppressing mRNA of p38 MAP kinase (RI = 0.01), p21-activated kinase and Nef-related CDK9 (RI = 0.014) at 18 h, and adenovirus E1A-related cyclin A1 (RI = 0.01), CDK6 and Vpr-related CDC25 (RI = 0.013) at 48 h. The levels of transcription factor TBP1, interacting with the long terminal repeat (LTR) basal/core promoter mRNA, were predominantly reduced (RI = 0.01) at 48 h. The mRNA of calcium-binding, filament (RI = 34.5) and heat shock proteins (RI = 20.0) increased at 48 h. These assays were conducted in triplicates, and similar results were obtained. The recombinant virus-infected cells were 98.2 ± 2.0% Nef positive, as scored under microscope by staining with anti-Nef monoclonal antibody. Although very few studies have used microarrays to examine the expression system of Nef recombinant adenoviruses, the results of suppression on host gene transcriptional machinery by Nef in HeLa cells here differed from a recent report using human Jurkat T cell lines lacking the T cell receptor (TCR) and expressing Nef by transient transfections . It is suggested that the state of host transcription for gene expression enforced by transfection may differ from that in viral infection, where de novo DNA methylation is epigenetically increased as a genome defence mechanism of gene silencing against foreign invaders .
Changes in localization of Nef protein in recombinant adenovirus infected cells
The possible link between Nef and NR in the nucleus was initially studied by following the localization of Nef in the nucleus using the Nef-IRES-EGFP recombinant adenovirus and cell-free Nef protein. HeLa cells were coinfected with NefNL43-IRES-EGFP operator plus Tet-Off adenoviruses in the presence or absence of 1 μg/ml Dox. Infected cells were subjected to SDS–PAGE and the cell lysates were immunoblotted with Nef-specific monoclonal antibody F3 (Fig. 1a). The blot showed a Nef-positive band only in lysates of cells infected with Nef-IRES-EGFP operator plus the Tet-Off viruses at 12 h after Dox. Infected cells were stained with monoclonal antibody F3 at 18, 24 and 48 h after Dox. In Fig. 1b, Nef can be seen in the margin of the plasma membrane at 18 h, surrounding the nucleus at 24 h and staining the nucleoplasm at 48 h. The nuclei were stained with propidium iodide (data not shown). Localization of Nef in the nucleus increased from 18 to 48 h after Dox treatment. The number of Nef-positive cells in the nucleus increased from approximately 5% at 12 h to 70% at 48 h (Fig. 1c). In flow cytometry, approximately 98% EGFP-positive cells were observed at 48 h after Dox in these recombinant adenovirus-infected cells.
Subcellular distribution of soluble Nef protein after incorporation into target cells
Previous studies have shown that Nef binds to the cell surface and penetrates cells [17,18]. Therefore, it was interesting to examine the intracellular topology of soluble Nef proteins using subcellular fractionation (Fig. 2). Cells were exposed to various recombinant Nef proteins and subsequently fractionated. Immunoblot assays with specific antibodies detected Nef (from strains NL43, SP, IP and RP)–GST fusion proteins of ca. 53 kDa on the membrane and in cytosolic fractions as well as in the nuclear fractions of a Molt-4 cell line (Fig. 2). To exclude an influence of the GST tag on subcellular distribution of the Nef fusion protein in E. coli, full-length and myristoylated Nef was used. After baculovirus recombinant HIV-1NL43 Nef was added to the culture, the cells were incubated and the lysates were fractionated; each fraction was subjected to SDS–PAGE. In immunoblotting with anti-Nef monoclonal antibody F3, bands of approximately 27 kDa were also detected in the cytosolic and nuclear fractions. The purity of each fraction was determined by immunoblotting with anti-transferrin receptor, anti-actin and anti-histone H1 monoclonal antibodies. Bands of approximately 95 kDa (transferrin receptor) and 42 kDa (actin) were detected in the plasma membrane and the cytosolic fractions, respectively. In the nucleus fraction, a dominant band of ca. 22 kDa was detected. Although several bands were also detected by the anti-histone H1 monoclonal antibody, it appeared that H1 may make complexes with degraded nucleosomes. Similar results were obtained using anti-Nef monoclonal antibody F3 (amino acids 128–137 epitope) and/or U937 cells for immunoblotting (data not shown). Taken together, these data agree with previous studies [19,20] and indicate that Nef may move to the nucleus after its penetration into cells and/or following its expression in cells.
The suppressive effects of Nef in adipogenesis
PPARγ is expressed and localized in the nucleus of adipocytes and monocyte/macrophage cells . In addition, PPARγ agonists inhibit HIV replication in macrophages by transcriptional and post-transcriptional effects . To assess any changes in adipogenesis associated with Nef, COS cells were cotransfected with the NefNL43-IRES-EGFP operator plus the Tet-Off plasmids and pSV-PPARα (NR1C1) or pSV-PPARγ (NR1C3) plus the CAT reporter plasmids containing the PPAR response element. In this experimental system, the CAT reporter did not respond at all to PPAR in HeLa cells. Therefore, COS cells were used and CAT activities were measured 40 h after transfection (Fig. 3a). Nef reduced induction of CAT activities by PPARγ, but not that induced by PPARα. The CAT assays were repeated at least three times, and the results were reproducible. These data suggest that this is an acceptable experimental system for examining the interaction between Nef and PPAR. In the same experiment, expression of PPARγ protein was also examined by immunoblotting (Fig. 3b). Nef reduced expression of PPARγ in two separate experiments (Fig. 3b), but not that of PPARα (data not shown).
Suppression of fatty acid accumulation by Nef in HIV-1-infected cells
To evaluate the above results in human cells, accumulation of lipids was examined using Oil Red O staining in HeLa cells (Fig. 4a). Positive spots in a dish (×1) and cells (×400) were increased in PPARγ gene-transfected cells. In cells cotransfected with HIV-1SF2 nef and PPARγ, positively stained spots and cells were decreased. The HIV-1NL43 Nef-expressing plasmid pCD3Nef was used as a positive control and Nef suppressed the effects of PPARγ. However, cells cotransfected with the EGFP operator plus the Tet-Off and pSV-PPARα plasmids did not show any change in fatty acids (data not shown). Similar results were obtained with COS cells (data not shown). Since endogenous lipid accumulation was not suppressed by Nef, because only 30% transfection efficiency could be achieved, lipids were directly extracted and quantified from human cells and fatty acids in infected, uninfected or Nef-transfected haematopoietic cell lines. Although upregulation of PPARγ expression has been reported to induce triglyceride synthesis, thus inhibiting release of fatty acids from cells , total fatty acids were reduced after HIV-1 infection of Molt-4 or phorbol myristate acetate (PMA)-stimulated HL60, or after transfection of nef and PPARγ into HL60 cells (Fig. 4b). These results demonstrate that Nef suppressed PPARγ activities in human cell lines and suppressed accumulation of fatty acids including endogenous ones, which suggests that Nef protein may be a PPARγ agonist.
The trans-action of Nef protein for viral transcription
The U3 region in HIV-1 LTR contains a nuclear receptor-responsive element (nucleotidest −356 to −320) and the heterodimer of PPARα and retinoid X receptor-α (RXRα; NR2B1) suppresses the LTR  and inhibits the host's trans-activators of HIV LTR, such as AP-1 and NF-κB. Absence of PPARγ is lethal for the host . Our data, together with these reports, suggest that host NR may participate in modulation of viral gene expression, or vice versa. The relationship between Nef and NR for HIV-1 replication was investigated further by examining the effect of Nef and/or PPARγ on LTR trans-activation (Fig. 5). Since endogenous expression of PPAR and Nef was shown in MT(−)4 T and U937 cell lines persistently infected by HIV-1IIIB (data not shown), each cell was cotransfected with Nef- and/or PPAR-expressing plasmids plus the LTR–Luc plasmid as a reporter of HIV-1 replication. MT(−)4 persistently infected with HIV-1 showed approximately 75-fold higher Luc activities with LTR–Luc plasmid alone (Mock) than infected U937 did. Overexpression of Nef, PPARγ or RXRα suppressed luc expression in MT(−)4 cells, but this was not observed in U937 cells. Nef alleviated the suppression with PPARγ or RXRα in MT(−)4 cells (Fig. 5a). PPAR inhibits HIV-1 transcription factors, such as AP-1 and NK-κB . These data indicate that the balance of expression of Nef and PPARγ may be responsible for trans-activation of viral replication in T cells; however, in macrophages, Nef and PPARγ may not be related to viral transcription because of the properties of PPARγ in induction of apoptosis .
Further, to confirm the relationships among HIV-1 LTR, NR and Nef in T cells, these expression vectors were cotransfected to uninfected MT(−)4 cells (Fig. 5b). Nef, PPARγ or RXRα reduced HIV-1 LTR in the presence of Tat but did not do so in its absence. Addition of a Nef-expressing plasmid restored the reduced transcriptional activity. With uninfected MT(−)4 and U937 cells, the reporter control activities (Luc basic plasmid) were low. Similar results were obtained in the presence of clofibric acid or ciprofibric acid. The transfection efficiency was 30.5 ± 5.5%. A 142 bp fragment of DNA from the gag region of HIV mRNA in infected MT-4 and U937 cells was amplified by RT-PCR. Viability of the cells was 89.8 ± 0.5% in the transfected cell culture. In the presence of 10 μmol/l zidovudine as a negative control, Luc activity was completely diminished. Plasmids expressing the viral accessory proteins Vpr, which can modestly trans-activate LTR, and Vif did not altered these results (data not shown). Expression of each protein was determined by quantitative RT-PCR (data not shown).
Nef protein has been reported to localize in the plasma membrane, the cytoplasm and the nucleus of nef-transduced  and acutely infected T cells . The present study has confirmed that Nef incorporated into cells also localized to the plama membrane and cytoplasm. In addition, Nef was also shown to accumulate in the nucleus. The accumulation of Nef in the nucleus was confirmed with the transfection of a Nef-expressing plasmid construct. Therefore, the phenomenon was neither a consequence of recombinant adenovirus infection nor an experimental artifact. Although it was hypothesized that Nef might have some relationship with the severe weight loss that is observed in AIDS patients, the nuclear localization of Nef supports the possibility that Nef could regulate a genomic transcriptional programme via PPAR in host cells. In our experimental system, the Nef-dependent suppression of PPARγ-stimulatory lipogenetic activity was specific for PPARγ, not for PPARα. With regard to virally encoded transcriptional events, the findings of Murphy et al. , and Ilyinski and Desrosiers  suggested that Nef increases HIV-1 and SIV LTR expression, respectively. In contrast, PPARα suppressed transcription of HIV-1 . In our data, Nef alleviated the suppressive effect of PPARγ or RXRα on HIV-1 LTR expression but did not affect PPARα-dependent suppression (unpublished data). Although we have no direct evidence of an interaction between Nef and PPARγ or PPARα, our preliminary experiments showed that HIV-1SF2 Nef bound with a high affinity (KD = 380 nmol/l) to PPARγ in surface plasmon resonance measurements. In contrast, the mutated HIV-1SP Nef had a KD value of 2.16 μmol/l and the affinity of HIV-1SF2 Nef for PPARγ was 10 times higher than for PPARα (unpublished data). The difference between these affinities may reflect the specific activities of PPARγ, but not PPARα, to Nef involving cofactors, such as the sterol-regulatory-element binding protein. It is well documented that PPAR binds to ligands within the cytoplasm and these complexes then localize in the nucleus . It appears that Nef may complex with PPARγ and that this complex may then get transported to the nucleus. Since quasispecies of nef and polymorphisms of PPAR have been shown [5,14,26], a balance between qualitative differences of the binding avidity and quantitative/molecular alterations of the expressed proteins and of natural ligands of PPAR may partly be responsible for onset of post-integration latency or transcriptional activation. Interestingly, PPAR remains an attractive therapeutic target for the development of drugs used in the treatment of chronic inflammatory diseases such as atherosclerosis .
Although Nef suppressed the effects of PPARγ on lipid accumulation, the suppressive effects were only in the range 15–50%. This may have significant implications in that the severe weight loss and muscle wasting associated with AIDS occurs very slowly. Intriguingly, severe weight loss and muscle wasting have also been observed in transgenic mice expressing the entire HIV-1 coding sequence ; additionally, the phenotype for AIDS development does not develop after mutation of the nef coding region . Nef has been reported to induce oxidation of fatty acids through activation of thioesterase II ; therefore, it is possible that Nef may have some role in the weight loss associated with AIDS. Here, we have shown that Nef reduces lipogenesis; if soluble Nef can incorporate into adipocytes in liver or muscle, the dyslipidaemia of AIDS may be causally related to the in vivo virulence factor Nef. Hence, the putative mechanism might reflect a nutritional background that can alter the onset of AIDS in HIV-1-positive individuals; it might also alter the risk of infection. As activators by ligand binding, PPAR are known to bind to promoters in target genes and modulate gene expression, and they have been implicated in regulation of cellular differentiation, glucose metabolism and tumour suppression. The data described here cumulatively support HIV-1 Nef interaction with PPAR and Nef as a PPAR agonist.
The authors gratefully acknowledge Dr J. A. Levy for donating HIV-1SF2 and HIV-1SF162 proviral DNA. We would like to thank Drs A. Thelen, A. Fukamizu and M. Imagawa for providing pRSVCAT119, PPARα and RXRα DNA, respectively. We also sincerely thank Dr T. Takemori for agreement to the use of recombinant adenoviruses, and Drs H. Hayashi and T. Takii for helpful discussion and technical advice. We also much appreciate Dr Y. Yokota's financial support and the technical support of M. Mizuguchi and Y. Murase.
1. Svoboda J, Hejinar J, Geryk J, Elleder D, Vernerova Z. Retroviruses in foreign species and the problem of provirus silencing. Gene 2000, 261:181–188.
2. Safrin S, Grunfeld C. Fat distribution and metabolic changes in patients with HIV infection. AIDS 1999, 13:2493–2505.
3. Lenhard JM, Croom DK, Weiel JE, Winegar DA. HIV protease inhibitors stimulate hepatic triglyceride synthesis. Arterioscler Thromb Biol 2000, 20:2625–2629.
4. Smith KJ, Skelton HG. Peroxisomal proliferator activity legand therapy for HIV lipodiestrophy. Clin Exp Dermatol 2001, 26:155–161.
5. Hayes MM, Lane BR, King SR, Markovitz DM, Coffey MJ. Peroxisome proliferator-activated receptor gamma agonists inhibit HIV-1 replication in macrophages by transcriptional and post-transcriptional effects. J Biol Chem 2002, 277: 16913–16919.
6. Deacon NJ, Tsykin A, Solomon A, Smith K, Lundford-Menting M, Hooker DJ, et al. Genomic structure of an attenuated quasi-species of HIV-1 from a blood transfusion donor and recipients. Science 1995, 27:988–991.
7. Learmont JC, Geczy AF, Mills J, Ashton LJ, Raynes-Greenow CH, Garsia RJ, et al. Immunologic and virologic status after 14 to 18 years of infection with an attenuated strain of HIV-1 - A report from the Sydney Blood Bank Cohort. N Engl J Med 1999, 340:1715–1722.
8. Beer B, Baier M, Megede JZ, Norley S, Kurth R. Vaccine effect using a live attenuated nef-deficient simian immunodeficiency virus of African green monkeys in the absence of detectable vaccine virus replication in vivo. Proc Natl Acad Sci USA 1998, 94:4062–4067.
9. Sawai ET, Hamza MS, Ye M, Shaw KE, Luciw PA. Pathogenic conversion of live-attenuated SIV vaccines is associated with expression of truncated nef. J Virol 2000, 74:2038–2045.
10. Jamieson BD, Aldovandi GM, Planelles V, Jowett JBM, Gao L, Bloch LM, et al. Requirement of human immunodeficiency virus type 1 nef for in vivo replication and pathogenicity. J Virol 1994, 6:3478–3485.
11. Simmons A, Aluvihare V, McMichael A. Nef triggers a transcriptional program in T cells imitating single-signal T cell activation and inducing HIV virulence mediators. Immunity 2001, 14:763–777.
12. Shaheduzzaman S, Krishnan V, Petrovic A, Bittner M, Meltzer P, Trent J, et al. Effects of HIV-1 Nef on cellular gene expression profiles. J Biomed Sci 2002, 9:82–96.
13. Otake K, Fujii Y, Nakaya T, Nishino Y, Zhong Q, Fujinaga K, et al. The carboxyl-terminal region of HIV-1 Nef protein is a cell surface domain that can interact with CD4+ T cells. J Immunol 1994, 153:5826–5837.
14. Shockett P, Difilippantonio M, Hellman N, Schatz D. A modified tetracycline-regulated system provides autoregulatory, inducible gene expression in cultured cells and transgenic mice. Proc Natl Acad Sci USA 1995, 92:6522–6526.
15. Yamamoto T, Omoto S, Mizuguchi M, Mizukami H, Okuyama H, Okada N, et al. Double-stranded nef RNA interferes with human immunodeficiency virus type 1 replication. Microbiol Immunol 2002, 46:809–817.
16. Fang J-Y, Mikovits JA, Bagni R, Petrow-Sadowski CL, Ruscetti FW. Infection of lymphoid cells by integration-defective human immunodeficiency virus type 1 increases de novo methylation. J Virol 2001, 75:9753–9761.
17. Otake K, Ohta M, Minowada J, Hatama S, Takahashi A, Ikemoto A, et al. Extracellular Nef of HIV-1 can target CD4 memory T population. AIDS 2000, 14:1662–1664.
18. Quaranta MG, Camponeschi B, Straface E, Malorini W, Viora M. Induction of interleukin-15 production by HIV-1 Nef protein: a role in the proliferation of uninfected cells. Exp Cell Res 1999, 250:112–121.
19. Murti KG, Brown PS, Ratner L, Garcia V. Highly localized tracks of human immunodeficiency virus type 1 Nef in the nucleus of cells of a human CD4+ T-cell line. Proc Natl Acad Sci USA 1993, 90:11895–11899.
20. Ranki A, Lagerstedt A, Ovod V, Aavik E, Krohn KJ. Expression kinetics and subcellular localization of HIV-1 regulatory proteins Nef, Tat and Rev in acutely and chronically infected lymphoid cell lines. Arch Virol 1994, 139:365–378.
21. Orchard K, Perkins N, Chapman C, Harris J, Emery V, Goodwin G, et al. A novel T-cell protein which recognizes a palindromic sequence in the negative regulatory element of the human immunodeficiency virus long terminal repeat. J Virol 1990, 64:3234–3239.
22. Kersten S, Desvergne B, Wahli W. Roles of PPARs in health and disease. Nature 2000, 405:421–424.
23. Chinetti G, Griglio S, Antonucci M, Torra I, Delerive P, Majd Z, et al. Activation of proliferator-activated receptors α and γ induces apoptosis of human monocyte-derived macrophages. J Biol Chem 1998, 273:25573–25580.
24. Murphy KM, Sweet MJ, Ross IL, Hume DA. Effects of the tat and nef gene products of human immunodeficiency virus type 1 (HIV-1) on transcription controlled by the HIV-1 long terminal repeat and on cell growth in macrophages. J Virol 1993, 67:6956–6964.
25. Ilyinskii PO, Desrosiers RC. Efficient transcription and replication of simian immunodeficiency virus in the absence of NF-kappaB and Sp1 binding elements. J Virol 1996, 70:3118–3126.
26. Barroso I, Gurnell M, Crowley VEF, Agostini M, Schwabe JW, Soos MA, et al. Dominant negative mutations in human PPARγ associated with severe insulin resistance, diabetes mellitus and hypertension. Nature 1999, 402:880–883.
27. Duval C, Chinetti G, Trottein F, Fruchart J, Staels B. The role of PPARs in atherosclerosis. Trends Mol Med 2002, 8:422–425.
28. Hanna Z, Kay DG, Cool M, Jothy S, Rebai N, Jolicoeur P. Transgenic mice expressing human immunodeficiency virus type 1 in immune cells develop a severe AIDS-like disease. J Virol 1998, 72:121–132.
29. Hanna Z, Kay DG, Rebai N, Guimond A, Jothy S, Jolicoeur P. Nef harbors a major determinant of pathogenicity for an AIDS-like disease induced by HIV-1 in transgenic mice. Cell 1998, 95:163–175.
30. Cohen GB, Rangan VS, Chen BJ, Smith S, Baltimore D. The human thioesterase II protein binds to a site on HIV-1 Nef critical for CD4 down-regulation. J Biol Chem 2000, 275: 23097–23105.
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