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*Division of Nephrology and Division of Infectious Diseases, Department of Medicine, Mount Sinai School of Medicine, New York, New York; Laboratory of Virology, Istituto Superiore di Sanità, Rome, Italy.
Correspondence to Dr. Mohammad Husain, Box 1243, Division of Nephrology, Mount Sinai School of Medicine, One Gustave L Levy Place, New York, NY 10029. Phone: 212-241-8041; Fax: 212-987-0389; E-mail: mohammad.husain{at}mssm.edu
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Abstract |
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G/P-EGFP). This construct was then pseudotyped with VSV.G envelope to infect podocytes that were conditionally immortalized with SV-40 T antigen. In addition, mutated constructs were engineered with premature stop codons in the HIV-1 env, vif, vpr, vpu, nef, or rev genes. The parental construct and all the other mutated constructs, with the exception of nef, induced proliferation under nonpermissive conditions and anchorage-independent growth (colony formation in soft agar) under permissive conditions. In contrast, deletion of nef markedly reduced proliferation and colony formation. Although tat alone, or tat plus rev induced marginal levels of anchorage-independent growth, coexpression with nef significantly increased colony formation. Finally, stable expression of Nef in a retroviral vector, pBabe-puro, was sufficient to induce increased proliferation and colony formation. Moreover, nef induced saturation density and loss of contact inhibition. These data indicate that Nef induces multiple proliferative effects in podocytes in culture and that nef may therefore be an important gene in the pathogenesis of HIVAN in vivo. |
Introduction |
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HIV-1 encodes three structural genes (gag, pol, env), two essential regulatory genes (tat and rev), and four accessory genes (vif, vpr, vpu, and nef) (9). An HIV-1 plasmid construct deleted for gag and pol (pNL4–3: d1443) had earlier been used to generate a Tg mouse model of HIVAN (10,11). These animals present with renal disease that is clinically and pathologically identical to that observed in patients with HIVAN. Thus, this construct expressing Env and the accessory proteins (Vif, Vpr, Vpu, Nef, Tat, and Rev) without Gag/Pol can induce HIVAN. Previously, the lack of an in vitro podocyte culture system prevented a detailed analysis of the effects of HIV-1 gene expression on renal podocytes. With, the establishment of conditionally immortalized murine podocytes by SV-40 T antigen (12,13), it is possible to study in vitro the HIV-1 genes responsible for cellular phenotypic changes. We have previously shown that conditionally immortalized nontransgenic podocytes are growth-inhibited at confluence, whereas HIV-1 Tg podocytes continued to proliferate after confluence, suggesting that HIV-1 Tg podocytes are not subject to contact inhibition (13). Furthermore, the growth rate of podocytes, as observed by 3H thymidine uptake, was threefold higher in HIV-1 Tg podocytes when compared with nontransgenic podocytes. The HIV-1 Tg podocytes also form colonies in soft agar, whereas nontransgenic podocytes do not (13). These findings served as the rationale to identify the HIV-1 genes responsible for the growth of cultured podocytes in soft agar. Here, we show that the expression of Nef is sufficient to induce proliferation and anchorage-independent growth of nontransgenic podocytes.
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Materials and Methods |
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(rIFN-; Life Technologies) to induce the H-2Kb promoter driving synthesis of the temperature-sensitive (tsA58) SV-40 T antigen (TAg). At 37°C (nonpermissive temperature [NPT]), the TAg is inactivated and podocytes show differentiated morphology with multiple foot processes.
Generation of Mutant HIV-1 Constructs Using pNL4-3 Provirus
To monitor infection efficiency of HIV-1, a fragment containing the EGFP reporter gene (from pEGFP-C1; Clontech, Palo Alto, CA) was inserted in place of the gag/pol deletion in HIV-1 proviral construct, pNL4–3: d1443 (Figure 1) (11). The resulting construct (pNL4–3: G/P-EGFP) was used to delineate the contribution of individual HIV-1 genes by mutating single or multiple genes. The mutations were made by in vitro site-directed mutagenesis using GenEditor (Promega, Madison, WI) or QuickChange mutagenesis kit (Stratagene, La Jolla, CA). HIV-1 env and/or accessory genes (vif, vpr, vpu, rev, and nef) were individually mutated specifically by inactivating the start codon without affecting the reading frame of other viral proteins that use the same transcript (Table 1). All mutations were confirmed by sequencing, and all the mutated constructs were tested for loss of gene expression by western blotting (15–18). Western blots, however, revealed that the construct mutated for vif and another construct mutated for nef could use downstream ATG codons. Therefore, vif was further mutated by altering the downstream ATG codons at positions 5086 and 5125. The nef downstream ATG was interrupted by digesting with XhoI, filling in with Klenow enzyme and religating with T4 DNA ligase (New England Biolabs, Beverly, MA). Each subsequent construct of vif and nef mutations showed loss of expression by Western blot analysis. To explore a role for tat and rev, tat and nef, or tat alone, these genes were left intact for expression while all the remaining genes were mutated in the vector backbone (Table 1).
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B vector (a gift of Dr. James C. Mulloy, Memorial Sloan Kettering Cancer Center, New York, NY). The genes were amplified by PCR using premix Taq (TaKaRa Biomedicals, PanVera Corp., Madison, WI) and cloned at BamH I-Sal I site or BamH I- EcoR I sites of the vector. The correct orientation of the gene was checked by sequencing, and the expression was confirmed by Western blot analysis. In spite of repeat cloning of nef and examining each time that it was cloned in correct orientation with correct sequences, it was not expressed in podocytes. Therefore, we used pBabe-puro retroviral expression vector (19) and cloned nef at BamH I-Sal I sites. The Nef was expressed in podocytes by this vector as confirmed by Western blotting.
Production of Pseudotyped Retroviral Supernatant
The HIV-1 parental construct (pNL4-3: G/P-EGFP), mutated HIV-1 plasmid constructs, and single HIV-1 gene constructs were used to produce VSV.G pseudotyped viruses to provide pleiotropism and high-titer virus stocks. Infectious viral supernatants were produced by transient transfection of 293T cells using Lipofectamine 2000 (Life Technologies) according to the manufacturer’s instructions. The HIV-1 gag/pol and VSV.G envelope genes were provided in trans using pCMV R8.91 and pMD.G plasmids, respectively (gifts of Dr. Didier Trono, Salk Institute, La Jolla, CA) (20). The Moloney murine leukemia virus gag/pol genes were provided using REP/GP plasmid to produce pseudotyped virus from pBabe-puro. As a negative control, virus was also produced from pHR-CMV-IRES2-GFP-B, which contained HIV-1 LTRs and EGFP as well as pBabe-puro empty expression vectors. The viral stocks were titrated by infecting 293T cells with tenfold serial dilutions. The reciprocal of the lowest dilution showing expression of green fluorescence protein (GFP) was defined as GFP-expressing units (GEU) per ml. Viral stocks ranging from 105 to 108 GEU/ml were obtained. Some low-titer viral stocks were further concentrated by ultracentrifugation.
Infection of Podocytes and Soft Agar Analysis
The podocytes at a concentration of 50,000 cells per plate were seeded in a 6-cm dish in the medium described above but without IFN. The cells were then washed twice the next day with serum-free RPMI 1640 and infected with MOI of 1 to 5 GEU in the presence of 5 µg/ml polybrene. The cells were trypsinized at day 7, and approximately 40,000 cells were suspended in 0.3% soft agar containing 1X RPMI, 1X PenStrep, 10 mM Hepes, pH 7.0, 10% FBS, and 13.2 mM NaHCO3 and then plated on a 6-cm dish before incubation at 33°C for 4 wk. Every 5 d, 1.0 ml of media was replenished.
Cell Growth Assay
Podocytes were infected with a panel of viruses (Table 2) to monitor the growth under NPT and PT. To study growth at NPT, the cells were first allowed to grow at PT on type-1 collagen–coated surface to 50% confluence and then transferred to NPT for a week to inactivate temperature-sensitive TAg. Thereafter, the cells were counted using hemocytometer by trypan blue dye exclusion.
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Results |
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G/P-EGFP (Figure 1) was used as the parental construct, and the following genes, env or one of the accessory genes (vif, vpr, vpu, nef, or rev) was mutated to abolish their expression to screen their precise role in podocyte proliferation. The loss of gene expression was confirmed by Western blot analysis. Virus supernatants were produced and titrated on the basis of GFP expression and then used to infect podocyte cells. Estimates of virus titers showed approximate 80% infection efficiencies as monitored by GFP expression at day 5 after infection (Figure 2, A and D). Western blotting of infected podocyte lysates was performed to confirm that recombination did not occur and to exclude reversion of the mutated gene. In another set of experiments, podocytes were infected with Babe-puro/Nef virus and selected for stable growth in 1.5 µg/ml puromycin antibiotic (Sigma-Aldrich, Inc.). Expression of Nef was confirmed by Western blot analysis (Figure 5A).
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G/P-EGFP) or vector (HR-CMV-IRES2-EGFP) viruses. The wild-type or vector-infected (Table 2) podocytes showed robust proliferation under permissive conditions for TAg function (33°C plus IFN), whereas they growth arrested after 3 to 5 d at the NPT (37°C without IFN) and adopted a differentiated morphology marked by extensive cell flattening and the productions of typical foot processes. In contrast, HIV-infected podocytes proliferated and exhibited dedifferentiation marked by an elongated morphology and the loss of foot processes. Additionally, whereas control podocytes grown to 50% confluence at the PT and then shifted to the NPT showed restricted growth, the HIV-infected cells continued to grow to a higher saturation density and produced foci (data not shown) (Table 2). Interestingly, HIV-infected cells formed colonies in soft agar at PT (Figure 2, B and C) but failed to form colonies at NPT. Taken with the inability of vector-infected podocytes to induce colony formation in soft agar at PT (Figure 2, E and F), this indicates that anchorage-independent growth in our podocytes requires TAg plus at least one HIV-1–encoded product. Most significantly, the ability of gag/pol–deleted pNL4–3 to induce proliferation and dedifferentiation in the absence of TAg correlated directly with the ability to induce contact- and anchorage-independence.
Mapping the HIV-1 Genes that Are Responsible for Anchorage-Independence
A relatively high frequency of colony formation was observed in the cells infected with env, vif, vpr, vpu, or rev viruses (Figure 3, A, B, D, and E), suggesting that colony-forming activity is independent of these genes. In contrast, very few colonies were observed after infection with either vector or nef virus (Figure 3, C and F). This strongly suggests that nef is necessary for inducing anchorage-independence. Because tat is required for robust LTR-derived transcription, all of the above deletion constructs contained functional tat. Therefore, to identify HIV-1 genes required for anchorage-independence, we performed identical experiments using viruses expressing Tat alone, Tat and Rev, or Tat and Nef (constructs described in Table 1). Very low colony-formation activity with a smaller colony size was observed with the expression of Tat alone or Tat plus Rev, whereas strong colony formation was restored when Nef was coexpressed with Tat (Figure 4).
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Growth Parameters Induced by Nef
The results above suggested that Nef is required and sufficient to induce the podocyte anchorage-independence encoded by gag/pol–deleted pNL4–3. We thus investigated whether Nef could induce other morphologic and growth parameters attributable to the HIV-parental virus. At the NPT, Babe-puro/Nef podocytes exhibited a similar dedifferentiated morphology and proliferative ability to that of HIV-infected cells; conversely, both vector and nef virus–infected cells growth-arrested and exhibited a flattened, differentiated morphology at NPT (Table 2). At PT, Babe-puro/Nef and Babe-puro vector podocytes proliferated at a similar initial rate; although the latter cells became contact-inhibited, the Babe-puro/Nef cells continued to proliferate (Figure 6), producing foci in confluent cultures incubated for 2 wk (Figure 7). Nef expression was sufficient to induce focus formation at NPT (Sunamoto M, Husain M, Gelman IH, Schwartz EJ, Ross MD, Klotman PE; unpublished results), although the efficiency was roughly a third of that at PT. These data confirm that Nef can induce loss of contact inhibition in podocytes as was shown previously in NIH 3T3 cells by SIV Nef (21).
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Discussion |
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G/P-EGFP). Specifically, inactivation of the Nef coding sequence in a pNL4–3 viral background ablated proliferation and dedifferentiation in the absence of TAg and anchorage-independent growth in the presence of TAg. Podocytes expressing Nef alone gained the ability to proliferate, dedifferentiate, and form foci independent of TAg and form soft agar colonies in conjunction with TAg. Our data are consistent with earlier studies showing that Nef induces proliferation of other cell types in vitro and in vivo. For example, Nef induces focus formation in NIH 3T3 cells (21) and focus- and soft agar colony-formation in astrocytes in culture (23) and is required for the development of papillomatous skin lesions in HIVd1443 Tg mice (22). Thus, Nef may play similar roles in inducing proliferative lesions associated with HIVAN as well as other HIV-associated sequelae.
Nef has been postulated to be critical for the development of renal disease. Jolicoeur et al. (24) and Hanna et al. (25) reported that Nef was the major determinant of pathogenicity in Tg mice expressing pNL4–3 under the control of a CD4 promoter. Renal disease was reported in these mice, but the specific manifestations of HIVAN were unclear, and there was no evidence of renal expression of HIV-1. Dickie (23) and Kajiyama et al. (26) reported development of HIVAN-like disease but much less severe in Tg mice containing HIVd1443 provirus with an inactivated Nef coding sequence. The decreased severity of HIVAN-like disease in these mice may, however, relate to the complex disease phenotype that contributes to HIVAN. This includes dysregulation of multiple cell types in both tubulointerstitial and glomerular compartments, wherein podocytes are the main contributors to the latter compartment. Taken together with previous reports, our findings suggest that Nef plays a critical role in altering the glomerular phenotype in vivo in HIVAN.
Nef is also a critically important gene product for viral infectivity, host pathogenesis, and evasion of immune surveillance. Rhesus macaques infected with Nef-deleted SIV have much lower viral loads and slower disease progression compared with animals infected with Nef-competent viruses (27). Nef encodes multiple functions capable of modulating cellular signaling pathways. Nef interacts with Src family tyrosine kinases, protein kinase C, mitogen-activated protein kinases (MAPK), serine/threonine kinases, and p21-activated kinases (28). The most dramatic signaling abnormality induced by Nef results from its interaction with the Hck protein tyrosine kinase, resulting in oncogenic transformation of Rat-2 fibroblast (29). Nef induces resistance to tumor necrosis factor–
–induced apoptosis in U251MG astrocyte cell line through interaction with MAPK and consequently promotes cell growth (30). In our study, the presence of Nef clearly triggers podocyte proliferation and dedifferentiation, growth in soft agar, and focus formation. Studies are currently underway to identify the molecular pathways by which Nef induces anchorage-independent growth in podocytes.
Other HIV proteins have also been reported to induce changes in growth properties of cells. Tat promotes aggregation and adhesion of primary rodent cerebellar neurons and astrocytes (31,32). These changes are independent of the ability of Tat to transactivate HIV gene expression. The presence of an arginine-glycine-aspartic acid (RGD) sequence motif in Tat commonly found in the extracellular matrix protein, fibronectin, is essential for the formation of aggregates. Tat-induced aggregation is not due to the induction of proliferation but rather possibly due to the interaction of Tat RGD and basic domains with integrins on the cell surface. Additionally, gp120 at low concentrations has been shown to induce proliferation of human glomerular epithelial cells in culture (33). In our study, however, deletion of Env or expression of Tat alone did not have a significant effect on anchorage-independent growth of podocytes.
Podocytes are thought to be terminally differentiated cells that perform specialized functions, including the maintenance of the glomerular filtration barrier and glomerular capillary architecture (34). Podocytes may proliferate, however, in response to certain types of injuries such as those associated with cellular forms of focal segmental glomerulosclerosis (35,36), collapsing glomerulopathy in HIV-seronegative patients (8,37,38), and HIV-associated nephropathy (39). Additionally, studies such as Nagata et al. (40) have suggested that proliferation of parietal epithelial cells, rather than podocytes, contributes to the collapsing glomerulopathy in HIVAN. It is also possible that the proliferating cells in vivo represent parietal epithelium that has migrated to a new location. The in vitro data presented here are, however, supportive of the hypothesis that the visceral epithelial cells are proliferating in response to HIV-1 gene expression.
A concern in the present study involves the use of temperature-sensitive TAg to facilitate the conditional immortalization of podocytes, presumably through its binding and downregulation of the cellular tumor suppressor p53 and pRb (41). The introduction of TAg alone is insufficient to induce focus formation in our cell system as well as in other cell types (42), although it is clearly required for immortalization. In addition, we observed no significant change in TAg expression after infection with gag/pol-deleted HIV virus expressing or deficient in Nef. However, although Nef could induce podocyte proliferation and contact-independence in the absence of TAg, anchorage-independent growth only occurred in the presence of TAg, suggesting that Nef is not sufficient to induce anchorage-independence in the absence of p53 and/or pRb downregulation. There is no evidence of p53 loss or mutation in HIVAN podocyte; this may therefore explain why the HIVAN lesion is hyperplastic and not neoplastic.
In summary, podocytes undergo dramatic changes in differentiation, lose contact inhibition, and proliferate after expression of HIV-1 as a transgene in mice or after infection in humans. The present data suggest that Nef is required and sufficient to induce proliferation in vitro in podocytes, and we speculate that Nef may have a critical role in the proliferative changes observed in our Tg mouse model of HIVAN and in patients with HIVAN. Further in vivo studies in mice are currently underway to understand the molecular mechanism for Nef-induced proliferation in podocytes.
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) in cell culture for 15 d under permissive conditions. Initially, 10,000 cells suspended in 1.0 ml of growth medium were seeded in 24-well plates. The cells were counted at 3-d intervals in quadruplicate wells after trypan blue dye exclusion. The mean of cells per well ± SD was plotted for each group of cells. The arrow indicates the time at which cells reached to confluence. No statistically significant difference in cell count was observed before confluence (P > 0.47), whereas it was significant after confluence (P < 0.001).
