DEC-205–Mediated Internalization of HIV-1 Results in the Establishment of Silent Infection in Renal Tubular Cells
Ikusuke Hatsukari*,,
Priyanka Singh*,,
Naoko Hitosugi*,,
Davorka Messmer*,
Elsa Valderrama,
Saul Teichberg*,
Wayne Chaung*,
Eleanore Gross*,
Helena Schmidtmayerova* and
Pravin C. Singhal*,
* Center for Immunology and Inflammation, The Feinstein Institute for Medical Research, Manhasset, and Department of Medicine, Long Island Jewish Medical Center, New Hyde Park, New York
Address correspondence to: Dr. Pravin C. Singhal, Department of Medicine, Long Island Jewish Center, 410 Lakeville Road, New Hyde Park, NY 11042. Phone: 516-465-5260; Fax: 516-488-0459; E-mail: singhal{at}lij.edu; and Dr. Helena Schmidtmayerova, The Feinstein Institute for Medical Research, 350 Community Drive, Manhasset, NY 11030. Phone: 516-562-3408; Fax: 516-562-1022; E-mail: hschmidt{at}nshs.edu
Received for publication December 1, 2006.
Accepted for publication December 29, 2006.
HIV-1 infection of renal cells has been proposed to play a rolein HIV-1–associated nephropathy. Renal biopsy data furthersuggest that renal tubular cells may serve as reservoir forHIV-1. The mechanism by which HIV-1 enters these cells has notbeen identified. Renal tubular cells do not express any of theknown HIV-1 receptors, and our results confirmed lack of theexpression of CD4, CCR5, CXCR4, DC-SIGN, or mannose receptorsin tubular cells. The aim of this study, therefore, was to determinethe mechanism that enables viral entry into renal tubular cells.An in vitro model was used to study the HIV-1 infection of humankidney tubular (HK2) cells and to identify the receptor thatenables the virus to enter these cells. Results of these studiesdemonstrate that the C-type lectin DEC-205 acts as an HIV-1receptor in HK2 cells. Interaction of HIV-1 with DEC-205 resultsin the internalization of the virus and establishment of a nonproductiveinfection. HIV-1–specific strong-stop DNA is detectedin the infected HK2 cells for at least 7 d, and the virus canbe transmitted in trans to sensitive target cells. HIV-1 entryis blocked by pretreatment with specific anti–DEC-205antibody. Moreover, expression of DEC-205 in cells that lackthe DEC-205 receptors renders them susceptible to HIV-1 infection.These findings suggest that DEC-205 acts as an HIV-1 receptorthat mediates internalization of the virus into renal tubularcells, from which the virus can be rescued and disseminatedby encountering immune cells.
HIV-associated nephropathy (HIVAN) is one of the clinical manifestationsof AIDS and is the single most common cause of chronic renaldisease in HIV-1–seropositive patients (1–3). HIVANis characterized by proteinuria with rapidly developing azotemiaand histologically by focal glomerulosclerosis (often a collapsingtype) and microcystic dilation of tubules. Other lesions, includingIgA nephropathy, immune complex glomerulonephritis, membranousnephropathy, and proliferative glomerulopathies, which are notpart of HIVAN, are seen in some HIV-infected patients. Recently,Szczech et al. (4) categorized these non-HIVAN renal lesionsas HIV-related renal diseases. The molecular mechanisms thatare involved in these non-HIVAN renal lesions are expected tobe different because these renal lesions bear no resemblanceto the histologic features that are seen in HIVAN. The glomerularlesion–collapsing glomerulopathy has also been reportedin association with other diseases; however, the tubular lesion–microcysticdilation of tubules has not been reported in association withany type of glomerular lesions.
For a long time, it was not clear whether the pathogenesis ofHIVAN was due to HIV infection in the renal cell or to an indirecteffect of the systemically dysregulated immune system (5,6).Studies that were designed to address this issue showed thatHIVAN can occur at any point during AIDS progression with noapparent correlation with either viral burden or CD4+ T cellnumber (7). Using elegant experimental approaches, Bruggemanet al. (8) demonstrated that expression of the HIV transgenein renal cells was necessary and sufficient for the developmentof HIVAN. These observations suggest that a direct effect ofHIV-1 infection is necessary and sufficient for the developmentof HIVAN in patients. In addition, other evidence supports arole for HIV-1 infection of renal epithelium in the pathogenesisof HIVAN (9–12). Using in situ hybridization, Marras etal. (12) showed HIV-1 gag and nef mRNA in renal epithelial cellsof patients with HIVAN. The same group showed that phylogeneticanalysis of the DNA sequence from infected renal epithelialcells as well as the corresponding sequences from peripheralblood mononuclear cells of the same patients reveal evidenceof tissue-specific viral evolution (12).
The mechanism by which HIV-1 enters the renal epithelium remainsunresolved (10,12,13). Renal cells do not express any knownHIV-1 receptors. Although the presence of CD4, CCR5, and CXCR4in tubular cells was reported in one study (10), others failedto confirm these data (13). The lack of conventional HIV-1 receptorshas prompted many investigators to propose unconventional modesof viral entry, including passive transfer of receptors fromone cell to another or fusion of tubular cells with HIV-1–infectedperipheral blood mononuclear cells and subsequent viral transmission(14,15).
Recently, dendritic cells (DC) have been shown to act as anHIV-1 reservoir (16–18), in which the virus is endocytosedafter binding to the C-type lectin receptor DC-SIGN (19–21).The family of C-type lectins is expressed in many cell types,including macrophages and dendritic cells, which internalizevarious glycoproteins and microbes for the purposes of clearanceand antigen presentation to T lymphocytes (22). In this, studywe focused on a C-type lectin receptor, DEC-205, which is abundantlyexpressed not only on DC but also on many epithelial cells (23–26).This 205-kD protein contains 10 external, contiguous, C-typelectin domains and is homologous to macrophage mannose receptor(MMR), previously shown to bind HIV-1 (27). Adsorptive endocytosisreceptors such as MMR, FcR, and B cell antigen receptor areused by immune cells to facilitate antigen capture and presentationof peptides to T cells (22). Both the MMR and DEC-205 mediateadsorptive uptake, and both have cytosolic domains with requisitecoated pit localization sequences (28). Despite that DEC-205shares structural homology with mannose receptor, the specificityof ligand binding is different. Here, we report that renal tubularcells do not express either DC-SIGN or MMR; however, DEC-205is expressed in renal tubular cells and serves as a novel receptorfor HIV-1, which mediates virus internalization and persistentlatent infection of tubular cells.
Cells and Viruses
HK2 and 293T cells were obtained from ATCC. Primary culturesof human renal proximal tubular cells (HRPT) were obtained fromScienCell (San Diego, CA). Primary macrophages and lymphocyteswere isolated from peripheral blood mononuclear cells as describedpreviously (29). 293T cells were transfected with plasmid expressinghuman DEC-205 (gift from Dr. Ralph Steinman, The RockefellerUniversity, New York, NY) or green fluorescence protein (GFP;Clontech, Mountain View, CA) using FuGENE (Roche, Indianapolis,IN) transfection. HK-2 and 293T cells were infected overnightwith primary HIV-1 strains, R5 strain HIV-1ADA or X4 strainHIV-1HT/92/599. Afterward, noninternalized virus was removedby incubation with 0.05% trypsin at 37°C for 10 min, followedby extensive washing. Viral stock of HIV-1ADA was prepared inprimary macrophages that were cultivated in the presence ofmacrophage colony stimulating factor. Viral stock of HIV-1HT/92/599was prepared in phytohemagglutinin-activated primary lymphocytesthat were cultivated in the presence of IL-2. Before infection,viral stocks were treated with 200 U of RNase-free DNase perml (1 h at room temperature) to eliminate contamination withviral DNA. In co-cultivation studies, HK2 and 293T cells weredetached and added to macrophages or lymphocytes in a ratioof 1:2. Target cells were maintained with infected HK2 and 293Tcells for 3 d. Afterward, target cells were transferred to anew plate, and virus replication in collected supernatants wasanalyzed by p24gag antigen assay according to the manufacturer’sinstruction (ZeptoMetrix Corp., Buffalo, NY). In blocking studies,cells were pretreated with 5 µg/ml of anti–DEC-205antibody (clone MG38) or isotype control (both from Serotec,Raleigh, NC) for 30 min before HIV-1 infection.
Reverse Transcriptase–PCR
Total RNA was isolated using RNeasy Kit (Qiagen, Valencia, CA).To eliminate DNA contamination, extracted RNA was subjectedto digestion using RNase free DNase. Reverse transcription wasperformed with 1 µg of RNA using Moloney murine leukemiavirus reverse transcriptase (Life Technologies BRL, Carlsbad,CA) in a reaction mix that contained 5 mM MgCl2, 50 mM KCl,10 mM Tris-HCl, 1 mM of dNTP, 2.5 µM random hexamers,and 2.5 U/µl Moloney murine leukemia virus. The resultingcDNA was amplified, using primers that were specific for CCR5(sense 5'-GGTGGAACAAGATGGATTAT-3'; antisense 5'-ATGTGCACAACTCTGACTG-3'),CXCR4 (sense 5'-AGCGAGGTGGACATTCATC-3'; antisense 5'-ACGTGATTCACTACAGCTC-3'),CD4 (sense 5'-TTGGAGTCGCAAGCTGAACTAGCG-3'; antisense 5'-CCAGGAAGTTGAGGCTGCAGTGAA-3'),DC-SIGN (sense 5'-GGGGGCCCAGCTCGTCGTAATC-3'; antisense 5'-ACCCCCAAAGGCATCCCACACC-3'),mannose receptor (sense 5'-GCAGGGGGCTTATGGGATGTT-3'; antisense5'- TTGGCTCAGGTTTTGGTGTTTGTC-3'), DEC-205 (sense 5'-ACTTCTGGACTGGCCTGAGA-3';antisense 5'-TCGTTCAGCTTCTTCCCAGT-3'), and -tubulin (sense 5'-GTTGGTCTGGAATTCTGTCAG-3';antisense 5'-AAGAAGTCCAAGCTGGAGTTC-3').
Detection of HIV-1-Specific DNA by PCR
Before preparation of cell lysates for PCR, infected cultureswere treated with trypsin at 37°C for 10 min, followed byextensive washing. Cell lysates were subjected to PCR analysisusing HIV-1–specific primers, amplifying LTR RU5 and poltranscripts as described previously (29). Amplified DNA wasanalyzed by Southern blot hybridization using 32P-labeled probesand quantified using an Instant Imager System (Packard, Meriden,CT). Amplification of the -tubulin gene was used to controlfor the amount of DNA in each sample. Serial dilutions of 8E5/LAIcells, which contained one HIV-1 genome per cell, were includedin each amplification reaction to standardize the results.
Electron Microscopy
HIV-1–infected cells were fixed in 2% glutaraldehyde,buffered with 0.05 M Na cacodylate (pH 7.3). Fixed cells wererinsed in cold buffer that contained 7% sucrose, scraped, andpelleted. The cell pellet was postfixed in osmium tetroxideand prepared for electron microscopic (EM) studies by routineprocedures.
Flow Cytometric Analysis
Cells were washed with PBS and blocked with PBS that contained20% human serum for 20 min at room temperature. Afterward, cellswere stained with anti–DEC-205 mAb, directly labeled withFITC (Clone MG38; eBiosciences, San Diego, CA), which was previouslyshown to bind specifically DEC-205 receptor (30), or with correspondingmouse isotype antibody IgG2b-FITC (eBiosciences) for 30 minat room temperature. After washing, cells were fixed with 2%buffered formalin. Staining was analyzed on FACS Calibur (BectonDickinson, San Jose, CA) using Cell Quest software.
Immunofluorescence Microscopy
Human renal tissue specimens, obtained from the nephrectomizedkidney for renal cell carcinoma, were fixed with 4% paraformaldehydefor 8 h, transferred to PBS for overnight incubation at 4°C,embedded in OCT, and stored at –80°C. Frozen sectionswere cut (5 µm) and blocked with 2% BSA in PBS, followedby incubation with anti–DEC-205 mAb, directly labeledwith FITC (Clone MG38; eBiosciences), or with correspondingmouse isotype antibody IgG2b-FITC (eBiosciences) for 60 minat room temperature. After staining, sections were washed withPBS, mounted with fluorescence mounting media, and examinedunder a fluorescence microscope.
HIV-1 Infection of HK2 Cells
For determination of susceptibility of HK2 cells to HIV-1 infection,cells were infected with one of two primary strains, R5 HIV-1ADAor X4 HIV-1HT/92/599. Immediately after infection, cells weretrypsinized and washed extensively to eliminate noninternalizedvirus. Three and 7 d later, cell culture supernatants were collectedand analyzed for the HIV-1–specific p24gag protein. Underthese conditions, no significant production of p24gag in HIV-1–infectedHK2 cultures (data not shown) was detected. Therefore, for determinationof whether the virus might enter HK2 cells and establish nonproductiveinfection, equal numbers of HIV-1ADA–or HIV-1HT/92/599–infectedHK2 cells were co-cultivated with primary macrophages or lymphocytes,respectively, for 3 and 7 d. Subsequently, viral replicationin primary macrophages and lymphocytes was analyzed by p24gagproduction. Although HIV-1–specific p24gag levels wereundetectable in the supernatants of infected HK2 cells (datanot shown), both viruses, HIV-1ADA and HIV-1HT/92/599, wererescued from 3 d-infected HK2 cells by co-cultivation with macrophagesand preactivated lymphocytes, respectively (Figure 1). The markeddifference between HIV-1 strains’ ability to persist inHK2 cells has been detected at day 7 after infection. Whereasco-cultivation of HIV-1HT/92/599–infected HK2 cells withlymphocytes yielded positive results with similar kinetics ofvirus replication in lymphocytes as detected after 3 d of infection,co-cultivation of HIV-1ADA–infected HK2 cells with macrophages7 d after infection yielded negative results (Figure 1). Thesedata suggest that although both HIV-1 strains enter HK-2 cellsand HIV-1HT/92/599 can persist in HK2 cells for at least 7 d,the infection is not productive.
Figure 1. Recovery of replication-competent virus from HIV-1–infected HK2 cells. HK2 cells were infected overnight with R5 strain HIV-1ADA (left) or X4 strain HIV-1HT/92/599 (right). After infection, cells were treated with 0.05% trypsin followed by extensive washing to eliminate noninternalized virus. Cells that were infected with R5 and X4 HIV-1 strains were co-cultivated with primary macrophages and lymphocytes, respectively, 3 and 7 d after infection. Viral replication was analyzed in macrophage and lymphocyte supernatants that were collected at the indicated time points.
HIV-1 Establishes Nonproductive Infection in HK2 Cells
For determination of whether viral infection results in pre-or postintegration latency, cell lysates from HIV-1ADA–andHIV-1HT/92/599–infected HK2 cells were prepared at differenttimes after infection and analyzed for the HIV-1–specificproducts of reverse transcription by PCR using HIV-specificprimers that detect early strong-stop DNA (LTR RU5) and late(pol) transcripts. As shown in Figure 2A, HIV-1–specificstrong-stop DNA was detected in both HIV-1ADA–and HIV-1HT/92/599–infectedHK2 cells during a 7-d follow-up period, although the levelsdecreased between days 3 and 7 in HIV-1ADA–infected cells.However, infection with neither strain yielded positive poltranscripts. Because nascent synthesis of HIV-1–specificstrong-stop DNA can occur within the viral particle (31), wewished to ascertain that detected strong-stop DNA was associatedwith internalized virus. Each sample for PCR was treated withtrypsin twice: After infection and again before preparationof cell lysates. Complete removal of noninternalized virus bytrypsinization has been verified after the incubation of cellswith the virus at 4°C, when the virus binds but does notenter cells. The results of these experiments showed that trypsinizationeliminated positive HIV-specific nascent strong-stop DNA signalin samples that were incubated at 4°C. In contrast, samplesprepared from cells that were incubated at 37°C showed positivestrong-stop DNA also after trypsinization (Figure 2B). In fact,trypsinization resulted in increased intracellular strong-stopDNA levels.
Figure 2. HIV-1 enters HK2 cells without establishing productive infection. (A) HK2 cells were infected with HIV-1ADA and HIV-1HT/92/599. After the infection, noninternalized virus was eliminated by trypsinization. At the indicated times after infection, cells were trypsinized again, lysed, and analyzed by PCR using primers that are specific for the HIV-1 strong-stop DNA (LTR RU5) and pol gene. Amplification of the -tubulin gene was used to control for the amount of DNA. (B) HK2 cells were incubated with the virus for 6 h at either 4 or 37°C. Afterward, half of the cultures were trypsinized, and remaining cells were left untrypsinized. After washing, cell lysates were prepared and analyzed by PCR using primers that are specific for the HIV-1 strong-stop DNA (LTR RU5). Amplification of the -tubulin gene was used to control for the amount of DNA. (C) Dilutions of 8E5/LAI cells that contained one HIV-1 genome per cell were used as PCR standards (HIV-1 copies).
Expression of HIV-1 Receptors in Renal Tubular Cells
Next, we focused our attention on the receptors that mediateHIV-1 internalization in HK2 cells. Previous studies showedlack of the expression of classical HIV-1 receptors in renalcells (13). However, because one study showed expression ofCD4, CCR5, and CXCR4 in renal cells (10), we wished to verifythe expression of known HIV-1 receptors, conventional as wellas C-type lectins. We analyzed mRNA expression of these receptorsin human renal tubular cells by reverse transcriptase–PCR(RT-PCR). In agreement with previously published data (13),we did not find any detectable mRNA levels of CD4, CCR5, orCXCR4 receptors (Figure 3A). Furthermore, neither HK2 cellsnor primary HRPT expressed DC-SIGN or a mannose receptor, previouslyshown to bind HIV-1 (19,27). However, they both expressed relativelyhigh levels of DEC-205 mRNA (Figure 3B), although surface expressionof DEC-205 was low (Figure 3C).
Figure 3. Analysis of the expression of classical HIV-1 receptors and C-type lectins in renal tubular cells. (A) Total RNA, extracted from HK2 cells and from primary lymphocytes that were activated with phytohemagglutinin/IL-2 (a positive control), was subjected to reverse transcriptase–PCR (RT-PCR) amplification using primers that are specific for CD4, CCR5, and CXCR4. -Tubulin amplification served as a control for equal efficiency of amplification. (B) RT-PCR amplification of C-type lectin receptor transcripts in HK2 cells and in primary human renal tubular cells (HRPT). Immature dendritic cells (DC) were used as a positive control. -Tubulin amplification served as a control for equal efficiency of amplification. (C) DEC-205 expression in HK-2 cells that were analyzed by flow cytometry after staining with anti–DEC-205 antibodies.
Anti–DEC-205 Antibody Blocks HIV-1 Entry into HK2 Cells
To determine whether DEC-205 mediates HIV-1 entry in tubularcells, we carried out blocking studies. In these studies, HK2cells were pretreated with anti–DEC-205–specificantibody before infection with HIV-1ADA or HIV-1HT/92/599. Threedays after the infection, HIV-1–specific strong-stop DNAwas analyzed by PCR. Pretreatment with matching isotype antibodyserved as a control. Obtained results show that pretreatmentwith anti–DEC-205 antibody markedly decreases entry ofboth viruses into HK2 cells (Figure 4), suggesting that DEC-205facilitates HIV-1 entry into the tubular cells.
Figure 4. Anti–DEC-205–specific antibody inhibits HIV-1 entry into HK2 cells. HK2 cells were pretreated with anti–DEC-205 or an isotype control before infection with HIV-1ADA and HIV-1HT/92/599. Cells were trypsinized immediately after the infection and again before preparation of cell lysates at day 3 after the infection. Presence of HIV-1–specific strong-stop DNA (LTR RU5) was determined by PCR. Amplification of -tubulin gene was used to control for the amount of DNA.
Expression of DEC-205 in Cells That Lack the DEC-205 Receptors Renders Them Susceptible to HIV-1 Infection
For verification that DEC-205 binds and mediates internalizationof HIV-1, 293T cells, which do not express DEC-205, were transfectedwith DEC-205–expressing plasmid. Approximately 50% oftransfected cells expressed DEC-205 3 d after transfection,whereas no DEC-205 was detected in control cells (Figure 5A).Control, GFP-transfected, and DEC-205–transfected cellswere infected with HIV-1ADA or HIV-1HT/92/599 and analyzed forsynthesis of HIV-1–specific strong-stop DNA. Similar toHK2 cells, DEC-205–transfected cells showed positive HIV-specificstrong-stop DNA but negative pol transcripts, whereas no signalwas detected in GFP-transfected cells (Figure 5B, top). We furtherverified complete removal of noninternalized virus by trypsinizationafter the incubation of DEC-205–transfected cells withthe virus at 4°C. The results showed similar effect of trypsinizationas detected in HK2 cells, an increase in intracellular strong-stopDNA levels in cells that were incubated at 37°C and completeremoval of the virus after the incubation at 4°C (Figure 5B,bottom left). Finally, both HIV-1ADA and HIV-1HT/92/599 wererescued from DEC-205–transfected 293T cells by co-cultivationwith human macrophages and lymphocytes, respectively, whereasno p24gag was detected in supernatants of cells that were co-cultivatedwith HIV-1–infected GFP-transfected 293T cells (Figure 5C).It is interesting that high expression of DEC-205 in 293T-transfectedcells (Figure 5A) enabled extended persistence of HIV-1ADA,and the virus was transmitted to macrophages even after 7 dof infection (Figure 5C).
Figure 5. DEC-205 mediates HIV-1 entry into target cells. (A) Expression of DEC-205 in transfected 293T cells. Cells were transfected with plasmid that expressed DEC-205 and stained 3 d later with FITC-labeled anti–DEC-205 antibody (right). (Left) DEC-205 expression in control cells. (B) DEC-205 expression renders 293T cells susceptible to HIV-1 infection. DEC-205 or green fluorescence protein (GFP)-transfected 293T cells were infected with HIV-1ADA and HIV-1HT/92/599, trypsinized after the infection and before preparation of cell lysates, and analyzed for HIV-1–specific strong-stop DNA and pol transcripts by PCR. Amplification of -tubulin gene was used to control for the amount of DNA (top). (Bottom left) Removal of noninternalized virus by trypsinization. Analysis was performed as described in the legend to Figure 2. (Bottom right) dilutions of 8E5/LAI cells that contained one HIV-1 genome per cell used as PCR standards. (C) Transmission of HIV-1 from DEC-205–transfected 293T cells to primary macrophages and lymphocytes. 293T cells that were transfected with DEC-205 or control GFP-expressing plasmid were infected with HIV-1ADA and HIV-1HT/92/599, trypsinized after the infection, and cultivated for 3 and 7 d before co-cultivation with primary macrophages (left) or lymphocytes (right).
Intracellular Localization of Virus by EM Studies
To ascertain further the internalization of the virus, we performedEM studies. HK2 cells and DEC-205–transfected 293T cellswere incubated with the virus for 6 h. Afterward, cells werewashed, fixed, and analyzed by EM. Results of these studiesrevealed the presence of intracellular virus-like particleswithin early multivesicular bodies in HK2 cells (Figure 6A)and DEC-205–transfected 293T cells (Figure 6B). Thesedata support results that are presented in Figures 2 and 5 indicatingthat HIV-1 virions are internalized and persist inside HK2 andDEC-205–transfected cells.
Figure 6. Detection of virus-like particles within intracellular multivesicular bodies in HIV-1–infected cells by electron microscopy. A virus-like particle (small arrow) seen within a multivesicular body (large arrow) in HIV-1–infected HK2 cells (A) and DEC-205–transfected 293T cells (B). Magnifications: x59,900 in A; x31,000 in B.
DEC-205 Expression in Human Renal Tissue
Our data strongly support the role of DEC-205 in HIV-1 internalizationand establishment of latent infection in HK2 cells. To ascertaina potential relevance of these data to the in vivo condition,we examined the expression of DEC-205 by immunofluorescencestaining of human renal tissues specimens that were obtainedfrom the nephrectomized kidney. Results from this study showedthat renal tissue expressed relatively high levels of DEC-205receptors following the pattern of tubular cell localization(Figure 7, right), whereas no staining was detected in the sectionthat was incubated with an isotype antibody (Figure 7, left).
Figure 7. Immunofluorescence analysis of DEC-205 expression in human renal tissues. Frozen sections from human renal tissues were stained with FITC-labeled anti–DEC-205–specific antibody (right) or FITC-labeled mouse IgG2b isotype control (left) and examined under the fluorescence microscope.
The data presented in this study demonstrate that primary R5and X4 HIV-1 strains enter renal tubular cells via DEC-205–mediatedinternalization. The internalized virus does not proceed throughreverse transcription; therefore, the infection is nonproductive.However, internalized replication-competent virus can persistinside the cells and ignite viral replication upon transmissionto sensitive targets, despite data from EM studies showing onlya low number of intracellular virus-like particles. As expected,only a low level of HIV-1–specific strong-stop DNA isdetected in infected cells. Still, the infectious virus canbe recovered from these cells after several days, suggestinga high efficacy of viral transmission from tubular cells toprimary macrophages and lymphocytes and substantial replicativecapacity of the internalized viruses. These results are in agreementwith previously published data showing a high efficiency ofviral transmission in trans. It has been shown that C-type lectinsDC-SIGN and DC-SIGNR capture and internalize HIV-1 particlesand promote enhanced trans infection of T cells (19,20). Evenundetectable levels of viral particles that are captured byDC can initiate vigorous infection upon dissemination to T cells(32). Similarly, a low amount of infectious virions that aretransmitted from macrophages to lymphocytes in trans can initiaterapid and efficient viral replication in lymphocytes (33,34).
Renal biopsy data suggested that renal cells may serve as areservoir for HIV-1 (12); however, the mechanism by which HIV-1enters these cells has not been identified. Results presentedhere suggest that virus that is internalized after the interactionwith DEC-205 may persist inside cells and establish silent infection,thereby enabling renal cells to serve as a reservoir for HIV-1.However, studies presented here were performed only during arelatively short-term period, up to 7 d after the infection.To determine whether virus that is internalized via DEC-205may persist inside tubular cells for an extended period of timeand establish long-term latent reservoirs would require furtherstudies.
This study indicates that trypsin treatment increases synthesisof viral nascent strong-stop DNA. This is an interesting observationthat might have an implication in increased infectivity andreplicative capacity of the virus upon transmission to targetcells, as previously suggested by Zhang et al. (35). The mechanismthat is responsible for this phenomenon has not been established.However, on the basis of published data, it is plausible toenvision that trypsin activates protease-activated receptor-1(PAR-1) and/or PAR-2 receptors (36), both expressed on renalcells (37). The activation of PAR in tubular cells by thrombin,another ligand of PAR, has been shown to stimulate DNA synthesis(37). During chronic renal disease, including HIVAN, fibrindeposition is frequently observed within tubulointerstitiumand suggests the presence of components of the coagulation pathway,including thrombin in a close proximity of renal tubular cells.Therefore, it is plausible to envision implication of this observationin the enhanced infectivity of the virus under in vivo conditions.However, further studies are necessary to determine the exactmechanism(s) responsible for this phenomenon and to validatetheir in vivo implication.
Besides CD4, which is the main HIV-1 receptor, members of thechemokine receptor family act as the essential co-receptorsrequired for virus entry into susceptible cells (38,39). Generally,expression of these molecules determines the cell permissivenessto productive HIV infection. According to a majority of publishedstudies, renal cells do not express these classical HIV-1 receptors(12,13). Furthermore, renal biopsy studies had also failed todemonstrate the expression of the conventional HIV-1 receptorby intrinsic renal cells in parenchyma of kidney in healthyand diseased states (40,41). In agreement with these publisheddata, our results from in vitro studies show lack of the expressionof CD4, CCR5, or CXCR4 mRNA in renal tubular cells. Furthermore,neither HK2 cells nor primary HRPT expressed DC-SIGN or a mannosereceptor that were previously shown to bind HIV-1 (19,27). However,both HK2 and HRPT cells expressed relatively high levels ofDEC-205 mRNA. We cannot exclude completely that low levels ofclassical receptors, which are under the detection limit ofRT-PCR, are expressed in renal cells. However, the blockingstudies with anti–DEC-205 antibody strongly support therole of DEC-205 in HIV-1 internalization. The role of DEC-205as an HIV receptor is further strengthened by DEC-205 transfectionstudies, which clearly demonstrate that upon DEC-205 expression,transfected cells acquire the ability to internalize and harborreplication-competent virus. It is possible to envision thatDEC-205 serves as an HIV receptor also under in vivo condition,because we detected a relatively high expression of DEC-205in renal tissue (Figure 7). Presumably, DEC-205 that is expressedon the basolateral surface of tubular cells might come to thecontact with virus that arrives from peritubular capillariesand mediate viral internalization.
DEC-205 is a professional endocytic receptor that plays an importantrole in antigen presentation. DEC-205–captured antigensare targeted into late endosomes or lysosomes that are richin MHC II products, where antigen is processed for presentationin the context of MHC II to T cells (26). The virus that isinternalized by DEC-205 therefore should be targeted to lateendosomes or lysosomes for degradation. However, our resultsshow persistence of replication-competent virus in tubular cells,suggesting that either a fraction of the incoming virus mayescape degradation and persist in endosomal compartments, orthe virus actively redirects intracellular trafficking. In eithercase, intravesicular localization of the virus raises the questionof how the virus persists in these acidic intracellular compartments.It was suggested previously that DC-SIGN–mediated internalizationinto a mildly acidic compartment may result in stabilizationof the virus and preservation of its infectivity (42). However,another study suggested that the majority of DC-SIGN–capturedvirions are targeted for degradation and lose infectivity rapidlywithin several hours (43). The latest results would suggestdifferent pathways for DC-SIGN–and DEC-205–mediatedvirus internalization. However, further work would be necessaryto clarify this question and determine the mechanisms that enablepersistence of the virus in tubular cells.
Our data show that under in vitro conditions, tubular cellsmay harbor the virus and transmit infection to encounteringimmune cells. Previously, many studies tried to determine whetherrenal cells are infected with HIV-1 by analyzing renal biopsiesfrom HIV-infected patients. The results of these studies arecontroversial (40,41,44–46). Several studies reportedthe presence of HIV-1 proteins in tubular and glomerular epithelialcells in renal biopsy specimens from patients with HIVAN (6,44–46).However, other studies failed to substantiate these findings(40,41). One plausible explanation for these discrepancies isthe use of antibodies with different sensitivity or specificityfor HIV-1 proteins. As discussed here, although a productiveinfection of tubular cells is plausible, the putative viralreplication must be very low, because, up to now, no publisheddata from EM studies have shown production of the viral particlesin renal cells. The assumption that HIV replicates in renalcells is based on immunohistochemical studies mentioned previouslyand on the detection of viral mRNA by RT-PCR and in situ RT-PCRamplification in microdissected glomeruli, tubules, interstitialcells, and infiltrating inflammatory cells in renal biopsies(46). In addition, Marras et al. (12) demonstrated productiveviral infection in renal tubular cells that were obtained froma patient with HIV infection. Contrary to these studies, ourdata suggest that under in vitro conditions, infection of renaltubular cells is nonproductive. One possible explanation forthis discrepancy is that under in vivo conditions, environmentin a kidney that has infiltrated T cells and macrophages, whichproduce a variety of cytokines, provides signals that enableviral "escape" from endosomal compartments followed by the establishmentof a low level of productive infection. However, further studieswill be necessary to test this hypothesis.
Our data demonstrate that in human kidney tubular cells, C-typelectin DEC-205 acts as an HIV-1 receptor, which mediates internalizationof the virus and the establishment of silent infection. Internalizedvirus does not proceed through reverse transcription; however,replication-competent virus persists inside cells for at least7 d and can be transmitted to encountering immune cells in trans.These data support the hypothesis that renal tubular cells mayharbor infectious virus.
This work was supported by National Institutes of Health grantDA12111 (P.C.S.).
This work was presented at the 39th Annual Renal Week Meetingof the American Society of Nephrology; November 14 through 19,2006; San Diego, CA.
We thank Dr. Ralph Steinman for hDEC-205–expressing plasmid.HIV-1ADA and HIV-1HT/92/599 were obtained through the AIDS Researchand Reference Reagent Program, Division of AIDS, National Instituteof Allergy and Infectious Disease.
Footnotes
Published online ahead of print. Publication date availableat www.jasn.org.
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Abstract
Introduction
-tubulin (sense 5'-GTTGGTCTGGAATTCTGTCAG-3'; antisense 5'-AAGAAGTCCAAGCTGGAGTTC-3'). 
