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Human immunodeficiency virus (HIV) infection results in a functional impairment of CD4+ T cells long before a quantitative decline in circulating CD4+ T cells is evident. The mechanism(s) responsible for this functional unresponsiveness and eventual depletion of CD4+ T cells remains unclear. Both direct effects of cytopathic infection of CD4+ cells and indirect effects in which uninfected “bystander” cells are functionally compromised or killed have been implicated as contributing to the immunopathogenesis of HIV infection. Because T-cell receptor engagement of major histocompatibility complex (MHC) molecules in the absence of costimulation mediated via CD28 binding to CD80 (B7-1) or CD86 (B7-2) can lead to anergy or apoptosis, we determined whether HIV type 1 (HIV-1) virions incorporated MHC class I (MHC-I), MHC-II, CD80, or CD86. Microvesicles produced from matched uninfected cells were also evaluated. HIV infection increased MHC-II expression on T- and B-cell lines, macrophages, and peripheral blood mononclear cells (PBMC) but did not significantly alter the expression of CD80 or CD86. HIV virions derived from all MHC-II-positive cell types incorporated high levels of MHC-II, and both virions and microvesicles preferentially incorporated CD86 compared to CD80. CD45, expressed at high levels on cells, was identified as a protein present at high levels on microvesicles but was not detected on HIV-1 virions. Virion-associated, host cell-derived molecules impacted the ability of noninfectious HIV virions to trigger death in freshly isolated PBMC. These results demonstrate the preferential incorporation or exclusion of host cell proteins by budding HIV-1 virions and suggest that host cell proteins present on HIV-1 virions may contribute to the overall pathogenesis of HIV-1 infection.
The envelope of human immunodeficiency virus type 1 (HIV-1) is comprised of host cell membrane-derived proteins and lipids incorporated into the envelope when the virion buds from an infected cell (reviewed in references 34 and 48). More than 20 different host cell-derived proteins have been identified in the HIV-1 envelope, including major histocompatibility complex class I (MHC-I) and MHC-II; the adhesion molecules CD44; LFA-1, -2, and -3; and ICAM-1 and ICAM-3 (2, 4, 21, 33). These virion-associated, host cell-derived proteins can serve as markers by which to identify the type of cell from which a virion budded (4, 6, 15). The molecular phenotype of the HIV virion envelope has been used to determine whether HIV virions produced in vivo budded from a macrophage (MΦ) or an activated T cell (27, 38). Incorporation of host cell-derived proteins into virions is not random or simply a function of expression level or density on the cell surface, since proteins that are highly expressed on infected cells, such as CD4, CD45, and the coreceptors CXCR4, CCR3, and CCR5, are not incorporated into virions (7, 15, 21, 25, 29).
Many cellular proteins incorporated into HIV-1 virions retain their biological function. For example, CD44 on the virion has been shown to bind hyaluronic acid (20) and CD55 (decay-accelerating factor) or CD59 present in the virion envelope can provide resistance to complement-mediated lysis (42, 43). The HIV virion envelope is enriched for HLA-DR but not DP or DQ (2, 6, 18, 45), and virion-associated MHC-II can bind and present the superantigen Staphylococcus enterotoxin B to resting T cells, resulting in T-cell activation (39). These observations demonstrate that virion-associated host cell proteins are functional and may play a role in HIV pathogenesis.
Normally, T cells require two signals to become fully activated. Signal one is antigen (Ag) specific and is generated by binding of the T-cell receptor (TCR) to Ag-MHC complexes on the Ag-presenting cell (APC). The second signal, a costimulatory signal, is generated by CD28 on the T cell interacting with CD80 (B7-1) or CD86 (B7-2) on an APC (reviewed in reference 19). We have previously reported that microvesicles and HIV-1 virions incorporate high levels of MHC-I and MHC-II upon budding (2, 5) and have hypothesized that virion- or microvesicle-associated MHC-I or MHC-II, with or without bound antigenic peptides, could bind to and signal through the TCR on responding T cells. It has not been previously determined whether CD80 and CD86 are incorporated into budding HIV-1 virions or microvesicles. Because TCR signaling in the absence of costimulation can lead to anergy or apoptosis, we examined whether microvesicles and/or HIV-1 virions incorporate CD80 or CD86 into their membranes. Here we report that HIV infection of cell lines, MΦ, and primary peripheral blood mononuclear cells (PBMC) upregulates cell surface expression of MHC-II and that virions derived from all of these cells incorporated MHC-II. CD86 was detected on virions produced from 17 of 21 sets of different virus isolates propagated on different cells, whereas CD80 was detected on virions from only 3 of the same 21 viruses produced from CD80- and CD86-expressing cells. Microvesicles were also enriched for CD86, whereas CD80 was excluded. CD45 was identified as a protein that was highly expressed on microvesicles but not on HIV-1 virions.
These data suggest that HIV has evolved to preferentially incorporate some immunoregulatory proteins, such as MHC-II and CD86, but to exclude other proteins like CD45 and CD80. The host cell molecules incorporated into virions influenced the biological effects of the virus. Noninfectious, MHC-containing HIV virions derived from the CEMX174/T1 cell line triggered cell death in resting PBMC, whereas noninfectious, MHC-negative virions derived from the matched CEMX174/T2 cell line did not. These findings suggest that HIV has evolved to preferentially incorporate certain immunoregulatory proteins into virions, potentially contributing to the ability of the virus to evade the immune system and contribute to pathogenesis.
Uninfected cell lines H9 (13), CEMX174/T1, CEMX174/T2 (44), and TBLCL-CD4 (30) were cultured in RPMI 1640 medium with 5% heat-inactivated fetal bovine serum, 2 mM l-glutamine, penicillin G at 100 U/ml, and streptomycin sulfate at 100 μg/ml (complete medium). Chronically HIV-1-infected cell lines MN/H9, NL4-3/H9, NL4-3/CEMX174/T1, NL4-3/CEMX174/T2, and NL4-3/TBLCL-CD4 were also cultured in complete medium. All cell lines were split twice weekly at 3 × 105 cells/ml, were mycoplasma negative (PCR Mycoplasma Detection Kit; American Type Culture Collection, Manassas, Va.), and were cultured in complete medium.
H9, CEMX174/T1, CEMX174/T2, and TBLCL-CD4 cells chronically infected with HIV-1NL4-3 were cultured as described previously (35). For experiments involving the induction of cell death, conformationally authentic noninfectious HIV-1 virions were prepared as previously described (1, 40). Concentrated (1,000×) virus preparations were produced by sucrose density gradient banding in a continuous-flow centrifuge (1, 5, 40). For the virion precipitation experiments, different HIV-1 isolates were examined, including patient isolates PI08-436, P2-285, P419, and P115 derived from ex vivo expansion of primary PBMC (a generous gift from Antonio Valentin, National Cancer Institute [NCI] at Frederick). Clade B, R5 patient isolates HIV-191US054, HIV-192US727, and HIV-192US657 (49), grown in PBMC activated with phytohemagglutinin (PHA) plus interleukin-2 (IL 2; 10 U/ml; Hoffman-La Roche, Nutley, N.J.), were acquired from the National Institute of Allergy and Infectious Diseases AIDS Research and Reference Reagent Program. HIV-1SF162 (R5) (10), HIV-189.6 (X4 and R5 dual tropic) (12), and HIV-1NL4-3 (X4) were also grown in PBMC activated with PHA-plus-IL-2 (47). HIV-1Adn-M (17) and HIV-1Ba-L (16) were produced from primary monocyte-derived MΦ (MDM) cultures (see below). Microvesicles, used as a control reagent, were isolated from supernatants of uninfected cell cultures in a manner identical to that used for virus preparation from infected cells (5). All virus and microvesicle stocks were stored at −70°C or in vapor phase liquid nitrogen until use.
PBMC were isolated by density centrifugation (Ficoll-Hypaque; Pharmacia, Uppsala, Sweden) from citrate-anticoagulated peripheral blood obtained from healthy, HIV-1-seronegative donors at the NCI at Frederick. PBMC were cultured in AIM-V medium (Gibco, Gaithersburg, Md.) with 2% human AB serum (Sigma, St. Louis, Mo.). Elutriated monocytes from HIV-negative donor leukopacs were grown at 2 × 106 cells per well on ultralow-attachment six-well Costar plates in RPMI 1640 medium (Biosource International, Camarillo, Calif.) supplemented with penicillin, streptomycin, gentamicin, amphotericin B, l-glutamine (Quality Biological, Gaithersburg, Md.), HEPES buffer (Sigma), and 10% fetal bovine serum (Biosource International). The monocytes were incubated at 37°C under 7% CO2 and 90% humidity for 7 days to generate MDMs. MDMs were infected with 10 50% tissue culture-infective doses of either HIV-1Ba-L or HIV-1ADA for 2 h, washed with phosphate-buffered saline (PBS; Biosource International) to remove free virus, and refed with culture medium. The infected MDMs were incubated for an additional 18 days with medium changes every 5 days. MDMs were stained on day 18 for intracellular HIV-1 core antigen using the KC57 monoclonal antibody (MAb; Beckman-Coulter, Miami, Fla.) and determined to be greater than 80% infected (data not shown). Culture supernatants were found to be positive for HIV-1 p24 by enzyme-linked immunosorbent assay (Beckman-Coulter) at 14 days postinfection. At day 18 postinfection, culture supernatants were harvested and the MDMs were recovered by centrifugation for flow cytometric analysis.
Total cell numbers and viability were determined by trypan blue analysis. Cells were counted on a hemocytometer in triplicate, and the percentage of dead cells was determined by the formula [dead/(live + dead)] × 100. Error bars represent 1 standard deviation of the mean. P values were calculated by using a one-tailed, equal-variance Student t test of experimental measurements versus a PBS control. Statistical analysis was performed with Microsoft Excel (Microsoft, Redmond, Wash.).
Immunofluorescent staining of PBMC and MDMs (3 × 105 per condition) was performed at 4°C for 30 min by using isotype immunoglobulin G1 (IgG1) (X40), IgG2a (X39), and V4 (non-gp120-interacting domain on CD4) and HLA-DR (L243) MAbs from Becton Dickinson Immunocytometry Systems (San Jose, Calif.). MAbs reactive with CXCR4 (12G5), CCR5 (3A9), CD45 (HI30), CD55 (IA10), CD80 (L307.4), CD86 (IT2.2), and MHC-I (G46-2.6) were all purchased from Pharmingen (San Diego, Calif.). All antibodies were phycoerythrin coupled. Following antibody staining, cells were washed three times with 250 μl of staining buffer and fixed with 2% paraformaldehyde overnight at 4°C prior to data acquisition on a FACS Calibur flow cytometer using CellQuest software (Becton Dickinson Immunocytometry Systems). Samples were gated on viable cells by forward and 90° light scatter, and at least 15,000 live-cell events were acquired for each sample. Acquired data were analyzed by using FlowJo software (Tree Star, Inc., San Carlos, Calif.).
Cells, virions, and microvesicles were solubilized in lysate buffer (1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate (SDS), 0.05 M Tris hydrochloride buffer [pH 7.5], 0.15 M NaCl, 1 mM EDTA, 1% aprotinin, 1 mM phenylmethylsulfonyl fluoride). Cell lysates were cleared by microcentrifugation at 12,000 × g for 5 min at 4°C. HIV-1 virions and microvesicles (50 μg of total protein equivalents per lane) for electrophoresis were run separately on discontinuous SDS-polyacrylamide (4 to 20% gradient) gels under nonreducing or reducing conditions. Proteins were transferred onto Immobilon-P membranes by a semidry blotting technique (Millipore, Bedford, Mass.), and specific proteins were detected by immunoblot analysis with a MAb against CD45 (HI30; Pharmingen), a rabbit polyclonal Ab to CD55 (H-319; Santa Cruz Biotechnology, Santa Cruz, Calif.), a goat polyclonal IgG against CD80 (N-20; Santa Cruz Biotechnology), a mouse MAb to CD86 (IT2.2; Pharmingen), a mouse MAb to MHC-I (a generous gift from Hidde Ploegh, Massachusetts Institute of Technology, Cambridge), or a mouse MAb to MHC-II (L243; American Type Culture Collection). Primary antibodies were detected with horseradish peroxidase-conjugated, species-specific goat secondary antibodies (Bio-Rad, Hercules, Calif.) and enhanced-chemiluminescence reagents (Amersham, Arlington Heights, Ill.).
A whole-virion immunoprecipitation assay (VPA) was performed essentially as previously described (2, 40), except that it was performed with a 96-deep-well (2.2 ml) plate (Marsh Biomedical Products, Inc., Rochester, N.Y.) or microcentrifuge tubes. Comparable input amounts of infectious or Aldrithiol-2-inactivated virus preparations (p24CA at 10,000 pg/ml or reverse transcriptase equivalents at 2,500 pg/ml) were incubated overnight at 4°C on a rocker with each MAb at 10 μg/ml in PBS plus 3% bovine serum albumin (BSA) in a total volume of 500 μl in deep-well plates sealed with aluminum plate sealers (Beckman, Fullerton, Calif.). Pansorbin cells (formalin-fixed Staphylococcus aureus strain Cowan; 25 μl; Calbiochem, La Jolla, Calif.) were incubated with PBS–3% BSA or with rabbit anti-mouse IgG (Sigma) under saturating conditions and washed three times in PBS plus 3% BSA. Pansorbin-Ab complexes were added directly to virus complexed with the mouse MAbs, and after incubation at 20°C for 30 min with rocking, virion Ab-Pansorbin complexes were precipitated by centrifugation (2,000 × g, 30 min). The residual virus content of the supernatant after immunoprecipitation was determined by p24 capture immunoassay (AIDS Vaccine Program, NCI at Frederick) or reverse transcriptase assay (Cavidi). The MAbs used in the VPA were the same MAbs used in the flow cytometry experiments. Clearance by a particular Ab in this assay is indicative of the presence of immunoreactive antigens on the virion surface (2). It is likely that a threshold density of a host cell-derived protein in the virus envelope is required to precipitate the virus and that the amount of virus precipitated depends in part on the density of a given protein in the envelope of the virus. However, because the VPA readout involves quantitation of a viral protein, this assay measures how many virions have been precipitated by the Ab-Pansorbin complex and not the number of host cell-derived proteins on a virion. Adding a rabbit anti-mouse secondary Ab to the Pansorbin cells allowed us to detect CD80 on virions that appeared to be CD80 negative when precipitated with the anti-CD80 MAb alone (D.G., unpublished observation). Error bars represent 1 standard deviation of the mean of triplicate measurements. P values were calculated by using a one-tailed, equal-variance Student t test of experimental measurements versus isotype control measurements. Statistical analysis was performed by using Microsoft Excel. Proteins for which immunoprecipitation with a specific MAb yielded a value statistically significantly greater than the value for the isotype control were considered to be incorporated into the virions at significant levels.
HIV preferentially incorporates or excludes different host cell proteins when budding from an infected cell. We have hypothesized that the presence of MHC molecules or the costimulatory protein CD80 or CD86 in the HIV-1 virion envelope could contribute to HIV pathogenesis (14). HIV incorporates MHC-I and MHC-II upon budding from infected T cells or macrophages in vitro (2, 6, 9) and in vivo (26, 27, 41), but it had previously not been determined whether the costimulatory proteins CD80 and CD86 are also incorporated into the HIV-1 virion envelope. By using a sensitive, Ab-based VPA, we performed an initial survey of seven primary HIV isolates derived from PBMC, two MΦ-derived isolates, and three laboratory isolates to determine whether CD80, CD86, MHC-I, and MHC-II were incorporated into the virions. All of the virions incorporated MHC-II, except the virions derived from the MHC-II-negative CEMX174/T2 cell line (Fig. (Fig.1).1). None of the virions incorporated significant levels of CD80, and 9 of the 12 viruses incorporated CD86 (Fig. (Fig.1).1). There was variable incorporation of MHC-I into the virions, depending on the virus and the cells from which the virus was produced (Fig. (Fig.1).1). These data suggested that, depending on the virus and the cell from which it was derived, there could be differential incorporation of CD80, CD86, MHC-I, and MHC-II into the virion envelope and that CD86 was more readily incorporated into virions than was CD80.
We attempted to determine the basis for the differential presence of different host cell proteins in different virus preparations produced from different cell types. To determine whether the presence or absence of CD80, CD86, MHC-I, and MHC-II on virions is directly related to the levels of these molecules on the surface of the cells from which the virus was produced, we examined the levels of these molecules on the surface of uninfected cells and that of the HIV-1-infected cells from which the virus we studied was produced. In addition to measuring the levels of CD80, CD86, MHC-I, and MHC-II on the uninfected and infected cells, we also examined the levels of CD45 and CD55. CD45 is one of the most highly expressed proteins on the surfaces of lymphocytes and monocytes and is reportedly excluded from virions produced from the Jurkat T-cell line (29). CD55 is a glycosylphosphatidylinositol-linked protein that is localized to cholesterol-rich regions in the plasma membrane, termed rafts, and its incorporation into virions has been used as evidence for virion budding through rafts (28). We therefore characterized the cell surface expression of CD4, CXCR4, CCR5, CD45, CD55, CD80, CD86, MHC-I, and MHC-II on uninfected and HIV-1-infected MΦ (see Table Table1),1), PBMC (see Table Table2),2), and cell lines (see Table Table3)3) and characterized the incorporation of CD45, CD55, CD80, CD86, MHC-I, and MHC-II into HIV-1 virions derived from MΦ (see Fig. Fig.2),2), PBMC (see Fig. Fig.3),3), and cell lines (see Fig. Fig.44 and and5),5), respectively.
Monocyte-derived MΦ expressed low to moderate levels of CD4 and both coreceptors CXCR4 and CCR5 (Table (Table1).1). MΦ infected with Ada-M, Ba-L 98-4, and Ba-L 98-7 showed increased expression of CD45, CD55, CD86, MHC-I, and MHC-II (Table (Table1).1). The uninfected and infected MΦ expressed low levels of CD86 and low to undetectable levels of CD80 (Table (Table1).1). Characterization of the proteins incorporated into the MΦ-derived virions revealed that CD80 was detectable on the Ba-L 98-7 and Ada-M 98-3 virions and CD86 was detectable on the Ba-L 98-4, Ba-L 98-7, and Ada-M 98-3 virions. Interestingly, the MΦ-derived virions did not incorporate detectable amounts of CD45 or CD55 (Fig. (Fig.2),2), despite moderate levels of CD45 and CD55 expression on the MΦ (Table (Table1).1). Lastly, all three MΦ tropic viruses incorporated significant levels of MHC-I and MHC-II (Fig. (Fig.2).2). These data support the premises that MHC-II is preferentially incorporated into MΦ-derived virions and that CD55 and CD45 are preferentially excluded.
We next characterized the cell surface expression and incorporation of cell surface proteins with immunoregulatory function into representative X4, R5, and dual-tropic HIV-1 virions produced from primary PBMC. The levels of CD4, CXCR4, CCR5, CD45, CD55, CD80, CD86, MHC-I, and MHC-II on activated PBMC revealed that the majority of the cells were CD4 and CXCR4 positive and CCR5 negative (Table (Table2).2). The majority of the cells expressed low levels of CD80 and moderate levels of CD55, CD86, and MHC-I. As observed with the MΦ, CD45 was the most highly expressed molecule and cell surface MHC-II expression was increased by HIV-1 infection (Table (Table2).2).
Characterization of the immunoregulatory proteins incorporated into the representative R5-tropic (SF162), dual-tropic (89.6), and X4-tropic (NL4-3) virions produced from PBMC revealed that CD80 was present on the SF162 virions but not on the 89.6 or NL4-3 virions (Fig. (Fig.3).3). All three PBMC-derived viruses incorporated significant levels of CD55, CD86, and MHC-II (Fig. (Fig.3).3). The SF162 virions and the NL4-3 virions incorporated significant levels of MHC-I, but the 89.6 virions did not (Fig. (Fig.3).3). As observed for the MΦ-produced virions, CD45 was not incorporated into the PBMC-derived virions (Fig. (Fig.3),3), despite being the most highly expressed molecule on the surface of the HIV-infected PBMC (Table (Table2).2). These data further supported the hypotheses that HIV infection upregulates MHC-II cell surface expression and that MHC-II is preferentially incorporated into budding virions whereas CD45 is preferentially excluded.
We next sought to characterize and compare the immunoregulatory proteins incorporated into HIV-1 virions and microvesicles. Microvesicles are nonviral membrane vesicle particles of unknown biological and immunological significance that bud from the surface of cells (5, 18). Identification and quantitation of cellular proteins associated with HIV-1 virions have been complicated by the presence of these microvesicles that inevitably copurify with HIV virions (5, 18). We have previously shown that microvesicles contain high levels of β2-microglobulin, MHC-I, and MHC-II (5), but it had not been previously determined whether microvesicles contain CD45, CD55, CD80, or CD86.
To determine if these immunoregulatory molecules are incorporated into microvesicles or HIV-1 virions, we first examined their cell surface expression on four different cell lines used to produce HIV-1NL4-3. Flow cytometric analysis of uninfected cultures of the T1, T2, TBLCL-CD4, and H9 cell lines and parallel infected cultures revealed that the four cell lines expressed CXCR4, but not CCR5, and expressed moderate to high levels of CD4, CD45, CD55, MHC-I, and MHC-II (Table (Table3).3). The H9 T-cell line did not express CD80 or CD86, and as expected (44), the T2 cell line did not express MHC-II and expressed very low levels of MHC-I (Table (Table3).3). CD80 and CD86 were expressed at higher levels on the T1, T2, and TBLCL-CD4 cell lines than on MΦ or freshly isolated PBMC (Table (Table3).3). Cell surface MHC-II expression was increased by HIV infection on the H9 and TBLCL-CD4 cell lines but not on the T1 cell line.
We next examined matched microvesicle and virion preparations by Western blot analysis to determine whether CD45, CD55, CD80, CD86, MHC-I, or MHC-II was present in the preparations. Microvesicles and HIV-1 virions derived from the T1, T2, TBLCL-CD4, and H9 cell lines were purified by sucrose banding density centrifugation and quantitated for total protein and p24 capsid levels. TBLCL-CD4 cell lysate served as a positive control because the TBLCL-CD4 cell line expressed moderate to high levels of CD45, CD55, CD80, CD86, MHC-I, and MHC-II (Table (Table3).3). Immunoblot analysis revealed that both the virion and microvesicle preparations contained large amounts of CD45, CD55, CD86, MHC-I, and MHC-II but not CD80 (Fig. (Fig.4).4). CD80 was readily detected in as little as 5 μg of total TBLCL-CD4 cell lysate but was barely detectable in 50 μg of the T1, T2, or TBLCL-CD4 virion or microvesicle preparations, suggesting that CD80 was excluded from both virions and microvesicles (Fig. (Fig.4).4). In contrast to CD80, CD86 was weakly detected in the TBLCL-CD4 cell lysate but easily detected in virion and microvesicle preparations, suggesting that CD86 was preferentially incorporated into virion and microvesicle preparations (Fig. (Fig.4).4). Neither CD80 nor CD86 was detected in the H9 virion or microvesicle preparations due to the fact that the H9 cell line did not express CD80 or CD86 (Table (Table3).3). Per microgram of total protein, there was more MHC-I and MHC-II in the microvesicle and virion preparations than in the cell lysate, suggesting that both microvesicle and virion preparations were enriched for MHC-I and MHC-II (Fig. (Fig.4).4). Importantly, CD45 was present at high levels in both the microvesicle and virion preparations. These findings demonstrate that microvesicle and virion preparations contained high levels of CD45, CD55, CD86, MHC-I, and MHC-II but that CD80 was excluded or present at very low levels.
As noted previously, even sucrose-banded HIV-1 virion preparations still contain copurifying microvesicles (5, 18). Because immunoblot analysis of the virion preparations (Fig. (Fig.4)4) cannot distinguish between virion-associated and microvesicle-associated host cell-derived molecules, we determined whether CD45, CD55, CD80, CD86, MHC-I, or MHC-II was incorporated into HIV-1NL4-3 virions derived from the T1, T2, TBLCL-CD4, and H9 cell lines by using the VPA. In this assay format, antibodies to host cell proteins incorporated into virions immunoprecipitate the virions while antibodies to host cell proteins present in virion preparations, but not physically incorporated into viral particles, for example, in copurifying microvesicles in the preparations, do not immunoprecipitate virions. Based on this immunoprecipitation assay, CD55 was significantly present on virions from all four sources (Fig. (Fig.5).5). MHC-I and MHC-II were significantly detected on virions derived from T1, TBLCL-CD4, and H9 cells but not on those from T2 cells (Fig. (Fig.5).5). CD86, but not CD80, was detected on virions derived from CD80 and CD86-expressing cells (Fig. (Fig.5),5), despite equivalent levels of CD80 and CD86 on the surfaces of the T1, T2, and TBLCL-CD4 cells (Table (Table3).3). Additionally, the anti-CD45 MAb did not precipitate virions derived from any of the cell lines (Fig. (Fig.5),5), although CD45 is the most highly expressed protein on the surfaces of all four cell lines (Table (Table3).3). These data extend the previous finding that there can be preferential incorporation of MHC-II and CD86 and preferential exclusion of CD80 and CD45. Importantly, these data reveal that the CD45 detected on the virions by Western blot analysis was present on the copurifying microvesicles and not incorporated into the virions.
As described in this report and elsewhere, HIV incorporates MHC molecules when it buds from infected cells. We have postulated that virion-associated, host cell-derived proteins might play a role in HIV pathogenesis (2), but previously it has been difficult to distinguish between cell death due to the direct effects of viral replication and lysis from indirect effects due to noninfectious virions. Specifically, we have proposed that MHC molecules incorporated into the HIV virion can interact with the TCR and other receptors on the surface of a T lymphocyte to induce anergy or apoptosis (2, 39). We have recently developed a procedure by which to inactivate HIV infectivity without affecting the conformational integrity of the virion surface proteins (1, 40). These conformationally and functionally intact but noninfectious virions interact authentically with target cells and provide a powerful tool with which to evaluate the role host cell-derived proteins present on the HIV-1 virion play in pathogenesis, independently of productive infection.
To better understand the effect of virion-associated host cell-derived proteins in HIV pathogenesis, we examined the effects of microvesicles and conformationally authentic, noninfectious HIV-1NL4-3-AT2 virions produced from T1, T2, TBLCL-CD4, and H9 cells on freshly isolated PBMC from a healthy, HIV-seronegative donor. Microvesicles derived from the four cell lines did not induce cell death in the cultures (Fig. (Fig.6).6). CD86-positive, MHC-positive, noninfectious virions derived from the CEMX174/T1 cell line triggered cell death, whereas CD86-positive, MHC-negative, noninfectious virions derived from the matched, MHC-II-negative CEMX174/T2 cell line did not (Fig. (Fig.6).6). Because these two cell lines differ only in MHC expression, these data strongly suggest that virion-associated MHC molecules can impact HIV pathogenesis. The CD86-positive, MHC-positive, noninfectious virions derived from the TBLCL-CD4 cell line also triggered cell death (Fig. (Fig.6).6). However, the MHC-positive, CD86-negative, noninfectious virions derived from the H9 cell line did not trigger cell death (Fig. (Fig.6).6). The differential killing effect of noninfectious HIV-1NL4-3 virions derived from different cell lines suggests that immunoregulatory proteins incorporated into the HIV virion, such as CD86, MHC-I, and MHC-II, may contribute importantly to indirect mechanisms of HIV pathogenesis.
A hallmark of HIV infection is the functional impairment of CD4+ T lymphocytes that precedes an eventual decline in circulating CD4+ T cells. The mechanism(s) behind this HIV-induced unresponsiveness or “anergy” and eventual apoptosis of CD4+ T cells remains unclear. Here we propose that host cell-derived immunoregulatory proteins present in the envelope of noninfectious virions could impact HIV pathogenesis. Specifically, binding of gp120 to CD4, virion-associated MHC molecules to TCRs, and virion-associated CD86 to CD28 on T lymphocytes could lead to T-cell activation, differentiation, anergy, or apoptosis. T cells normally require two signals to become fully activated. Signal one is Ag specific and is initiated by TCR binding to Ag-MHC complexes on the APC. The second, or costimulatory, signal is generated by CD28 on the T cell binding to CD80 or CD86 on the APC (reviewed in reference 19). We therefore determined whether HIV-1 virions or microvesicles incorporate CD80 or CD86. When a sensitive immunoprecipitation procedure was used, CD80 was detected on only 3 of 21 viruses derived from CD80-expressing cells whereas CD86 was detected on 17 of 21 viruses derived from CD86-expressing cells (Fig. (Fig.1,1, ,2,2, ,3,3, and and5).5). Additionally, virions and microvesicles derived from the T1, T2, and TBLCL-CD4 cells preferentially incorporated CD86 compared to CD80 (Fig. (Fig.44 and and5),5), despite approximately equivalent levels of CD80 and CD86 expression on the three cell lines (Table (Table3).3). These results suggest that CD86 is generally incorporated into budding virions and microvesicles, whereas CD80 is generally excluded. The molecular mechanisms behind the preferential incorporation of CD86 and exclusion of CD80 remain to be elucidated, but this phenomenon could be mediated by the cytoplasmic domains, which bear no similarity to one another (3).
The immunological significance of microvesicles enriched for MHC-I, MHC-II, and CD86 but not CD80 is also unclear. However, in some experiments, microvesicles have suppressed virus-specific T-cell responses (M. T. Esser, unpublished observation). CD80 and CD86 do not simply play redundant roles in the immune system (19). Antibodies that bind CD86 block the development of Th2 T cells and can exacerbate inflammation, whereas Abs that bind CD80 can reduce the severity of inflammation in certain models of autoimmunity (24, 37). These and other studies raise the possibility that interactions with CD86 present on virions and microvesicles may help differentiate naive T cells into Th2-like effectors (11, 46). Interestingly, there is an increase in the percentage of CD86-expressing CD4+ T lymphocytes in HIV-infected individuals (A. Valentin, personal communication). Microvesicles may be a mechanism the immune system uses to down-regulate ongoing inflammatory responses. HIV and other viruses may have exploited this microvesicle secretion pathway as a way to enhance virion assembly and as a mechanism to suppress a T-cell-mediated immune response.
We also undertook these studies in the hope of identifying a protein present on microvesicles that was not present on HIV-1 virions. As mentioned previously, microvesicles can be roughly the same size as HIV virions and band at the same density (1.13 to 1.16 g/ml) as HIV-1 virions in a sucrose gradient and are an inevitable contaminant of all HIV-1 preparations (5, 18). Toward this end, we identified CD45 as a molecule that was present at high concentrations on microvesicles but was not detected on virions (Fig. (Fig.1,1, ,2,2, ,3,3, and and5),5), despite being the most highly expressed protein on all of the cells examined (Tables (Tables1,1, ,2,2, and and3).3). These results extend the findings of Nguyen and Hildreth that CD45 was not incorporated into HIV-1RF derived from the Jurkat T-cell line (29). Importantly, the presence of CD45 on microvesicles but not on virions may provide a way in which to purify HIV-1 virions of contaminating microvesicles. Microvesicle-free HIV-1 preparations would have practical applications for biochemical analyses. The ability to remove microvesicles from purified virus preparations may also be advantageous for the production of inactivated HIV-1 vaccines.
The mechanism(s) that determines which proteins are incorporated into the budding HIV-1 virion is not well understood. The incorporation of immunoregulatory proteins into virions was not random, since some highly expressed proteins, like CD45, were excluded from virions while others, like MHC-II, appeared to be specifically incorporated (Fig. (Fig.1,1, ,2,2, ,3,3, and and5).5). Nguyen and Hildreth have proposed that HIV-1 buds selectively from glycolipid-enriched membrane domains called lipid rafts (29). Supporting this hypothesis, we found the lipid raft marker CD55 on T-cell-derived virions, suggesting that the T-cell-tropic virions budded from these rafts whereas the CD55-negative, MΦ-tropic virions may have budded via a different mechanism. In this regard, it is worth noting that in HIV-1-infected MΦ virions accumulate in intracellular vacuoles and are rarely seen budding from the plasma membrane (32) whereas T-cell-derived viruses bud predominantly from the plasma membrane (22, 23). CD55 may be a useful marker with which to dissect the different pathways that MΦ-tropic and T-cell-tropic virions use to egress from an infected cell. It is also possible that HIV-1-encoded proteins directly bind to host cell proteins to facilitate their incorporation into the mature virion. We have previously reported that a 43-amino-acid region in the cytoplasmic tail of gp41 is required for the efficient incorporation of MHC-II, but not MHC-I, into HIV-1 virions derived from T-cell lines and PBMC (36). These results suggest that HIV-1 may have evolved to specifically incorporate MHC-II into the virion as a mechanism of immune evasion.
Regardless of the specific mechanism that HIV uses to incorporate host cell-derived proteins, it is clear from the experiments with HIV-1NL4-3-AT2 virions that host cell-derived proteins can dramatically affect viral pathogenicity (Fig. (Fig.6).6). The HLA-DR genotype of the T1, T2, TBLCL-CD4, and H9 cell lines; the phenotype of the virion envelope; and whether the HIV-1NL4-3-AT2 virions triggered cell death are summarized in Table Table4.4. MHC-containing, T1-derived virions triggered cell death, whereas MHC-negative, T2-derived virions did not (Fig. (Fig.66 and Table Table4),4), strongly suggesting that virion-associated MHC molecules contribute to HIV pathogenesis. Importantly, the T1- and T2-derived virions differed only in MHC expression (Fig. (Fig.55 and Table Table4)4) due to the fact that the T2 cell line has a deletion in chromosome 6 (44). Interestingly, AT2-inactivated NL4-3/H9 virions did not trigger cell death despite containing MHC-II (Fig. (Fig.6).6). This may have been due to the fact that H9-derived virions did not contain CD86, whereas the T1- and TBLCL-CD4-derived virions did (Fig. (Fig.55 and Table Table4),4), or it may have been due to the fact that the H9-derived virions contained HLA-DRβ 0400, whereas the T1-derived virions contained HLA-DRβ 0701 and the TBLCL-CD4-derived virions contained HLA-DRβ 1501, 1104 (Table (Table4).4). Future studies will determine whether the HLA-DR phenotype of the virus or the responder PBMC affects HIV-1-triggered apoptosis.
Virion-associated MHC molecules could play several roles in HIV pathogenesis. The natural ligands for MHC-II are the TCR and CD4, and virion-associated MHC-II could enhance the avidity of the virion to increase infectivity as reported by Cantin et al. (6). Alternatively, virion-associated MHC-I and MHC-II could bind to TCRs on CD8+ and CD4+ T lymphocytes, respectively, to trigger apoptosis. Because the AT2-inactivated virions were not infectious, our data favor the second interpretation. Noninfectious-virion-triggered cell killing is especially relevant in the light of recent data from Lawn and Butera demonstrating that virions isolated from patient plasma during primary viremia did not contain MHC-II molecules whereas virions isolated late in infection or from patients with opportunistic infections contained high levels of MHC-II (26). Our current evidence supports the hypothesis that HIV has evolved specific strategies by which to acquire MHC-II as a way to thwart the host immune response.
In summary, this study demonstrated that the incorporation of host cell proteins into virions and microvesicles was not random. HIV-1 infection of MΦ, PBMC, and cell lines increased cell surface expression of MHC-II, and all of the viruses examined incorporated MHC-II. CD86, but not CD80, was preferentially incorporated into both microvesicles and virions. CD45 was identified as a molecule that was highly expressed on microvesicles but excluded from virions. Studies with noninfectious HIV-1NL4-3-AT2 virions revealed that host cell-derived proteins can dramatically affect the pathogenicity of HIV-1 virions. Dissection of the mechanisms by which HIV acquires host cell immunoregulatory proteins and the role virion-associated host cell proteins play in triggering cell death will advance our understanding of HIV pathogenesis.
We thank Mike Grimes and Bill Bohn for the production and characterization of sucrose-banded, purified HIV-1NL4-3, Antonio Valentin and Jim Turpin for the generous gifts of selected HIV-1 isolates, and Darlene Marti and Mary Carrington for HLA-DR genotyping of the PBMC donor and cell lines. We also thank Tom Parks and Jeff Rossio for critical review of the manuscript.
This project was funded in whole or in part with funds from the NCI, under contract NO1-CO-560000, and utilized reagents provided by the AIDS Reagent Repository of the National Institute of Allergy and Infectious Diseases.