A novel virus capture assay reveals a differential acquisition of host HLA-DR by clinical isolates of human immunodeficiency virus type 1 expanded in primary human cells depending on the nature of producing cells and the donor source

Réjean Cantin1, Geneviève Martin1 and Michel J. Tremblay1

Centre de Recherche en Infectiologie, Hôpital CHUL, Centre Hospitalier Universitaire de Québec and Département de Biologie Médicale, Faculté de Médecine, Université Laval, Ste-Foy (Québec), Canada1

Author for correspondence: Michel J. Tremblay. Mailing address: Laboratoire d’Immuno-Rétrovirologie Humaine, Centre de Recherche en Infectiologie, RC709, Hôpital CHUL, Centre Hospitalier Universitaire de Québec, 2705 boul. Laurier, Ste-Foy (Québec), Canada G1V 4G2. Fax +1 418 654 2212. e-mail Michel.J.Tremblay{at}crchul.ulaval.ca


   Abstract
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Abstract
Introduction
Methods
Results
Discussion
References
 
Previous findings indicated that HLA-DR is probably one of the most abundant cellular constituents incorporated within the human immunodeficiency virus type 1 (HIV-1) envelope. Given that the life-cycle of HIV-1 has been reported to be modulated by virion-bound host HLA-DR, an improved version of a virus capture technique was developed to assess the degree of HLA-DR incorporation in several clinical isolates of HIV-1 derived from primary human peripheral blood mononuclear cells (PBMCs) and monocyte-derived macrophages (MDM). Analysis of virus stocks purified from PBMCs and MDM indicated that primary isolates of HIV-1 bearing distinct tropism (i.e. T-, macrophage-, and dual-tropic) all incorporate host cell membrane HLA-DR protein. The amount of incorporated HLA-DR varies among the primary HIV-1 isolates tested. Propagation of some clinical HIV-1 isolates in either autologous PBMCs or MDM resulted in differential incorporation of virion-bound cellular HLA-DR depending on the nature of the virus producer cells. Differences in the degree of HLA-DR incorporation were also noticed when macrophage-tropic isolates of HIV-1 were produced in MDM from different donors. Altogether these data show that the efficiency of HLA-DR incorporation into the envelope of primary isolates of HIV-1 is a multifactorial phenomenon since it is affected by the virus isolate itself, the nature of host cells (i.e. PBMCs or MDM) and the donor source.


   Introduction
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Abstract
Introduction
Methods
Results
Discussion
References
 
The final stage of human immunodeficiency virus (HIV) assembly is budding, an event ultimately resulting in release of the virion from the host plasma membrane. During this process, HIV structural proteins Pr55gag and Pr160gag–pol assemble at the host plasma membrane (Gelderblom, 1991 ). Underneath the lipid bilayer, these precursor proteins tend to assemble in patches where they form virus-like particles (Freed, 1998 ). As the newly formed virus entities adopt their spherical shape, lipids from the cell cytoplasm membrane wrap such viruses. This host cell-derived lipid bilayer then forms the viral envelope that surrounds the nucleocapsid or core (McKeating & Willey, 1989 ).

Although the formation of the viral membrane is thought to be a process of stringent protein sorting during which most cellular proteins are displaced by virus-specific envelope proteins, some viruses may be less selective in the assembly of their envelope. Over the years, an increasing number of studies reported that indeed an impressive number of host plasma membrane proteins are integral constituents of retroviruses such as HIV-1, HIV-2 and simian immunodeficiency virus (Tremblay et al., 1998 ). Among the reported virion-anchored cellular constituents are adhesion molecules (Capobianchi et al., 1994 ; Frank et al., 1996 ). This observation led to the hypothesis that adhesion molecules, once located onto the exterior of retroviruses, could in theory interact with their respective cognate ligands on the target cell (Tremblay et al., 1998 ). These additional interactions between the virion and the cell surface could affect the attachment, a process now recognized as being crucial for the virus life-cycle.

Initial studies aimed at defining the propensity of HIV-1 to incorporate cellular membrane proteins were focused on HLA-DR (Arthur et al., 1992 ; Henderson et al., 1987 ). In addition to its normal interaction with the T cell receptor, HLA-DR binds also to CD4 on the T cell to strengthen cell adhesion and complement intracellular signalling (Gay et al., 1987 ). It was thus thought that HLA-DR exposed on the surface of HIV-1 could still interact with CD4 on the target cell. The validity of this postulate was confirmed by the observation that the physical presence of host-encoded HLA-DR proteins was found to enhance HIV-1 infectivity (1·6–2·3-fold increase) and accelerate the kinetics of virus entry (Cantin et al., 1997a , b ). Virus infectivity is increased in a more important manner when HIV-1 particles bearing host-derived ICAM-1 molecules interact with LFA-1-expressing target cells (6–10-fold increase) (Fortin et al., 1997 ). It was further shown that if the target cell expresses LFA-1 in its high avidity state, HIV-1 infectivity and virus-mediated syncytium formation are more remarkably enhanced (Barbeau et al., 1998 ; Fortin et al., 1998 ; Paquette et al., 1998 ). Incorporation of CD28 and CD44, two other cell membrane proteins, can also augment HIV-1 infectivity for target cells expressing the appropriate counter-ligands (J. F. Giguère, J. S. Paquette, R. Cantin & M. J. Tremblay, unpublished data; Guo & Hildreth, 1995 ).

A previous study revealed the existence of quantitative differences with respect to the acquisition of cellular HLA-DR for HIV-1 expanded on established human lymphoid cell lines (Cantin et al., 1996 ). Indeed, the capture efficiency of HLA-DR-bearing virus was found to vary depending of the virus isolate and the producer cell. This work also suggested that the nature of HLA-DR alleles of the host cell could also affect the amount of virion-anchored cellular HLA-DR. A recent study demonstrated that host-encoded HLA-DR, CD19, CD25 and CD26 can be detected in different HIV-1 subtypes produced in mitogen-stimulated human peripheral blood mononuclear cells (PBMCs), demonstrating that the incorporation process is a conserved phenomenon (Roberts & Butera, 1999 ).

The differential acquisition of host cell membrane proteins on clinical isolates of HIV-1 expanded on primary human PBMCs needs to be more extensively addressed. Indeed, studies aimed at defining the incorporation efficiency of virion-bound host proteins are important considering that such cellular molecules retain their functionality and can even modify the biological properties of HIV-1 (Cantin et al., 1997b ; Fortin et al., 1997 ; Rossio et al., 1995 ; Saifuddin et al., 1995 ). In an attempt to better measure the degree of host antigen found embedded in the envelope of clinical isolates of HIV-1 derived from primary human cells, we have developed a modified version of a previously published method based on the capture of virus entities with immunomagnetic beads (Cantin et al., 1996 ). With this highly sensitive method, we focused our study on the detection of cellular HLA-DR in the HIV-1 envelope. We were thus able to semi-quantitatively monitor the extent of cellular HLA-DR incorporated in several clinical isolates expanded in autologous PBMCs and monocyte-derived macrophages (MDM). In this work, we present evidence that clinical isolates of HIV-1 incorporate host HLA-DR protein with different efficiency when propagated in either PBMCs or MDM from the same healthy donor. Moreover, significant variations in the amounts of virion-anchored cellular HLA-DR were detected when a macrophage-tropic HIV-1 isolate was produced in MDM from several donors. These results suggest that the process of HLA-DR incorporation is a complex phenomenon.


   Methods
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Abstract
Introduction
Methods
Results
Discussion
References
 
{blacksquare} Cells.
RAJI-CD4 is a CD4 stable transfectant derived from the RAJI EBV-transformed B cell line (Tremblay et al., 1994 ). RAJI-CD4 cells were maintained in RPMI 1640 (GIBCO BRL) supplemented with 10% foetal bovine serum (FBS) (GIBCO BRL), glutamine (2 mM), penicillin G (100 U/ml) and streptomycin (100 µg/ml). 293T human embryonic kidney cell line was kindly provided by Warner C. Green (J. Gladstone Institutes, San Francisco, CA, USA) and maintained in DMEM (GIBCO BRL) supplemented with 10% FBS, glutamine (2 mM), penicillin G (100 U/ml) and streptomycin (100 µg/ml). PBMCs were isolated by Ficoll–Hypaque gradient from venous blood samples and were stimulated for 3 days with PHA-P (3 µg/ml; Sigma) and recombinant IL-2 (30 U/ml). MDM were obtained by adherence. Briefly, freshly isolated PBMCs were seeded in 75 cm2 flasks at 3x106 cells/ml in RPMI 1640 supplemented with 10% human serum type AB, 20% FBS, glutamine (2 mM), penicillin G (100 U/ml) and streptomycin (100 µg/ml). Cells were left for 5 days at 37 °C in a 5% CO2 atmosphere to allow monocyte adherence. Cultures were then washed four times with warmed PBS (pH 7·4) to remove non-adherent cells. At this step, MDM were ready to be infected by HIV-1 as described below.

{blacksquare} Virus production.
ADA (macrophage-tropic) and 89.6 (dual-tropic) isolates of HIV-1 were used in our studies. Various clinical isolates of HIV-1 were also used in this work and have all been obtained through the NIH AIDS Repository Reagent Program. Characteristics of these isolates are summarized in Table 1. Virus stocks were prepared in mitogen-stimulated PBMCs as follows. PBMCs (5x106) were mixed with similar amounts of each virus strain, and incubated for 4 h at 37 °C in a final volume of 1 ml. Next, cells were washed once with complete RPMI and resuspended at 106 cells/ml in the presence of 30 U/ml of recombinant IL-2. Infected PBMCs were cultured for a period of about 10 days at 37 °C, and IL-2 was added every 2–3 days. Harvested virus preparations were clarified using a 0·45 µm filter to eliminate cell debris, aliquoted and stored at -80 °C until use. For MDM infection, a fixed amount of virus was left to adsorb on adherent cells in a final volume of 5 ml RPMI supplemented with 20% FBS. After a 4 h incubation period at 37 °C, the virus inoculum was removed and 20 ml of RPMI containing 20% FBS was added. Culture media was changed twice a week over a period of 25–30 days after infection. Virus preparations were harvested during the plateau phase, i.e. usually between days 20 and 30 after infection. Supernatants were clarified as stated above, aliquoted and stored at -80 °C. Virus stocks were titrated in terms of viral p24 antigen by using a commercial p24 ELISA kit (Organon Teknika).


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Table 1. Phenotypic characteristics of primary isolates of HIV-1 used in this study

 
{blacksquare} Antibodies and FACS analysis.
L243 hybridoma (IgG2a) secretes an antibody specific for a monomorphic epitope of the {alpha} and {beta} chain of HLA-DR and was obtained from the ATCC. The anti-CD45RO UCHL-1 monoclonal antibody (IgG2a) was a generous gift from Dr Rafick-P. Sékaly (Institut de Recherches Cliniques de Montréal, Montréal, Québec, Canada). Purified antibody from the above-mentioned clones was derived from hybridoma culture supernatant and purification was achieved with the HiTrap antibody screening test kit (Pharmacia Biotech) following the manufacturer’s instructions. Biotinylation of antibody was performed using NHS-LC-biotin (Pierce). FACS analyses were performed with 106 cells co-incubated with 100 µl of PBS containing a saturating amount of the L243 monoclonal antibody for 30 min on ice. After washing the cells with cold PBS, cells were labelled for 30 min on ice with 100 µl of a saturating amount of R-phycoerythrin-conjugated goat anti-mouse (Caltag). Finally cells were washed and analysed on a cytofluorometer (EPICS Elite ESP, Coulter Electronics).

{blacksquare} Virus capture assay.
We have developed a modified version of our previously described virus precipitation assay based on the capture of HIV-1 particles using immunomagnetic beads (Cantin et al., 1996 ). For this new virus capture assay, we took advantage of the very strong streptavidin–biotin interaction. In brief, commercially available streptavidin-coated magnetic beads (2·5x108) (Caltag Laboratories) were mixed with 2 µg of biotinylated L243 (anti-HLA-DR antibody) in a final volume of 1 ml PBSA (1 M PBS, pH 7·4, 0·1% BSA). This mixture was left on a rocking plate for 1 h at room temperature to allow formation of the complex. Preliminary experiments were performed with different amounts of magnetic beads and biotinylated antibody to ensure that experimental parameters were optimal in precipitating HIV-1 particles. Immunomagnetic beads were next washed three times with 0·5 ml PBSA with the use of a magnet support (Dynal), and resuspended in 50 µl PBSA. Various amounts of virus standardized in terms of viral p24 core protein were incubated for 16 h at 4 °C on a rocking plate with 50 µl of the biotinylated L243/streptavidin-coated magnetic beads mixture and the sample was made up to a final volume of 1 ml with PBSA. Immunomagnetic beads were thereafter washed three times in 0·5 ml PBSA to get rid of uncaptured virus particles, and finally resuspended in 100 µl of PBSA. Viruses trapped on magnetic beads were disrupted by adding 25 µl of lysis buffer (1 M PBS, pH 7·4, containing 2·5% Triton X-100), and incubated for 30 min at room temperature under gentle mixing. Magnetic beads were then pelleted by centrifugation (5 min, 300 g) and supernatants were carefully removed. Quantitative evaluation of precipitated viruses was finally performed by measuring the amount of p24 antigen in lysed supernatants with a commercial p24 ELISA kit (Organon Teknika).


   Results
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Abstract
Introduction
Methods
Results
Discussion
References
 
Capture of HIV-1 particles when using a combination of biotinylated antibodies and streptavidin-coated magnetic beads
It was previously shown that the presence of cellular proteins on the exterior of the virion renders these host-derived constituents accessible to reagents such as specific antibodies directed against the cellular protein of interest (Cantin et al., 1996 ). This experimental approach was based on the capture of HIV-1 particles with magnetic beads coated with an antibody specific for a given host-encoded protein. We developed an improved version of this virus capture test that semi-quantitatively measures the number of virions recovered from the starting virus preparation. This new assay takes advantage of the strong natural interaction between streptavidin and biotin molecules. This methodology allows for a higher density of biotinylated antibody. The final result is an assay that is highly sensitive, being able to capture very low levels of whole virus particles (see below).

The ability of this new technical strategy to capture virus entities was initially tested using HIV-1NL4-3 particles bearing (i.e. NL4-3 DR/POS) or not (i.e. NL4-3 DR/NEG) host cell membrane HLA-DR molecules on their surface. HLA-DR-bearing virions were made by acute infection of RAJI-CD4, a cell line that expresses high levels of HLA-DR (Cantin et al., 1997a ), with NL4-3. Viruses devoid of cellular HLA-DR were produced by transient transfection of 293T cells (HLA-DR-negative) with the infectious molecular clone pNL4-3 as described previously (Cantin et al., 1997b ). Virions were captured by using a biotinylated form of L243, a mouse monoclonal antibody that reacts with a non-polymorphic region of human HLA-DR. As depicted in Fig. 1(A), significant levels of viral p24 protein were captured when using NL4-3 DR/POS particles. Interestingly, a dose-dependent increase in the amount of captured viruses was observed with viruses bearing cellular HLA-DR on their surface. The virus capture efficiency in this set of experiments ranged between 15·0 and 31·9% of the total initial p24 input. In comparison, the virus capture efficiency ranged between 7·5 and 13·1% of the total initial p24 input when our previous virus precipitation assay was used instead (i.e. L243 in combination with magnetic beads coated with a goat anti-mouse antibody). A positive signal-to-noise ratio (NL4-3 DR/POS over NL4-3 DR/NEG virus preparations) is obtained even when using a virus input as low as 25 pg of p24 with the new capture test. Based on earlier calculations that have indicated that 100 pg of virus-encoded p24 is equivalent to 106 viruses, we estimated that this virus capture assay allowed detection of host cell membrane HLA-DR on as few as 2·5x105 virus entities (Bourinbaiar, 1994 ). It should be stated that higher amounts of virus may be necessary to detect incorporation of HLA-DR for HIV-1 particles prepared from cells bearing levels of surface HLA-DR lower than in RAJI-CD4 cells.



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Fig. 1. Immunocapture of NL4-3/NEG and NL4-3/POS virus preparations. (A) HLA-DR/POS virions were produced in HLA-DR-expressing RAJI-CD4 B lymphoid cells while HLA-DR/NEG virions were generated following transient transfection of HLA-DR-negative 293T cells with the infectious molecular clone pNL4-3. Immunocapture of such virus preparations was achieved using either our previous or the new virus capture assay (VCA) as described in Methods ({square}, NL4-3 DR/NEG and previous VCA; {blacksquare}, NL4-3 DR/POS and previous VCA; {circ}, NL4-3 DR/NEG and new VCA; and {bullet}, NL4-3 DR/POS and new VCA). Precipitated virus entities were quantified using a virus p24 antigen capture assay according to the manufacturer’s instructions. (B) Comparative analysis of capture of HLA-DR/NEG and HLA-DR/POS viruses with streptavidin-coated magnetic beads coupled with either an anti-HLA-DR antibody (clone L243; {blacksquare}) or a control isotype-matched anti-CD45RO antibody (clone UCHL-1; {square}). Data shown are the mean (±SD) of triplicate samples and are representative of at least two independent experiments.

 
To ensure that the signal obtained with the new virus capture assay results from a specific binding of viruses to antibody-coated beads, we used an anti-CD45RO antibody (UCHL-1) as a control. This is based on the previous observation indicating that CD45 is excluded from the HIV-1 envelope (Orentas & Hildreth, 1993 ). It should be noted that the UCHL-1 antibody is of the same isotype as L243 (i.e. IgG2a). As shown in Fig. 1(B), the levels of NL4-3/POS viruses captured with the control antibody are comparable with the amounts of NL4-3/NEG viruses precipitated with either the anti-HLA-DR or the control antibody, thereby demonstrating the specificity of this test.

Cellular HLA-DR proteins are present on the exterior of clinical isolates of HIV-1 bearing distinct phenotypes when grown on primary human cells
The capacity of the virus to incorporate host cell-derived HLA-DR was previously investigated for clinical isolates of HIV-1 that were grown on either human established or primary cells (Cantin et al., 1996 ; Capobianchi et al., 1994 ; Roberts & Butera, 1999 ). Such investigations deserve to be performed with a larger number of clinical HIV-1 isolates bearing distinct phenotypes (i.e. T-, macrophage and dual-tropic) and expanded on natural cellular reservoirs of HIV-1 (i.e. PBMCs and MDM). We thus initially investigated the efficiency of incorporation of host HLA-DR in different field isolates of HIV-1 produced from human PBMCs using the currently described virus capture assay. All clinical isolates of HIV-1 used in the present work are listed in Table 1. First, we assessed the capacity of the virus precipitation assay to capture different amounts of either an X4 T-tropic (92HT599) or an X5 macrophage-tropic (92US657) clinical isolate of HIV-1 grown on PBMCs from the same healthy donor. Efficient precipitation of both virus isolates was observed when using total virus inputs ranging from 312 to 2500 pg of p24 (Fig. 2A, B).



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Fig. 2. Detection of cellular HLA-DR protein on the surface of clinical isolates of HIV-1 produced in primary human cells. Several primary isolates of HIV-1 were first expanded in PHA-stimulated PBMCs from the same healthy donor. Increasing concentrations of the X4 T-tropic (92HT599) (A) or the R5 macrophage-tropic (92US657) (B) isolate of HIV-1 were subjected to the capture assay using streptavidin-coated beads bearing either a biotinylated isotype-matched anti-CD45RO antibody (clone UCHL-1) ({square}) or a biotinylated anti-HLA-DR antibody (clone L243) ({blacksquare}). In addition, four X4 T-tropic (92UG046, 92UG024, 92HT599 and 92UG070) and one R5X4 dual-tropic (92US151) clinical isolates of HIV-1 (1000 pg of p24) were also captured (C). Numbers shown above each bar in (C) represent the percentages of precipitated viral p24 from the initial viral input. Data shown are the mean (±SD) of three determinations and are representative of at least two independent experiments.

 
We then examined the ability of the anti-HLA-DR antibody to capture four clinical X4 T-tropic (92UG046, 92UG024, 92HT599 and 92UG070) and one R5X4 dual-tropic (93US151) isolates of HIV-1 propagated in PHA-stimulated PBMCs originating from the same healthy donor. As shown in Fig. 2(C), virion-anchored cellular HLA-DR protein was detected on the exterior of all field isolates of HIV-1 tested. Moreover, the degree of incorporation was found to be slightly different for the virus isolates tested as ratios of immunocaptured viruses with magnetic beads coated with the L243 antibody had a 6–10-fold increase over the controls made of beads bearing isotype-matched UCHL-1 antibody. The recovery of p24 antigen in the experiments varied between 0·8 and 5·8%. Although these values are low, our results are nonetheless in accordance with previously published data of experiments using antibodies against host HLA-DR to precipitate HIV viruses grown on PBMCs (Cantin et al., 1996 ; Lawn et al., 2000 ; Saarloos et al., 1997 ). These results support the idea that primary isolates of HIV-1 do acquire substantial amounts of host-encoded HLA-DR when using a natural cellular reservoir such as PBMCs as host cells.

The degree of acquisition of cellular HLA-DR by primary isolates of HIV-1 is affected by the nature of host cells
Previous experiments showed that the nature of producer cells used for the preparation of virus stocks influences the incorporation process of virion-bound cell-derived HLA-DR glycoproteins (Cantin et al., 1996 ). This previous work was performed mainly with laboratory and clinical isolates of HIV-1 prepared from different human continuous lymphoid cell lines. We thus used the present virus capture assay to compare the capacity of the virus to incorporate host HLA-DR within four different primary isolates of HIV-1 that were expanded in autologous blood-derived PBMCs and MDM. Briefly, PBMCs and MDM from the same healthy donor were used as host cells for the preparation of 92TH026 (an R5 macrophage-tropic isolate), ADA (a molecular clone of an R5 macrophage-tropic primary isolate), 92RW009 (a dual-tropic isolate) and 89.6 (a molecular clone of a dual-tropic primary isolate of HIV-1). We did not use X4 T-tropic primary isolates of HIV-1 for this type of comparative study because such viruses could not, as expected, productively infect MDM (data not shown). Virus stocks were made in primary cells from the same donor to guarantee that virus producer cells possess the same genetic background. This is based on the idea that HLA-DR is highly polymorphic and the possibility that some specific HLA-DR alleles may potentially be preferentially incorporated within budding HIV-1 particles. Extensive variations in the degree of HLA-DR-containing virions precipitated were noticed when the clinical isolates of HIV-1 tested were produced in either autologous PBMCs or MDM (Fig. 3). The ratios of captured virus were similar for 92TH026 (compare 6·3- and 10·4-fold increase over control anti-CD45 antibody) and 89.6 (compare 155- and 91·7-fold increase over control anti-CD45 antibody) when produced in either autologous PBMCs or MDM. However, the virus recovery rates varied extensively between virus stocks produced in MDM (recovery rates ranging between 3·4 and 92·4%), whereas the recovery rates for virus preparations derived from PBMCs varied only between 1·9 and 8·6%. Interestingly, the ADA strain was found to be more easily precipitated with an anti-HLA-DR antibody when produced in MDM rather than in autologous PBMCs (compare recovery rates of 49·7 and 8·6%). In addition, a greater difference in the amounts of precipitated virus was observed for 92RW009 virus stock expanded in MDM rather than in PBMCs (compare recovery rates of 92·4 and 2·3%). Such differences in recovery rates for viruses produced in PBMCs and MDM do not seem to be related to changes in levels of surface HLA-DR expression (Table 2).



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Fig. 3. Comparative analysis of the degree of HLA-DR incorporation for clinical isolates of HIV-1 derived from autologous PBMCs and MDM. Two R5 macrophage-tropic (92TH026 and ADA) and two R5X4 dual-tropic (92RW009 and 89.6) primary isolates of HIV-1 were expanded in autologous blood-derived PBMCs (A) and MDM (B) from the same healthy donor. Virus preparations (1000 pg of p24) were subjected to the capture assay using streptavidin-coated beads bearing either a biotinylated isotype-matched anti-CD45RO antibody (clone UCHL-1) ({square}) or a biotinylated anti-HLA-DR antibody (clone L243) ({blacksquare}). The amount of virus captured by each antibody was estimated with the use of a virus p24 antigen capture assay. Numbers shown above each bar represent the percentages of precipitated viral p24 from the initial viral input. Data shown are the mean (±SD) of triplicate samples and are representative of at least two independent experiments.

 

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Table 2. Cell surface expression of HLA-DR on PBMCs and MDM from four healthy donors

 
To define whether the noticed finding is a generalized phenomenon, the capture efficiency of 92TH026 and 92RW009 produced in MDM was monitored using an antibody recognizing ICAM-1, another cell surface molecule known to be incorporated within HIV-1. A lesser variation in the virus recovery rates was detected with the anti-ICAM-1 antibody than with the antibody specific for HLA-DR (recovery rates of 7·8 and 12·1% for 92TH026 and 92RW009, respectively, when using the anti-ICAM-1 antibody as compared with 28·0 and 92·4% with the anti-HLA-DR antibody).

Acquisition of virion-anchored host HLA-DR in a primary macrophage-tropic strain of HIV-1 produced in MDM from different individuals is influenced by the donor source
Tsai et al. (1999) reported that relatively few or no cellular HLA-DR molecules were acquired by HIV-1ADA derived from MDM. In contrast to these findings, noticeable amounts of host-encoded HLA-DR molecules were incorporated in the primary isolate ADA grown from MDM in the present study (Fig. 3B). In an attempt to shed light on such discrepant results, HIV-1ADA was expanded in MDM originating from six healthy donors. The anti-HLA-DR L243 antibody efficiently captured viruses from all six individuals tested. ADA virus stocks expanded on donors 1 and 3 gave the highest virus recovery values (i.e. 25·0 and 22·0%, respectively) (Fig. 4).



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Fig. 4. Comparative analysis of the degree of HLA-DR incorporation for HIV-1ADA produced in MDM isolated from six donors. Virus stocks were prepared by inoculating MDM from six healthy donors with ADA. Virus preparations (1000 pg of p24) were subjected to the capture assay using streptavidin-coated beads bearing either a biotinylated isotype-matched anti-CD45RO antibody (clone UCHL-1) ({square}) or a biotinylated anti-HLA-DR antibody (clone L243) ({blacksquare}). The amount of virus captured by each antibody was estimated with the use of a p24 antigen capture assay. Numbers shown above each bar represent the percentages of precipitated viral p24 from the initial viral input. Data shown are the mean (±SD) of triplicate samples and are representative of at least two independent experiments.

 

   Discussion
Top
Abstract
Introduction
Methods
Results
Discussion
References
 
The present study was prompted by the observations that virion-anchored host cell membrane HLA-DR contributes to virus infectivity, and that the level in virion-bound host protein is in linear correlation with HIV-1 infectivity (Cantin et al., 1997b ; Paquette et al., 1998 ). To date there are only a limited number of reported studies addressing the acquisition of cellular proteins into the envelope of primary isolates of HIV-1. We have therefore assessed the incorporation of host cellular HLA-DR found within several primary isolates of HIV-1 prepared from autologous blood-derived PBMCs and MDM using a new immunomagnetic capture technique based on streptavidin-coated beads. The present virus precipitation assay is simple and also very sensitive since it can capture even minute amounts of virus. Although this technique does not directly assess the amount of cellular HLA-DR associated with the virions, we can presume that the capacity to recover the virus by precipitation is correlated with the level of the host protein on the virus. Recent data obtained in our lab using HIV-1 stocks recovered from B7-2-transfected 293T cells illustrated that precipitation of the virus with magnetic beads coated with an anti-B7-2 antibody was a direct reflection of the cellular surface expression of B7-2 (Bounou et al., 2001 ). In addition, previous experiments indicated that the level of ICAM-1 proteins on the surface of the host cell directly influences the amount of cell-derived ICAM-1 found embedded onto purified HIV-1 particles (Paquette et al., 1998 ).

Initially, we used PBMCs as host cells to expand four X4 T-tropic and one R5X4 dual-tropic primary isolates of HIV-1. Although no major differences were noticed in the degree of virion-bound cellular HLA-DR between these viruses, all isolates tested were found to have incorporated detectable amounts of host-derived HLA-DR protein. Our investigations were also aimed at studying a putative link between virus tropism and the incorporation of host cell membrane HLA-DR protein. This feature of HIV-1 could influence the magnitude of virion-bound cellular HLA-DR by directing replication of HIV-1 in specific cell types or tissues that express different surface levels of HLA-DR. Our observations suggest that the viral phenotype does not seem to modulate the degree of incorporation of host-encoded HLA-DR in clinical HIV-1 strains produced in primary human PBMCs and MDM. We were unable to define a general trend when comparing the recovery rates of HLA-DR-bearing virus when using four X4 T-tropic (92UG046, 92UG024, 92HT599 and 92UG070), two R5 macrophage-tropic (92TH026 and ADA) and two R5X4 dual-tropic (92RW009 and 89.6) primary isolates of HIV-1. The idea that HLA-DR incorporation does not appear to be affected by virus tropism is logical based on the notion that HIV-1 tropism is primarily conferred by the V3 loop located within the external envelope gp120 protein (Hwang et al., 1991 ), a viral constituent that is not responsible for HLA-DR incorporation into budding HIV-1 (see below).

The successful capture of two R5 macrophage-tropic (92TH026 and ADA) and two R5X4 dual-tropic (92RW009 and 89.6) primary isolates of HIV-1 purified from autologous blood-derived PBMCs and MDM by antibodies to HLA-DR is consistent with virus replication occurring in macrophages and/or activated T lymphocytes. Although the recovery rates of 89.6 with an anti-HLA-DR were not affected by the nature of the virus producer cells, striking differences in precipitation rates were observed for 92TH026, 92RW009 and ADA isolates of HIV-1 expanded in MDM as compared to similar isolates produced in homologous PBMCs. Furthermore, in contrast to the previous report by Tsai et al. (1999) , we have observed that higher precipitation levels of HLA-DR-containing virus were obtained with ADA particles derived from MDM than with ADA expanded in autologous PBMCs. This observation is somewhat unclear since MDM have been shown to express on their surface substantial levels of MHC-II molecules, including HLA-DR (Auger, 1992 ). In an attempt to solve this puzzling issue, ADA was allowed to grow in MDM isolated from six healthy donors. All six ADA stocks were found to incorporate detectable amounts of host-encoded HLA-DR, although the magnitude of virus recovery differed depending on the donor (recovery rates ranging between 2·7 and 25·0%). This last set of data provides a logical explanation for the previous observations made by Tsai et al. (1999) . In fact, it can be proposed based on data from Table 2 and Fig. 3 that cell surface levels of HLA-DR cannot account for the noticed variation in the degree of HLA-DR-bearing virus when MDM from different donors are used as host cells for ADA stocks. For example, a virus recovery rate of 49·7% is reached when ADA is produced in MDM, while only 8·6% of the total virus input is precipitated when ADA is expanded in PBMCs, despite the fact that MDM do express lower levels of HLA-DR as compared to homologous PHA-stimulated PBMCs. The observations made with HLA-DR cannot be extrapolated to all cell surface constituents inserted within the virus envelope. In fact, our results showed a lesser variation in the degree of incorporation of host ICAM-1 as compared to HLA-DR when testing two clinical isolates of HIV-1. The explanation(s) for this difference is currently under investigation.

The process through which host proteins are incorporated into budding HIV-1 particles is not well understood. Data from recent studies have provided new information on this phenomenon. Some enveloped viruses have the ability to bud through specific microdomains of the host cell cytoplasmic membrane (Manie et al., 2000 ; Zhang et al., 2000 ). These areas, or so-called glycolipid-enriched membrane (GEM), are very rich in cholesterol and sphingolipids. They form patches, or rafts, on the cytoplasmic membrane and they tend to aggregate some specific membrane proteins, particularly glycosylphosphatidylinositol-linked proteins (Jacobson & Dietrich, 1999 ). Nguyen et al. (2000) reported that HIV-1 tends to bud through these specific GEM domains on the cell surface. A previous report has demonstrated that the HLA-DR glycoprotein has been found to be associated with GEM at the cell surface level as well (Huby et al., 1999 ). The study carried out by Nguyen and co-workers used human T lymphoid Jurkat as host cells for the preparation of virus stocks. It can be postulated that the cytoplasmic membranes of MDM may also contain, as is the case for Jurkat cells, such glycolipid-enriched raft microdomains or similar structures. The most compelling evidence that HIV-1 was actively incorporating cellular MHC-II proteins, including the HLA-DR determinant, came from Poon et al. (2000) . Mutagenic analysis of the viral envelope protein revealed that the transmembrane gp41 cytoplasmic region (i.e. between aa 708 and 750) is required for MHC-II incorporation into the HIV-1 envelope. Some clinical isolates may harbour transmembrane gp41 envelope proteins with a different affinity/avidity for HLA-DR, thereby affecting the degree of HLA-DR incorporation. A specific HLA-DR allele could also interact with a higher affinity/avidity with gp41, thereby influencing the extent of HLA-DR incorporation. This hypothesis may partly explain the results obtained in the present study. Further studies are needed to solve this issue.

The exact molecular mechanism(s) underlying the greater incorporation rates of host HLA-DR when clinical isolates of HIV-1 are expanded in MDM is still a matter of speculation. However recent findings provide new insights on this phenomenon. Earlier reports aimed at studying the infection of MDM with HIV-1 denoted a differential budding process as compared to T lymphocytes. In fact, it was found that the majority of HIV-1 particles were located inside intracellular vesicles (Blom et al., 1993 ; Orenstein et al., 1988 ). Such a distinct budding process might qualitatively and/or quantitatively influence the cellular proteins that will be part of the released virions. It can thus be proposed that virus particles produced by cells of the mononuclear phagocyte series (i.e. monocyte/macrophages) will egress through an intracellular lipid bilayer that is enriched in specific molecules. This is perhaps the case for HLA-DR, as antigen-presenting cells possess some intracellular structures that are enriched in membrane HLA-DR protein (e.g. MIIC, CIIV) (Neefjes, 1999 ).

The incorporation of different cellular constituents in laboratory-adapted isolates of HIV-1 produced by human transformed cell lines has been the focus of several in vitro studies. The present work scrutinized the incorporation process of host-derived HLA-DR within several HIV-1 clinical isolates derived from primary cells using a new HIV-1 capture technique. We found that the presence of virion-anchored cellular HLA-DR on the virus is affected by the virus strain, the nature of host cells (PBMCs or MDM) and the donor source. These findings do carry a physiological significance because the degree of HLA-DR incorporation on the virion surface might influence HIV-1 pathogenesis. Indeed, the presence of HLA-DR alone was reported to augment HIV-1 infectivity (Cantin et al., 1997b ). Although the reported increase in virus infectivity conferred by HLA-DR was modest, it should be kept in mind that even a faint upregulating effect on virus infectivity can have dramatic issue in infected individuals where an extensive number of rounds of replication is occurring.


   Acknowledgments
 
This study was supported by a grant to M.J.T. from the Canadian Institutes of Health Research HIV/AIDS Research Program (grant #HOP-14438). M.J.T. is the recipient of a Canada Research Chair in Human Immuno-Retrovirology.


   References
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Abstract
Introduction
Methods
Results
Discussion
References
 
Arthur, L. O., Bess, J. W. J., Sowder, R. C.II, Benveniste, R. E., Mann, D. L., Cherman, J.-C. & Henderson, L. E. (1992). Cellular proteins bound to immunodeficiency viruses: implication for pathogenesis and vaccines. Science 258, 1935-1938.[Medline]

Auger, M. J. & Ross, J. A. (1992). The biology of the macrophage In The Macrophage: The Natural Immune System , pp. 1-57. Edited by C. E. Lewis & J. O’D. McGee. Oxford:IRL Press at Oxford University Press.

Barbeau, B., Fortin, J.-F., Genois, N. & Tremblay, M. J. (1998). Modulation of human immunodeficiency virus type 1-induced syncytium formation by the conformational state of LFA-1 determined by a new luciferase-based syncytium quantitative assay. Journal of Virology 72, 7125-7136.[Abstract/Free Full Text]

Blom, J., Nielsen, C. & Rhodes, J. M. (1993). An ultrastructural study of HIV-infected human dendritic cells and monocytes/macrophages. APMIS 101, 672-680.[Medline]

Bounou, S., Dumais, N. & Tremblay, M. J. (2001). Attachment of human immunodeficiency virus-1 (HIV-1) particles bearing host-encoded B7-2 proteins leads to nuclear factor-{kappa}B- and nuclear factor of activated T cells-dependent activation of HIV-1 long terminal repeat transcription. Journal of Biological Chemistry 276, 6359-6369.[Abstract/Free Full Text]

Bourinbaiar, A. S. (1994). The ratio of defective HIV-1 particles to replication-competent infectious virions. Acta Virologica 38, 59-61.[Medline]

Cantin, R., Fortin, J.-F. & Tremblay, M. (1996). The amount of host HLA-DR proteins acquired by HIV-1 is virus strain- and cell type-specific. Virology 218, 372-381.[Medline]

Cantin, R., Fortin, J.-F., Lamontagne, G. & Tremblay, M. (1997a). The acquisition of host major histocompatibility complex class II glycoproteins by human immunodeficiency virus type 1 accelerates the process of virus entry and infection in human T-lymphoid cells. Blood 90, 1091-1100.[Abstract/Free Full Text]

Cantin, R., Fortin, J.-F., Lamontagne, G. & Tremblay, M. (1997b). The presence of host-derived HLA-DR1 on human immunodeficiency virus type 1 increases viral infectivity. Journal of Virology 71, 1922-1930.[Abstract]

Capobianchi, M. R., Fais, S., Castilletti, C., Gentile, M., Ameglio, F. & Dianzani, F. (1994). A simple and reliable method to detect cell membrane proteins on infectious human immunodeficiency virus type 1 particles. Journal of Infectious Diseases 169, 886-889.[Medline]

Fortin, J.-F., Cantin, R., Lamontagne, G. & Tremblay, M. (1997). Host-derived ICAM-1 glycoproteins incorporated on human immunodeficiency virus type 1 are biologically active and enhance viral infectivity. Journal of Virology 71, 3588-3596.[Abstract]

Fortin, J.-F., Cantin, R. & Tremblay, M. (1998). T cells expressing activated LFA-1 are more susceptible to infection with human immunodeficiency virus type 1 particles bearing host-encoded ICAM-1. Journal of Virology 72, 2105-2112.[Abstract/Free Full Text]

Frank, I., Stoiber, H., Godar, S., Stockinger, H., Steindl, F., Katinger, H. W. D. & Dierich, M. P. (1996). Acquisition of host cell-surface-derived molecules by HIV-1. AIDS 10, 1611-1620.[Medline]

Freed, E. O. (1998). HIV-1 gag proteins: diverse functions in the virus life cycle. Virology 251, 1-15.[Medline]

Gay, D. P., Maddon, P. J., Sékaly, R.-P., Talle, A., Godfrey, M., Long, E., Goldstein, G., Chess, L., Axel, R., Kappler, J. & Marrack, P. (1987). Functional interaction between human T-cell protein CD4 and the major histocompatibility complex HLA-DR antigen. Nature 328, 626-629.[Medline]

Gelderblom, H. R. (1991). Assembly and morphology of HIV: potential effect of structure on viral function. AIDS 5, 617-638.[Medline]

Guo, M. M. L. & Hildreth, J. E. K. (1995). HIV acquires functional adhesion receptors from host cells. AIDS Research and Human Retroviruses 11, 1007-1013.[Medline]

Henderson, L. E., Sowder, R., Copeland, T. D., Oroszlan, S., Arthur, L. O., Robey, W. G. & Fischinger, P. J. (1987). Direct identification of class II histocompatibility DR proteins in preparations of human T-cell lymphotropic virus type III. Journal of Virology 61, 629-632.[Medline]

Huby, R. D., Dearman, R. J. & Kimber, I. (1999). Intracellular phosphotyrosine induction by major histocompatibility complex class II requires co-aggregation with membrane rafts. Journal of Biological Chemistry 274, 22591-22596.[Abstract/Free Full Text]

Hwang, S. S., Boyle, T. J., Lyerly, H. K. & Cullen, B. R. (1991). Identification of the envelope V3 loop as the primary determinant of cell tropism in HIV-1. Science 253, 71-74.[Medline]

Jacobson, K. & Dietrich, C. (1999). Looking at lipid rafts? Trends in Cell Biology 9, 87-91.[Medline]

Lawn, S. D., Roberts, B. D., Griffin, G. E., Folks, T. M. & Butera, S. T. (2000). Cellular compartments of human immunodeficiency virus type 1 replication in vivo: determination by presence of virion-associated host proteins and impact of opportunistic infection. Journal of Virology 74, 139-145.[Abstract/Free Full Text]

McKeating, J. A. & Willey, R. L. (1989). Structure and function of the HIV envelope. AIDS 3, s35-s41.[Medline]

Manie, S. N., Debreyne, S., Vincent, S. & Gerlier, D. (2000). Measles virus structural components are enriched into lipid raft microdomains: a potential cellular location for virus assembly. Journal of Virology 74, 305-311.[Abstract/Free Full Text]

Neefjes, J. (1999). CIIV, MIIC and other compartments for MHC class II loading. European Journal of Immunology 29, 1421-1425.[Medline]

Nguyen, D. H. & Hildreth, J. E. (2000). Evidence for budding of human immunodeficiency virus type 1 selectively from glycolipid-enriched membrane lipid rafts. Journal of Virology 74, 3264-3272.[Abstract/Free Full Text]

Orenstein, J. M., Meltzer, M. S., Phipps, T. & Gendelman, H. E. (1988). Cytoplasmic assembly and accumulation of human immunodeficiency virus types 1 and 2 in recombinant human colony-stimulating factor-1-treated human monocytes: an ultrastructural study. Journal of Virology 62, 2578-2586.[Medline]

Orentas, R. J. & Hildreth, J. E. K. (1993). Association of host cell surface adhesion receptors and other membrane proteins with HIV and SIV. AIDS Research and Human Retroviruses 9, 1157-1165.[Medline]

Paquette, J. S., Fortin, J. F., Blanchard, L. & Tremblay, M. J. (1998). Level of ICAM-1 surface expression on virus producer cells influences both the amount of virion-bound host ICAM-1 and human immunodeficiency virus type 1 infectivity. Journal of Virology 72, 9329-9336.[Abstract/Free Full Text]

Poon, D. T., Coren, L. V. & Ott, D. E. (2000). Efficient incorporation of HLA class II onto human immunodeficiency virus type 1 requires envelope glycoprotein packaging. Journal of Virology 74, 3918-3923.[Abstract/Free Full Text]

Roberts, B. D. & Butera, S. T. (1999). Host protein incorporation is conserved among diverse HIV-1 subtypes. AIDS 13, 425-427.[Medline]

Rossio, J. L., Bess, J., Henderson, L. E., Cresswell, P. & Arthur, L. O. (1995). HLA class II on HIV particles is functional in superantigen presentation to human T cells: implications for HIV pathogenesis. AIDS Research and Human Retroviruses 11, 1433-1439.[Medline]

Saarloos, M.-N., Sullivan, B. L., Czerniewski, M. A., Parameswar, K. D. & Spear, G. T. (1997). Detection of HLA-DR associated with monocytotropic, primary, and plasma isolates of human immunodeficiency virus type 1. Journal of Virology 71, 1640-1643.[Abstract]

Saifuddin, M., Parker, C. J., Peeples, M. E., Gorny, M. K., Zolla-Pazner, S., Ghassemi, M., Rooney, I. A., Atkinson, J. P. & Spear, G. T. (1995). Role of virion-associated glycosylphosphatidylinositol-linked proteins CD55 and CD59 in complement resistance of cell line-derived and primary isolates of HIV-l. Journal of Experimental Medicine 182, 501-509.[Abstract]

Tremblay, M., Meloche, S., Gratton, S., Wainberg, M. A. & Sékaly, R. P. (1994). Association of p56lck with the cytoplasmic domain of CD4 modulates HIV-1 expression. EMBO Journal 13, 774-783.[Abstract]

Tremblay, M. J., Fortin, J.-F. & Cantin, R. (1998). The acquisition of host-encoded proteins by nascent HIV-1. Immunology Today 19, 346-351.[Medline]

Tsai, W. P., Kung, H. F. & Nara, P. L. (1999). The presence and absence of histocompatibility antigens in HIV type 1 produced by autologous blood-derived macrophages and peripheral blood lymphoblasts. AIDS Research and Human Retroviruses 15, 33-41.[Medline]

Zhang, J., Pekosz, A. & Lamb, R. A. (2000). Influenza virus assembly and lipid raft microdomains: a role for the cytoplasmic tails of the spike glycoproteins. Journal of Virology 74, 4634-4644.[Abstract/Free Full Text]

Received 30 April 2001; accepted 14 August 2001.