Department of Immunology/Microbiology, Rush University, 1653 West Congress Parkway, Chicago, IL 60612, USA1
Author for correspondence: Mohammed Saifuddin. Fax +1 312 942 2808. e-mail msaifudd{at}rush.edu
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Abstract |
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Introduction |
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Human immunodeficiency virus type 1 (HIV-1) is an enveloped virus that encodes gp120/gp41, a receptor/fusion protein complex on the virion surface. HIV-1 gp120 is extensively glycosylated, with N-linked carbohydrate accounting for nearly half of the molecular mass (reviewed in Fenouillet et al., 1994 ), whereas gp41 has a lower carbohydrate to protein ratio with only four or five potential carbohydrate sites. Leonard et al. (1990)
determined that all 24 potential carbohydrate attachment sites of the HIV-1IIIB strain gp120 produced in CHO cells are utilized, including 13 sites that contain complex-type oligosaccharides and 11 sites that contain either high-mannose type or hybrid oligosaccharides. This endows gp120 with an unusually high number of high-mannose oligosaccharides for a mammalian glycoprotein (Fenouillet et al., 1994
). Many of these glycosylation sites were found to be conserved among HIV isolates, even though some are found in hypervariable regions of gp120 (Geyer et al., 1988
; Leonard et al., 1990
; Mizouchi et al., 1990
).
Several recent studies provide structural and experimental evidence that the gp120 carbohydrates on HIV and simian immunodeficiency virus (SIV) are involved in protection of virus from reactivity with neutralizing antibodies and may also help prevent the host from establishing effective neutralizing antibody responses. The crystal structure of gp120 showed that many of the N-linked sites are arrayed to cover variable regions of gp120 which would otherwise be exposed to neutralizing antibody (Kwong et al., 1998 ). Additionally, mutation of specific glycosylation sites in SIV gp120 increased the antibody response of infected macaques and increased the susceptibility of mutated virus to neutralization by antibody (Reitter et al., 1998
). While these studies indicate that the carbohydrate of gp120/gp41 on virions provides protection from antibodies, they further suggest a structure that could be a prime binding site for MBL.
Despite the fact that HIV-1 appears to be an excellent target for interaction with MBL due to the extensive high-mannose glycosylation of gp120, there are no studies that have investigated the interaction of MBL with HIV primary isolates (PI). A previous study investigated the interaction of MBL with T cell line-adapted (TCLA) HIVIIIB and showed that MBL bound to and neutralized this strain (Ezekowitz et al., 1989 ). However, there are significant differences between TCLA and PI viruses. The goal of the current study was to determine if MBL binds to intact virions of HIV-1 PI and whether this binding is dependent on gp120/gp41 expression. A virus capture assay was developed to test both CCR5-tropic and CXCR4-tropic PI for binding to MBL.
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Methods |
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Cells and virus.
The human primary embryonic kidney cell line (293; contributed by Andrew Rice, Baylor College of Medicine, Houston, TX, USA) and the T-lymphoblastic H9 cell lines were obtained through the AIDS Research and Reference Reagent Program (NIH, Rockville, MD, USA). The promonocytic U937 (ATCC CRL 1593) cell line was obtained from the ATCC (Manassas, VA, USA). The 293 cells were grown in DMEM with 10% foetal bovine serum (FBS) (Whittaker Bioproducts), while the other cell lines were grown in RPMI-1640 medium (Whittaker Bioproducts) with 10% FBS.
TCLA HIV-1MN was obtained through the AIDS Research and Reference Reagent Program and PI of virus used in this study were prepared as previously described (Takemfan et al., 1998 ). Briefly, PI GP (X4), TH (R5) and TA (R5) were isolated from HIV-1-infected patients and grown in phytohaemagglutinin (PHA; Sigma)-stimulated peripheral blood mononuclear cells (PBMC) in the presence of IL-2 for 714 days. The macrophage-tropic Ada strain of HIV-1 was obtained through the AIDS Research and Reference Reagent Program and was passaged in primary macrophage culture as described previously (Gendelman et al., 1988
). In brief, Ficoll Hypaque-isolated PBMC from normal donors were incubated in tissue culture flasks and then plastic-adherent cells were cultured for 5 days in the presence of macrophage colony stimulating factor (5 ng/ml; R&D Systems). Cells were then infected and cultured for an additional 14 days.
The gp120/gp41 envelope-positive (env+) and the corresponding envelope-negative (env-) viruses were prepared by transiently transfecting 293 cells with plasmids pNL4-3 and pNL4-3(E-) obtained through the AIDS Research and Reference Reagent Program, contributed by Malcolm Martin (NIH) and Nathaniel Landau (Aaron Diamond AIDS Research Center, New York, NY, USA), respectively. Cells were transfected using Lipofectamine reagent (GIBCO BRL) according to the manufacturers protocol. At 45 days after transfection, cell supernatants were collected, centrifuged to remove cellular debris and then purified by ultracentrifugation (140000 g) over 20% glycerol (Saifuddin et al., 1997 ). The amount of virus was determined by p24 antigen ELISA (Coulter) after lysis with 0·5% Triton X-100.
Binding of HIV-1 to MBL.
Ninety-six-well, flat-bottom polystyrene tissue culture plates (Costar) were coated with 100 µl of either MBL (10 µg/ml in most experiments, see Results) or 1% BSA diluted in veronal-buffered saline (5 mM veronal, pH 7·5 and 0·145 M NaCl) containing 10 mM CaCl2 (VBSCa). In some experiments, wells were coated with human MAb IgG1b12 (Burton et al., 1994 ) at 10 µg/ml in VBSCa. After overnight incubation at room temperature, wells were blocked with BSA (1%) for 60 min at room temperature, washed with VBSCa and then incubated for 28 h with 100 µl of different isolates of HIV-1. In most experiments, virus was adjusted to 1040 ng p24 antigen/ml by dilution with VBSCa. In some experiments, wells were incubated with virus diluted in VBS containing 10 mM EDTA (VBSEDTA). The plates were washed, and bound virus was lysed with 1% Triton X-100 and detected by p24 ELISA (Coulter). The percentage of HIV bound was calculated by dividing the total input of virus (100%) into the amount of bound virus detected by ELISA. A control experiment showed that when free p24 was incubated with immobilized MBL and wells were then treated with detergent, no p24 was detected by ELISA (not shown). Experiments were also performed which showed that detection of p24 in the ELISA was not affected by MBL (not shown).
In some experiments, the MBL-coated wells were preincubated with 100 µl Saccharomyces cerevisiae mannan (Sigma) at 100 µg/ml in the presence of VBSCa before addition of virus. To determine whether MBL can interact with HIV in solution, virus was preincubated with different concentrations of MBL before addition to MBL-coated microtitre wells.
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Results |
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Previous studies have shown that the binding of MBL to carbohydrates is Ca2+-dependent. To determine whether the binding of HIV to MBL-coated wells was also Ca2+-dependent, HIV-1MN was incubated in wells with either 10 mM Ca2+ or EDTA. The amount of virus binding to MBL-coated wells was reduced to background levels in the presence of EDTA indicating that the interaction of MBL with HIV was Ca2+-dependent (Fig. 2A). Binding of the GP PI was also reduced to background levels in the presence of EDTA (not shown). Preincubation of MBL-coated wells with mannan at 100 µg/ml (the highest amount tested) also substantially reduced HIV binding (Fig. 2A
), although there remained a small amount of residual binding to MBL. These experiments provide evidence that HIV bound to MBL-coated wells via its carbohydrate-recognition domain (CRD) and that binding to MBL was dependent on carbohydrates on the virus surface.
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To confirm that other PI also bind to MBL, the TA strain (R5) produced in PHA-stimulated PBMC and the Ada strain (R5) produced in macrophages were compared with the GP isolate (X4) for binding to MBL. All three virus strains bound to MBL-coated wells at similar levels (Fig. 3) indicating that binding to MBL is a common characteristic of HIV PI.
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Discussion |
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A previous in vitro study showed that the IIIB strain of HIV produced in H9 T cells bound MBL since incubation with MBL resulted in virus neutralization (Ezekowitz et al., 1989 ). The current study confirms that TCLA virus strains interact with MBL since HIV-1MN bound to immobilized MBL. However, since there are many differences between cell line-derived and PI virus, especially with regard to reactivity with anti-viral antibodies (Moore & Ho, 1995
), it was important to determine if PI also reacted with MBL. Additionally, PI are usually produced in primary cells rather than cell lines and studies have shown that N-linked glycosylation of proteins can vary depending on the type of cell (Fukuda, 1985
; Galvan et al., 1998
). The current study is the first to show that X4 and R5 PI produced in PHA-stimulated PBMC, as well as a macrophage-tropic virus strain produced in primary macrophages, bind MBL. Binding of virus was shown to occur to immobilized MBL as well as to MBL in solution since preincubation of virus with soluble MBL inhibited binding of virus to immobilized MBL. Binding was also shown to occur through the CRD of MBL since it was inhibited by EDTA as well as by soluble mannan.
Interestingly, this study also shows that expression of gp120/gp41 on virions substantially increased the amount of virus attachment to MBL indicating that most of the MBL/virus binding occurred via carbohydrates located on the virus-encoded envelope protein. This was the first study to compare the contribution of host cell- and virus-encoded glycoproteins to MBL binding. This observation helps to localize which carbohydrates are most important for the MBL/virus interaction since many host cell-derived glycoproteins are incorporated into virus at high levels and some are expressed on virions at amounts comparable to that of gp120 (Arthur et al., 1992 ; Saarloos et al., 1997
; Saifuddin et al., 1997
, 1995
). Our data support the possibility that the high-mannose glycosylation sites of gp120 contribute largely to the MBLHIV interaction.
A number of studies found that the ratio of infectious to non-infectious virus is low with less than 1 in 1000 particles being infectious. Since our studies show that MBL can capture as much as 10% of virus particles, MBL must bind at least some of the non-infectious virus. Previous studies by Ezekowitz et al. (1989) show that HIV is also neutralized by MBL suggesting that MBL can capture both infectious and non-infectious particles. Thus, it is not unreasonable to conclude that both infectious and non-infectious virus particles have gp120/gp41 that is glycosylated in such a way as to bind to MBL.
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Acknowledgments |
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References |
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Received 25 October 1999;
accepted 4 January 2000.