HIV-1 Env associates with HLA-C free-chains at the cell membrane modulating viral infectivity

Abstract

HLA-C has been demonstrated to associate with HIV-1 envelope glycoprotein (Env). Virions lacking HLA-C have reduced infectivity and increased susceptibility to neutralizing antibodies. Like all others MHC-I molecules, HLA-C requires β₂-microglobulin (β₂m) for appropriate folding and expression on the cell membrane but this association is weaker, thus generating HLA-C free-chains on the cell surface. In this study, we deepen the understanding of HLA-C and Env association by showing that HIV-1 specifically increases the amount of HLA-C free chains, not bound to β₂m, on the membrane of infected cells. The association between Env and HLA-C takes place at the cell membrane requiring β₂m to occur. We report that the enhanced infectivity conferred to HIV-1 by HLA-C specifically involves HLA-C free chain molecules that have been correctly assembled with β₂m. HIV-1 Env-pseudotyped viruses produced in the absence of β₂m are less infectious than those produced in the presence of β₂m. We hypothesize that the conformation and surface expression of HLA-C molecules could be a discriminant for the association with Env. Binding stability to β₂m may confer to HLA-C the ability to preferentially act either as a conventional immune-competent molecule or as an accessory molecule involved in HIV-1 infectivity.

Figure 1. Cytofluorimetric analysis of A3.01 and ACH-2 cell lines.

(a) Dashed line: negative control (secondary antibody fluorescence); thin line: unstimulated cells; thick line: cells stimulated with TNF-α for 24 h. 2G12, DT9 and L31 antibodies detect HIV-1 Env, HLA-C heterotrimers and HLA-C free chains at the cell membrane, respectively. Following TNF-α stimulation, ACH-2 cells express HIV-1 Env glycoprotein (2G12 antibody); HLA-C heterotrimers are upregulated in both cell types (DT9 antibody); HLA-C free chains are upregulated only in ACH-2 cells (L31 antibody). (b) ○: unstimulated A3.01 cells; ●: TNF-α stimulated A3.01 cells; △: unstimulated ACH-2 cells; ▲: TNF-α stimulated ACH-2 cells. Data from 6 independent experiments are shown as mean fluorescence intensity (MFI) of L31 staining. L31 fluorescence results to be significantly different between TNF-α stimulated ACH-2 cells (chronically infected by HIV-1) and A3.01 cells (parental cell line, uninfected) (p = 0.01), and between TNF-α stimulated and unstimulated ACH-2 cells (p = 0.002). p values were calculated by two-way ANOVA comparing ACH-2-TNF-α with A301-TNF-α and ACH-2-TNF-α with ACH-2-unstimulated.

Figure 2. Time course expression analysis of HIV-1 Env, MHC-I and β₂m in TNF-α stimulated and unstimulated A3.01 and ACH-2 cells.

Cells were stained after 3, 24, 48 and 72 h of culture. Solid line: ACH-2 HIV-1 infected cells; dotted line: parental A3.01 cells. RFI (Relative Fluorescence Intensity) = (Mean Flourescence Intensity (MFI) sample − MFI control)/MFI control. Data are shown as fluorescence fold change between RFI of TNF-α stimulated and unstimulated cells. The presence of HLA-C free chains at the cell surface appears to be dependent on the presence of HIV-1 Env; expression of both Env and HLA-C free chains decreases at later times (48 and 72 h post TNF-α stimulation, panel 2G12 for Env and panel L31 for HLA-C free chains). No changes in the expression of total HLA-C (L31 AW panel, after an acid wash to remove β₂m), HLA-C heterotrimers (DT9 panel), MHC-I (W6/32 panel) or β₂m (NAMB-1 panel) are observed.

Figure 3. HIV-1 Env expression correlates with HLA-C free chains upregulation.

(a) Black line: negative control (secondary antibody fluorescence); blue line: PM1 unstimulated cells; red line: PM1 cells stimulated with TNF-α for 48 h. In the absence of stimulation, HIV-1 (IIIB strain) infection (2G12 staining) induces the downregulation of MHC-I molecules (W6/32 staining) and a slight increase of HLA-C free chains molecules at the cell surface (L31 staining), compared to the uninfected cells. After TNF-α stimulation, the HIV-1 replication is increased and the expression of MHC-I molecules at the cell membrane is restored. The increased HIV-1 Env expression correlates with a higher presence of HLA-C free chains molecules at the cell surface of the infected cells, compared to the uninfected ones. (b) Shaded curve: negative control (secondary antibody fluorescence); black line: 293T transfected with pcDNA3 (Mock); blue line: 293T transfected with pSG3^Δenv; red line: 293T transfected with HIV-1 QHO-Env plasmid. L31 staining indicates an increase of HLA-C free chains at the cell membrane of cells transfected with HIV-1 Env, compared to the the transfection with pcDNA3 or pSG3^Δenv plasmids. On the contrary, after acid wash (L31 AW) no differences in the amount of total HLA-C molecules were detected.

Figure 4. Cytofluorimetric analysis of β₂m negative 293T cells, after transfection with different HIV-1 genes.

Shaded curve: negative control (secondary antibody fluorescence); red line: L31 labelled cells. After transfection with plasmids encoding different HIV-1 proteins, cells were labelled with the L31 antibody, after acid wash. As a positive control cells were transfected with β₂m. Cytofluorimetric analyses indicate that only β₂m transfection is able to restore HLA-C at the cell membrane of β₂m negative cells. Neither the presence of Env, nor the presence of other HIV-1 proteins is sufficient to translocate HLA-C to the cell surface.

Figure 5. The presence of HLA-C free-chains at the cell membrane is dependent on β2m expression.

(a) L31 labelling, after acid wash, indicates that both HeLa and HeLa-Lai β₂m negative cells (β₂m−) are unable to express HLA-C molecules at the plasma membrane while the parental cell lines (β₂m+) normally translocate HLA-C to the cell surface. The error bars represent standard errors from the mean calculated from quadruplicate determinations. (b) Shaded curve: negative control (secondary antibody fluorescence); dashed line: β₂m− cells; solid line: β₂m+ cells. HLA-C membrane expression is detected only in the parental β₂m positive cells.

Figure 6. BiFC assay between Env and HLA-C in HeLa cells.

(a) Nuclear complementation between Jun-Yn and Fos-Yc was assessed as a positive control (green signal). (b) Complementation between Env-Yn and Fos-Yc was assessed as a negative control. (c,e) Transfection of Env-Yn and Env-Yc leads to the BiFC signal (green), indicating Env oligomerization at the cell membrane. (d,f) Transfection of Env-Yn and HLA-C-Yc leads to the BiFC signal (green) indicating the Env/HLA-C interaction at the cell membrane. In panels c and d β₂m is labelled in red.

Figure 7. Env and HLA-C are associated at the cell surface.

(a) HeLa and HeLa-Lai cells were treated with the cell membrane insoluble DTSSP cross-linker. Proteins were purified on GN lectin columns by elution with increasing concentrations (from 0.25 to 1 M) of methyl α-D-mannopyranoside. Purified complexes heavier than 100 kDa were analysed by western blot. Env purification is evident in HeLa-Lai cells and not in control HeLa cells. HLA-C molecules, containing few mannoses, are eluted in the first fractions from HeLa cells, while they are co-purified in higher amounts in the presence of Env (HeLa-Lai). No differences in flotillin-1 purification, used as negative control, were detected. (b) Membrane protein complexes from HeLa and HeLa-Lai cells were immunoprecipitated with 2G12 (anti-Env), DT9 (anti-HLA-C) and a-β₂m (anti-β₂m) antibodies. Western blot analysis of immunoprecipitated samples shows that HLA-C is associated either with β₂m or with Env. HLA-C and α/β-tubulin expression were detected as loading control.

Figure 8. Luciferase-based HIV-1 pseudoviruses infectivity assay on TZM-bl cells.

Pseudoviruses produced in parental 293 T β₂m+ cells (solid line) are significantly more infectious than those produced in 293 T β₂m− cells (dotted line). This is observed for two HIV-1 Env pseudotyped viruses (QHO and pRHPA) (p < 0.0001) and not for an unrelated pseudotyped virus (VSV-G). Data are expressed as Relative Luminescence Units (RLU). The error bars represent standard deviations from the mean calculated from quadruplicate determinations. p values were calculated by two-way ANOVA, comparing β₂m+ and β₂m− pseudoviruses.