Interaction between complement receptor gC1qR and hepatitis C virus core protein inhibits T-lymphocyte proliferation - PubMed (original) (raw)
Interaction between complement receptor gC1qR and hepatitis C virus core protein inhibits T-lymphocyte proliferation
D J Kittlesen et al. J Clin Invest. 2000 Nov.
Abstract
Hepatitis C virus (HCV) is an important human pathogen that is remarkably efficient at establishing persistent infection. The HCV core protein is the first protein expressed during the early phase of HCV infection. Our previous work demonstrated that the HCV core protein suppresses host immune responses, including anti-viral cytotoxic T-lymphocyte responses in a murine model. To investigate the mechanism of HCV core-mediated immunosuppression, we searched for host proteins capable of associating with the core protein using a yeast two-hybrid system. Using the core protein as bait, we screened a human T cell-enriched expression library and identified a gene encoding the gC1q receptor (gC1qR). C1q is a ligand of gC1qR and is involved in the early host defense against infection. Like C1q, HCV core can inhibit T-cell proliferative responses in vitro. This core-induced anti-T-cell proliferation is reversed by addition of anti-gC1qR Ab in a T-cell proliferation assay. Furthermore, biochemical analysis of the interaction between core and gC1qR indicates that HCV core binds the region spanning amino acids 188 to 259 of gC1qR, a site distinct from the binding region of C1q. The inhibition of T-cell responsiveness by HCV core may have important implications for HCV persistence in humans.
Figures
Figure 1
Physical association between HCV core protein and the gC1qR. (a) Identification of interaction between core and gC1qR. Structure of gC1qR is drawn as a box, and the C1q-binding site is indicated as a filled box. Six independent clones interacting with core are represented as a line with a location within the gC1qR. (b) Quantification of interaction between HCV core and the gC1qR. Yeast cells of strain Y187 were cotransformed with plasmids encoding the DNA-binding and -activation domains of the GAL4 transcriptional complex as fusion proteins with the following bait-prey protein combinations: (first bar) pAS2.1 parental vector (GAL4 DNA-binding domain without any carboxy-terminal fusion construct) + pGAD10/gC1qR (GAL4 transcriptional activation domain as the amino-terminal end of a fusion protein with the human gC1q receptor); (second bar) pAS2.1/core 1–124 (GAL4 DNA-binding domain as the amino-terminal end of a fusion protein with the HCV core protein amino acids 1 to 124) + pGAD10/gC1qR; (third bar) pAS2.1/core 1–124 + pGAD10; (fourth bar) pAS2.1/core 1–124 + pGAD10/CRAT1 (a parental GAL4 activation domain vector encoding an irrelevant prey protein as a carboxy-terminal fusion construct); and (fifth bar) pAS2.1/HE4Z (GAL4 DNA-binding domain as amino-terminal end of fusion protein construct with an irrelevant bait protein) + pGAD10/gC1qR. Yeast cells were grown in media maintaining selection for each of the plasmid pairs. Cells were harvested and tested for GAL4 gene activity, indicating association between bait and prey fusion constructs, using a standard method (26). (c) Binding assay for core and gC1qR interaction. Yeast two-hybrid was performed as described above using gC1qR (bait) and core (prey) constructs: (first bar) pAS2.1/gC1qR + pACT2; (second bar) pAS2.1 + pACT2/core; and (third bar) pAS2.1/gC1qR + pACT2/core. aa, amino acid.
Figure 2
In vitro association between HCV core protein and the gC1qR. GST alone (lane 2), GST/core 1–124 (lane 3), or GST/core 1–194 (lane 4) were incubated with in vitro translated and 35S-methionine–labeled gC1qR (lane 1) protein. After binding to glutathione resin and subsequent washing, samples were separated on SDS-PAGE and detected using autoradiography. The arrow indicates the location of gC1qR (39 kDa).
Figure 3
Identification of core-binding region to the gC1qR. (a) Diagram for deletion constructs. The COOH-terminal deletion constructs of HCV core protein (amino acids 1–152, 1–124, 26–124, 125–192) were generated by site-directed mutagenesis and inserted into the GST fusion vector pGEX4T.3. (b) GST-binding assay. The truncated forms of core protein as shown in the diagram were partially purified from bacteria transformed with the GST fusion plasmids. Purified GST fusion proteins expressing truncated forms of core protein were then examined for their binding ability to gC1qR, as described above. Lane 1: GST alone (29 kDa); lane 2: GST/core 125–192 (33.8 kDa); lane 3: GST/core 26–124 (37.7 kDa); lane 4: GST/core 1–124 (40.7 kDa); lane 5: GST/core 1–152 (43.4 kDa); lane 6: GST/core 1–192 (47.8 kDa). (c) Coomassie blue staining of the purified GST-core fusion proteins. Samples for each lane are same as in b.
Figure 4
Identification of gC1qR-binding region to HCV core protein. (a) Diagram for deletion constructs. The COOH-terminal deletion constructs of gC1qR (amino acids 46–259, 46–187, 46–115) were generated by site-directed mutagenesis. The resulting plasmids were inserted into the pCI:neo vector to allow the in vitro transcription and translation. (b) GST-binding assay. The truncated forms of gC1qR as shown in the diagram were labeled with 35S-methionine by in vitro transcription and translation reaction. Asterisks indicate the correct size of truncated form of gC1qR. Minor bands present in the left panel represent the preterminated protein during in vitro transcription and translation. 35S-methionine–labeled truncation forms of gC1qR were examined for their binding ability to the GST-core fusion protein. Lane 1: radiolabeled gC1qR 46–115 (17.5 kDa); lane 2: radiolabeled gC1qR 46–187 (25.5 kDa); lane 3: radiolabeled gC1qR 46–259 (33.8 kDa); lane 4: radiolabeled gC1qR 1–282 (39 kDa); lane 5: GST-core/gC1qR 46–115; lane 6: GST-core/gC1qR 46–187; lane 7: GST-core/gC1qR 46–259; lane 8: GST-core/gC1qR 1–282. (c) GST-binding assay with GST-truncated forms of gC1qR fusion protein and 35S-methionine–labeled core protein. Lane 1: radiolabeled core (21 kDa); lane 2: GST alone; lane 3: GST-gC1qR 1–282/core; lane 4: GST-gC1qR 46–259/core; lane 5: GST-gC1qR 46–187/core; lane 6: GST-gC1qR 46–115/core. (d) GST binding assay with GST-core and 35S-methionine–labeled gC1qR 188–259. Lane 1: radiolabeled gC1qR 188–259 (8.4 kDa); lane 2: GST alone; lane 3: GST-core/gC1qR 188–259.
Figure 5
Inhibition of T-lymphocyte proliferation by HCV core protein. (a) Effect of HCV core protein on T-cell proliferation. Standard one-way mixed lymphocyte reactions were performed in the presence of C1q (50 μg/ml), the indicated concentration of purified HCV core protein, or core buffer (control response). After 6 days of coculture, proliferation was measured as 18-hour 3(H)-thymidine incorporation. (b) Effect of HCV NS3 protein on T-cell proliferation. HCV NS3 protein was tested for its ability to modulate the proliferation of T lymphocytes as described above. (c) Effect of HCV core protein on conA-stimulated T-cell proliferation. MLR was performed in the presence of conA (2 μg/ml) and the indicated amount of HCV core protein or control protein, β-gal.
Figure 6
Cell-surface expression of gC1qR on human PBMCs and MOLT-4 cell lines. Cell-surface expression of gC1qR in MOLT-4 T-cell line (a), and human PBMCs (b). MOLT-4 T-cell line or human PBMCs were incubated with media alone (left panel) or anti-gC1qR Ab, 74.5.2 (right panel), and stained cells were examined for FACS analysis. Value (%) represents the cell population gated with gC1qR-positive cells. (c) Analysis of surface-biotinylated gC1qR. Cell-surface proteins of human PBMCs were biotinylated. Solubilized membrane proteins were subjected to SDS-PAGE before (lane 1) or after (lane 2) immunoprecipitation with anti-gC1qR mAb 60.11 and 74.5.2. The separated proteins were transferred to PVDF membranes and developed using streptavidin. MFI, mean fluorescence intensity; 1°, primary; 2°, secondary.
Figure 7
Reversal of core-induced inhibition of T-cell proliferation by anti-gC1qR and anti-core Ab’s. (a) Inhibition of core-induced anti–T-cell proliferation by the addition of anti-gC1qR Ab’s in cultures. To examine the effect of anti-gC1qR Ab on reversing the core-induced anti–T-cell proliferation, anti-gC1qR Ab’s (60.11, 74.5.2) were cocultured in the T-cell proliferation assay in the presence of core protein (13 nM) as described. (b) The effect of anti-gC1qR Ab’s on recovery of core-induced anti–T-cell proliferation. The T-cell proliferation assay was performed in the presence of anti-gC1qR Ab’s. (c) The reversed effect of anti-core Ab on core-induced anti–T-cell proliferation. The indicated amount of anti-core Ab or an isotype-matched control Ab was added to the conA-stimulated lymphocyte culture.
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