MAbs to CD81 have been shown to induce cell-cell aggregation and an antiproliferative effect in B-cell lines (54
). Similarly, ligation of CD81 by glycoprotein E2661
was shown to induce B-cell aggregation and inhibit cell proliferation (22
). Studies done with other lymphoid cells showed that CD81 engagement by truncated E2 provides a costimulatory signal for human T cells, whereas it inhibits natural killer functions (14
To determine whether purified E1FLAG
E2 specifically interacts with cell surface-expressed CD81, we used the U937 cell line, which does not express CD81 (Fig. ), and engineered U937 cells expressing CD81. U937 cells were transfected with the mouse ecotropic viral receptor as detailed previously (2
), and a single-cell clone was isolated for subsequent infection by retroviral vectors. Human CD81 was amplified with the FOR primer GGTATGAATTC
GGAGTGGAGGG and the REV primer GGTACTCGAG
GTACACGGAGCTGTTC (italicized are the Eco
RI and Xho
I sites, and underlined are the start and stop codons). The amplified CD81 DNA was digested with Eco
RI and Xho
I and inserted into the respective sites in the multicloning region of the retroviral vector pBMN-IRES-GFP. This retroviral vector encodes an IRES downstream of the multicloning region, followed by the green fluorescent protein (GFP), and was kindly provided by G. Nolan, Stanford University. Production of high-titer ecotropic retrovirus in θNX-Eco cells and infection with a retrovirus encoding GFP alone or CD81 and GFP was performed as detailed at http://www.stanford.edu/group/nolan/retroviral_systems/phx.html
. Isolated U937 subclones that constitutively express CD81 (U937-CD81) or GFP (U937-GFP) were tested by flow cytometry of cells washed twice with PBS-2% BSA and incubated with an anti-CD81 MAb (5A6) (41
) or an immunoglobulin G1 (IgG1) isotype control. After two washes with PBS-2% BSA, cells were incubated with phycoerythrin-conjugated anti-mouse IgG1 (Becton Dickinson, San Jose, Calif.). Flow cytometry data were acquired with a Becton Dickinson FACScalibur. U937-GFP cells, like the parental uninfected cells, do not express CD81 (Fig. , left side), whereas U937-CD81 cells express CD81 on their surface (Fig. , right side). These cell lines were used to determine if the binding of E1FLAG
E2 is CD81 dependent.
FIG. 4. E1FLAGE2 binding is dependent on cell surface-expressed CD81 and induces cell-cell aggregation. (A) Flow cytometry analysis of binding of anti-CD81 MAb 5A6 (shaded curve) or a mouse IgG1 isotype control (dashed curved) to U937 cells expressing GFP (left (more ...)
We have previously shown that anti-CD81 MAbs induce cell aggregation (54
). To test whether E1FLAG
E2 could have a similar effect, we used an additional step to isolate and separate the noncovalently linked heterodimers from the disulfide-linked E1E2 aggregates. This was done by immunoaffinity on conformation-sensitive MAb H53. Magnetic beads precoated with goat anti-mouse IgG (Dynabeads; Dynal, Lake Success, N.Y.) were washed in accordance with the manufacturer's instructions and incubated with 10 μg of purified anti-CD81 MAb 5A6 or anti-E2 MAb H53 for 1 h at room temperature. Following incubation, the beads were washed twice in PBS supplemented with 0.1% bovine serum albumin (PBS-BSA) by exposure to a hand-held magnet. H53 beads were then incubated or not overnight at 4°C with purified E1FLAG
E2 and washed twice in PBS-BSA supplemented with 0.1% Triton X-100. Magnetic beads immobilized with 5A6, with H53, or with E1FLAG
E2 bound to H53 were resuspended in PBS-BSA supplemented with 0.1% Triton X-100. Magnetic beads were added to U937 cell lines suspended in 96-well flat-bottom plates (1.5 × 105
cells/well, 10:1 bead-to-cell ratio), and cell aggregation was observed after 8 h of incubation.
Purified E1FLAGE2 heterodimers, isolated this way, induced aggregation of U937-CD81 cells but not of U937-GFP cells (Fig. B, right side). As expected, immobilized anti-CD81 MAb induced U937-CD81, but not U937-GFP, cell aggregation (Fig. , left side). Neither cell reacted to beads immobilized with H53 alone (Fig. , middle). These experiments indicate that binding of E1FLAGE2 heterodimers is CD81 dependent and leads to cell aggregation. Unlike U937 cells, which are among the few human cell lines that do not express CD81, human B-cell lines express this molecule. Immobilized E1FLAGE2 heterodimers induced the aggregation of all of the B-cell lines tested (data not shown). Taken together, these data demonstrate that E1FLAGE2 complexes are biologically functional and induce a biological effect upon binding to cells.
Because engagement of B cells by the anti-CD81 MAb was previously shown to induce an increase in protein tyrosine phosphorylation (52
), we next questioned whether E1FLAG
E2 can induce a similar effect. For this purpose, OCI-LY8 cells were washed and resuspended in RPMI medium-1% FCS (RPMI-FCS). Magnetic beads coated with 5A6, H53, or H53-E1FLAG
E2 were prepared as described above, washed, and resuspended in RPMI-FCS. Samples of 4 × 106
OCI-LY8 cells were preincubated with beads for 15 min at 4°C. Rosetted cells were then washed and resuspended in 0.4 ml of RPMI-FCS. Samples of 0.1 ml were incubated for the indicated times at 37°C. The cells were lysed in 0.1 ml of lysis buffer (PBS [pH 7.4], 1% NP-40, 0.5% deoxycholic acid, 0.1% SDS, phosphatase inhibitor cocktail [Sigma], protease inhibitor cocktail) for 30 min on ice. Clarified cell lysates were then loaded onto 10% acrylamide gel, and tyrosine phosphoproteins were analyzed by Western blotting for the presence of tyrosine-phosphorylated proteins with an antiphosphotyrosine antibody (4G10; Upstate Biotechnology, Lake Placid, N.Y.).
Incubation of OCI-LY8 cells with E1FLAGE2 or with anti-CD81 MAb 5A6 induced a similar increase in the level of protein tyrosine phosphorylation similar to that in control cells (Fig. ). The increase in tyrosine phosphorylation of several protein bands was seen after as little as 1 min of incubation (Fig. , lanes 2 and 6). Maximal tyrosine phosphorylation was seen after 15 min of exposure of the cells to the beads-E1FLAGE2 or beads-5A6 (lanes 4 and 8).
FIG. 5. Engagement of B cells by E1FLAGE2 complexes induces protein tyrosine phosphorylation. Human OCI-LY8 B cells were exposed to magnetic beads precoated with H53-captured E1FLAGE2 (middle), anti-CD81 MAb 5A6 (left side), or anti-E2 MAb H53 (control, right (more ...)
Taken together, our results show that engagement of B cells by E1FLAGE2 heterodimers induces cell aggregation and an increase in protein tyrosine phosphorylation, a hallmark of B-cell activation, suggesting that the interaction of B cells with HCV may lead to their activation.
HCV infection is associated with B-cell lymphoproliferative disorders (reviewed in reference 58
) and is the major cause for mixed cryoglobulinemia (MC), a disorder characterized by serum immunoglobulin complexes that precipitate in the cold. It is therefore of note that in B cells, CD81 is a component of a multimeric protein complex, which includes the signaling molecule CD19, complement receptor 2 (CD21), and the interferon-inducible Leu-13 (CD225) protein (4
). Coengagement of this CD19-CD21-CD81 complex together with the B-cell antigen receptor reduces the threshold of B-cell activation (7
). Binding of HCV to CD81 and at the same time to a specific B-cell receptor could similarly reduce their activation threshold. Indeed, we have previously shown that the B-cell receptor expressed by a B-cell lymphoma of an HCV-infected patient had specificity for the E2 glycoprotein (47
). Such dual binding by HCV to B-cell signaling complexes would lower their activation threshold, which in turn may promote cell proliferation, resulting in B-lymphocyte proliferative disorders (58
). Other lymphoid cells could also be activated by the virus; thus, binding of CD81 on human T cells by anti-CD81 MAbs or by the truncated E2 glycoprotein provided a costimulatory signal to the engagement of CD3 (57
). This binding led to a sustained increase in interleukin-2 production and enhanced gamma interferon production (57
). Although HCV affects the immune system, the liver is the organ most damaged by the virus. Future studies may benefit from the use of functional E1E2 heterodimers for the study of virus-hepatocyte interactions.