Soluble receptor-induced retroviral infection of receptor-deficient cells

Abstract

Current models of retroviral entry hypothesize that interactions between the host cell receptor(s) and viral envelope protein induce structural changes in the envelope protein that convert it to an active conformation, allowing it to mediate fusion with the membrane. Recent evidence supporting this hypothesis is the demonstration that Tva, the receptor for subgroup A avian sarcoma and leukosis virus (ASLV-A), induces conformational changes in the viral envelope protein. These changes include conversion of the envelope protein to an active, membrane-binding state likely representing a fusogenic conformation. To determine whether binding of the soluble Tva (sTva) receptor was sufficient to activate fully the fusogenic potential of the ASLV-A envelope protein, we have evaluated the ability of ASLV-A to infect receptor-deficient cell lines in the presence of sTva. Soluble receptor efficiently mediated infection of cells devoid of endogenous Tva in a dose-dependent manner, and this infection was dependent absolutely on the addition of sTva. The infectivity of the virus was enhanced dramatically in the presence of the polycationic polymer Polybrene or when centrifugal forces were applied during inoculation, resulting in viral titers comparable to those achieved on cells expressing endogenous receptor. sTva functioned to mediate infection at low concentrations, approaching the estimated binding constant of the receptor and viral envelope protein. These results demonstrate that receptor binding can activate the ASLV-A envelope protein and convert it to a fusogenic conformation competent to mediate the fusion of the viral and cellular membranes.

FIG. 1

sTva-induced infection of receptor-deficient cells. Approximately 10⁴ AP⁺ IU of RCAS (A)-AP was incubated with or without sTva at the indicated concentrations as described in Materials and Methods. Receptor-deficient 293T cells were inoculated with the virus-receptor complexes and stained for AP activity 40 to 48 h p.i. AP-positive cells were identified and enumerated microscopically. Results are representative of multiple independent experiments.

FIG. 2

Centrifugation of virus-receptor complexes increases viral infectivity. RCAS (A)-AP (approximately 10⁴ AP⁺ IU) was incubated with or without sTva at the indicated concentrations as described in Materials and Methods. Preformed virus-receptor complexes were added to chilled 293T cells. Plates were returned to a 37°C incubator (filled circles) or centrifuged before being returned to the incubator (open circles) as described in Materials and Methods. Cells were stained for AP activity 40 to 48 h p.i., and viral titers were determined. In the absence of sTva, no infected cells were detected. The data reflect results typical of multiple independent experiments.

FIG. 3

Polybrene and/or centrifugation enhances sTva-induced infection. RCAS (A)-AP was incubated with or without sTva. 293T cells were inoculated with virus-receptor complexes in the presence or absence of 4 μg of Polybrene per ml. Plates were immediately returned to a 37°C incubator or centrifuged as described in Materials and Methods. Forty to 48 h p.i., cells were stained and viral titers were determined. In the absence of sTva, no infected cells were detected. The data reflect results typical of multiple independent experiments employing approximately 10⁴ AP⁺ IU.

FIG. 4

Infection of 293T cells was nonlinear in response to sTva concentration. RCAS (A)-AP was incubated without or with increasing concentrations of sTva from 5 × 10⁻⁷ to 10 nM, followed by inoculation of 293T cells in the presence of Polybrene (4 μg/ml) and centrifugation. AP-positive cells were enumerated 40 to 48 h p.i., and viral titers were calculated. No AP-positive cells were detected in the absence of sTva. The data reflect results typical of multiple independent experiments using 100 μl of RCAS (A)-AP (approximately 10⁴ AP⁺ IU).

FIG. 5

sTva-induced infection is highly efficient. RCAP (A)-AP (10⁴ AP⁺ IU in 100 μl) was incubated in the absence (−) or presence (+) of sTva (5 nM) as described in Materials and Methods. 293T cells were inoculated in the presence of Polybrene (4 μg/ml) and centrifuged. Tva-expressing 293T cells (Tva-293 or QT6 cells) were inoculated with virus alone (without sTva) or with virus preincubated with 5 nM sTva for 30 min at 37°C. Tva-expressing cells were inoculated in the absence of Polybrene and without centrifugation. Infected cells were enumerated 48 h p.i., and the viral titers were determined. The data reflect results typical of multiple independent experiments.