FIG. 3.

JSRV TM oligomerization induced by sHyal2 and low pH. (A) Purified JSRV Env pseudovirions were incubated with or without 1.5 μg of sHyal2, followed by treatment with a pH 7.4 or pH 5.0 buffer. Samples were either cross-linked by 25 μM DSP or left untreated, and TM oligomerization was analyzed by Western blotting using an anti-FLAG (α-FLAG) antibody. (B) The membrane was then stripped and reblotted using an anti-His (α-His) tag antibody to detect sHyal2. (C) Effect of sHyal2 on TM oligomerization. Purified virions were incubated with the indicated amounts of sHyal2 or were treated with a neutral or a low pH, which served as negative and positive controls, respectively. Samples were cross-linked and analyzed by Western blotting. (D) Purified JSRV pseudovirions bearing F-Jenv-F or F-Jenv were either treated with 1.5 μg sHyal2, alone or in combination with pH 5.0, or left untreated. Samples were either cross-linked by DSP (lanes 1 to 3 and 5 to 7) or boiled without cross-linking (lanes 4 and 8). Note that fivefold more virus was used for F-Jenv than for F-Jenv-F, in order to enhance the detection of SU. (E and F) Purified pseudovirions bearing F-Jenv-F were either left untreated or treated with 1.5 μg sHyal2 plus pH 5.0; then they were cross-linked with DSP. The cross-linked virus samples (indicated as “37°C”) and boiled virions and cell lysates (“Boiled”) were resolved by SDS-PAGE and analyzed by Western blotting using an anti-FLAG antibody (E) or an anti-JSRV SU (α-SU) antibody (F).