FIG 3.

Determination of the mutation responsible for ERV-DC14/F. s. silvestris Env dysfunction. (A) Amino acid sequence alignment of Env proteins of ERV-DC14 and ERV-DC4/F. s. silvestris. SU, surface subunit; PRR, proline-rich region; TM, transmembrane subunit. RXRR is the cleavage motif. CXXC and CX₆CC are sites of covalent interaction. Arrows indicate the positions of amino acids 148 and 273, which differ between these two ERV-DC14 proviruses. (B) Assessment of Env-pseudotyped viruses based on the ERV-DC14/F. s. silvestris wild type (WT), Mutant1 (K148E), and Mutant2 (S273P) or on ERV-DC14. GPLac cells were transfected with the indicated Env expression plasmids. The corresponding Env-pseudotyped viruses were used to infect fresh HEK293T cells. The viral titers are illustrated as the log number of infectious units (IU) per milliliter with standard deviations. *, P < 0.0001 (one-way ANOVA). (C) Western blotting of GPLac cells expressing ERV-DC14/F. s. silvestris Env (K148E, S273P, or WT) or ERV-DC14 Env. The cell lysates and viral pellets from culture supernatants were analyzed. A goat polyclonal anti-FeLV SU (gp70) antibody was used to detect ERV-DC14 SU, and a mouse monoclonal anti-FeLV TM protein (p15E) antibody was used to detect the ERV-DC14 TM protein. The black arrow indicates immature SU; the gray arrow indicates mature SU. Precursor Gag (Pr65) and Gag CA (p30) were both detected with a goat anti-Raucher MLV CA antibody.