Figure 3

Synthesis, release, and processing of WT and mutant M-PMV CA-NTD. HEK 293 T cells were transfected with WT or mutant M-PMV CA-NTD proviral DNA. Viral proteins were metabolically labeled with a [³⁵S] cysteine-methionine mix for 30 min and chased for 16 h. M-PMV CA-related proteins were then immunoprecipitated from the cells and culture media and analyzed by SDS-PAGE: (A) Intracellular M-PMV proteins Gag (Pr78), Gag-Pro (Pr95), and Gag-Pro-Pol (Pr180) immunoprecipitated from cell lysate after 30 min pulse, and (B) after 16 h chase. (C) CA-derived proteins of released M-PMV particles were immunoprecipitated from the culture media 16 h after the chase. (D) Quantification of WT and mutant M-PMV particle release from HEK 293 T cells. Band intensities of [³⁵S] pulse-labeled Gag (Pr78) and released CA were calculated. The relative percent of CA released into the culture media was corrected for intracellular expression of individual samples. (E) Western blot analysis of released WT and mutant M-PMV CA-NTD particles. VLPs from the culture media were collected by centrifugation through a 20% sucrose cushion 48 h post-transfection. The viral proteins were analyzed by SDS-PAGE, blotted onto a nitrocellulose membrane, and detected with rabbit antibodies raised against M-PMV CA. (F) Amino acid sequence of 80 N-terminal amino acids of M-PMV CA. The arrow indicates the internal cleavage between A⁴³ and I⁴⁴ identified by N-terminal amino acid sequencing.