Figure 3.

[³H]NEM has access to the viral core proteins. In A, isolated and purified IMVs were labeled for 20 min on ice with [³H]NEM (³H). Infected cells were labeled for 3 d with [³⁵S]methionine and the labeled virions isolated and purified (³⁵S). ³⁵S-labeled virions were disrupted by incubation for 30 min at 37°C with 1% SDS and subsequently diluted in lysis buffer to a final concentration of 0.1% SDS. From these lysed particles the protein 4a (4a), p25 (p25), and p11 (p11) were immunoprecipitated. Analysis was on 15% SDS-PAGE followed by autoradiography. M-¹⁴C-labeled marker proteins of 14, 30, 45, 69, 93, and 200 kD. In B, infected cells were treated (NEM +) or not treated (NEM −) with 20 mM NEM for 20 min on ice before the preparation of PNS in buffer with or without NEM. The virions were concentrated by pelleting through a sucrose cushion at 24 krpm in a SW40 rotor for 30 min. Pellets were resuspended in buffer without NEM. Equal amounts of pelleted virions were incubated with 5 μCi of [³H]NEM and the labeled virions analyzed on 15% SDS-PAGE followed by autoradiography. In the left lane (of + or − NEM) 3 μg of total protein, in the right lane 8 μg of protein was used for [³H]NEM labeling. ³⁵S virus control lane consisting of [³⁵S]methionine-labeled, purified virions. M-¹⁴C-labeled marker proteins of 69, 45, 30, and 14 kD. In C, the same amounts of virions as used for the [³H]NEM labeling (3 μg of protein [left] and 8 μg [right], respectively) isolated from cells that were pretreated (NEM +) or not pretreated (NEM −) with NEM were run on 15% SDS-PAGE and the proteins were detected by silver staining.