Figure 6
Characterization of the processing at the Pr74Gag C-terminus. (A) Alignment of the amino acid sequences of oricoHERV-K113, MPMV (AAC82573) and MMTV (AAC82557.1) starting from the N-terminus of the NC domains. The red arrow indicates the NC-p4 cleavage site in MPMV [24]. Identical amino acids in different sequences are indicated in yellow. Black boxes span the RNA-binding zinc finger region. The alignment was generated using BLOSUM 62 (Clone Manager) and was subsequently adjusted by hand. (B) MALDI-TOF analysis of the NC domain of oricoHERV-K113. The first major peak represents doubly charged NC (z = 2) and the second major peak NC with a single charge. (C) Confirmation of the C-terminal NC cleavage site by mutagenesis. HERV-K113 VLPs and F624D mutants were loaded on an 18% SDS-PAGE and protein bands visualized by silver nitrate staining. (D) MALDI-TOF analysis of wt NC and the F624D mutant. The NC subdomains were purified by RP-HPLC and trypsin digested before MALDI-TOF analysis. Peaks of the wt and of the F624D mutant are shown in the upper and lower spectra respectively. The major peak of 1228.58 Da (framed) is unique for wt and the 3705.17 Da peak (framed) is unique for the F624D mutant. These peaks match with the sequences "GQPQAPQQTGAF" in the wt NC digest and "GQPQAPQQTGADPIQPFVPQGFQGQQPPLSQVFQG" in the F624D mutant. The peaks of approximately 1199 and 1303 Da visible in both spectra match with the expected trypsin fragments "QNITIQATTTGR" and "NGQPLSGNEQR" from internal NC regions. Additional peaks could be assigned to trypsin generated NC peptides (not shown).