PMC

Histogram of height of ordered Ty3 wt and PR and RT mutant VLPs. VLP fractions were prepared from cells expressing wt Ty3 as well as cells expressing either the PR or RT mutant of Ty3. For wt, 150 particle diameters were measured according to particle heights above background; for PR mutants, 154 particles; and for RT mutants, 149 particles.

PR and RT mutant Ty3 VLPs imaged by AFM. Aliquots of the VLP-enriched fraction of sucrose step gradient interface fractions of cells expressing the Ty3 PR and RT mutants were prepared and imaged as described for wt Ty3 VLPs in the legend of Fig. . A and B, Ty3 wt VLPs; C and D, Ty3 PR mutant VLPs; and E and F, Ty3 RT mutant VLPs. Dimension of scan is indicated at lower right of each panel.

Ty3 wt VLPs imaged by AFM. VLP fractions were prepared from cells grown under inducing conditions, and aliquots of the enriched sucrose step gradient 30/70% interface fractions were fixed and imaged directly as described in Materials and Methods. Dimension of scan is indicated at lower right of each panel. Panels A and B, extracts from cells transformed with vector only; panels C and D, extracts from cells induced to express wt Ty3 from pDLC201.

Icosahedral structure of wt Ty3 VLP imaged by AFM. (A) Left and right panels, wt Ty3 particle. Scale of scan is shown. Right panel, hexagonal net overlay corresponding to T=7 showing pentagonal vertices in red. Center, diagram of triangular face of icosahedron showing positions of pentagonal and hexagonal capsomeres for T=7. (B) As described above for panel A, but illustrating the relationship of pentons in a T=4 arrangement of capsomeres. Because the entire particle is not imaged, these relationships illustrate local features of particles rather than proving them to be T=7 and T=4.

Immunoblot analysis of Ty3 VLP preparations. Whole-cell extracts of cells expressing wt Ty3 (pDLC201) and PR (pJK776) and RT (pTy3RTD213N) mutant Ty3 elements and vector alone (pYES2.0) (left panel, 5 μg protein per lane), and Ty3 VLPs prepared from sucrose step gradients from the same whole-cell extract (WCE) (right panel, 0.2 μg protein per lane) were fractionated on a SDS-10% polyacrylamide gel. Proteins were transferred to Immobilon membrane and reacted with anti-CA and secondary antibody as described in Materials and Methods.

Coverage of Gag3, p27, and CA by tryptic peptides. The presumptive primary protein structures for Gag3, p27, and CA are depicted as heavy black lines beginning on the left at amino acid 2 (aa 2). Numbering of amino acids is according to the theoretical Gag3 ORF beginning with the first Met codon. The positions of trypsin cleavage sites (arginine residues) are shown throughout each protein as slashes, with the numbers below indicating the aa positions of the arginine residues. Actual tryptic peptides matched to Gag3, p27, and CA are shown above each protein as hatched boxes (MS peptides). Mass data for the tryptic peptides are shown in Table .

Ty3 Gag3 processing. (A) Diagram indicating inferred processing sites for Ty3 Gag3 and Gag3-Pol3. Open boxes indicate Ty3 polyprotein precursors Gag3 and Gag3-Pol3. Amino-terminal processing of Gag3 and Gag3-Pol3 and processing to create p34 (aa 2 to 290), p27 (aa 2 to 233), and CA p24 (aa 2 to 207) were as determined in this study. Amino termini of PR, RT, and IN were determined previously (). (B) Hydrophobicity plot of Ty3 cleavage sites P4 (amino terminal) to P4′ (carboxyl terminal) flanking the scissile bond. Five previously described Ty3 PR cleavage sites were assigned hydrophobicity scores at positions P4 and P4′ according to Kyte and Doolittle (). These scores were averaged and plotted (triangles). The average is compared to the hydrophobicity of residues flanking the newly described CA-P3 site (circles).