Zhadina et al. 10.1073/pnas.0708002104. |
Fig. 7. Surface filament formation by murine leukemia virus (MLV) Gag. DF-1 cells were cotransfected with a plasmid expressing dsRED fluorescent protein and an MLV Gag-Pol expression plasmid bearing mutations in the PPXY motif. (A) A combined fluorescent/phase-contast image to identify transfected cells before sample preparation for scanning electron microscopy (SEM). (B) Shows the same field of cells as A but imaged by SEM. (C) An expanded portion of the images in A and B indicated by the dashed boxes. Note the short filamentous projections (compare with Fig. 2B).
Supporting Methods
Plasmid Construction.
The PFVDEnv proviral plasmid and the PFV Env expression plasmid have been described previously, and were a gift from Axel Rethwilm (1). The wild-type and mutant (PSAP284-287AAAT) PFV Gag ORFs were amplified from corresponding proviral PFV plasmids, which have been described previously (2), by using primers directed to the 5′ and 3′ termini of the PFV Gag-coding sequence and appended with NcoI and XhoI restriction enzyme sites, respectively. These amplicons were inserted into an NcoI/XhoI-digested pCAGGS expression vector to generate pCAGGS/Gag and pCAGGS/Gag(PTAP-), respectively. Membrane-targeted Gag expression plasmids were derived from pCAGGS/Gag by inserting annealed oligonucleotides encoding the peptides MyrR (MGARASGSGRRRGSGRRR), Fyn (MGCVQCKDKE), FynR (MGCVQCRDRE), and Lck (MGCGCSSHPE) into the NcoI site at the 5′ end of PFV Gag. Lysine-free PFV Gag proteins were generated by Lys-396 to Arg by PCR-based mutagenesis. pCAGGS-based plasmids expressing Lck-Gag-PY or Lck-Gag-PY-3K were constructed by inserting the MLV late domain with or without three appended lysine residues (LLTEDPPPYRD [KKK]) at the 3′ end of Lck-Gag(K396R) by using long PCR primers directed to the C terminus of Gag. Plasmids expressing cyan-fluorescent fusions of PFV Gag proteins were constructed by inserting cerulean fluorescent protein encoding sequence into the 3′ XhoI site of all pCAGGS/PFVGag derivatives. pCR3.1/YFP-based plasmids expressing YFP-WWP1, and derivatives have been previously described (3).1. Heinkelein M, Rammling M, Juretzek T, Lindemann D, Rethwilm A (2003) J Virol 77:11855-11858.
2. Patton GS, Morris SA, Chung W, Bieniasz PD, McClure MO (2005) J Virol 79:6392-6399.
3. Martin-Serrano J, Eastman SW, Chung W, Bieniasz PD (2005) J Cell Biol 168:89-101.