A Capsid-Encoded PPxY-Motif Facilitates Adenovirus Entry

Wodrich, Harald; Henaff, Daniel; Jammart, Baptist; Segura-Morales, Carolina; Seelmeir, Sigrid; Coux, Olivier; Ruzsics, Zsolt; Wiethoff, Christopher M.; Kremer, Eric J.

o the protein VI ORF without affecting its amino acid sequence. The homology region attached to the reverse primers carried the same ClaI site and overlapped with the PPSY motif. Two different reverse primers were generated: one carried an unaltered PPSY motif and another that encoded the amino acids PGAA instead of PPSY and introduced an additional Pst I site by the new coding sequence. The PCR products were transformed into the SW102 bacteria harbouring the Ad5-BAC following heatshock to induce red-recombination. Chloramphenicol and kanamycin double resistant clones were selected and BAC DNA was prepared from individual clones. The isolated BAC DNA was digested with ClaI and subsequently religated. This procedure eliminated the kanamycin cassette and reconstituted the protein VI ORF concomitant to the recircularisation of the ClaI treated BACs, because there was no other ClaI site present in the rest of the pAd5-FRT. If the reverse primer with an intact PPSY motif was used for amplification, the wild type protein VI amino acid sequence was reconstituted with a silent genetic tag introducing a ClaI site. If the reverse primer with PGAA motif was used for amplification, the protein VI coding sequence was modified at two positions i) a silent genetic tag was inserted introducing a ClaI site as above and ii) the PPSY motif was replaced by the PGAA motif introducing a new PstI site. After the ClaI treatment and re-ligation the modified genomes were retransformed in E. coli DH10B. Kanamycin negative colonies were identified by replica plating and the resulting mutants were analysed by restriction digestions and verified by sequencing. The mutant Ad genomes were released from the respective BACs by Pac I digestion and transfected into 293 cells. Following the appearance of cytopathic effects the virus was amplified and purified by double CsCl banding, dialyzed into PBS/10% Glycerol and snap frozen. B) To verify the identity of the purified virions and analyse whether detectable reversion occurred during reconstitution and propagation of the virus stocks viral DNA was extracted from purified virions and was PCR amplified with protein VI specific primers. The PCR products were digested with Pst I (left) and Cla I (right) to identify the recombinant viruses with or without the altered protein VI sequences. To insert a GFP expression cassette we used bacterial Flp-recombination using the FRT site in the E1-deleted region of the Ad5-VI-wt and Ad5-VI-M1 BACs. We cloned the left end of the Ad5 (nt 1-341) flanked by a Pac I restriction site into the plasmid pOriR6K-ie [55] ; GenBank Acc. AY700022) upstream of its FRT site. We also replaced its zeocin resistance marker with an pGPS1.1(NEB) derived kanamycin cassette and cloned an EGFP ORF from pEGFP-N1 (Clontech) in its expression locus and termed this plasmid pO6-A5-gfp. The pOriR6Kie derived plasmids can only be maintained in special E. coli strains such as PIR1 (Invitrogen) because they are dependent on the presence of R6Kc phage replicase [55] . To carry out the recombination, E. coli strain DH10B (Invitrogen) was co-transformed with pAd5-FRT derived BACs and pCP20 encoding the Flp-recombinase [56] and cultured at 30uC. The Flp recombinations were carried out as described in Bubeck et al. [55] . Briefly, the E. coli cell carrying the target BACs and the Flp expression plasmid pCP20 were transformed with the pO6-A5-GFP and selected for chloramphenicol and kanamycin resistance upon induction of the