Expression of chimeric IN proteins.

<p><b>A.</b> Composition of chimeric subdomains. <b>B.</b> Protein expression. Proteins were expressed in <i>E. coli</i>, purified by nickel affinity chromatography, fractionated on SDS 4–20% gradient polyacrylamide gels under denaturing conditions, and stained with Coomassie brilliant blue.</p

Alignment of CCD domain for Ty3, PFV, and HIV-1 IN proteins.

<p>PFV and HIV-1 sequence and secondary structure alignments were adapted from Hare, et al. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0063957#pone.0063957-Hare1" target="_blank">[13]</a> and Valkov, et al. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0063957#pone.0063957-Valkov1" target="_blank">[11]</a>. Ty3 sequence and secondary structure alignments are the consensus of several methods as described in the text. “H” denotes both 3–10 and alpha helices. “E” denotes extended (beta strand). “cons TP” denotes conservation between Ty3 and PFV, and “cons TPH” conservation between Ty3, PFV, and HIV-1, using the ClustalW conservation groups <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0063957#pone.0063957-Thompson1" target="_blank">[32]</a>. Note that PFV sequence numbering is according to NCBI but the actual chimeric IN starts at the fourth residue in the PFV sequence. Conserved D,D X₃₅E catalytic residues as numbered in the alignment are Ty3 IN: D164, D225, E 261; PFV: D128, D185, E221; HIV-1: D64, D116, E152.</p

Strand-transfer assay of chimeric IN proteins.

<p><b>A. Strand-transfer assay strategy.</b> HIV-1, PFV, and Ty3 IN proteins and chimeras were assayed and products were detected by PCR using primers annealed to the donor substrate (P1) and the target plasmid pLY1855 (P2) to control DNA per reaction (C1,C2), as described in Materials and Methods. <b>B. PCR products of strand-transfer assays.</b> Reactions were done under conditions where native Ty3 IN targets the strand transfer in a sequence specific mode (Mn²⁺, left panel) or in a position-specific mode (Mg²⁺ plus host factor TFP, right panel). Donor substrates represent preprocessed U5 ends of the HIV-1-, PFV-, and Ty3-cDNAs. Assay products were extracted and fractionated as described in Materials and Methods. The assays were performed a minimum of two times and representative products are shown. P, positive control PCR of Ty3 integrated in target plasmid; N, negative control <i>in vitro</i> reaction with no IN processed for PCR. Abbreviations are the same as in the Fig. 2 legend.</p

Production of CODA assembled chimeric IN coding sequences.

<p><b>A. Example of crossover oligonucleotide-directed production of chimeric HTT IN-coding sequence.</b> Parental recoded HIV-1 and Ty3 IN cassettes in pCRII-Blunt-TOPO plasmids provided full-length, methylated “parental” DNA templates. Step 1, the bipartite Ty3-NTDxHIV1-CCD crossover oligonucleotide was extended by polymerization on the Ty3 template to produce a 5′-truncated coding strand. The complement (noncoding strand) of this DNA was produced by DNA polymerase extension of primer 1 annealed to the Ty3 downstream end. This truncated noncoding strand annealed to the HIV-1 template and a polymerase extension reaction yielded the full-length HTT noncoding strand. Step 2, primer 2 annealed to the noncoding HTT template and was extended to yield the full-length HTT coding strand. Terminal primers were present at 0.2 µM and the crossover primer at 0.04 µM. The full-length HTT chimeric product was amplified with the upstream HIV-1 primer 1 and downstream Ty3 primer 2. In cases where the NTD and CTD domains were from the same gene, <i>Dpn</i>I was added to the second reaction to remove the methylated parental DNA. B. Chimeric IN gene products. Final extension products from the assembly described in A were isolated by electrophoresis on 1% agarose gels. Chimeric sequences are identified using three letter codes: H, HIV-1; P, PFV; and T, Ty3; in the NTD-, CCD-, and CTD-coding regions respectively.</p