Figure 1

Tools for characterization and analysis of the I5 protein. (A) A Kyte-Doolittle hydrophilicity plot of the I5 protein was generated using the Protean module of the Lasergene software (DNASTAR, Inc.). (B) An alignment of the I5 homologs encoded by several chordopoxviruses is shown; sequences were obtained from http://www.poxvirus.org and aligned using the Megalign module of the Lasergene software. Vaccinia virus (VV, GenBank ID 29692238), camelpox virus (CMLV, GenBank ID 18640364), ectromelia virus (EV, GenBank ID 22164721), variola virus (VAR, GenBank ID 439035), monkeypox virus (MPV, GenBank ID 179750543), Yaba-like disease virus (YLDV, GenBank ID 12085087), myxoma virus (MYX, GenBank ID 18426922), molluscum contagiosum virus (MCV, GenBank ID 1492060), Shope fibroma virus (SFV, GenBank ID 18448493), lumpy skin disease virus (LSDV, GenBank ID 151505037), sheeppox virus (ShPV, GenBank ID 21492557), and swinepox virus (SPV, GenBank ID 18640187). (C) The key genomic features of three recombinant viruses used in this study are shown. In the vI5V5 virus, the endogenous locus has been modified such that the I5 protein is fused to a C-terminal V5 epitope tag. In the inducible vΔind I5V5 recombinant, the endogenous I5 ORF has been replaced by a NEO cassette, and the tetR gene and an inducible copy of the I5V5 ORF has been inserted into the non-essential thymidine kinase (TK) locus of the genome. The inducible I5V5 ORF is under the regulation of the I5 promoter and the TET operator. The vΔI5 virus was generated by replacing the I5V5 locus of vI5V5 with the NEO cassette; this virus is deleted for the I5 locus. The primers and strategy used for the generation of these viruses are shown in Table 1.