Construction of recombinant viruses

All viruses used in this study expressed the A5-GFP fusion protein, which was introduced into a WT VACV Western Reserve strain [5] and its ∆F12 and ∆A36 mutants [44]. A v∆F12 was constructed by introducing a 1207 bp internal deletion [26] but for the purposes of this study another v∆F12 was constructed that deleted more of the F12 ORF. A BamHI fragment was subcloned from p∆F12 [26] into the BamHI site of pUC13-EcoGPT-mCherry [43]. A DNA fragment encompassing the last 43 bp of the F12L ORF and 365 bp downstream from VACV WR was generated by PCR using oligonucleotides F12-DnStrF1 (5′-CACCCCCGGGTAAAGATATTAATGAA-TCCATGAGTCAGATG-3′) and F12-DnStrR1 (5′-CACCGAATTCCAGCATCTAACTTGATGTCAGG-3′) and cloned into the XmaI-EcoRI sites of the same plasmid to generate pEcoGPT-mCherry-∆F12 mkII. This plasmid was used to generate the v∆A36∆F12-A5GFP virus by transient dominant selection [62, 63]. Briefly, CV-1 cells were transfected with the plasmid and infected with the parent virus. Recombinant viruses that incorporated the plasmid by recombination and expressed the EcoGPT-mCherry fusion protein were selected by serial passage and plaque purification on monolayers of BS-C-1 cells under selection by mycophenolic acid supplemented with hypoxanthine and xanthine. After isolation of an intermediate clone, the virus was plaque purified three additional times without selection to isolate clones that had undergone a second recombination event and lost expression of EcoGPT-mCherry. Viruses that had incorporated the new ∆F12 mkII allele were differentiated from those that had reverted to a parental genotype by PCR and sequence analysis. The virus lacking both F12 and A36 (double deletion virus) was constructed by using v∆A36-A5GFP as parental virus. In parallel, the ∆F12 mkII allele was used to replace the ∆F12 allele in v∆F12-A5GFP to generate viruses with identical deletions at the F12 locus. Analysis of this new v∆F12, used for the rest of this study, confirmed it had the same phenotype as the original parental virus.

The pEcoGPT-mCherry-A36rev plasmid was generated by amplifying the A36 locus by PCR, including about 350 bp upstream and downstream of the ORF, using oligonucleotides A36 UP F (5′-GATCAAGCTTTGGCTAGATTCAACATT TATAGCATTTGTG-3′) and A36 DN R (5′-GA-TCGAATT CACGACACATTTACTCAGTGGGGATATG-3′) and cloning the product into the HindIII-EcoRI sites of pUC13-EcoGPT-mCherry. The pEcoGPT-mCherry-A36YdF plasmid was generated by amplifying two DNA fragments by PCR using VACV WR genomic DNA template and oligonucleotides A36 UP F with A36YdF R (5′-ACTGTAG-TGTTCTGAAAAATAGTCTGTTCATTACGATCATTATTTATTA GCAGCGTGCTTCCAGCAACACTATCGAA-3′), and A36 DN R with A36YdF F (5′-AGCACAGAACACATTTTCGATAGTGTTGCTGGAAGCACGCTGC-TAATAAATAATG ATCGTAATGAACAGACTATTTTTC-3′). The two fragments incorporating the YdF mutations were spliced by overlap extension and inserted into the HindIII-EcoRI sites of pUC13-EcoGPT-mCherry. The pEcoGPT-mCherry-A36WEWD and pEcoGPT-mCherry-A36dm plasmids were generated by splicing two DNA fragments generated by PCR using pEcoGPT-mCherry-A36rev as template and oligonucleotides A36atgKpnIF (5′-ATGATGCTGGTACCTCTTATCAC-3′) with A36NarI-WD/AA R (5′-GATGGGCGCCATGACATTGGATTCG-TTAGCCGCTATTAAACTACC-3′) and A36-WE/AA SOE F (5′-TCAGACGCTGCAGATCACTGTAGTGCTATGGAAC-3′) with A36-WE/AA SOE R (5′-GTGATC-TGCAGCGTCTGATTCGCTATCAGTTGATTT AC-3′) and inserting the product into pEcoGPT-mCherry-A36rev and pEcoGPT-mCherry-A36YdF, respectively.

These plasmids were all used to generate recombinant viruses as described above using v∆A36-A5GFP as the parental virus to generate recombinants expressing both F12 and a mutant form of A36, and v∆A36∆F12-A5GFP as the parental virus to generate recombinants expressing the mutant A36 proteins but lacking F12 expression.

Viruses were analysed by PCR to identify recombinants. PCRs were used to distinguish the WT F12 and ∆F12 alleles (oligonucleotides F12RevDC1 5′-CATCTTTGATCTCGA-TGGAATGCA-3′ with F12ForDC1 5′-ATGTTAAACA-GGGTACAAATCTTGATGA-3′), the presence or absence of the EcoGPT cassette (oligonucleotides EcogptDCF1 5′-CGTCACCTGGGACATGTTG-3′ with EcogptDCR1 5′-GACGAATACGACGCCCATAT C-3′), the full length and ∆A36 alleles [oligonucleotides A36 UP F see above (green arrow in Fig. 2a) with DCA36seqR1 5′-ACCGTTTCAT-CCATCTGTCTATTG-3′ (blue arrow in Fig. 2a)], and the full length A36 using oligonucleotide A36deltestF (5′-CTTA-TCACGGTGACCGTAG-3′, red arrow in Fig. 2a) with DCA36seqR1. To verify that the correct A36 mutations had been incorporated into the final recombinant viruses PCR fragments were sequenced.