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. 2011 Nov 9;6(11):e25674. doi: 10.1371/journal.pone.0025674

Figure 1. Generation and characterization of NYVAC-C-ΔB19R.

Figure 1

A) Scheme of construction. The plasmid transfer vector pGem-RG-B19R wm was obtained by sequential cloning of selectable markers dsRed2 and rsGFP and B19R recombination flanking sequences into the plasmid pGem-7Zf. For the generation of NYVAC-C-ΔB19R, BSC-40 cells were infected at an MOI of 0.01 with NYVAC-C and transfected 1 hour later with 6 µg DNA of plasmid pGem-RG-B19R wm. 48 hours post-infection, the cells were harvested, lysed by freeze-thaw cycling, sonicated and used for recombinant virus screening. The deletion mutant was selected from progeny virus by consecutive rounds of plaque purification in BSC-40 cells during which plaques were screened for Red2/GFP fluorescence. B) PCR analysis. Viral DNA was extracted from BSC-40 cells mock-infected or infected at an MOI of 5 with NYVAC wt, NYVAC-C or NYVAC-C-ΔB19R. Primers LFB19R-AatII-F and LFB19R-BamHI-R spanning B19R flanking regions were used for PCR analysis of B19R locus. C) Expression of HIV-1 antigens gp120 and GPN. BSC-40 cells were mock-infected or infected at an MOI of 5 with NYVAC wt, NYVAC-C or NYVAC-C-ΔB19R. At 48 hours post-infection, cells were lysed in Laemmli buffer, cells extracts were fractionated by 10% SDS-PAGE and analyzed by Western blot using rabbit polyclonal anti-gp120 antibody or polyclonal anti-gag p24 serum. D) Replication in CEF cells. CEF cells were infected at an MOI of 0.01 with NYVAC wt, NYVAC-C or NYVAC-C-ΔB19R. At different times post-infection (hpi, 0, 24, 48 and 72 hours), cells were harvested, freeze-thawed three times and sonicated. Virus titers in cell lysates were determined by crystal violet staining in BSC-40 cells.