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VOL. 76 (3), 327-342, 2010 ANTITUMORAL ACTIVITY OF ONCOLYTIC VACCINIA VIRUS...

Figure 1. Characteristics of vaccinia virus recombiant vectors expressing PKR.
A. Scheme of the recombinant vaccinia virus vectors. The Western Reserve strain of
vaccinia virus was used to generate the recombinant viruses expressing the human
wild type (WT) and the mutant PKR proteins. The recombinant genes were inserted
in the TK locus of viral genome. PKR expression is regulated by the lac I repressor
gene that is under the control of VV early-late promoter p7.5. The mutant PKR, K296R
has lost the catalityc activity of PKR due to substitution of the lysine 296 by argi-
nine. B. TRAMP-C1 cells were infected with 5 pfu/cell of vaccinia virus recombinants
VV-PKR and VV-K296R in presence or absence of the inductor IPTG (5 mM). The
PKR expression and PKR catalytic activity was analyzed at different time post-infec-
tion using specific antibodies against total PKR and the phosphorylated form of
eIF2a. C. TRAMP-C1 cells were infected with 5 pfu/cell of vaccinia virus recombi-
nants VVLUC and VV-PKR in presence or absence of the inductor IPTG (5 mM). The
vaccinia virus proteins at 24 hpi were detected using specific polyclonal antibody to
VACV proteins. D. TRAMP-C1 cells were infected with 0.001 pfu/cell of vaccinia virus
recombinants VVLUC and VVPKR in presence or absence of the inductor IPTG
(5 mM). Virus yields were measured at 0 and 48 hpi after titration by plaque assay
in monkey BSC-40 cells.

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