Figure 6.

Expression of exogenous ASS1 from a recombinant MYXV improves viral replication in ASS1^−/− tumors: (A) genomic schematic of recombinant viruses used in this study including MyxASS1 and MyxASS1^FS. (B) Expression of murine ASS1 in BSC40 cells infected with either MyxASS1 or MyxASS1^FS for 24 hours. (C) The indicated cells were incubated in complete media, −Arg media, or −Arg media supplemented with citrulline for 24 hours and then infected with either MyxASS1^FS (left) or MyxASS1 (right) at an MOI=5. Twenty-four hours post infection, cells were harvested and the amount of infectious virus present was quantified using viral titration assays. Statistical significance was determined by unpaired Student’s t-test (n=3 per group, per cell line). (D) ASS1^WT and ASS1^KO (KO#1 and KO#4) tumors were established in C57Bl/6 J mice. Tumors were then treated with a single bolus of 1×10⁶ FFU of either MyxASS1 or MyxASS1^FS. Six days post infection, tumors were harvested and the abundance of infectious virus was quantified using viral titer assay (n=3–6 tumors per group). Statistical significance determined by unpaired Student’s t-test (specific p values given). (E) A9F1 tumors were established in C57BL/6J mice and treated (as in D). Statistical significance was determined by unpaired Student’s t-test (n=5–6 tumors per group). ***P<0.001. Arg, arginine; ASS1, argininosuccinate synthetase 1; FFU, focus-forming unit; MyxASS1, MYXV encoding full-length murine ASS1; MyxASS1^FS, MYXV encoding a non-functional, frame-shifted murine ASS1; MYXV, myxoma virus; n.s., no significance; WT, wild type.