REPLICATION OF AUTOGRAPHA-CALIFORNICA NUCLEAR POLYHEDROSIS-VIRUS IN A THYMIDINE KINASE DEFICIENT SPODOPTERA-EXIGUA CELL-LINE (SE-UCR-1A)

Cultivation of a Spodoptera exigua cloned cell line (SE-UCR-1A) for 8 to 9 mo. in a medium containing increasing amounts of bromodeoxyuridine (BUdr) resulted in the selection of a BUdr-resistant subline unable to grow in TNMFH medium supplemented with HAT (hypoxanthine, 10-4 M; aminopterin, 10-7 M; thymidine, 10-3 M). Subsequent assay of this subline revealed an absence of thymidine kinase (TK) activity. The specific activity of the wild-type (wt) cells was 878±192 counts per min (cpm)/μg supernatant protein compared to 9 cpm/μg for the BUdr-resistant, HAT-sensitive subline. In addition the wt activity was inhibited >90% by addition of BUdr to the assay, indicating that the activity is predominantly due to TK and not to a nonspecific nucleoside phosphotransferase. The morphology of the TK-deficient (-) cells was indistinguishable from that of wt cells. The doubling time for wt cells in TNMFH was 16 h; however, in TNMFH-HAT it was 36 h. In comparison the TK(-) cells in TNMFH had a doubling time of 61 h. Cultivation of TK(-) cells in nonselective TNMFH for 14 mo. to date has not changed the TK(-) characteristic of the subline. The host-cell TK was not required for development of progeny virus from Autographa californica NPV inoculum. Although similar numbers of cells were infected (80 to 90%) in the wt and TK(-) lines and extracellular virus was generated at similar rates to similar titers in both media, the initial appearance of virus in the medium of TK(-) cell was delayed 10 to 20 h compared to wt cells. In addition, polyhedra appearance was similarly delayed in TK(-) cells and only 20 to 25% of the cells contained >10 polyhedra per cell compared to 75 to 90% for wt cells. Also, during infection of wt cells, specific activity of TK increased twofold peaking at 20 to 30 h postinfection, yet there was no stimulation of TK activity in infected TK(-) cells. © 1990 Tissue Culture Association.