The biological properties of cowpox virus (CPXV) mutants with target deletion of 4 of the 6 BTB/kelch genes (D11L, C18L, G3L, and A56R) were examined in CV-1 cell cultures. There were changes in mutant temperature sensitivity and a reduction in a viral cytopathic effect. The mutant-infected culture yielded a smaller number of cells with actin-related long cellular protrusions (63 of 300 cells) as compared with wild CPXV (127 of 300). The length of the protrusions was 20-60 and 40-120 microm, respectively). Confocal microscopy revealed the formation of large globed structures containing both actin and CPXV antigens in the cells infected with quadruple mutants. These globed structures were recognized as incomplete protrusions. The findings show that the formation of long protrusions in the cells infected with wild type CPXV represents a type of specific viral potency related to the activity of BTB/kelch genes whose deletion results in cellular insufficiency to form full-fledged protrusions.