In Spodoptera frugiperda (SF IPLB-21) cells productively infected with S. frugiperda multicapsid nuclear polyhedrosis virus (SfMNPV), we observed the synthesis of infected-cell-specific polypeptides (ICSPs) using the protein synthesis inhibitor cycloheximide (CX) and the DAN synthesis inhibitor cytosine arabinoside (Ara-C) in an attempt to assess whether a temporal cascade and viral DNA synthesis are involved in the gene expression program of SfMNPV. Inhibition of protein synthesis with CX at 0 h postinfection resulted in the active synthesis of a group of ICSPs immediately after removal of CX, suggesting that these polypeptides, designated α-ICSP, did not require de novo protein synthesis for their production. A second group of ICSPs (designated β-ICSPs), requiring a prior interval of protein synthesis, was detected when CX was added at later times. A third group of ICSPs also needed an interval of ICSP synthesis, but differed from β-ICSPs in that they required a longer interval of protein synthesis. Moreover, the synthesis of most of these ICSPs also required DNA synthesis, which was demonstrated by addition of Ara-C before and after viral DNA replication; these ICSPs were therefore designated γ-ICSPs. In conclusion, two kinds of regulatory proesses are involved in SfMNPV infection: the first process controls the sequential synthesis of the three ICSP groups, that is α→β→γ; a second process represses the synthesis of ICSP groups, first of the α-ICSPs and subsequently of β-ICSPs. Thus, we propose temporally regulated as well as negative control circuits in SfMNPV gen