Analysis of the lytic step in the herpes simplex virus antibody-dependent cellular cytotoxicity system

Abstract

An antibody-dependent cellular cytotoxicity (ADCC) system in which herpes simplex virus-infected Chang liver cells are used was assessed for its dependency on cellular energy, ribonucleic acid and protein synthesis, and cytoskeletal structures such as microfilaments and microtubules. The cytotoxic reaction was only slightly inhibited when glycolysis was blocked in a glucose-free medium containing 10(-2) M 2-deoxy-d-glucose. It was more substantially inhibited when respiration was blocked with 10(-2) M sodium azide. The reaction was totally ablated, however, only when both glycolysis and respiration were suppressed. This inhibitory effect of energy deprivation was mediated solely at the level of the effector cell. Ribonucleic acid synthesis or protein synthesis by the effector cells was not required, as shown by the fact that neither actinomycin D, cycloheximide, nor emetine significantly inhibited ADCC. The ADCC reaction was partially inhibited by cytochalasin B, whose inhibitory effect was rapidly reversible, and was completely and irreversibly inhibited by cytochalasin A. Cytochalasin A acted on the effector cells rather than the target cells. The reaction was also partially inhibited by colchicine, whose inhibitory effect was directed solely against the effector cells and was slowly reversible. The inhibitory effects of cytochalasin B and colchicine, when used in tandem at submaximal inhibitory concentrations, were slightly more than additive. The results suggest a cooperative role for effector cell microfilaments and microtubules in mediating ADCC. Kinetic studies of ongoing herpes simplex virus ADCC reactions after initial centrifugation showed that the lytic step requires expenditure of metabolic energy as well as intact function of both microfilaments and microtubules. These findings, in concert with previous data, indicate that the ADCC process against herpes simplex virus-infected Chang liver cells can be resolved into adhesion and lytic steps. The lytic step can be readily distinguished from the adhesion step by its increased sensitivity to low ambient temperature or metabolic energy deprivation, its sensitivity to thermal inactivation, its requirements for extracellular divalent cations, and its dependence on normal function of both microfilaments and microtubules.