Isolation and Characterization of Tubules and Plasma Membranes from Cytophaga columnaris

Arildone (also known as Win 38020), a novel aryl diketone, inhibited replication of herpes simplex virus type 2 in tissue culture by interfering with an event that occurs prior to 6 h postinfection. The inhibition could be partially reversed by washing. Although the exact mechanism of action is unknown, neither viral deoxyribonucleic acid nor viral proteins were synthesized in the presence of arildone. Plaque assay. HSV-2 was quantitated by plaque assay. Serial 10-fold dilutions were made in Leibovitz medium (L-15), and 2-ml portions of the dilutions were pipetted onto 3-or 4-day-old monolayers of BSC1 cells grown in 25-cm2 plastic flasks. After 1 h at 330C, the inoculum was removed and replaced with 5 ml of L-15 supplemented with 5% calf serum (Reheis, Chicago, Ill.) and 0.65% Ionagar (Colab, Chicago Heights, Ill.). Infected cultures were incubated for 3 days at 330C and flooded with a solution of crystal violet stain (0.25%), prepared with 10% Formalin containing 2% sodium acetate. Plaques were counted after the agar was removed from the flasks and expressed as plaqueforming units per flask.Cell viability. showed that DMSO had no effect on HSV-2 replication at the concentrations employed. Three-or 4-dayold monolayers of BSC, cells in test tubes or Falcon flasks were used to determine the effect of arildone when added at various times after infection. HSV-2 Curtis strain, containing from 5 x 105 to 3 x 10' plaque-forming units per ml, was used to inoculate the cells, usually at a multiplicity of infection of about 1. Virus dilutions were prepared in L-15, and inoculum volumes were 0.2 ml and 2 ml for tubes and flasks, respectively. Cultures were incubated at 33°C for 1 h, the inoculum was removed by aspiration, and the monolayers were washed twice with 2-ml or 5-ml volumes of L-15. Fresh L-15 containing 5% calf serum and various concentrations of arildone was added, and incubation was continued at 33°C. In all experiments, 0 h postinfection (PI) refers to the time of addition of the fresh medium. For time of addition experiments, medium without arildone was added at 0 h PI and replaced with medium containing 3 ug of arildone per ml at the indicated time. Reversal of drug action was initiated by removing the culture fluid containing arildone, washing the monolayers twice with 1 or 2 ml of L-15, and finally continuing the incubation with fresh medium devoid of arildone. Virus yield was determined by discarding the culture fluid from monolayers, replacing it with 0.1% EDTA, and then assaying the contents after two cycles of freezing and thawing. Pilot studies had shown that prior to lysis the culture fluid contained less than 2% of the total virus.

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Isolation and Characterization of Tubules and Plasma Membranes from Cytophaga columnaris

Cited by 3 publications

References 34 publications

The Order Cytophagales

The Order Cytophagales

The Order Cytophagales

Preliminary Studies of the Mode of Action of Arildone, a Novel Antiviral Agent

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