Viral inactivation by disulfide bond reducing agents

Disulfide bonds tie different peptide chains together and lock folded dipeptide chains into particular configurations. It is of interest to learn whether there is a differential susceptibility of viruses to inactivation by disulfide bond reduction. This may lead to information concerning differences in virion structures and may help in grouping them in different morphological categories. Dithiothreitol (DTT) is a highly water-soluble compound prepared by Evans et al. As noted by Cleland (6), DTT is thermodynamically capable of reducing the disulfide bonds found in nature, since it has a low redox potential (-0.33 v at pH 7). Thus, DTT is well-suited for use in studies of viral inactivation by disulfide bond reduction. Previous reports (1, 2, 4, 10-12) indicated that viruses exhibit a differential susceptibility to inactivation by sulfhydryl-binding reagents. In some cases, viral reactivation could be accomplished with the reducing reagents cysteine and reduced glutathione. We are unaware of any publication concerning viral inactivation by reduction of virion disulfide groups other than a recent report by Hare and Chan (8); these investigators used the reducing reagents DTT and mercaptoethanol to enhance the destructive effects of urea on polyoma, as measured by loss of infectivity and hemagglutination. Stocks of Sindbis, West Nile, and vaccinia viruses were prepared in chick embryo cell (CEC) monolayers. Poliovirus type 2, echovirus 6, and coxsackievirus B5 stocks were prepared in a line of green monkey kidney cells (MA134) obtained from Microbiological Associates, Inc. (Bethesda, Md.). These viruses were harvested when cytopathic changes appeared, between 24 and 48 hr after infection. Since all of the virus stocks grown in tissue culture were harvested in attachment solution (AS) medium, they all contained the same concentration of extraneous nonviral proteins. Stocks of Newcastle disease virus (California strain) were prepared from egg allantoic fluid as described by Marcus and Puck (9). A stock of Mayaro virus prepared in suckling mouse brains was obtained from Nicola Tauraso. Titers [plaque-forming units (PFU) per ml] of the virus stocks used were as follows: Sindbis, 108; Mayaro, 2 X 106; West Nile, 7 X 105; vaccinia, 2 X 105; Newcastle disease, 108; coxsackie B5, 4 x 105; type 2 polio, 3 X 107; and echo 6, 2 x 106. CEC monolayers were prepared in 60-mm plates as previously described (3) and were used when the cells first became confluent. Baby hamster kidney line cells (BHK-21; originally obtained from Microbiological Associates) and green monkey kidney line cells (MA134 and vero; obtained from Microbiological Associates) were kept in continuous culture, and monolayers of cells were prepared in 60-mm plastic petri dishes. These cells were used when they first became confluent. AS was used as medium for all of the cell cultures used. Plaquing procedures on all of the monolayers were performed as previously described (3). Sindbis virus was plaqued on CEC, BHK-21, and vero cells. West Nile, Newcastle disease, and vaccinia virus were plaqued on chick embryo cells. Coxsackievirus B5 and poliovirus type 2 were plaqued on vero cells. Mayaro virus was plaqued on MA134 cells. DTT, oxidized glutathione, reduced glutathione, mercaptoethanol, and cysteine were obtained from Calbiochem (Los Angeles, Calif.) and were all freshly dissolved in phosphatebuffered saline (PBS) before each experiment. The solutions were sterilized by passage through a swinnex filter (Millipore Corp., Bedford, Mass.) before use. Viral inactivation with DTT was done by adding 1.0 ml of stock virus to 1.0 ml of DTT solution and agitating the mixture at 37 C for 3 hr, except where otherwise stated. For controls, 1.0 ml of stock virus was added to 1.0 ml of PBS before agitation at 37 C for 3 hr. Serial 10-fold dilutions of virus in the test and control solutions were made with AS after the 3-hr agitation period. In experiments in which oxidized glutathione was added to the DIT before viral inactivation, the concentration of the reagents was adjusted so that the final concentration ofDTT in the solution was 3 X 10-3 M, as it was in the solution not containing oxidized glutathione. Treatment of viruses with cysteine and mercaptoethanol was done in the same manner as with DTT. Hemagglutination tests with goose cells were