Recently I began experiments to understand how zinc inhibits rhinovirus replication, and I promised to document my findings on the pages of this blog. Here are the results of the second plaque assay.
In the last experiment I confirmed the finding that 0.1 mM ZnCl2 inhibits plaque formation by rhinovirus type 1A. Based on the results of that plaque assay, shown in the figure at left, I’ve decided that this concentration of zinc isn’t sufficient to completely inhibit viral replication. Although 0.1 mM ZnCl2 blocked plaque formation when 20 or 200 pfu were inoculated on cells, many plaques arose on plates inoculated with 2000 pfu. These cannot be viral mutants resistant to zinc – there are too many of them. If there are 2000 plaques on the untreated plate, and 200 on the plate with zinc, that would mean that resistance to zinc arises at a frequence of 200/2000 = 0.1 or one in ten viruses. I would expect a mutation rate for an RNA virus to be more in the range of 1/1000 – 1/10,000.
I decided to repeat the plaque assay with higher levels of zinc in the agar overlay – 0.2, 0.3, and 0.4 mM. The results of that experiment are shown below.
Unfortunately, the cells did not like the higher concentrations of ZnCl2! All the plates with zinc had very lightly staining monolayers – compare with the original experiment shown above – and no plaques were visible.
I was surprised that slight increases in the concentration of ZnCl2 would have such a dramatic effect on cell viability. The cells I used are HeLa cells, which are quite sturdy. Two other variables were changed in this experiment. I made a new stock of ZnCl2 – I took the 1 M stock I made for the first experiment and diluted it to 0.1 M. I doubt this made any difference. Second, the cell monolayers were less confluent than in the first experiment – about 70% of the surface of the cell culture dish was covered with cells. In the previous experiment, the monolayers were 100% confluent.
I repeated the experiment with HeLa cell monolayers that are 100% confluent. The results will be posted here in a few days.