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. 2022 Sep 8;2(9):100287. doi: 10.1016/j.crmeth.2022.100287

Figure 2.

Figure 2

CM treatment generates replication-defective HSV-2

(A) Growth curve analysis of CM-treated HSV-2-GFP in the ARPE-19 cells. HSV-2-GFP was treated with different concentrations of CM for 2 h at RT, followed by washing using 20% sucrose, and added to ARPE-19 cells at an MOI of 0.2. The experiment was performed in triplicates. A standard plaque assay was performed to determine the growth curves of HSV-2-GFP using samples collected at indicated time points. At 100 μM CM, the virus was inactive and unable to infect the cells. HSV-2-GFP treated with 10 μM CM could not grow in infected cells due to defective replication. At 1 μM CM, the virus was able to infect cells and grew at a reduced rate. Data are presented as mean ± SEM.

(B) Fluorescence image of HSV-2-GFP treated with different concentrations of CM. GFP represents the HSV-2-GFP-infected cells, DIC represents the cells in the bright phase, and merge represents the merge of both images. The images were taken at 40× objective post 36 h of virus infection. HSV-2-GFP treated with 10 μM CM was able to infect cells, but the virus cannot further grow due to defective replication produced by CM-mediated alkylation of viral DNA. The untreated virus grows efficiently. HSV-2-GFP treated with 100 μM CM was completely inactive. Treatment of virus with 1 μM CM leads to host cell infection and a slow spread to neighboring cells. The alkylation of viral DNA can lead to defective replication and transcription of genes.