Figure 1.

Centanamycin treatment generates attenuated replication-defective human cytomegalovirus

(A) In vitro growth curve analysis of Toledo-Luc treated with different concentrations of centanamycin (CM). Toledo-Luc was added to MRC-5 cells after treatment with CM for 2 h at RT, followed by washing. Luciferase activity was measured on the indicated days using IVIS-50. Growth curve lines for 10 and 100 μM CM are hidden under a 1 μM growth curve. The virus was not able to grow in 100, 10, and 1 μM concentrations. At 0.1 μM CM, Toledo-Luc could infect the cells but was unable to replicate, as suggested by the luciferase activity of infected cells. At low concentrations such as 0.01 μM CM, the virus grows initially but did not reach its peak, which may be due to damage to viral DNA caused by CM. Random alkylation of viral gDNA can lead to defective replication of the virus. RLU, relative light units. Data are presented as mean ± SEM. Each data point represents the average triplicate value.

(B) Fluorescence image of ARPE-19 cells infected with CM-treated AD169-GFP. The representative images of infected cells were acquired on the 8th day post-infection. GFP represents the AD169-GFP-infected cells, DIC represents the cells in the bright phase, and merge represents the merge of both images. At 10 μM CM, AD169-GFP was not able to infect the cell, and the virus was inactive. AD169-GFP treated with 1 μM CM was able to infect the cells but unable to replicate. Due to defective replication, the GFP signal of the virus could not spread to adjacent cells and was limited to only 1 or 2 infected cells. These results confirm replication-defective viruses that can infect cells but not replicate. At 0.01 μM CM, AD169-GFP was able to infect and spread to adjacent cells but at a reduced rate.