# The Impact of the S-Adenosylmethionine Analogue Sinefungin on Viral Life Cycles
## 3 Results
### Antiviral Activity
At 50 and 25 \(\mathrm{\SIUnitSymbolMicro g}\)/mL, the titers were 7.2 \(\times\) 10\({}^{4}\) and 1.0 \(\times\) 10\({}^{5}\) PFU/mL, respectively (\(\mathrm{log_{10}}\) fold reduction \(<\) 0.35). At 12 h, the control titers increased to 1.53 \(\times\) 10\({}^{9}\) PFU/mL. Sinefungin at 200 and 100 \(\mathrm{\SIUnitSymbolMicro g}\)/mL reduced the titers to 8.0 \(\times\) 10\({}^{4}\) and 7.2 \(\times\) 10\({}^{6}\) PFU/mL, respectively (\(\mathrm{log_{10}}\) fold reduction = 4.28 and 2.33). At 50 and 25 \(\mathrm{\SIUnitSymbolMicro g}\)/mL, the titers were 9.04 \(\times\) 10\({}^{6}\) and 1.53 \(\times\) 10\({}^{9}\) PFU/mL, respectively (\(\mathrm{log_{10}}\) fold reduction = 2.23 and 0). At 24 h, the control titers further increased to 1.71 \(\times\) 10\({}^{12}\) PFU/mL. Treatment with 200 and 100 \(\mathrm{\SIUnitSymbolMicro g}\)/mL resulted in titers of 1.12 \(\times\) 10\({}^{6}\) and 6.51 \(\times\) 10\({}^{6}\) PFU/mL (\(\mathrm{log_{10}}\) fold reduction = 6.18 and 5.42). At 50 and 25 \(\mathrm{\SIUnitSymbolMicro g}\)/mL, the viral titers were 5.9 \(\times\) 10\({}^{10}\) and 1.5 \(\times\) 10\({}^{12}\) PFU/mL, with modest or no reduction (\(\mathrm{log_{10}}\) fold reduction = 1.46 and 0.06). Overall, the data demonstrate the potent antiviral activity of sinefungin against HSV-1, particularly at 200 \(\mathrm{\SIUnitSymbolMicro g}\)/mL, with increasing effectiveness over time. Against SARS-CoV-2, sinefungin also exerted significant antiviral activity in a time- and dose-dependent manner, though this was slightly less pronounced than that for HSV-1. To confirm the results obtained from the plaque reduction assay, qPCR was performed (Figure 5). For HSV-1, the expression of the _UL27_ gene, a late gene encoding the structural glycoprotein B (gB), was evaluated. For SARS-CoV-2, the expression levels of the \(S\) gene, which encodes the spike protein involved in viral entry into host cells, were analyzed. A virus-sinefungin co-treatment assay was conducted for the molecular analysis. In the SARS-CoV-2-infected cells, treatment with sinefungin at 200 \(\mathrm{\SIUnitSymbolMicro g}\)/mL resulted in an approximately 50-fold reduction in the \(S\) gene expression compared to that in the virus control. At 100 \(\mathrm{\SIUnitSymbolMicro g}\)/mL and 50 \(\mathrm{\SIUnitSymbolMicro g}\)/mL, the reductions were 2.36-fold and 0.98-fold, respectively, indicating a loss of antiviral efficacy at lower concentrations. For HSV-1, the reduction in the _UL27_ gene expression was even more pronounced, further confirming the plaque reduction assay findings observed in the infected monolayers. Treatment with 200 \(\mathrm{\SIUnitSymbolMicro g}\)/mL of sinefungin led to a 96.1-fold decrease in gene expression, while reductions of 8.47-fold and 1.26-fold were observed at 100 \(\mathrm{\SIUnitSymbolMicro g}\)/mL and 50 \(\mathrm{\SIUnitSymbolMicro g}\)/mL, respectively. Figure 3: Antiviral activity of sinefungin against HSV-1 in a virus yield reduction assay. VERO-76 cells infected with HSV-1 (MOI 0.01) were treated with sinefungin (25–200 \(\mathrm{\SIUnitSymbolMicro g}\)/mL). The viral titers were measured at 10 (**A**), 20 (**B**), and 30 (**C**) hours post-infection. Sinefungin induced a dose- and time-dependent reduction in the viral yield, with a maximal effect at 200 \(\mathrm{\SIUnitSymbolMicro g}\)/mL, resulting in a \(\mathrm{log}_{10}\) reduction of 0.48 at 10 h (**A**), 1.82 at 20 h (**B**), and 6.3.05 at 30 h (**C**). The data represent the mean \(\pm\) standard deviation (SD) of three independent experiments: ****\(p\)-value \(\leq\) 0.0001; ****\(p\)-value \(\geq\) 0.0002; ns \(p\)-value \(\geq\) 0.1975. Figure 2: Antiviral activity of sinefungin against HSV-1 (**A**) and SARS-CoV-2 (**B**) in a plaque reduction assay. VERO-76 cell monolayers were co-treated with sinefungin (25–200 \(\mathrm{\SIUnitSymbolMicro g}\)/mL) HSV-1/SARS-CoV-2 (MOI 0.01).