GA inhibition of enveloped viruses correlates with inhibition of viral DNA and protein synthesis
To validate the PRA observations, we tested whether there is an effect on downstream viral DNA synthesis in HCMV infected cells when treated with GA. HFF were inoculated with HCMV for 3 hours, then washed and incubated with different concentrations of GA or vehicle. DNA was extracted from 4 pooled wells of HFF at 7 days post inoculation, and then analyzed by qPCR targeting the viral polymerase gene. DNA copies were reduced in a dose dependent manner by the addition of GA to culture medium (Fig. 7A). Although HCMV DNA copy numbers were reduced by GA, this could be secondary to inhibition of virus entry rather than direct inhibition of DNA replication.
Figure 7 GA inhibits HSV-1 replication by inhibition of viral protein synthesis and HCMV by DNA synthesis. (A) Effect of GA on HCMV DNA synthesis. Viral replication is inhibited in the presence of increasing concentration of GA, as determined by decreasing viral DNA copies. DNA was extracted from 4 pooled wells of HFF at 7 dpi, then analyzed by qPCR targeting the viral polymerase gene. DNA copies were reduced in a dose dependent manner by the addition of GA to culture medium as determined by ANOVA analysis with pairwise post-hoc t-test analysis in SPSS. Data are presented as mean percent of control. (*5 µM p = 0.014, 8 µM p = 0.003, 10 µM p = 0.002). 6 well cultures of HEp-2 (B) or 293T (C) were infected with HSV-1 strain F at an MOI of 1 for 2 hours, then washed with 199V and supplemented with 10 µM GA (Lanes 1–4) or vehicle (Lanes 5–8). Mock (M), 5, 11 and 24 hours after inoculation, cells were collected and a fraction of the total cell lysate was subject to Western Blot analysis using antibodies directed against ICP8, ICP27, β-Actin and US11. Normalized ratios of protein expression are in the bar graphs.
Viral protein synthesis was determined in HSV-infected cells following GA treatment. Untreated HEp-2 and 293T cells were infected with HSV-1 F’ at an MOI of 1 for 2 hours, allowing the virions to internalize into the cells, then washed with 199V medium and supplemented with 10 µM GA or vehicle. The viral replication was evaluated by Western Blot detection of HSV-1 proteins. Although the infection of HEp-2 and 293T cells had been initiated, the addition of 10 µM GA to the infected HEp-2 and 293T cells inhibited the virus replication from the point of exposure to GA (Fig. 7B,C). The observed effect could be the result of inhibition of secondary infections. However, the early down regulation of HSV proteins, occurring prior to a complete viral replication cycle, suggests that there could be a secondary inhibitory mechanism targeting viral protein synthesis.