Journal:

Article Title: Human Cytomegalovirus Up-Regulates the Phosphatidylinositol 3-Kinase (PI3-K) Pathway: Inhibition of PI3-K Activity Inhibits Viral Replication and Virus-Induced Signaling

doi: 10.1128/JVI.75.13.6022-6032.2001

Figure Lengend Snippet: Antiviral effects of LY294002 on HCMV lytic life cycle. HEL fibroblasts were grown to confluence, serum starved for 48 h, infected with HCMV, and harvested at the indicated times postinfection. (A) Viral titers are reduced in the presence of LY294002. Confluent, serum-starved fibroblasts were pretreated with LY294002 and then were infected with HCMV in the presence of LY294002. Six days after infection, supernatant was harvested and a standard HCMV plaque assay was performed to determine the infectious virus titer. The graph shows the relative number of plaques in each sample, where 100% is the infectivity of supernatant harvested from cells infected in the absence of LY294002. Each sample was done in triplicate, and the entire assay was performed three times. Error bars represent the standard deviation for the experiment. (B to D) Effect of LY294002 on viral DNA replication. Confluent, serum-starved fibroblasts were infected with HCMV in the presence of the indicated chemical compounds. Where indicated, LY294002 was removed from the culture media at 96 hpi, and the infection was allowed to continue for an additional 24 h. Cells were harvested at 96 hpi (B and D) or 120 hpi (C) and were analyzed for total viral DNA by dot blot hybridization. DHPG was used as a positive control. PI-3,4-P2 and PI-3,4,5-P3 are the lipid products of active PI3-K and were used to demonstrate that the inhibition of viral DNA replication by LY294002 is due to inhibition of PI3-K activity. (A to C) Each experiment was performed a minimum of three times, and representative results are shown. (D) Results from two separate experiments are shown. Dot blot analysis was viewed on X-ray film. DHPG, ganciclovir; inf, infection; mock, mock-infected cells.

Article Snippet: Ganciclovir (1,3-dihydroxy-2-propoxymethylguanine [DHPG]), a nucleotide analog that specifically inhibits HCMV viral DNA replication ( 48 ), was from Syntex Inc. (Palo Alto, Calif.).

Techniques: Infection, Plaque Assay, Standard Deviation, Dot Blot, Hybridization, Positive Control, Inhibition, Activity Assay