anisomycin and Cell-Transformation--Viral

The time course of accumulation of herpes simplex virus immediate-early (IE) mRNA and the requirement for infected cell protein synthesis for mRNA transcription and accumulation were compared. Measurements of transcription in nuclear run-on assays, accumulation of cytoplasmic mRNA by Northern (RNA) blot hybridization, and rates of infected cell protein synthesis by pulse-labeling did not indicate differences among the five IE gene, consistent with previous studies. However, as a result of varying the amount of de novo protein synthesis after infection, at least three patterns of maximal expression of the IE genes were revealed. Addition of the protein synthesis inhibitor anisomycin to cells coincident with infection resulted in maximal rates of transcription and accumulation of functional ICP0 mRNA, while 0.5 h of infected cell protein synthesis prior to addition of the drug was required for maximal expression of ICP22/47 and ICP27 mRNAs. Maximal expression of ICP4 mRNA occurred only when 1 h of de novo protein synthesis occurred prior to the addition of the drug. These results are discussed in the context of alternative mechanisms for regulating IE gene expression.

Topics: Animals; Anisomycin; Blotting, Northern; Cell Nucleus; Cell Transformation, Viral; DNA Probes; Gene Expression; Gene Expression Regulation, Viral; Genes, Viral; Kinetics; Protein Biosynthesis; Restriction Mapping; RNA, Messenger; Simplexvirus; Transcription, Genetic; Vero Cells

We studied the accumulation of viral mRNA in the presence of inhibitors of protein synthesis in an adenovirus type 5-transformed cell line (line 293 cells). An analysis of the endogenous viral mRNA's and proteins revealed that only early regions 1A and 1B were expressed in uninfected 293 cells. However, viral mRNA's from early regions 2, 3, and 4, as well as mRNA's from early regions 1A and 1B, accumulated in 293 cells after infection with adenovirus type 2. Cells treated with anisomycin before infection showed a drastic enhancement of mRNA from early region 4 compared with drug-free controls, This increase in viral mRNA was detected by using filter hybridization, S1 endonuclease mapping, and in vitro translation. The rate of transcription of early region 4 nuclear RNA also increased significantly in the presence of anisomycin. In contrast to the results with early region 4, the levels of cytoplasmic mRNA's from early regions 2 and 3 did not increase in cells treated with inhibitors. These results suggested that multiple virus encoded controls operate on the early regions of the adenovirus genome.

Topics: Adenoviruses, Human; Anisomycin; Cell Line; Cell Transformation, Viral; Gene Expression Regulation; Genes, Viral; Humans; Kidney; Protein Biosynthesis; RNA Splicing; RNA, Messenger; RNA, Viral; Transcription, Genetic; Viral Proteins