Frog Virus 3 Replication: Induction and Intracellular Distribution of Polypeptides in Infected Cells

Abstract

The synthesis of the polypeptides induced in frog virus 3-infected cells was analyzed by high-resolution sodium dodecyl sulfate-polyacrylamide gel electrophoresis of radiolabeled cell extracts. Purified frog virus 3 contained 22 polypeptides, with molecular weights in the range 9 x 10(3) to 114 x 10(3). All of the structural and an additional seven nonstructural polypeptides were detected in infected cell lysates. The following three classes of induced polypeptides (under temporal control) were observed in BHK cells: at 2 h, four alpha polypeptides; at 4 h, 13 beta polypeptides; and at 6 h, the remaining 12 gamma polypeptides. The total molecular weight of the infected cell-specific polypeptides (ICPs) was approximately 1.5 x 10(6), which accounts for about 30% of the coding capacity of the viral genome. At least 10 of the induced polypeptides were phosphorylated, but none was glycosylated or sulfated. No evidence for posttranslation cleavage of polypeptides in pulse-chase and inhibition experiments was obtained. The synthesis of gamma polypeptides was not detected in the presence of the viral DNA replication inhibitors cytosine arabinoside and hydroxyurea, but halogenated nucleotides apparently had no effect. These results suggest that alpha and beta polypeptides are "early" events and that detectable gamma polypeptide synthesis is dependent on the production of progeny viral DNA. The regulation of frog virus 3-induced polypeptide synthesis in infected BHK cells was examined by using inhibitors of protein and RNA synthesis and amino acid analogs. These experiments confirmed the existence of three sequentially synthesized, coordinately regulated classes of polypeptides, designated alpha, beta, and gamma. The requirements for the synthesis of each class were as follows: (i) alpha polypeptides did not require previous cell protein synthesis; (ii) beta polypeptides required a prescribed period of alpha polypeptide synthesis and new mRNA synthesis; and (iii) gamma polypeptides required prior synthesis of functional beta polypeptides and new mRNA synthesis. alpha polypeptide synthesis was controlled by beta and gamma polypeptides, and alpha and beta polypeptides were involved in the suppression of host cell polypeptide synthesis. Indirect evidence was obtained for the temporal regulation of frog virus 3 transcription. The intracellular distribution of virus-induced polypeptides in cells infected with frog virus 3 was investigated by using standard cell fractionation techniques. Most of the 29 induced polypeptides were bound to structures within the nucleus, and only two ICPs were not associated with purified nuclei. When isolated nuclei were incubated in an infected cell cytoplasm preparation, all of the nuclear ICPs were incorporated in vitro. All of the ICPs were associated with ribosomal and rough endoplasmic reticulum fractions of infected cells, and a number of ICPs were found on smooth intracellular membranes. Most of the ICPs were also associated with purified plasma membranes of infected cells, and one polypeptide (ICP 58) was highly enriched in the plasma membrane compared with whole cell extracts or purified frog virus 3.