Regulation of Herpesvirus Macromolecular Synthesis I. Cascade Regulation of the Synthesis of Three Groups of Viral Proteins

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Abstract
Based on evidence that 50% of herpes simplex 1 DNA is transcribed in HEp-2 cells in the absence of protein synthesis we examined the order and rates of synthesis of viral polypeptides in infected cells after reversal of cycloheximide- or puromycin-mediated inhibition of protein synthesis. These experiments showed that viral polypeptides formed three sequentially synthesized, coordinately regulated groups designated α, β, and γ. Specifically: (i) The α group, containing one minor structural and several nonstructural polypeptides, was synthesized at highest rates from 3 to 4 h postinfection in untreated cells and at diminishing rates thereafter. The β group, also containing minor structural and nonstructural polypeptides, was synthesized at highest rates from 5 to 7 h and at decreasing rates thereafter. The γ group containing major structural polypeptides was synthesized at increasing rates until at least 12 h postinfection. (ii) The synthesis of α polypeptides did not require prior infected cell protein synthesis. In contrast, the synthesis of β polypeptides required both prior α polypeptide synthesis as well as new RNA synthesis, since the addition of actinomycin D immediately after removal of cycloheximide precluded β polypeptide synthesis. The function supplied by the α polypeptides was stable since interruption of protein synthesis after α polypeptide synthesis began and before β polypeptides were made did not prevent the immediate synthesis of β polypeptides once the drug was withdrawn. The requirement of γ polypeptide synthesis for prior synthesis of β polypeptides seemed to be similar to that of β polypeptides for prior synthesis of the α group. (iii) The rates of synthesis of α polypeptides were highest immediately after removal of cycloheximide and declined thereafter concomitant with the initiation of β polypeptide synthesis; this decline in α polypeptide synthesis was less rapid in the presence of actinomycin D which prevented the appearance of β and γ polypeptides. The decrease in rates of synthesis of β polypeptides normally occurring after 7 h postinfection was also less rapid in the presence of actinomycin D than in its absence, whereas ongoing synthesis of γ polypeptides at this time was rapidly reduced by actinomycin D. (iv) Inhibitors of DNA synthesis (cytosine arabinoside or hydroxyurea) did not prevent the synthesis of α, β, or γ polypeptides, but did reduce the amounts of γ polypeptides made.