Baculoviruses are currently used as vectors for the transient high-level expression of foreign gene products in insect cells. In this study, we demonstrate that baculoviruses can also be made to continuously express a foreign gene product by using the promoter from IE1, an immediate early viral gene, to produce stably-transformed insect cells. This approach gave levels of foreign gene expression lower than those usually obtained with the lytic baculovirus expression vector system. Expression, however, was continuous and stable, and a complex human glycoprotein (tissue plasminogen activator) was processed more efficiently. We conclude that stable transformation is a feasible approach for baculovirus-mediated foreign gene expression in lepidopteran cells, particularly for products that are relatively poorly-expressed and/or processed in lytically infected cells.

The baculovirus Autographa californica multicapsid nuclear polyhedrosis virus expresses two immediateearly genes from the HindIII-G region (map units 90.4 to 96.8) of the genome. During the early phase of infection, nonspliced 1.9-kb and spliced 2.1-kb transcripts are expressed which encode the IEl and IEO (spliced IEI) gene products, respectively. These two gene products differ only in that IEO contains an additional 54 amino acids at the amino terminus. RNA analysis of these two genes during infection revealed that they were differentially expressed. IE1 was expressed early and late, whereas IEO was expressed only early in infection. The regulation of these two immediate-early genes was analyzed by transient expression assays. The IEl gene product stimulated expression of IEI promoter-directed expression but down-regulated expression from the IEO promoter. The IEO gene product also transactivated the IEl promoter but did not affect expression from its own promoter. Unlike IEl, which transactivates the delayed early 39K gene in the presence and absence of the homologous region (hr) enhancers, IEO transactivated the 39K promoter only in the presence of cis-linked hr5 enhancer. The results of this study in conjunction with previous results suggest that the IEl gene encodes a multifunctional gene product that may be involved in (i) repression of immediate-early gene expression, (ii) continued expression of its own gene product during infection, and (iii) transactivation of the delayed early and late classes of genes.

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Autographa califomica multicapsid nuclear polyhedrosis virus-infected insect cells express a viral immediateearly transcriptional regulatory protein, IEl, that has been shown by transient-expression assays to stimulate the expression of certain baculovirus delayed-early (DE) promoters and to inhibit the expression of other immediate-early (IE) genes. It is believed that certain DE promoters are activated, in part, by direct interactions between IEl and enhancer elements located in regions adjacent to these genes. We have used transient cotransfection and DNA-binding assays to examine the function of mutant forms of IEl. Our results indirectly show that IEl has at least two separable domains that are essential for its role in the modulation of baculovirus gene expression. A domain rich in acidic residues and essential for transactivation is located within the N-terminal 145 amino acids of the polypeptide. A second domain, located in the C-terminal 437 amino acids

The B1 gene of vaccinia virus encodes a serine/threonine protein kinase that is expressed early after infection. Under nonpermissive conditions, temperature-sensitive mutants (ts2 and ts25) that map to B1 fail to efficiently replicate viral DNA. Studies on the regulation of intracellular processes involved in eukaryotic nucleic acid metabolism have pointed to protein phosphorylation as playing a key role. Poxviruses have acquired analogs of a number of host cell genes to maintain their autonomy from the cell, including two serine/threonine protein kinases (B1 and F10) and a dual-specificity protein phosphatase (H1). The F10 kinase (22) and H1 phosphatase (15) are expressed late in infection, are incorporated into virions, and play major roles in virion morphogenesis (11,24,38). Interestingly, the H1 phosphatase has been implicated in early gene transcription (24). Mutant virus particles devoid of H1 phosphatase were unable to transcribe early genes either in vivo or in vitro. This finding led us to investigate the role that phosphorylation might play in the transcription of the other gene classes. The B1 kinase is expressed exclusively early in infection; it localizes to viral replication factories and appears to be a minor component of the virion (3,23,34