**5. Expression of early genes**

At the time the virus reaches the nucleus, the template DNA for transcription as well as proteins of the virion that are carried over and may activate transcription are at their lowest levels. Therefore, the success of the infection depends on the ability of the virus to efficiently redirect the cellular system to express early genes encoded in its genome. Baculovirus DNA is infectious without any accompany protein, as proved by the infection that follows after transfection of permissive cells with viral DNA. This indicates that baculovirus early promoters are respon‐ sive to the RNA pol II and accordingly, their structural organization resembles that of the host genes which are transcribed into mRNAs.

Promoters of baculovirus early genes consist in a core region and regulatory proximal sequences that may be recognized for specific binding of transcription factors from either the host or the virus. The core promoter includes two characteristic elements: a TATA-box-like sequence and a transcription initiator (INR), although one or both are absent in some early promoters [12]. The TATA box is the site for assembly of the preinitiation transcription complex by first binding of TATA binding protein (TBP). After the RNA pol II is recruited to the complex, transcription starts about 30 nucleotides downstream of the position of the TATAbox. The INR determines the starting site for transcription (nucleotide +1 of the primary transcript), and ensures proper initiation when there is no TATA-box present. CAGT is the most conserved INR sequence motif in baculovirus early promoters. Other activating sequen‐ ces can be found either upstream of the core promoter or downstream, in the 5´UTR of the regulated gene.

Besides the sequences within the promoter region that modulate expression of a gene through the binding of regulatory proteins, there are sequences that enhance transcription from promoters even if they are located at a long distance. In baculoviruses there are non-coding regions known as homologous regions (*hr*) that play the role of enhancers [13] (see below).

Baculovirus early genes can be subdivided into two categories: immediate-early (*ie*) and delayed-early genes. Expression of *ie*-genes does not require viral factors, whereas the transcription of delayed-early genes was shown to need activation by *ie*-genes in transient expression assays. The major transactivator of AcMNPV early genes is the product of *ie1*, a gene that is present in all lepidopteran baculoviruses [14].

Other *ie-*genes known to regulate the expression of early genes are *ie2* and *pe38*. *ie2* is conserved only in group I alphabaculoviruses, one the two phylogenetic lineages in which members of this genus can be separated. In transient expression experiments it was determined that *ie2* increases IE1-mediated transactivation of early promoters when *ie1* is present at low concentrations [15]

#### **5.1. Immediate early transactivator IE1**

**Figure 1.** Kinetics of transcription of baculovirus genes. The time courses of steady-state level of transcripts (y axis) of the three AcMNPV temporal classes of genes, in Sf21 cells, are shown schematically. Solid lines represent mRNAs in wild-type (wt) infection; dotted lines represent idealized transcriptional profiles in infections in which a virus gene re‐ quired for either DNA replication or late transcription was silenced by RNAi (denoted by "-"). Early genes are transcri‐ bed by the host RNA polymerase II before virus DNA replication, which initiates 6 to 9 hours post infection (hpi). Transcripts of early genes are detectable within 1 hpi, reach a maximum between 6 to 12 hpi and diminish thereafter (shutoff). Transcription of late and very late genes depends on viral DNA replication and is performed by a virus-en‐ coded RNA polymerase. Transcription of late genes begins with the onset of DNA replication and continues mainly up to 24 hpi, while transcription of very late genes bursts around 18 hpi and continues through 72 hpi. The figure shows that late/very late gene transcription is abolish by silencing *p47*, a gene that encodes a subunit of the RNA polymer‐ ase, which is essential for the activity of the enzyme. A similar effect is shown as consequence of silencing *lef3*, a gene essential for virus DNA replication, since DNA replication is required for transcription of late/very late genes. However, the effects of silencing these genes on expression of early genes are different. When *p47* is silenced, early gene tran‐ scription declines as in wild-type infection; in contrast, silencing of *lef3* blocks the reduction of transcription, indicating that DNA replication, rather than late transcription, is a primary determinant of the shutoff of early genes (adapted

60 Current Issues in Molecular Virology - Viral Genetics and Biotechnological Applications

from [65]).

AcMNPV IE1 is a 582 aminoacids long protein exhibiting general characteristics of transcrip‐ tion factors (figure 2). It has a modular organization with domains associated to different functions: dimerization, nuclear import, DNA-binding, transactivation and replication [16-17]. Dimer formation is required for nuclear localization since the protein mutated in the dimeri‐ zation domain cannot be imported into the nucleus [18]. IE1 transactivates early promoters including its own. Two mechanisms are postulated by which IE1 is capable of activating an early promoter: one independent and one dependent on DNA-binding. In the first one, IE1 activates transcription by interaction with cell factors recruited to the promoter regulatory regions. In the DNA-binding-dependent mechanism, the activation depends on binding of IE1 to *hr* sequences which function as enhancers. The *hrs* contain a variable number of imperfect palindromic repeats in tandem, separated by non-palindromic sequences. The palindromes are conserved within a genome but differ widely between genomes. In AcMNPV the imperfect palindrome consensus sequence has a length of 28 nucleotides with a central *Eco*RI site. According to the current knowledge, each subunit of the IE1 dimer interacts with a corre‐ sponding hemipalindrome during binding to an *hr*. Binding of IE1 to the enhancer increases the effective concentration of IE1 molecules able to interact with cellular factors in the promoter region. It was found that *hrs* also bind cellular factors in sites overlapping palindromic repeats and within interpalindromic regions [19-20]. *Hrs* are enriched in sequence motifs similar to cAMP and TPA response elements known to interact with transcription factors of the bZIP family. This is consistent with the ability of *hrs* to enhance transcription from baculovirus early promoters even in the absence of viral factors (see [21]for a review).

It has been reported that AcMNPV IE1 down-regulates the expression of certain genes. Promoters of these genes contain a sequence motif similar to one half of a typical *hr* palindrome which still functions as a target for IE1 binding; however, IE1 bound to this sequence is no longer able to promote activation, instead it functions as a repressor [22].

IE1 is the only known baculoviral gene that is expressed as part of a product of alternative splicing designated IE0. Compared to IE1, AcMNPV IE0 contains 54 additional aminoacids at its N-terminus. Both protein species are required for an efficient infection, although each one is dispensable given the other is present [23].
