**4. Transcription program of baculovirus genes**

feature that differentiates baculovirus from other DNA viruses that replicate in the nuclei of infected cells is that they encode a novel DNA-dependent RNA polymerase. This enzyme, of uncertain evolutionary origin, is responsible for transcription of baculovirus late and very late genes. At this time of the infection the transcription of most cellular genes is shutoff and the synthesis of the polyhedrin/granulin becomes prominent to finally account for up to 95% of total cellular protein production. This high capacity of protein synthesis has been exploited for the development of baculoviruses as vectors for expres‐

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

**2. Two types of virus progeny serve at different steps of host invasion**

ruses and deltabaculoviruses are restricted to the midgut of their hosts.

Baculovirus infection causes cells to enlarge and stop dividing; the nucleus swells and forms the virogenic stroma (VS), which is the nuclear compartment where the viral DNA is replicated and the nucleocapsids of virus progeny are assembled. The host chromatin adopts a marginal distribution at 24 hours post infection (hpi), when the VS becomes evident. At this time, it was shown that histone H4 fused to fluorescent protein markers colocalizes with the chromatin in the periphery but not with the VS [5]. The new distribu‐ tion of the chromatin is determined by the replication of the virus and may have effects

**3. Nuclear events associated with infection**

Alphabaculoviruses and betabaculoviruses produce virions of two phenotypic classes: occlusion-derived virus (ODV) and budded-virus (BV). Nucleocapsids of both types of virus particles are assembled in the nucleus of the infected cell. During the late phase of infection, when proteins of the nucleocapsid are expressed, BVs are produced as the newly formed nucleocapsids exit the cell, acquiring their envelopes from the cell membrane during the budding process. BVs disseminate the infection within the host by entering other cells via a mechanism of receptor-mediated endocytosis. ODVs arise at very late times, when nucleocapsids are enveloped in membrane units derived from the nuclear envelope to finally be embedded in the OBs. OBs persist in the environment after liquefaction of the insect cadaver and are responsible for the horizontal transmission of the virus between hosts. Upon ingestion by a susceptible insect larva, OBs are dissolved in the alkaline environment of the midgut and ODVs are released. The ODVs move through the peritrophic membrane and nucleocapsids are delivered into midgut epithelial cells through a mechanism of membrane fusion mediated by specific viral proteins known as *per os* infectivity factors (PIFs). This primary infection in the midgut is followed by a secondary infection, consisting in the dissemination of BVs to other tissues. In contrast to this infection cycle, which is typical of lepidopteran-specific baculoviruses, infections of gammabaculovi‐

sion of foreign proteins.

In general, genes of DNA viruses are transcribed in a temporal sequence and the process is highly regulated by infection-derived mechanisms and proteins from both host and viral origin. This stepwise mode of gene expression ensures the availability of gene products required for the progression into the next phase of the infection. Baculoviruses express their genes in three successive phases designated as early, late and very late (figure 1). Early genes are transcribed by the host RNA polymerase II before virus DNA replica‐ tion, while late and very late genes are transcribed by a virus-encoded RNA polymerase, after starting of viral DNA replication. Products of a number of early genes are required for virus DNA synthesis and for expression of late/very late genes, and at least one late gene product is also needed for expression of very late genes. The progression of the infection into the late phase correlates with the transcriptional shutoff of cellular and early viral genes. Genes belonging to the different temporal classes are encoded in both DNA strands without any associative distribution in the genome. A number of baculovirus genes contains promoters with sequence elements characteristic of both early and late classes. Their transcription is regulated independently at each temporal phase which ensures their expression throughout the infection [8]. Most of our knowledge of baculovirus gene regulation comes from studies in *Autographa californica* nucleopolyhedrovirus (AcMNPV), which is the type species of the family. This alphabaculovirus has a wide host range and causes productive infections in permissive insect cell-lines Sf21 (and its clonal isolate Sf9), derived from *Spodoptera frugiperda* [9] and TN368, from *Trichoplusia ni* [10]. The genome of AcMNPV encodes 154 predicted open reading frames (ORFs) [11]. Roughly, one half corresponds to late genes, according to the sequence elements present in their promoters. About 25 gene products, mostly of early genes, have functions directly or indirectly related to gene expression.

**5. Expression of early genes**

genes which are transcribed into mRNAs.

gene that is present in all lepidopteran baculoviruses [14].

present at low concentrations [15]

**5.1. Immediate early transactivator IE1**

regulated gene.

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

Baculovirus Gene Expression http://dx.doi.org/10.5772/56955 61

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

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

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

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].

**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 from [65]).
