**6. Expression of late and very late genes**

Experiments using α-amanitin, an inhibitor of RNA pol II, showed that the synthesis of virusspecific mRNA becomes resistant to the drug with the progression of the infection, indicating that a novel RNA polymerase is induced in infected cells [24-25]. In AcMNPV-infected cells this viral encoded enzyme transcribes late genes mostly from 6 to 24 hpi and very late genes between 18 and 72 hpi. These times correlate with the production of BVs and ODVs, respec‐ tively. Expression of late genes depends on viral DNA replication. When replication is blocked with the DNA synthesis inhibitor aphidicolin, transcription of late genes is also inhibited [26]. In accordance to this dependency, whose nature is not known, genes involved in DNA replication are also required for transcription of late genes, and therefore, they are considered as a subset of the factors regulating late gene expression, collectively known as late expression factors (*lefs*). The remaining *lefs*, including those encoding the multi-subunit RNA polymerase, are considered to regulate transcription-specific events. AcMNPV *lefs* were discovered using

different approaches including temperature-sensitive mutations mapping and transient assays of plasmid DNA replication and late gene expression (for review see [8]). In transient expres‐ sion assays, a plasmid containing a reporter gene under control of a late promoter was

tion and dimerization, are indicated in lower panel with I, II and III, respectively (adapted from [37]

**Figure 2.** Functions of IE1 during the replicative cycle of AcMNPV. IE1 is targeted to the nucleus by a nuclear localiza‐ tion element (NLE) that becomes functional upon IE1 dimerization. The NLE is determined by a small basic domain (BDII) located at the C-terminus of the molecule adjacent to a helix-loop-helix dimerization domain. IE1 transactivates early promoters through interacting directly with transcription factors in the promoter (A) or via binding to *hr* enhanc‐ ers (B). The *hr*-mediated transactivation mechanism depends on the interaction of the basic domain I (BDI) of each IE1 monomer in the dimer with a corresponding hemipalindrome of an *hr* repeat. IE1 may bind sequences within a pro‐ moter that resemble an *hr* palindrome half-site and down-regulate rather than activate transcription, as observed in transient expression assays (C). With the onset of viral DNA replication, early transcripts are down-regulated and there is a switch to the viral-encoded RNA polymerase which takes over transcription of late genes. At this stage, IE1 is re‐ quired for the replication of virus DNA. *Hrs* may serve as origins of replication and it appears that binding of IE1 to *hrs* recruits requisite factors to assemble the replication complex (D). This IE1 function is associated with the N-terminal domain of the protein designated as the replication domain. This domain mediates phosphorylation of IE1 at the ini‐ tiation of DNA replication. It has been proposed that this event is timely regulated and determines the functional switch of IE1 from transcription- to replication-associated activities. IE1 functional domains for replication, transactiva‐

Baculovirus Gene Expression http://dx.doi.org/10.5772/56955 63

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

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

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

Experiments using α-amanitin, an inhibitor of RNA pol II, showed that the synthesis of virusspecific mRNA becomes resistant to the drug with the progression of the infection, indicating that a novel RNA polymerase is induced in infected cells [24-25]. In AcMNPV-infected cells this viral encoded enzyme transcribes late genes mostly from 6 to 24 hpi and very late genes between 18 and 72 hpi. These times correlate with the production of BVs and ODVs, respec‐ tively. Expression of late genes depends on viral DNA replication. When replication is blocked with the DNA synthesis inhibitor aphidicolin, transcription of late genes is also inhibited [26]. In accordance to this dependency, whose nature is not known, genes involved in DNA replication are also required for transcription of late genes, and therefore, they are considered as a subset of the factors regulating late gene expression, collectively known as late expression factors (*lefs*). The remaining *lefs*, including those encoding the multi-subunit RNA polymerase, are considered to regulate transcription-specific events. AcMNPV *lefs* were discovered using

promoters even in the absence of viral factors (see [21]for a review).

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

longer able to promote activation, instead it functions as a repressor [22].

is dispensable given the other is present [23].

**6. Expression of late and very late genes**

**Figure 2.** Functions of IE1 during the replicative cycle of AcMNPV. IE1 is targeted to the nucleus by a nuclear localiza‐ tion element (NLE) that becomes functional upon IE1 dimerization. The NLE is determined by a small basic domain (BDII) located at the C-terminus of the molecule adjacent to a helix-loop-helix dimerization domain. IE1 transactivates early promoters through interacting directly with transcription factors in the promoter (A) or via binding to *hr* enhanc‐ ers (B). The *hr*-mediated transactivation mechanism depends on the interaction of the basic domain I (BDI) of each IE1 monomer in the dimer with a corresponding hemipalindrome of an *hr* repeat. IE1 may bind sequences within a pro‐ moter that resemble an *hr* palindrome half-site and down-regulate rather than activate transcription, as observed in transient expression assays (C). With the onset of viral DNA replication, early transcripts are down-regulated and there is a switch to the viral-encoded RNA polymerase which takes over transcription of late genes. At this stage, IE1 is re‐ quired for the replication of virus DNA. *Hrs* may serve as origins of replication and it appears that binding of IE1 to *hrs* recruits requisite factors to assemble the replication complex (D). This IE1 function is associated with the N-terminal domain of the protein designated as the replication domain. This domain mediates phosphorylation of IE1 at the ini‐ tiation of DNA replication. It has been proposed that this event is timely regulated and determines the functional switch of IE1 from transcription- to replication-associated activities. IE1 functional domains for replication, transactiva‐ tion and dimerization, are indicated in lower panel with I, II and III, respectively (adapted from [37]

different approaches including temperature-sensitive mutations mapping and transient assays of plasmid DNA replication and late gene expression (for review see [8]). In transient expres‐ sion assays, a plasmid containing a reporter gene under control of a late promoter was cotransfected into cells with an overlapping clone library representing the AcMNPV genome [27]. Genomic DNA fragments containing *lefs* were identified as a consequence of the lack of reporter activity when they were omitted in cotransfections. Gradual shortening of those fragments led to the identification of each *lef*. Nineteen AcMNPV *lefs* were identified as required for activation of the late promoter in this system: *lef1* to *lef12*, *ie1*, *ie2*, *dnapol*, *p143*, *p35*, *p47* and *39k* [28] (Table 1). In addition to the *lefs*, a gene designated *vlf1* was found necessary to support expression from a very late promoter. Some other genes influence DNA replication or late gene expression either directly or indirectly, and may be considered as *lefs* also [8]. Differences in reports on the relative contribution of specific genes appear to be consequence of different experimental approaches utilized in studies.

**6.1. Replication lefs**

by phosphorylation [37].

Sf21 cells.

Among *lefs* involved in viral DNA replication [29-30], four baculovirus core genes are essential for this process: *lef1*, *lef2*, *dnapol* and *p143*; they were required in transient assays for plasmid replication as well as for late gene expression. *lef1* is a DNA primase that interacts with *lef2*, a primase accessory protein [31]. *dnapol* encodes a DNA polymerase with 3' to 5' exonuclease activity. The sequence of *dnapol* is the most conserved among baculovirus replication *lefs*; the phylogeny of the family Baculoviridae inferred from its sequence is congruent with that resulting from the analysis of all core genes altogether, suggesting that *dnapol* is an ancestral gene [32]. P143 is a helicase that requires ATP to unwind DNA. Two other AcMNPV replication *lefs*, *ie1* and *lef3*, which are present in all lepidopteran baculoviruses, are also essential as determined in transient assays. Neverthe‐ less, their absence in the genome of γ- and δ-baculoviruses suggests that there may be functional homologs of these genes involved in replication/gene expression in species belonging to these genera. LEF3 is a single stranded DNA-binding protein that promotes unwinding of DNA duplex and annealing of complementary strands [33]. LEF3 interacts

Baculovirus Gene Expression http://dx.doi.org/10.5772/56955 65

The actual function of IE1 in DNA replication is poorly understood, nonetheless it appears to depend on the *hr*-binding capacity of IE1. *Hr* regions function not only as enhancers of early genes but also serve as origins of DNA replication in plasmid replication assays [35]. It was shown that in the presence of an *hr* element, transiently expressed IE1 adopts a focal distribu‐ tion within the nucleus. When LEF3 and P143 are simultaneously expressed they localize to the *hr*-induced IE1 foci [36]. This suggests that IE1 functions by recruiting viral replication factors to the *hr* origin. The switch to the replication activity of IE1 seems to be timely regulated

Other genes have a stimulatory effect in transient replication/late expression assays. These are *ie2*, *lef7* and *p35*. IE2 is a transactivator involved in cell cycle arrest [38] and LEF7 has sequence similarity to single stranded-DNA binding proteins. Both are present in all genomes of group I α-baculoviruses and LEF7 is also present in some other α- and βbaculoviruses. P35 blocks the apoptotic response of cells triggered by DNA replication through inhibition of effector caspases [39]. The contribution of P35 in transient assays reflects its protective effect against apoptosis, and therefore represents an indirect require‐ ment for replication and late gene expression. Actually, it may be replaced in the assays by a member of the baculovirus *iap* (inhibitor of apoptosis) gene family with similar results. *ie2*, *lef7* and *p35* are dispensable for the infection of TN368 cells by AcMNPV [40]; hence, they are considered as host range factors able to extend the infectivity of AcMNPV towards

AcMNPV *lef11*, which was necessary for late gene expression in transient assays, was not required in transient plasmid replication assays. However, an AcMNPV *lef 11*-null bacmid was unable to replicate its DNA [41]. Therefore, *lef11* is essential for DNA replication in the context

of the virus infection and may be considered as a replication *lef*.

with p143 and shuttles this molecule to the nucleus [34].


§ Core genes have homologs in all sequenced baculoviruses. α-I, α-II: Group I and Group II of alphabaculoviruses, respectively. β: betabaculoviruses, γ: gammabaculoviruses.

\* There is at least one member in the indicated group having an homolog of the corresponding gene.

**Table 1.** Ac*M*NPV *lefs*
