**6.1. Replication lefs**

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

**Functional class LEF function**

, β\* Replication possible ssDNA binding protein

, β\* Replication apoptosis inhibitor

, β Transcription DNA binding protein

protein

cell-cycle control

of different experimental approaches utilized in studies.

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

*lef11* 37 112 α-I, α-II, β, γ Replication

*lef6* 28 173 α-I, α-II, β Transcription

*lef12* 41 181 α-I, α-II\* Transcription

**Homologs in baculovirus lineages§**

*lef1* 14 266 core Replication DNA primase *lef2* 6 210 core Replication primase accessory protein *dnapol* 65 984 core Replication DNA polymerase *p143* 95 1221 core Replication DNA helicase

*lef3* 67 385 α-I, α-II, β Replication ssDNA binding protein

*ie2* <sup>151</sup> <sup>408</sup> α-I Replication transactivator of early genes,

*lef8* 50 876 core Transcription RNA polymerase subunit *lef9* 62 490 core Transcription RNA polymerase subunit *p47* 40 401 core Transcription RNA polymerase subunit *lef5* 99 265 core Transcription transcription initiation factor

, α-II\*

*ie1* <sup>147</sup> <sup>582</sup> α-I, α-II, β Replication transactivator of early genes, *hr*-binding

*lef4* 90 464 core Transcription RNA polymerase subunit, capping enzyme,

, β\* Transcription

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

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

**Amino acid residues**

*lef7* 125 226 α-I, α-II\*

*p35* 135 299 α-I\*

*39k/pp31* 36 275 α-I, α-II\*

*lef10* 53A 78 α-I, α-II\*

respectively. β: betabaculoviruses, γ: gammabaculoviruses.

**ORF name**

\*

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

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

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 by phosphorylation [37].

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 Sf21 cells.

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*.
