**6.2. Transcription lefs**

The viral RNA polymerase is a complex of the products of four baculovirus core genes: *lef4*, *lef8*, *lef9* and *p47*. LEF8 and LEF9 have motifs present in the two large subunits of RNA polymerases from prokaryotes and eukaryotes, and are supposed to participate of the catalytic domain. Their sequences are the most highly conserved among LEFs*.* The role of LEF4 as a capping enzyme is discussed below, while the specific function of P47 remains to be elucidated. The polymerase complex was chromatographycally isolated as a fraction that was active in *in vitro* transcription assays using template DNA containing signals specific of baculovirus late gene promoters [42]. In the complex, the four subunits are present in an equimolar ratio.

*lef5* is a core gene that encodes a protein with sequence similarity to the eukaryotic transcription elongation factor TFIIS, however, *in vitro* assays evidenced the ability of LEF5 to increase the transcriptional activity of the viral RNA polymerase at the initiation step rather than to have any effect in the elongation process [43]. The remaining *lefs* have a distribution among species restricted to lepidopteran baculoviruses. *39k* (also known as *pp31*) and *lef10* are considered essential for late gene expression although their specific role in transcription is not known. 39K binds single- and double-stranded DNA and localizes to the virogenic stroma during infection. LEF10 is a small polypeptide without homology to known proteins. *lef6* and *lef12* are consid‐ ered auxiliary lefs because although they were shown necessary in transient assays (*lef12* is dispensable in TN368 cells), knockout mutants for these genes sustained late gene expression with minor deviations from wild type virus [44-45].

#### **6.3. Late and very late mRNAs synthesis and regulation**

Promoters of late genes contain a TAAG sequence motif from which transcription is initiated. There are less TAAG motifs in baculovirus genomes than expected by random occurrence, suggesting that the activity of this sequence as a late promoter selects negatively its random distribution. The integrity of this motif is strictly necessary for transcription, while adjacent sequences up to eighteen nucleotides may affect the level of expression [46]. There may be more than one functional TAAG over a variable distance upstream the translational start codon of the regulated gene [47].

nucleotide by an RNA cap 2'O-methyltransferase (MTase-I). Several alphabaculoviruses have a MTase-I gene. The gene of AcMNPV has been found to stimulate late gene expres‐ sion in transient assays [51]. Late transcripts are terminated by the polymerase at U-rich se‐ quences present in their 3'UTR, and subsequently the enzyme adds adenosine residues

**Figure 3.** Cascade of baculovirus gene transcription events. Diagram of the AcMNPV genome indicating the localiza‐ tion of the genes encoding key proteins involved in the regulatory network of transcription and DNA replication.

Baculovirus Gene Expression http://dx.doi.org/10.5772/56955 67

The most expressed very late genes in AcMNPV are those encoding polyhedrin and P10. Their transcription depends on a TAAG initiation promoter but their high level of expression depends on the presence of an AT-rich sequence known as the "burst" sequence, located between the TAAG and the translational start codon [53-54]. The burst sequence binds very late expression factor-1 (VLF1; [55]) originally identified in a temperature sensitive AcMNPV mutant defective in occluded virus production [56]. VLF1 is a baculovirus core gene that is

**7. Cellular responses to infection and changes in host gene expression**

Early in infection, baculoviruses produce cell cycle arrest at G2/M or S phase, prior to viral DNA replication [57]. The AcMNPV early transcription coactivator IE2 is considered to be

essential for the packaging of DNA into normal nucleocapsids.

independently of template [52].

Late transcripts usually span more than one ORF; likewise, one specific ORF may be repre‐ sented in transcripts with different 5' or 3' ends. The significance of these polycistronic mes‐ sages is not known and it is generally assumed that only the leading gene in the message is translated into protein. Late genes are encoded in both DNA strands, distributed over the genome, therefore there may be opposite late transcripts with complementary stretches. It is not known if this may play any regulatory role considering that baculovirus genes are sus‐ ceptible to silencing by double-stranded RNA [48]. Late transcripts are capped and polyade‐ nylated at their 5' and 3' ends, respectively. At least two enzymatic activities required for capping reside in the LEF4 subunit of the RNA polymerase. This protein functions as RNA triphosphatase and guanylyltransferase but lacks activity of N7-methyltransferase, which is required for methylation of the cap structure in position N7 of guanine [49-50]. A gene re‐ sponsible for this activity has not been identified in baculovirus. The structure of cap 1 mRNAs includes methylation of the 2'hydroxyl group of the ribose of the first transcribed

**6.2. Transcription lefs**

The viral RNA polymerase is a complex of the products of four baculovirus core genes: *lef4*, *lef8*, *lef9* and *p47*. LEF8 and LEF9 have motifs present in the two large subunits of RNA polymerases from prokaryotes and eukaryotes, and are supposed to participate of the catalytic domain. Their sequences are the most highly conserved among LEFs*.* The role of LEF4 as a capping enzyme is discussed below, while the specific function of P47 remains to be elucidated. The polymerase complex was chromatographycally isolated as a fraction that was active in *in vitro* transcription assays using template DNA containing signals specific of baculovirus late gene promoters [42]. In the complex, the four subunits are present in an equimolar ratio.

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

*lef5* is a core gene that encodes a protein with sequence similarity to the eukaryotic transcription elongation factor TFIIS, however, *in vitro* assays evidenced the ability of LEF5 to increase the transcriptional activity of the viral RNA polymerase at the initiation step rather than to have any effect in the elongation process [43]. The remaining *lefs* have a distribution among species restricted to lepidopteran baculoviruses. *39k* (also known as *pp31*) and *lef10* are considered essential for late gene expression although their specific role in transcription is not known. 39K binds single- and double-stranded DNA and localizes to the virogenic stroma during infection. LEF10 is a small polypeptide without homology to known proteins. *lef6* and *lef12* are consid‐ ered auxiliary lefs because although they were shown necessary in transient assays (*lef12* is dispensable in TN368 cells), knockout mutants for these genes sustained late gene expression

Promoters of late genes contain a TAAG sequence motif from which transcription is initiated. There are less TAAG motifs in baculovirus genomes than expected by random occurrence, suggesting that the activity of this sequence as a late promoter selects negatively its random distribution. The integrity of this motif is strictly necessary for transcription, while adjacent sequences up to eighteen nucleotides may affect the level of expression [46]. There may be more than one functional TAAG over a variable distance upstream the translational start codon

Late transcripts usually span more than one ORF; likewise, one specific ORF may be repre‐ sented in transcripts with different 5' or 3' ends. The significance of these polycistronic mes‐ sages is not known and it is generally assumed that only the leading gene in the message is translated into protein. Late genes are encoded in both DNA strands, distributed over the genome, therefore there may be opposite late transcripts with complementary stretches. It is not known if this may play any regulatory role considering that baculovirus genes are sus‐ ceptible to silencing by double-stranded RNA [48]. Late transcripts are capped and polyade‐ nylated at their 5' and 3' ends, respectively. At least two enzymatic activities required for capping reside in the LEF4 subunit of the RNA polymerase. This protein functions as RNA triphosphatase and guanylyltransferase but lacks activity of N7-methyltransferase, which is required for methylation of the cap structure in position N7 of guanine [49-50]. A gene re‐ sponsible for this activity has not been identified in baculovirus. The structure of cap 1 mRNAs includes methylation of the 2'hydroxyl group of the ribose of the first transcribed

with minor deviations from wild type virus [44-45].

of the regulated gene [47].

**6.3. Late and very late mRNAs synthesis and regulation**

**Figure 3.** Cascade of baculovirus gene transcription events. Diagram of the AcMNPV genome indicating the localiza‐ tion of the genes encoding key proteins involved in the regulatory network of transcription and DNA replication.

nucleotide by an RNA cap 2'O-methyltransferase (MTase-I). Several alphabaculoviruses have a MTase-I gene. The gene of AcMNPV has been found to stimulate late gene expres‐ sion in transient assays [51]. Late transcripts are terminated by the polymerase at U-rich se‐ quences present in their 3'UTR, and subsequently the enzyme adds adenosine residues independently of template [52].

The most expressed very late genes in AcMNPV are those encoding polyhedrin and P10. Their transcription depends on a TAAG initiation promoter but their high level of expression depends on the presence of an AT-rich sequence known as the "burst" sequence, located between the TAAG and the translational start codon [53-54]. The burst sequence binds very late expression factor-1 (VLF1; [55]) originally identified in a temperature sensitive AcMNPV mutant defective in occluded virus production [56]. VLF1 is a baculovirus core gene that is essential for the packaging of DNA into normal nucleocapsids.
