Functional dissection of the baculovirus late expression factor-8 gene: sequence requirements for late gene promoter activation

Jane S. Titterington{dagger}, Tamara K. Nun{ddagger} and A. Lorena Passarelli

Molecular, Cellular and Developmental Biology Program, Division of Biology, Kansas State University, 232 Ackert Hall, Manhattan, KS 66506-4901, USA

Correspondence
Lorena Passarelli
lpassar{at}ksu.edu


   ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
The late expression factor-8 gene (lef-8) of Autographa californica M nucleopolyhedrovirus encodes the largest subunit of the virally encoded DNA-directed RNA polymerase specific for the transcription of late and very late viral genes. The sequence of lef-8 predicts a C-terminal motif of 13 amino acids that is conserved in other polymerases. Detailed mutagenesis throughout lef-8 was performed, including this C-terminal motif, to define sequences required for late promoter activation. It was found that the conserved C-terminal motif was critical for late gene expression. In addition, regions throughout the entire lef-8-encoding sequence were important for optimal function, suggesting complex protein–protein and protein–DNA interrelationships in the late gene-specific viral transcriptosome.

{dagger}Present address: University of Kansas Medical Center, Kansas City, KS 66160, USA.

{ddagger}Present address: University of North Carolina, Chapel Hill, NC 27599, USA.


   INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Baculoviruses belong to the family Baculoviridae, a diverse family of invertebrate-specific viruses with large, enveloped, double-stranded circular DNA genomes (Burgess, 1977; Schafer et al., 1979). Autographa californica M nucleopolyhedrovirus (AcMNPV), the best-studied baculovirus, has a biphasic replication cycle producing two forms of the virus with identical genetic makeup and nucleocapsid structure: the budded virus spreads systemically within the insect and the occluded virus spreads infection naturally in the wild when ingested by a host (reviewed by O'Reilly et al., 1992). Upon infection of permissive cells with AcMNPV, viral genes are transcribed during three phases, designated early, late and very late. Early genes are transcribed prior to viral DNA replication by the host RNA polymerase II and host transcription accessory factors and, in some cases, also require specific virus factors. Their promoter regions resemble those of host promoters, supporting their dependence on the host transcription apparatus (reviewed by Friesen, 1997). In contrast, expression of late and very late genes is dependent on viral DNA replication and requires viral late expression factor (lef) genes for optimal expression in transient reporter gene expression assays. A total of 22 late/very late expression factors have been identified (Li et al., 1999; Lu & Miller, 1995; Rapp et al., 1998; Todd et al., 1995) with differential requirements for late and very late promoters and in different cell lines. The lef genes include the subunits of the viral DNA-directed RNA polymerase, an {alpha}-amanitin- and tagetitoxin-resistant enzyme, and genes involved in viral genome replication (Glocker et al., 1993; Grula et al., 1981; Guarino et al., 1998b; Huh & Weaver, 1990; Li et al., 1999; Lu & Miller, 1995; Rapp et al., 1998; Todd et al., 1995). Unlike early genes, late and very late genes are transcribed from a transcription initiation and promoter sequence, basically consisting of only four nucleotides (TAAG) (Ooi et al., 1989; Rankin et al., 1988; Rohrmann, 1986).

Baculoviruses are the only nuclear-replicating DNA viruses that use a virally encoded multisubunit polymerase to transcribe late and very late genes (Guarino et al., 1998b). Poxviruses are other eukaryotic viruses with DNA genomes that encode a complex DNA-directed RNA polymerase but, unlike baculoviruses, they replicate in the cytoplasm of permissive cells and rely on their encoded products to carry out most of their DNA and RNA biosynthetic reactions (reviewed by Moss, 2001). African swine fever virus, a cytoplasmic double-stranded DNA virus with genetic similarities to poxviruses and iridoviruses, also encodes a DNA-directed RNA polymerase. Baculoviruses could have evolved this enzyme to preferentially and efficiently transcribe viral genes over host genes during the post-replication phase or to discriminate expression of early viral genes in a tightly regulated manner.

Four lef genes, lef-8, lef-4, lef-9 and p47, were found to be part of an RNA polymerase activity isolated from virus-infected cells specific for the transcription of late and very late promoter templates in vitro (Guarino et al., 1998b). This simple four-subunit enzyme has the ability to bind DNA (Guarino et al., 1998b) and, potentially, to modify the 5' (Gross & Shuman, 1998; Guarino et al., 1998a; Jin et al., 1998) and 3' ends (Jin & Guarino, 2000) of transcripts. Although the specific role of each subunit in transcription is not known, the product of lef-4 (LEF-4) has RNA 5'-triphosphatase, nucleoside triphosphatase and guanylyltransferase activities, predicting its role as a capping enzyme (Gross & Shuman, 1998; Guarino et al., 1998a; Jin et al., 1998). LEF-9 contains five of seven residues of the motif NADFDGD present in the large subunits of DNA-directed RNA polymerases (Broyles & Moss, 1986; Lu & Miller, 1994). This motif is part of the catalytic centre of RNA polymerases, but whether this motif is important for the function in LEF-9 is not known. Less is known about the role of p47 in transcription, but a virus with a temperature-sensitive p47 allele is defective in the production of late and very late proteins at non-permissive temperatures (Carstens et al., 1994; Partington et al., 1990).

The AcMNPV lef-8 gene was first discovered as one of 19 genes required for the expression of late and very late genes in SF-21 insect cells (Passarelli et al., 1994). It encodes a protein, LEF-8, that predicts a 102 kDa product with a conserved C-terminally positioned sequence motif of 13 amino acids, GXKX4HGQ/NKGV/I, also found at the C terminus of the {beta} or {beta}' subunits of DNA-directed RNA polymerases in animals, plants, eubacteria and archaeobacteria (Passarelli et al., 1994). However, LEF-8 has no other obvious sequence similarities to known RNA polymerases. Furthermore, this 13-residue motif is located within the Escherichia coli RNA polymerase conserved region H and has been proposed to be part of the catalytic site of the E. coli enzyme complex (Schultz et al., 1993).

In this study, we performed a detailed deletion analysis of LEF-8, the largest subunit of the AcMNPV DNA-directed RNA polymerase complex, in order to define domains important for expression from a late viral promoter. Site-specific mutagenesis analysis of the C-terminal motif sequence within LEF-8, which is part of the catalytic pocket of homologous polypeptides, revealed the importance of this motif in activity. Mutations throughout lef-8 had negative effects on its function, suggesting the possibility that multiple surfaces of the protein are important for its function in transcription.


   METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Cells.
The lepidopteran cell line IPLB-SF-21 (SF-21) (Vaughn et al., 1977) was grown at 27 °C in TC-100 medium (Invitrogen) supplemented with 10 % foetal bovine serum (Invitrogen) and 0·26 % tryptose broth (O'Reilly et al., 1992).

lef library and reporter plasmid constructs.
The 19 plasmids, each encoding an AcMNPV lef gene (lef-1 to lef-12, ie-1, ie-2, p143, p47, 39K, p35 and dnapol) necessary for optimal late promoter gene expression, hereafter referred to as the lef library, have been described previously and shown to be expressed and able to activate late promoters (Rapp et al., 1998). The expression of each LEF is controlled by the Drosophila melanogaster heat shock protein 70 promoter, and the coding sequences are fused to both the influenza virus haemagglutinin (HA) epitope and polyhistidine tags at the N terminus. The plasmid pCAPCAT (Thiem & Miller, 1990), containing the late promoter of the major capsid gene, vp39, in front of the bacterial reporter gene encoding chloramphenicol acetyltransferase (CAT), was used to monitor activation of late genes.

Mutagenesis.
The plasmid described above containing lef-8, pHSEpiHislef8 (Rapp et al., 1998), was used as a template to make all of the mutations within lef-8. Although lef-8 has a dual tag at its N terminus, its activity is equivalent to that of the native lef-8 in transient gene expression assays (Rapp et al., 1998; data not shown) and this tagged version of lef-8 will be referred to as wild-type lef-8. All site-directed mutations and N-terminal deletions were confirmed by nucleotide sequence analysis and other deletions and mutations introducing EcoRI sites were confirmed by restriction endonuclease digestion. Mutations within the RNA polymerase homologous motif were created using the Transformer Site-Directed Mutagenesis kit (Clontech), whereas the remaining mutations were introduced using the Quik-Change Mutagenesis kit (Stratagene). Oligonucleotides used for mutagenesis are shown in Table 1, except for oligonucleotides with the complementary sequence needed for the Quik-Change mutagenesis method.


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Table 1. Oligonucleotides used for the mutagenesis of lef-8

 
C-terminal truncations within LEF-8 were made by introducing a BglII site every 50 amino acids (on average, at aa 482, 530, 581, 633, 684, 735, 776 and 825) followed by a premature stop codon, TGA, which is also found in the wild-type LEF-8 sequence, thus truncating the polypeptide from that amino acid to the last amino acid (aa 876). An arginine and a serine provided by the restriction site were thus inserted before the stop codon. Numbers in the plasmid names indicate the amino acids in LEF-8 that were deleted (e.g. Del 482–876). Oligonucleotides used for C-terminal truncations were lef8BglIITGA-482, lef8BglIITGA-530, lef8BglIITGA-581, lef8BglIITGA-633, lef8BglIITGA-684, lef8BglIITGA-735, lef8BglIITGA-776, and lef8BglIITGA-825, where the numbers correspond to the amino acid position in LEF-8 where the BglII restriction site was inserted (Table 1).

N-terminal deletions within LEF-8 were made by amplifying the lef-8 sequence with oligonucleotides lef8 : 879-900 and lef8 : 400-17BglII or lef8 : 604-21BglII between the XhoI site within the lef-8 ORF and either amino acid 66 or 134 that was preceded by a BglII site. This amplified sequence was then inserted, maintaining the correct reading frame, into pHSEpiHislef8 at the XhoI and BglII sites. The BglII site in pHSEpiHislef8 is positioned immediately downstream of the epitope tags and upstream of the lef-8 ORF.

Internal deletions were made by replacing two pairs of LEF-8 amino acids with a pair of EcoRI sites encoding the amino acids glutamic acid and phenylalanine approximately every 50 amino acids throughout the ORF and being careful to introduce these amino acids at the most conserved positions to cause the least disruption. The plasmids containing these EcoRI sites were digested with EcoRI and ligated, thereby creating a deletion. Thus, we generated a panel of single EcoRI substitutions every 50 amino acids and one containing two EcoRI substitutions spaced 50 amino acids apart throughout LEF-8. Oligonucleotides were designated lef8RI-X, where X is the LEF-8 amino acid number where the insertion occurred. Plasmid clone names denote where one (e.g. RI 99; one amino acid position noted) or two (e.g. RI 99,153; two amino acid positions noted and separated by a comma) EcoRI sites were introduced or whether a deletion between two EcoRI sites was made (e.g. Del 99–153; two amino acid positions noted and separated by a hyphen).

Co-transfections and CAT assays.
Approximately 1x106 cells in 35 mm dishes were co-transfected for 4 h at 27 °C using 3–6 µl of a 1·5 : 1 sonicated mixture of N-[2,3-(dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride salt (Avanti Polar Lipids) and L-{alpha}-phosphatidylethanolamine (Sigma) lipids, 2 µg pCAPCAT and 0·5 µg of each lef in the lef library (19 plasmids). Wild-type lef-8 in pHSEpiHislef8 was used in co-transfections or substituted for lef-8 carrying a mutation as indicated. The vector plasmid pBlueScript (Stratagene) was used in each co-transfection reaction to maintain a constant concentration of DNA in each reaction if needed. Cells were harvested 48 h after co-transfection, a protein lysate was then obtained and CAT activity was assayed, as described previously (Passarelli & Miller, 1993). Each transfection was performed between three and nine times and the average of these experiments is presented.

Immunodetection.
Approximately 1x106 cells were transfected with 2 µg of an HA- and histidine-tagged LEF-8, as described above. Due to difficulties in detecting LEF-8 expression, we routinely used N–CBZ–LEU–LEU–LEU–AL (MG 132, Sigma), a proteasome inhibitor, to increase the levels of accumulated protein available for detection. At 24 h post-transfection, the proteasome inhibitor MG 132 was added to a final concentration of 50 µg ml-1 and the cells were incubated for 30 min at 27 °C. Cells pre-treated with MG 132 were then incubated at 42 °C for 30 min to induce expression of lef-8 from the heat shock 70 promoter. LEF-8 was allowed to be expressed for 2 h at 27 °C before harvesting cells. Cells were washed twice with PBS and collected with 100 µl of Laemmli's loading buffer. Proteins were resolved by 8 % SDS-PAGE, transferred to a PVDF membrane (Pierce) and immunodetected with a 1 : 1000 dilution of HA.11 monoclonal antibody (Covance), 1 : 3000 dilution of goat anti-mouse IgG–horseradish peroxidase (Bio-Rad) and SuperSignal chemiluminescent substrate (Pierce). Images were prepared using Adobe Photoshop 7.0.


   RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Analysis of the predicted polypeptide sequence of lef-8 does not reveal overall homology to other known polypeptides except to other baculovirus lef-8 homologues. The most obvious similarity is in the C-terminal motif of 13 amino acids (aa 721–733 of LEF-8) and flanking residues to other RNA polymerases in several organisms; however, other domains found in these evolutionarily conserved enzymes are not obvious in lef-8. Thus, in order to begin studying the function of this simple viral RNA polymerase complex, we performed mutagenesis throughout the coding sequence of LEF-8 to map regions required for function.

The lef-8 sequence encodes 876 amino acids and predicts a 102 kDa polypeptide, the third largest predicted polypeptide in the AcMNPV sequence. To analyse regions critical for function, we created a panel of both terminal and internal deletions of approximately 50 amino acids in length. In the process of constructing these deletion sets, we substituted between one and four amino acids by inserting one or two EcoRI sites at one or two positions approximately 50 amino acids apart. In addition to being useful for generating deletions, these EcoRI mutants also effectively introduced point mutations throughout the lef-8 sequence and allowed us to evaluate the importance of single amino acids in late gene expression. A schematic of the mutagenesis strategy is illustrated in Fig. 1.



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Fig. 1. Schematic diagram showing deletion clones of lef-8. The top line represents wild-type lef-8 and the box indicates the approximate position of the LEF-8 region H homologous motif. N and C indicate the N- and C-termini of LEF-8, respectively. The next two lines represent N-terminal deletions of 66 and 134 amino acids, respectively. N-terminal deletions retain the initiation methionine codon as well as in-frame HA and polyhistidine tags. Numbering in the deletions refers to the amino acid numbers in the native LEF-8 sequence. An in-frame BglII site was used after the tags to bridge the N- and C-termini. The subsequent eight lines represent C-terminal deletions of decreasing size. The C terminus of each clone contains a BglII followed by the native stop codon. The last 17 lines represent internal in-frame lef-8 deletions of approximately 50 amino acids, each containing an in-frame EcoRI site. Del, deletion; RI, EcoRI; bp, base pairs.

 
Mutations in the LEF-8 RNA polymerase homologous motif
A C-terminal motif of 13 amino acids positioned within region H of RNA polymerases has been proposed to be part of the catalytic centre of the enzyme and crucial for transcription. However, the importance of this motif, GIKICGIHGQKGV, in LEF-8 function had not been determined. We made alanine substitutions at three conserved amino acids, K723, G729 and K731 (Fig. 2A). Altering either lysine reduced late gene expression to background levels (Fig. 2B, compare column 2 to columns 3 and 5). Surprisingly, the G729->A mutation reduced expression only 40 %. This glycine and the preceding residue, H728, are invariant in the subunits of all non-baculovirus RNA polymerases in the databases, except for that encoded by poxviruses. The vaccinia virus A24R (rpo132) gene product has a serine preceded by a tyrosine at these positions (Amegadzie et al., 1991). Changing G729 to serine introduces a longer side-chain to the general amino acid structure but maintains hydrophilicity. This change reduced expression to nearly background levels (Fig. 2B, compare columns 4–6). The importance of these invariant residues was demonstrated by introducing a conserved change. Changing K723 to arginine reduced expression 10-fold (Fig. 2B, compare columns 1 and 7). All LEF-8 mutants were expressed, as assessed by immunoblotting (Fig. 2C).



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Fig. 2. Mutagenesis of the LEF-8 region H homologous motif. (A) Amino acid sequence of the LEF-8 13-residue motif homologous to part of region H of the E. coli RNA polymerase {beta} subunit. Boldface letters highlight invariant residues in RNA polymerases. The first and last LEF-8 amino acids in this sequence motif are shown in parentheses. (B) The late major capsid promoter controlling the chloramphenicol acetyltransferase (cat) gene was transfected with the lef library (column 1) or the lef library lacking lef-8 (columns 2–7), as noted by the labelled line under the graph. A plasmid containing lef-8 was substituted for lef-8 containing mutations, as indicated below each column. The value of 100 % CAT activity has been assigned to the activity obtained from the activation of the late promoter by the complete lef library. (C) Wild-type LEF-8 and LEF-8 containing mutations in the motif shown in (A), as indicated below each lane, were expressed in SF-21 cells and detected by immunoblotting using the anti-HA.11 monoclonal antibody.

 
N- and C-terminal LEF-8 deletions
Deletion of LEF-8 amino acids 2–66 or 2–134 abolished expression of CAT from the late capsid, vp39, promoter (Fig. 3A). This is consistent with three internal mutations in this region deleting amino acids 3–53, 53–99 and 99–153 (see below and Fig. 4), and one or two amino acid substitutions in this region (see below and Fig. 5). A Western blot showing expression of each terminal deletion is shown in Fig. 3(B).



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Fig. 3. Effects of N- (A) and C-terminal (C) LEF-8 deletions on late gene promoter activation. The lef library, lef library lacking lef-8 or lef library lacking lef-8 but containing a lef-8-deletion plasmid were co-transfected into cells with pCAPCAT, as indicated beneath panels (A) and (C). The value of 100 % CAT activity has been assigned to the activity obtained from the activation of the late promoter by the complete lef library. Immunodetection of HA-tagged LEF-8 and deletion LEF-8 clones (B, D) with anti-HA.11 monoclonal antibody, as indicated below each lane, is shown.

 


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Fig. 4. Effects of internal LEF-8 deletions on late promoter activity. (A) The lef library (column 1) or lef library lacking lef-8 (column 2) but with a deletion clone (columns 3–19), as specified below each column, were transfected into SF-21 cells along with the late promoter reporter gene to monitor late promoter activation. Cells were harvested 48 h post-transfection and lysates were analysed for CAT activity and compared to the relative CAT activity in column 1 set at 100 %. (B) Expression from lef-8 deletion constructs was observed by immunoblotting with anti-HA.11 monoclonal antibody and compared to HA-tagged LEF-8, as indicated below each corresponding column. Each row corresponds to a separate immunoblot.

 


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Fig. 5. Effects of amino acid substitutions within LEF-8 on late gene expression. Glutamic acid and phenylalanine, encoded by the recognition site of EcoRI, were substituted for one to four amino acids in the LEF-8 sequence. One EcoRI site was introduced about every 50 amino acids (A) or two sites were introduced and spaced by about 50 amino acids (B) in LEF-8 at the amino acid noted below each lane. The lef library (columns 1) or the lef library lacking lef-8 (columns 2) and containing a construct with EcoRI mutations, as noted below the remaining columns, were tested for their ability to substitute for wild-type lef-8 in activation of the major capsid late promoter. The value of 100 % CAT activity has been assigned to the activity obtained from the activation of the late promoter by the complete lef library. Expression from each clone tested is shown below the columns. In panel (A), mutant constructs were tested for expression in three different experiments and compared to wild-type LEF-8, as illustrated by separate rows.

 
All of the plasmids tested containing deletions of the C terminus of LEF-8 did not stimulate the late promoter significantly above background (Fig. 3C) but were expressed (Fig. 3D). This is consistent with results obtained with EcoRI mutations in the region between amino acid 506 and 859 (Fig. 5). Each C-terminal deletion included at least one amino acid essential for function (Fig. 5A), except for the C-terminal deletions of amino acids 776–876 and 825–876, where other residues may be important for function. In the region between amino acids 776 and 876, we only changed one base pair but conserved the predicted amino acid sequence; both of these mutants, RI 809 and RI 859, exhibited wild-type levels of activity (Fig. 5A).

Internal LEF-8 deletions
Internal deletions of approximately 50 amino acids were made throughout LEF-8 by deleting and religating between EcoRI sites introduced at the deletion endpoints. All of the plasmids containing these deletions within lef-8 were unable to substitute for the wild-type lef-8 (Fig. 4A, compare column 1 to columns 3–19), suggesting there are regions important for function throughout the entire length of the polypeptide. All of the mutant proteins were expressed at levels similar to wild-type LEF-8, except for Del 652–701, which exhibited slightly lower levels of expression (Fig. 4B). Nevertheless, this region contained at least one important element for function, since RI 652 (Fig. 5A) was not functional.

Targeted amino acid substitutions within LEF-8
EcoRI mutations throughout lef-8 where one or two amino acids were substituted showed that there is very little room for change in the lef-8 sequence. All mutations decreased LEF-8 activity, except one at amino acid 553 and one at amino acid 701 (the last two mutations, RI 809 and RI 859, did not change the amino acid sequence; Fig. 5A). Both RI 553 and RI 701 are regions of LEF-8 that are not well conserved when aligned to other baculovirus LEF-8 sequences (see supplementary data Figs 1 and 2 at JGV Online, http://vir.sgmjournals.org). Three mutations gave over 50 % activity but did not fully substitute for wild-type lef-8 (RI 255, RI 306 and RI 408). One third of the mutations with one EcoRI abolished late gene expression to background levels (RI 153, RI 354, RI 456, RI 603, RI 652 and RI 759) and five other mutations resulted in 50 % or lower activity (RI 3, RI 53, RI 99, RI 202 and RI 506). Most of the substituted residues are conserved among LEF-8 sequences, while the least conserved are RI 53, RI 202 and RI 506 (see supplementary data Figs 3, 4 and 5, respectively, at JGV Online, http://vir.sgmjournals.org). All mutant LEF-8 proteins were expressed, except for RI 456 (Fig. 5A). However, it is likely that a mutation at this site would be deleterious, since the double EcoRI mutations RI 408,456 and RI 456,506 were inactive, even though mutations at RI 408 and RI 506 gave some activity above background (Fig. 5). Expression of RI 354 was very low but the double mutants containing an RI 354 mutation also confirmed the likelihood that this mutation would abolish function (Fig. 5). Finally, we could not detect any expression from RI 652,701 and cannot make any certain conclusions about this mutation (Fig. 5B).

Interestingly, all double EcoRI mutations had similar activity to that of the least active or inactive single EcoRI mutation, except for two double EcoRI mutations, RI 3,53 and RI 53,99 (Fig. 5). RI 53,99 had almost wild-type LEF-8 activity but neither RI 53 nor RI 99 gave more than 50 % of wild-type LEF-8 activity. It is possible that if these two regions interact in the same molecule, one mutation may compensate for the other. A reaction testing RI 53 and RI 99 on separate plasmids rather than the double mutation did not yield activity much higher than RI 53 alone (data not shown).


   DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
To explore the function of the AcMNPV RNA polymerase, a simple yet evolutionarily dissimilar enzyme, we began by performing detailed mutagenesis on its largest subunit, one which contains a putative catalytic motif conserved in region H of the E. coli {beta} RNA polymerase subunit. We individually altered three highly conserved RNA polymerase amino acids in LEF-8 that correspond to the active centre in region H of the E. coli {beta} subunit. This region has been cross-linked to a substrate probe on initiating NTPs (Mustaev et al., 1991). Changing K723 of LEF-8 to alanine completely abolished the ability of LEF-8 to activate late promoters. The conservative substitution of K723 for arginine still resulted in a drastic reduction of late gene expression. Surprisingly, the substitution of the corresponding amino acid in the bacterial enzyme, K1065, to an arginine did not inactivate the catalytic function leaving initiation complexes in the abortive initiation stage. Thus, K1065 in the E. coli RNA polymerase is critical for the transition between transcription initiation and elongation (Mustaev et al., 1991). This lysine residue is also conserved in monomeric RNA polymerases, highlighting the importance of this residue within the motif in both multimeric and monomeric enzymes. Substitution of K723 in LEF-8 for either alanine or another basic residue, arginine, also abolished function. Unexpectedly, a construct in which the highly conserved RNA polymerase motif residue G729 was substituted for alanine still retained over 50 % activity. However, its substitution for serine, which is functional in the vaccinia virus RNA polymerase subunit containing this motif, drastically reduced activity. Thus, there is very little room for change in this motif and this has been apparent by its high conservation in RNA polymerases from both unicellular and multicellular organisms and in baculovirus LEF-8 sequences, including the divergent Culex nigripalpus NPV (CuniNPV) (see supplementary data Fig. 2 at JGV Online, http://vir.sgmjournals.org).

Both termini of the protein were essential for function. Since the first two N-terminal deletions, amino acids 2–66 and 2–134, reduced late gene expression to background levels, we did not test any additional mutations from the N terminus. In addition, internal deletions and EcoRI mutations in that region also abolished function, suggesting that the N terminus of the polypeptide is important for function. The structure of the N-terminal domain of the T7 RNA polymerase undergoes a dramatic reorganization during elongation complex formation and this substrate-induced change may be a conserved mechanism in multisubunit RNA polymerases (Tahirov et al., 2002; Yin & Steitz, 2002). Likewise, deletions from the C terminus to approximately the middle of the polypeptide did not support late gene expression. It was anticipated that deletions that included the C-terminal H region RNA polymerase homologous motif would not be functional, since single amino acid mutations in that region abolished function. However, we also found three deletions that did not include this motif, starting at amino acids 825, 776 or 735 and extending to the end of the polypeptide, did not stimulate expression from the late gene promoter.

Four observations in other systems help understand the functional importance of the region downstream of region H. First, a number of residues in the N- and C-terminal regions flanking the RNA polymerase homologous motif of LEF-8 are conserved between AcMNPV LEF-8 and other RNA polymerases. This region of homology extends between amino acid 691 and 765 of LEF-8. Second, protein–RNA cross-linking interactions in the active centre of the transcription elongation centre of the E. coli RNA polymerase mapped a continuous region of over 30 amino acids between the beginning of region H (corresponding to the motif in LEF-8) and the C terminus of the {beta} subunit as a region participating in catalysis (Markovtsov et al., 1996). Third, H1237 in the E. coli {beta} subunit, which is about 174 amino acids downstream of the start of the motif in region H, affected the transition from initiation to elongation of transcription (Mustaev et al., 1991). Although the region downstream of the region H motif is not as long in the baculovirus polypeptide as it is in the bacterial enzyme, there are six histidines downstream of this motif that may be important for this function. Alternatively, other conserved residue(s) located at the C terminus of the LEF-8 polypeptide may be required for function. Fourth, it is known that lethal mutations in the Drosophila second largest RNA polymerase subunit map to the C terminus, further emphasizing that this region is crucial for function (Chen et al., 1993).

A total of 17 deletions of approximately 50 amino acids were made throughout LEF-8. In all cases, activity was under 10 % of that resulting when the wild-type lef-8 was present. Although smaller deletions could have been made, LEF-8 is among the largest baculovirus predicted polypeptides, and we chose to begin our studies by trying to scan arbitrary 50 amino acid regions through the length of the predicted polypeptide sequence. Instead of constructing smaller deletions, in the process of making these deletions, we substituted one to four amino acids throughout LEF-8 with the restriction endonuclease recognition cleavage sequence for EcoRI and all of these were tested for function. While all but two altered LEF-8 proteins were expressed in SF-21 cells, the possibility remains that the mutations affected the correct folding of the polypeptide rendering it inactive. However, judging from the accrued knowledge of how eukaryotic transcriptases operate, we know that RNA polymerases are usually multimeric and interact with a number of other factors in order to transcribe. This implies that regions throughout the polypeptide may be required for either protein–DNA or protein–protein interactions or association with polymerizing substrates at the active site and lack of one interaction may eliminate optimal function.

In the E. coli enzyme, it is known that {beta}-D, {beta}-H, {beta}'-D and {beta}'-G regions co-ordinate Mg2+ in the active centre where the initiating ribonucleotide chain is being synthesized. Mg2+ is a critical requirement for in vitro transcription of late AcMNPV promoter templates (Funk et al., 1998; Glocker et al., 1992, 1993; Guarino et al., 1998b; Mans & Knebel-Mörsdorf, 1998; Xu et al., 1995). We suggest that the baculovirus RNA polymerase has conserved the modular organization of the catalytic pocket where sequences in different subunits are proximal in space to form the active site. A modular organization may allow other regulatory factors the opportunity to interact with a subunit containing one catalytic motif (Mustaev et al., 1997). Also, this organization may be important for the reported stretching of the active centre during elongation (Mustaev et al., 1993). Thus, this modular arrangement may also allow for the divergence of other regions in the LEF-8 polypeptide that lie outside the catalytic centre. Furthermore, the protein sequences of most, but not all, {beta}-like RNA polymerase subunits in the database are larger than that of LEF-8 (Passarelli et al., 1994). Although the homology between LEF-8 and {beta}-like RNA polymerase subunits is not as obvious outside region H, viral LEF-8 sequences may have condensed their function in a smaller polypeptide chain allowing for little variation in sequence. It is possible that the structure of this viral polymerase is similar to that of other polymerases that are dissimilar in primary structure but have a common function. The crystal structures of the monomeric T7 RNA polymerase, Klenow and human immunodeficiency virus type 1 reverse transcriptase share a common structure and these structural analyses led to the identification of polymerase substrate specificity elements (Sousa et al., 1993). The recent structures of yeast RNA polymerase II and the bacterial Thermus aquaticus core polymerase also share architectural similarities (Cramer et al., 2000; Zhang et al., 1999). Thus, several polymerases studied to date have evolved conserved sequences, function and structure, thus preserving the nature of transcription in unicellular and multicellular organisms.

AcMNPV RNA polymerase is composed of at least four subunits, but the interactions among these subunits are not known. There is preliminary evidence that Bombyx mori NPV (BmNPV) LEF-8 and LEF-9 interact (Acharya & Gopinathan, 2002). This is not unexpected, since both polypeptides contain regions that may form the catalytic centre of the enzyme. As mentioned before, LEF-8 has a motif homologous to the {beta}-H region and LEF-9 has the short seven-residue stretch homologous to {beta}'-D, two regions at the heart of the enzyme. It is not known if the other transcription-specific LEFs co-operate via protein–protein interactions with the viral RNA polymerase.

BmNPV with a temperature-sensitive mutation within LEF-8 was defective in vivo and cell culture, affecting late and very late processes. The mutation was mapped to a single nucleotide that changed A542 to valine (Shikata et al., 1998). The corresponding amino acid in AcMNPV, amino acid 541, is conserved at the nucleotide and amino acid level. This amino acid is also conserved in all NPVs except for the divergent CuniNPV (see supplementary data Fig. 1 at JGV Online, http://vir.sgmjournals.org). Deletion of AcMNPV LEF-8 amino acids 506–553, which encompass this residue, resulted in an inactive protein.

Small alterations that changed one to four amino acids in LEF-8 tested in our assay all affected expression of late genes, except in three cases in which two amino acids were substituted (RI 553, RI 701 and RI 53,99; Fig. 5), again denoting the significance of each region for function. The lack of flexibility for introducing mutations in lef-8 suggests that the transcription-associated requirements of the polypeptide are dispersed throughout its sequence. In addition, alignment of the baculovirus LEF-8 sequences available shows sequence conservation blocks throughout the entire protein and this suggests there is very little room for variability. These functions may include DNA or protein interactions or tertiary structure requirements that are conserved in RNA and DNA polymerases of prokaryotic and eukaryotic organisms. This is the first detailed analysis of a key late gene-specific baculovirus RNA polymerase subunit. The panel of mutations that we have constructed will aid in detailing the steps necessary for late viral gene activation.


   ACKNOWLEDGEMENTS
 
We are very grateful to Erin Harvey for excellent technical assistance and Kimberly Krager for constructing a number of LEF-8 internal deletion clones. We thank Mandar Deshpande, Youping Deng and Erin Crouch for help with figures and Angela Iseli and Erin Crouch for providing cells. We are also thankful to Rollie Clem for critical reading of the manuscript. This work was supported by the Cooperative State Research, Education and Extension Service, National Research Initiative Competitive Grants Program, US Department of Agriculture, under agreement number 2001-35302-09983 and by the NIH COBRE awards IP20RR16433 and IP20RR15563. J. S. T. was supported, in part, by the Howard Hughes Medical Institute award number 52003057. This is contribution number 03-70-J from the Kansas Agricultural Experiment Station.


   REFERENCES
Top
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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Received 3 January 2003; accepted 13 March 2003.



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