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
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ABSTRACT |
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Present address: University of Kansas Medical Center, Kansas City, KS 66160, USA.
Present address: University of North Carolina, Chapel Hill, NC 27599, USA.
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INTRODUCTION |
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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
or
' 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.
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METHODS |
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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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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 99153; 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 36 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--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 NCBZLEULEULEUAL (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 IgGhorseradish peroxidase (Bio-Rad) and SuperSignal chemiluminescent substrate (Pierce). Images were prepared using Adobe Photoshop 7.0.
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RESULTS |
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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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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 319), 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 652701, 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).
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DISCUSSION |
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Both termini of the protein were essential for function. Since the first two N-terminal deletions, amino acids 266 and 2134, 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, proteinRNA 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 subunit as a region participating in catalysis (Markovtsov et al., 1996
). Third, H1237 in the E. coli
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 proteinDNA or proteinprotein 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 -D,
-H,
'-D and
'-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,
-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
-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
-H region and LEF-9 has the short seven-residue stretch homologous to
'-D, two regions at the heart of the enzyme. It is not known if the other transcription-specific LEFs co-operate via proteinprotein 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 506553, 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.
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ACKNOWLEDGEMENTS |
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Received 3 January 2003;
accepted 13 March 2003.
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