Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore 560012, India1
Author for correspondence: Karumathil Gopinathan. Fax +91 80 360 2697. e-mail kpg{at}mcbl.iisc.ernet.in
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Abstract |
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Introduction |
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In AcMNPV, transient expression with overlapping clones representing the entire genome identified a set of genes called late gene expression factors (lefs), essential for expression from late and very late promoters (Lu & Miller, 1994 , 1995
; Todd et al., 1995
; Li et al., 1999
). Their homologues have been identified in various baculoviruses whose genomes have been completely sequenced (Ayres et al., 1994
; Gomi et al., 1999
; Kuzio et al., 1999
; Ahrens et al., 1997
). Of these, the products encoded by lefs 4, 5, 6, 8, 9, 10, 11, 12, 38K and p47 are believed to regulate late gene expression at the level of transcription. In addition, very late gene expression factor 1 (encoded by vlf-1) was required for transcription from very late gene promoters (McLachlin & Miller, 1994
; Yang & Miller, 1998
, 1999
). Deletion analysis of all the lefs in BmNPV has demonstrated that except for four lefs, ie-2, 39K, lef-7 and p35, the others were essential for virus propagation in vitro (Gomi et al., 1997
). However, molecular characterization of only a few of the BmNPV lefs has been reported so far (Sriram & Gopinathan, 1998
; Mikhailov, 2000
).
The differences in promoter structure as well as the nature of polymerase capable of initiating transcription from late and very late promoters supported the notion of a virus-encoded/modified polymerase in baculoviruses. Besides being insensitive to -amanitin, the viral polymerase also differed from the host polymerase in cofactor requirements (Grula et al., 1981
; Fuchs et al., 1983
). Beniya et al. (1996)
purified the AcMNPV RNA polymerase that could accurately initiate transcription from late (p6.9) and very late (polh) promoters. The minimal constituents of the polymerase were subsequently identified to be LEF-4, LEF-8, LEF-9 and P47 (Guarino et al., 1998a
). LEF-8 shows homology to the second largest
-subunit of prokaryotic DNA-directed RNA polymerase, harbouring the conserved GXKX4HGQ/NKG motif (Passarelli et al., 1994
), whereas LEF-9 is homologous to the largest
'-subunit in harbouring the conserved NADFDGD sequence motif (Lu & Miller, 1994
). LEF-4 from AcMNPV has been characterized as the mRNA capping enzyme (Guarino et al., 1998b
; Jin et al., 1998
). The BmNPV counterpart of LEF-4 also carries out all the enzymatic functions related to mRNA capping activity (S. Sehrawat & K. P. Gopinathan, unpublished data). The function or presence of any recognizable motifs in P47 is not reported but the protein has been localized to the nucleus of infected cells (Carstens et al., 1993
, 1994
). Recently, 3' polyadenylation activity has also been demonstrated to be an inherent property of the viral polymerase (Jin & Guarino, 2000
). The transcriptional regulation of most of the lefs, however, has not been analysed in detail.
In this study, we describe the cloning and characterization of lef-9 and lef-8 from BmNPV. Detailed transcriptional analyses revealed that lef-9 transcripts initiated at multiple sites different from the consensus baculovirus early transcription start site motif CAGT and terminated downstream to a canonical polyadenylation sequence. Using antibodies raised against the bacterially expressed LEF-9, the synthesis of the protein in infected cells was also monitored. Preliminary studies on the transcriptional mapping of lef-8 as well as the possible interaction between LEF-8 and LEF-9 in BmNPV-infected BmN cells have also been carried out.
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Methods |
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Generation of plasmid constructs.
The synthetic oligonucleotides used as primers for PCR, cloning, sequencing and primer extensions are shown in Figs 3 and 5(c). Plasmid construct pRPU9, harbouring the N-terminal region and immediate 5' upstream sequences of lef-9 used in RNase protection analysis of lef-9 transcripts, was constructed by PCR amplification of a 210 bp fragment from BmNPV DNA using primers P3 and P4 and cloning at the EcoRV site of pBS-SK+. For expression of lef-9 ORF in Escherichia coli, lef-9 was amplified from viral genomic DNA using primers P1 and P2 and Pfu DNA polymerase and inserted into plasmid pET32a (harbouring a His-tag as well as a thioredoxin fusion sequence) as a 1·5 kb BamHIEcoRI fragment (clone designated pTrxALef-9). To study the transcription profile of lef-8, a genomic fragment encoding the C-terminal region of the gene and the downstream sequences encompassing the etm open reading frame (ORF) was amplified as a 566 bp fragment using primers P7 and P8 and cloned in pBS-KS+ (clone designated pCL8). For expression as a FLAG epitope-tagged protein in insect cells, the lef-8 clone was generated in three steps. The 5' region (480 bp) of lef-8, starting from +1 ATG of the ORF, was amplified using primers P9 and P10 and cloned as a BamHIXhoI fragment in pBS-SK+. The remaining part of lef-8 was derived from the clone pBmXJ (harbouring the 4·1 kb XhoI J fragment of BmNPV genomic DNA in pBS-SK+) as a 3·0 kb XhoIEcoRI fragment. The 3·5 kb full-length lef-8 gene together with downstream sequences was mobilized as a BamHIEcoRI fragment under the control of the BmNPV p10 promoter harbouring the FLAG epitope tag (clone designated pFLef-8). The BmNPV p10 promoter harbouring the FLAG tag was constructed in our laboratory (V. B. Palhan & K. P. Gopinathan, unpublished results) by inserting a 57-mer synthetic oligonucleotide (5' CATTTTATTTAACTATCATCTCATGGACTACAAAGACG-ACGACGACAAAGGATCCCG 3') encoding the FLAG peptide (NAspTyrLysAspAspAspAspLysC) downstream of the BmNPV p10 promoter.
RNase protection, primer extension, Northern blotting and 3' RACE.
Total RNA from uninfected- and BmNPV-infected BmN cells at various times post-infection (p.i.) was isolated by the guanidiniumisothiocyanate method (Chomczynski & Sacchi, 1987 ) and treated with RNase-free DNase I (10 U per 1x107 cells). Expression profiles of lef-9 and lef-8 were analysed by RNase protection assays using the appropriate complementary RNA probes. Antisense riboprobes of high specific activity were generated in vitro from the cloned BmNPV genomic DNA fragments in plasmid pBS-SK+ using either T3 or T7 RNA polymerase in the presence of radiolabelled [
-32P]UTP. Total RNA (2040 µg) was coprecipitated with the corresponding antisense riboprobes (1·5x105 c.p.m.) in the presence of 200 mM NaCl and 20 µg carrier DNA using 2·5 vols ethanol. Following hybridization at 50 °C for 16 h in the presence of 50% formamide, the RNase digestion mixture containing RNase A (2 U) and RNase T1 (1 U) was added and, after 1 h at 37 °C, the samples were precipitated in the presence of 10 µg yeast tRNA (added as carrier) and 2·5 vols ethanol. The RNase-protected samples were analysed by electrophoresis on 6% acrylamide gels containing 7 M urea and visualized by autoradiography.
Transcription start sites for lef-8 and lef-9 were determined by primer extension analysis. Total RNA (2040 µg) was annealed to 5 pmol of the appropriate primer and reverse transcription was performed using Superscript II Reverse Transcriptase (Gibco BRL) in the presence of [-32P]dATP (10 µCi; 3000 Ci/mmol) and 100 pmol each of dCTP, dGTP and dTTP for 5 min at 42 °C. This was followed by extension reactions for 5 min in the presence of vast excesses of all four dNTPs (200 µM each). The reaction was terminated using 80% formamide gel-loading dye containing 200 µM EDTA. The primer-extended products were analysed on 6% acrylamide gels containing 7 M urea together with the appropriate DNA sequencing ladders for sizing. Bands were detected by autoradiography.
Northern blotting was carried out as described by Sambrook et al. (1989) . Total RNA (40 µg) isolated from control as well as BmNPV-infected BmN cells was separated on a 1·2% MOPSformaldehyde agarose gel and transferred onto a N+ nylon membrane (Amersham Pharmacia). The blot was probed using a radiolabelled full-length lef-9-specific probe, which was generated by random priming. Following hybridization (16 h at 42 °C in the presence of 50% formamide), the blots were washed at a final stringency of 65 °C in 0·1x SSC and 0·1% SDS and autoradiographed.
The 3' end of the lef-9 transcript was mapped precisely by RACE. Reverse transcriptions were performed with total RNA using the 3' RACE adapter primer, 5' GGCCACGCGTCGACTAGTAC(T)17 3', and Superscript II Reverse Transcriptase at 42 °C for 1 h. Following RNase H treatment at 30 °C for 30 min, PCR amplification was carried out with one-tenth of the volume of the above reaction and Vent DNA polymerase using the lef-9 N-terminal primer P1 (encompassing the +1 ATG) and the 3' RACE anchor primer, 5' GGCCACGCGTCGACTAGTAC 3'. This was followed by two rounds of amplification with the lef-9 RACE forward primer P5 (50 nt upstream of the ORF stop codon) and the 3' RACE anchor primer. The amplified product was cloned in pBS-SK+ at the EcoRV site and sequenced with the lef-9 forward primer P5 to precisely map the transcription termination site.
Polyclonal antisera and Western blotting.
Rabbit polyclonal antiserum was raised against LEF-9 expressed as a thioredoxin fusion protein with a C-terminal His-tag (clone pTrxALef-9) in E. coli strain BL-21. The bacterially expressed protein was purified through affinity chromatography in an NiNTA agarose column. The purified protein (800 µg) was injected into a rabbit in the presence of Freunds complete adjuvant followed by three rounds of boosters, each with 500 µg of the purified protein (in Freunds incomplete adjuvant) administered at an interval of 10 days; the rabbit serum was checked for the presence of antibodies to LEF-9 by Western blot.
To analyse the temporal synthesis of LEF-9, uninfected as well as BmNPV-infected BmN cells (1x105) were suspended in SDS gel loading buffer [50 mM Tris (pH 6·8), 2% SDS, 1% -mercaptoethanol and 10% glycerol] and analysed on an 8% polyacrylamide gel containing 0·1% SDS. Following electrophoresis, the proteins were electrophoretically transferred onto a PVDF membrane at 1·0 mA/cm2 for 1 h. The membrane was blocked with 3% gelatin overnight and probed with a 1:1000 dilution of the anti-LEF-9 antiserum followed by incubation with secondary anti-rabbit goat antibody conjugated to horseradish peroxidase. After extensive washing, the blot was developed using the ECL+Plus Western Blot Detection kit (Amersham Pharmacia).
To study the interaction between LEF-8 and LEF-9 in vivo, the FLAG-tagged construct pFLef-8 was transfected into BmN cells, (2·5 µg DNA per 1x106 cells) in serum-free medium. After 8 h, the cells were infected with BmNPV (m.o.i. of 10) in TC-100 complete medium. At 48 h p.i., the cells were harvested, washed with PBS and lysed with 1% NP-40 in 10 mM Tris (pH 7·9), 10 mM NaCl, 1·5 mM MgCl2, 5 mM DTT and 10% glycerol. The nuclei were pelleted by centrifugation at 3000 r.p.m. for 10 min. The nuclear proteins were extracted in extraction buffer [50 mM Tris (pH 7·4), 500 mM NaCl, 5 mM DTT and 1 mM EDTA] containing 1% Triton-X-100. The sample was then diluted to 150 mM NaCl in TBS and bound to an anti-FLAG M2 affinity gel, previously equilibrated with TBS. After 1 h of binding at 4 °C, the matrix was washed with TBS and bound proteins were eluted using 0·1 M glycine or competitive elution with the FLAG peptide (25 nmol). This fraction was analysed by Western blotting using the FLAG- or LEF-9-specific antibodies and the ECL+Plus Western Blot Detection kit.
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Results |
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Immunodetection of LEF-9 in BmN cells
Synthesis of LEF-9 in BmNPV-infected BmN cells was analysed by Western blot using the polyclonal antibodies raised against the bacterially expressed purified protein (Fig. 4). A 52 kDa band, corresponding to LEF-9, appeared from 12 h p.i. and maximal levels were seen at 36 h p.i. in BmNPV-infected cells; levels of expression remained high until later times in infection. The protein showed slight anomalous mobility (52 kDa) as compared to the predicted size of the protein (56·4 kDa).
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Transcriptional mapping of the lef-8 region is presented schematically in Fig. 5(c). The major differences between AcMNPV and BmNPV in this region [e.g. complete deletion of AcMNPV ORF49 (pcna) and partial deletion of ORF48 (etm) from the BmNPV genome], the base changes in the transcription start site regions (circled) and the primers used for different purposes are all marked.
Interaction between BmNPV LEF-8 and LEF-9
Since both LEF-8 and LEF-9 have been demonstrated to be the subunits of the baculoviral RNA polymerase in the prototype AcMNPV (Guarino et al., 1998), the possible interaction between the counterparts of these two subunits from BmNPV was examined. In preliminary studies using the yeast two-hybrid system, we could not demonstrate any interaction between them. Therefore, a coimmunoprecipitation approach was attempted in which lef-8 was expressed as a FLAG-tagged protein, under the control of the strong viral very late p10 promoter, by transfection of pFlef-8 into BmN cells followed by infection with BmNPV. The transiently expressed FLAG-tagged LEF-8, together with interacting viral proteins (resulting from BmNPV infection), was purified on an anti-FLAG M2-affinity matrix. Western blotting of the eluates from the affinity matrix with monoclonal anti-FLAG and polyclonal anti-LEF-9 antibodies detected bands of 102 and 52 kDa, respectively (Fig. 6
, lanes 2 and 4, respectively). The signals corresponding to the FLAG-tagged LEF-8 and the viral LEF-9 proteins suggested that the two proteins either interacted directly or constituted part of a complex in vivo. In control samples (where the cells were not transfected with the FLAG-tagged lef-8 construct) when the nuclear proteins were passed through FLAG-specific antibody affinity column, no immunoreactive bands were detected with anti-FLAG or anti-LEF-9 antibodies (Fig. 6
, lanes 1 and 3, respectively).
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Discussion |
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LEF-8 and LEF-9 being constituents of the viral polymerase required for transcription of late and very late promoters should, ideally, be expressed early in infection and, presumably, by the host RNA polymerase. It is likely that their transcription also depends on the early virus transactivators or host factors. The transcription start site for lef-8 is located at -19 nt from the +1 ATG of the ORF, mapping to the first G residue of the sequence GTGCAAT. In AcMNPV, the transcription start site of lef-8 is not mapped but the sequence in the corresponding region is GCGCAGT (differing from BmNPV at two bases and thus harbouring the early transcription start site motif CAGT). The sequences downstream of lef-8 also show major differences between the two viruses. This region in AcMNPV (29·030·1 map units) encompasses three ORFs, encoded by etl, etm and ets, located immediately downstream of lef-8 (Ayres et al., 1994 ). The largest of these, etl, encodes a 28 kDa polypeptide expressed early in infection that shows homology to the eukaryotic DNA polymerase
processivity factor, PCNA. The disruption of etl had no effect on virus viability (Crawford & Miller, 1988
). The other two early ORFs, encoding ETM and ETS, have not been assigned any function. In BmNPV, this region harbours a 1·1 kb deletion resulting in the complete loss of etl and 150 nt from the 5' region of etm (Gomi et al., 1999
). The analysis of BmNPV lef-8 transcripts by RNase protection revealed that the transcripts extended to the remaining etm region. The first potential polyadenylation signal (AATAAA) after the two tandem termination codons of LEF-8 was 130 nt downstream and 132 nt upstream of the +1 ATG of the adjoining ets ORF. Our efforts to map the 3' end of lef-8 transcript were not successful due to the low abundance of the transcript and the limitations in electrophoretic resolution of RNase-protected fragments, which were larger when other primer combinations available to us were used. The large size of the lef-8 transcript and its possible instability were also responsible for the extensive degradation observed in Northern blots (data not shown).
The 5' end mapping of lef-9 transcripts revealed the presence of multiple transcription start sites. One of these transcription start sites, GCACT, differed from the consensus early motif CAGT by 1 nt, but the other, CTCTT, did not fall into any of the known consensus motifs. The sequences reported here were similar to those in AcMNPV (Guarino et al., 1998a ). The shorter transcripts initiating from the GCACT sequence were detected only at 12 h p.i., whereas the more distal transcription start site at CTCTT was preferentially utilized at later time-points. The significance of these multiple initiation sites is not clear at present. The precise sites of transcription termination and poly(A) addition of lef-9 transcripts mapped here demonstrated the utilization of the consensus polyadenylation signal, located 7 nt downstream of the first of the two tandem translation termination codons of the LEF-9 ORF. This consensus motif was followed by an immediate downstream U-rich sequence implicated in transcript processing in most AcMNPV mRNAs as well as other eukaryotic transcripts (Westwood et al., 1993
; MacLauchlan et al., 1985
; McDevitt et al., 1986
).
Although LEF-8 and LEF-9 harbour the conserved RNA polymerase subunit motifs, so far no independent functions have been identified. Being constituents of the virally encoded polymerase, a possible interaction or association between these subunits was predictable. Our attempts to demonstrate a direct interaction between LEF-8 and LEF-9 of BmNPV by yeast two-hybrid analysis did not show any interaction between them (data not presented). However, in the preliminary studies reported here, an association of these two proteins in vivo could be demonstrated by immunocoprecipitation exploiting a FLAG-tagged lef-8 construct. Our results suggest that, in vivo, a subcomplex of LEF-8 and LEF-9 may be weak or the association with the rest of the polymerase subunits is essential to form a stable complex.
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Acknowledgments |
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References |
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Received 2 January 2002;
accepted 28 March 2002.