Role of the 3' untranslated region of baculovirus p10 mRNA in high-level expression of foreign genes

Monique M. van Oers1, Just M. Vlak2, Harry O. Voorma1 and Adri A. M. Thomas1

Department of Molecular Cell Biology, University of Utrecht, Padualaan 8, 3584 CH Utrecht, The Netherlands1
Department of Virology, Wageningen Agricultural University, The Netherlands2

Author for correspondence: Monique van Oers.Fax +31 30 2542219. e-mail m.m.vanoers{at}bio.uu.nl


   Abstract
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Abstract
Introduction
Methods
Results
Discussion
References
 
The p10 gene of Autographa californica nucleopolyhedrovirus has two putative AATAAA polyadenylation signals. The downstream signal is used predominantly, as was determined by analysing 3' cDNA ends. This downstream motif is followed by a GT-rich sequence, known to be important for efficient polyadenylation in mammalian systems. To analyse the importance of polyadenylation for p10 gene expression, recombinant viruses with altered 3' untranslated regions (UTRs) were tested using chloramphenicol acetyltransferase (CAT) as a reporter. Surprisingly, after inactivation of the downstream AATAAA motif, CAT expression remained at the same high level as observed with a wild-type 3' UTR. Polyadenylation occurred 24–28 nucleotides further downstream, probably due to an ATTAAA sequence motif. Replacing the p10 3' UTR with the SV40 early terminator sequence as part of an hsp70–lacZ–SV40 gene cassette, which is commonly used in baculovirus expression vectors, resulted in a reduction in reporter gene expression. Polyadenylation occurred far more efficiently in the original p10 3' UTR than in the SV40 terminator sequence, as was shown by testing the SV40 terminator separately. These results indicate that in order to obtain high levels of foreign gene expression, vectors that provide a wild-type p10 3' UTR are to be preferred over those containing the hsp70–lacZ–SV40 gene cassette. Comparison of the p10 genes of various baculoviruses showed the presence of at least one AATAAA or ATTAAA motif in combination with a GT-rich sequence in the 3' UTR, suggesting an evolutionary conservation of these two elements, thereby maintaining the high level of p10 gene expression.


   Introduction
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Abstract
Introduction
Methods
Results
Discussion
References
 
Two proteins, polyhedrin and p10, accumulate to very high levels at the final stage of infection with a nucleopolyhedrovirus (NPV) (reviewed by Rohrmann, 1986 ; van Oers & Vlak, 1997 ). Since these proteins are not essential for virus replication in cultured insect cells, the p10 and polyhedrin genes can be modified; their promoters have therefore been exploited for the production of foreign proteins in insect cells (King & Possee, 1992 ). Transcription of the p10 and polyhedrin genes starts within a baculovirus late promoter motif TAAG (Blissard & Rohrmann, 1990 ) and requires a virus-induced RNA polymerase activity (Beniya et al., 1996 ). Mutational analyses have shown that the region between the TAAG motif and the ATG translational start codon, i.e. the 5' untranslated region (UTR), is necessary for efficient expression from both the p10 and polyhedrin promoters (Possee & Howard, 1987 ; Luckow & Summers, 1988 ; Weyer & Possee, 1988 ; Ooi et al., 1989 ; Qin et al., 1989 ). Furthermore, an intact 5' UTR is important for efficient translation initiation, as was shown by in vitro translation of p10 mRNA in insect cell lysates (Scheper et al., 1997 ). The collective data suggest that the high expression of the p10 and polyhedrin genes is determined both at the transcriptional and translational level.

The role of the 3' UTR in polyhedrin and p10 gene expression is less clear. Deletions in the 3' UTR did not affect p10 mRNA translation in vitro. In vivo, however, such deletions negatively influenced the transient expression of a reporter gene (Scheper et al., 1997 ). These deletions might affect the polyadenylation and, hence, the stability of p10 transcripts. In this paper, the role of the 3' UTR in the high-level expression of baculovirus major late genes was investigated in more detail, using the p10 gene of Autographa californica multicapsid NPV (AcMNPV) as a model. The AcMNPV p10 gene is transcribed into two major polyadenylated transcripts, approximately 2500 and 750 nucleotides in size, of which the latter is the more abundant (Rankin et al., 1986 ). One of two AATAAA putative polyadenylation signals can be used to generate this 750 nt transcript. To begin investigating which elements are important for high-level gene expression, the exact site of polyadenylation was determined by amplification and sequencing of 3' cDNA ends (3' RACE). The importance of polyadenylation for high-level gene expression was further examined by testing a modified 3' UTR, in which the active polyadenylation signal was mutated. The effect of a heterologous 3' UTR, as provided by the SV40 early terminator, was also investigated. This terminator is often used in p10-based expression vectors (López-Ferber et al., 1995 ). In the expression vector system described by Vlak et al., (1990) , the SV40 terminator is part of a gene cassette that enables easy visual screening of recombinant virus plaques. The experiments presented here show that the use of this gene cassette leads to suboptimal levels of foreign gene expression.


   Methods
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Abstract
Introduction
Methods
Results
Discussion
References
 
{blacksquare} Rapid amplification of 3' cDNA ends (3' RACE) and Southern blot analysis.
Total RNA was extracted with RNAzol (Campro Scientific) at 48 h post-infection (p.i.) from Sf21 cells (Vaughn et al., 1977 ) infected with wild-type AcMNPV, strain E2 (Smith & Summers, 1978 ) or the p10 deletion mutant AcAS3 (Vlak et al., 1990 ). First-strand cDNA was made by incubating 2 µg total RNA for 1 h at 37 °C in the presence of 2 mM dNTPs, 1·9 µM anchor primer (Table 1) and 15 U AMV reverse transcriptase in the supplied buffer (Amersham) in a total volume of 20 µl. A PCR was assembled with 1 µl first-strand cDNA, 0·25 µM anchor primer, 0·25 µM p10-specific primer (see Table 1), 200 µM dNTPs and 5 U Taq polymerase in 50 µl Taq buffer (Pharmacia Biotech) and the products were analysed in 2% agarose gels. Southern blot analysis was performed with a 32P-labelled probe of the p10 coding sequence, derived as a BamHI fragment from pAcMO15 (van Oers et al., 1993 ). To determine the exact poly(A)-addition site in p10 mRNA, the major 3' RACE product was digested at BamHI and PstI restriction sites originating from the p10-specific and anchor primers, respectively. The resulting fragment was cloned into pTZ18R and sequence analysis was performed on seven individual cDNA clones.


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Table 1. Oligonucleotides used for 3' RACE, mutagenesis and recombinant genome analysis

 
{blacksquare} Cloning strategies.
The construction of plasmid pAcCAT (Fig. 1A) has been described previously (Scheper et al., 1997 ). To generate plasmid pAcCAT{Delta}poly(A)2 (Fig. 1A), the DNA encoding the p10 3' UTR was isolated from pAcJJ1 (Scheper et al., 1997 ) as a 670 bp BamHI–HincII fragment and cloned into pTZ18R. Plasmid pAcJJ1 has the same 3' flanking region as pAcCAT. A PCR was assembled with the cloned 3' UTR as template, a mutagenic primer (Table 1) and the standard reverse sequencing primer, thereby changing the second AATAAA motif into AAGGTA, simultaneously introducing a KpnI restriction site. The 280 bp PCR product was used as a primer in a second PCR together with the universal sequencing primer to obtain a full-length p10 flanking sequence. The new product was cloned as a BamHI–HincII fragment into pAcCAT, thereby replacing the original 3' flanking region. To obtain pAcCAT-SV40-lacZ (Fig. 1A), the BamHI–EagI fragment of pAcCAT was replaced with that of pAcAS3 (Vlak et al., 1990 ; see Fig. 1B). In this way, a gene cassette consisting of the SV40 early terminator, the lacZ coding sequence and the Drosophila melanogaster hsp70 promoter was inserted downstream of the chloramphenicol acetyltransferase (CAT) coding sequence. To construct plasmid pAcCAT-SV40 (Fig. 1A), the CAT and SV40 terminator sequences as present in pAcCAT-SV40-lacZ were amplified by PCR with the upstream p10 primer and an SV40 primer, which introduces a BglII site at the end of the SV40 sequence (Table 1). The PCR fragment was cloned in plasmid pGEM-T (Promega). An NcoI–BglII fragment containing the CAT coding sequence and the SV40 terminator sequence was inserted between the NcoI and BamHI sites of pAcCAT. All DNA fragments obtained by PCR were verified by sequence analysis.



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Fig. 1. Schematic representation of plasmid constructs and parental viruses. (A) Plasmid pAcCAT contains the CAT coding sequence (670 nt) flanked by wild-type AcMNPV p10 flanking regions. TAAG indicates the late promoter motif in the p10 promoter. The asterisks in the 3' UTR represent the two AATAAA polyadenylation signals at positions +65 and +163. The pAcCAT construct used in this study does not contain the engineered EcoRI site in the 5' UTR, in contrast to the plasmid tested by Scheper et al. (1997) . Plasmid pAcCAT{Delta}poly(A)2 has a modified 3' UTR, in which the downstream polyadenylation signal at +163 was inactivated. In pAcCAT-SV40-lacZ, the CAT coding sequence is followed by the SV40 early terminator through insertion of an hsp70 promoter–lacZ–SV40 terminator gene cassette. The three polyadenylation motifs in the SV40 terminator are also indicated by asterisks: one AATAAA motif serves for p10 promoter-derived transcripts and two function in the other orientation to polyadenylate lacZ transcripts. In pAcCAT-SV40, only the SV40 terminator sequence is inserted downstream of the CAT coding sequence. (B) Schematic representation of the p10 locus in the genome of wild-type AcMNPV and the two p10-deletion mutants AcMO21 and AcAS3. AcMO21 (Martens et al., 1995 ) was used to generate the recombinant AcCAT-SV40-lacZ, and AcAS3 (Vlak et al., 1990 ) was used for the recombinants AcCAT, AcCAT{Delta}poly(A)2 and AcCAT-SV40. The genomes of both AcMO21 and AcAS3 contain a unique Bsu36I site used to linearize the viral genome prior to transfection. B, BamHI; Bs, Bsu36I; Ea, EagI; H, HindIII; HII, HincII; K, KpnI, N, NcoI, Nd, NdeI.

 
{blacksquare} Generation of recombinant viruses.
The recombinant viruses AcCAT, AcCAT{Delta}poly(A)2 and AcCAT-SV40 were generated by co-transfecting plasmids pAcCAT, pAcCAT{Delta}poly(A)2 and pAcCAT-SV40 (Fig. 1A) with viral DNA isolated from the p10 deletion mutant AcAS3 (Fig. 1B; Vlak et al., 1990 ). The viral DNA was linearized before transfection by virtue of a unique Bsu36I site in the lacZ sequence. Recombinant viral plaques were selected by their white phenotype upon X-Gal addition and then plaque-purified. To generate the recombinant virus AcCAT-SV40-lacZ, the p10 deletion mutant AcMO21 (Martens et al., 1995 ) served as parental virus. The genome of AcMO21 contains a unique Bsu36I site at the p10 locus (Fig. 1B), which was used to linearize the viral genome prior to transfection. AcCAT-SV40-lacZ recombinant viral plaques were selected by their blue phenotype. The presence of the CAT coding sequence at the p10 locus in the various recombinant viruses was verified by PCR on DNA isolated from budded virions using the upstream p10 primer and a 3' CAT-specific primer (Table 1). The introduction of the mutation in AcCAT{Delta}poly(A)2 was verified by the presence of a KpnI restriction site in the p10 3' UTR (Fig. 1A), as determined by PCR with the up- and downstream p10 primers (Table 1) followed by restriction enzyme analysis. The identity of recombinant AcCAT-SV40 was further verified by PCR with the 5' CAT and the downstream p10 primer.

{blacksquare} Analysis of reporter gene expression.
To test the effect of a mutated polyadenylation signal on reporter gene expression, Sf21 cells were infected either with AcCAT or AcCAT{Delta}poly(A)2 at an m.o.i. of 10 TCID50 per cell. Infected cells were harvested at 24, 48 and 72 h p.i. To determine the effect of a heterologous 3' UTR, cells were infected with AcCAT, AcCAT-SV40-lacZ or AcCAT-SV40 and harvested at 48 h p.i. To determine CAT activity, infected cells were lysed in 0·25 M Tris–HCl, pH 7·6, by three rounds of freeze–thawing. CAT activity was determined by acetylation of [14C]chloramphenicol (Gorman et al., 1982 ). Measurements were performed within the linear range of the assay. A phosphorimager was used to determine relative levels of enzyme activity.

{blacksquare} RNA analysis.
Sf21 cells (2x106) were infected with the various recombinant viruses at an m.o.i. of 10 TCID50 per cell and total RNA was extracted at 48 h p.i. Samples of 5 µg total RNA were glyoxylated and analysed in a 1·5% agarose gel in 15 mM sodium phosphate buffer, pH 6·5, as described previously (van Oers et al., 1993 ). RNA was transferred to Hybond membrane in 25 mM sodium phosphate buffer, pH 6·5. CAT-specific transcripts were visualized with a 32P-labelled CAT probe derived from the NcoI–BamHI fragment of pAcCAT (Fig. 1A). A phosphorimager was used to determine relative transcript levels. The AcMNPV HindIII-V fragment (Smith & Summers, 1978 ) was used as a polyhedrin probe to check for equal infection and loading, and to serve as a marker for transcript sizes.

3' RACE analysis was performed on total RNA isolated from infections with either AcCAT or AcCAT{Delta}poly(A)2, as described above for AcMNPV wild-type, by using the anchor primer and a 5' CAT-specific primer (Table 1). PCR products were analysed in a 2% agarose gel. The product obtained with RNA derived from the infection with AcCAT{Delta}poly(A)2 was digested with BamHI and XbaI and cloned into pTZ18R. Two of these cDNA clones were subjected to sequence analysis. 3' RACE analysis for AcCAT-SV40-lacZ was performed by using the anchor primer in combination with the 5' CAT primer.


   Results
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Abstract
Introduction
Methods
Results
Discussion
References
 
The poly(A)-addition site of the 750 nt p10 transcript
The AcMNPV p10 gene is transcribed as two major polyadenylated transcripts, about 750 and 2500 nt in length, that have a common 5' end (Rankin et al., 1986 ). For the more abundant, 750 nt transcript, two putative AATAAA polyadenylation signals (Birnstiel et al., 1985 ) are located at positions +65 and +163 relative to the translational stop codon TAA (Kuzio et al., 1984 ; see Fig. 1B). 3' RACE techniques were exploited to determine the exact site of poly(A) addition for the 750 nt transcript. To this aim, a p10-specific primer that hybridized to the 5' end of the p10 coding sequence and an anchor primer that recognized the poly(A) tail (Table 1) were used to amplify 3' cDNA ends. Analysis by agarose gel electrophoresis showed a 3' RACE product of approximately 500 bp (not shown, see also Fig. 4A), which would correspond to a transcript polyadenylated due to the second polyadenylation motif. The nature of this 3' RACE product was confirmed by Southern blot analysis with a p10-specific probe (Fig. 2A; indicated by `2'). This PCR product was absent when RNA from the p10 deletion mutant AcAS3 was analysed. A minor band of approximately 400 bp, corresponding to polyadenylation at the first AATAAA motif, was detected only upon Southern blot analysis (Fig. 2A; indicated by `1'). This experiment showed that the AATAAA motif at position +163 is used for polyadenylation of the majority of the p10 transcripts.



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Fig. 4. 3' RACE analysis of p10 transcripts generated after inactivation of the downstream polyadenylation signal. (A) Total RNA was isolated from Sf21 cells at 48 h p.i. with AcMNPV (lane 1), AcCAT (2) or AcCAT{Delta}poly(A)2 (3). The 3' cDNA ends were amplified by using an anchor primer in combination with a p10-specific primer for AcMNPV and a 5' CAT-specific primer for the two recombinant viruses (see Table 1). The products were analysed on a 1·5% ethidium bromide-stained agarose gel. Size markers are indicated in bp. The CAT coding sequence is 670 nt long, with the 5' CAT primer hybridizing at position 100. The p10 coding sequence is 282 nt long and the p10 primer hybridizes at the 5' end of the coding sequence. (B) Sequence analysis of two clones of the 3' RACE product obtained with recombinant AcCAT{Delta}poly(A)2. The upper line shows the sequence of the AcMNPV p10 gene, with the AATAAA motif at +163 and the accompanying GT-rich sequence (both shaded). The second line shows the genomic sequence of the recombinant AcCAT{Delta}poly(A)2, where the AATAAA motif has been changed to AAGGTA. The alternative signal ATTAAA is indicated, as is a GT-rich sequence further downstream (both shaded). The two lower lines show the sequence of two 3' cDNA ends found with this recombinant (designated types I and II). The poly(A) stretches are underlined.

 


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Fig. 2. Determination of the poly(A)-addition site in the 750 nt p10 transcripts. (A) Southern blot analysis of amplified 3' cDNA ends generated from total RNA isolated from Sf21 cells after infection with either wild-type AcMNPV or the p10-deletion mutant AcAS3, used as a negative control. The p10 coding sequence was used as a probe to visualize p10-specific PCR products. Size markers are indicates in bp. Arrow `2' indicates the major PCR product, corresponding to polyadenylation downstream of the AATAAA motif at +163, while arrow `1' points to a minor PCR product, which corresponds to transcripts polyadenylated at the AATAAA motif at +65. (B) Sequences of AcMNPV p10 3' cDNA ends based on the analysis of seven individual cDNA clones. The DNA sequence of the AcMNPV genome is shown in the upper line. Two types of cDNA clones were obtained (types I and II), which varied in length by one base. The sequences are shown from the AATAAA motif located at position +163 (shaded). A GT-rich sequence located downstream of the AATAAA motif is also indicated by shading. The poly(A) tail is underlined.

 
To allow further analysis, the major 3' RACE product was cloned into pTZ18R as a BamHI–XbaI fragment. Sequence analysis of seven individual clones revealed that the poly(A) tail started either 17 (Fig. 2 B; indicated by `cDNAI') or 18 nt (`cDNAII') downstream of the second AATAAA motif. This result is in good agreement with S1 nuclease experiments that have placed the 3' end roughly 240 nt downstream from the HindIII site in the p10 gene (Rankin et al., 1986 ; see Fig. 1B).

Inactivation of the second polyadenylation motif
To examine the importance of polyadenylation for the high-level expression of the p10 gene, a modified 3' UTR was constructed in which the AATAAA sequence at nucleotide residue +163, that used predominantly to polyadenylate p10 transcripts, was changed to AAGGTA (see Fig. 1A). The generation of a KpnI site marked this alteration. To allow the use of CAT as reporter gene, the 3' UTR of pAcCAT was replaced with the mutated 3' UTR, resulting in pAcCAT{Delta}poly(A)2. Recombinant viruses were generated to analyse the effect of a mutated poly(A)-addition site in the p10 3' UTR. Infection of Sf21 cells with recombinant AcCAT (Fig. 3A, lanes 2–4), which has a wild-type 3' UTR, gave the same level of CAT expression as infection with mutant AcCAT{Delta}poly(A)2 (lanes 5–7) at all time-points tested.



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Fig. 3. Effect of the inactivation of the second polyadenylation motif in the p10 gene on CAT activity and mRNA levels. (A) CAT activity in Sf21 cells that were either mock-infected (lane 1) or infected with the recombinant viruses AcCAT (lanes 2–4) or AcCAT{Delta}poly(A)2 (5–7) and harvested at 24, 48 or 72 h p.i. as shown. CAM, chloramphenicol; 1 Ac-CAM and 3 Ac-CAM, the two acetylated forms of chloramphenicol. (B) Northern blot analysis of 5 µg total RNA isolated from Sf21 cells at 48 h p.i. with either wild-type AcMNPV (lane 1), AcCAT (2) or AcCAT{Delta}poly(A)2 (3). The CAT coding sequence was used as probe to visualize CAT-specific transcripts. The AcMNPV HindIII fragment was used as a probe to check for equal loading (not shown) and to allow use of the polyhedrin transcript as a size marker, according to Smith et al. (1983) .

 
Northern blot analysis was performed to determine whether the transcription pattern was changed in cells infected with the AcCAT{Delta}poly(A)2 mutant as compared with AcCAT. Two major CAT-specific transcripts were detected upon infection with AcCAT (Fig. 3B, lane 2), approximately 1100 and 2900 nt in size, as expected when the p10 coding sequence is replaced with the CAT coding sequence. Although the poly(A)-addition motif used predominantly to polyadenylate the 1100 nt CAT transcript was mutated in AcCAT{Delta}poly(A)2 (Fig. 3B, lane 3), a transcript was present comparable in size and quantity to the 1100 nt transcript obtained upon infection with AcCAT, in good agreement with the similar levels of CAT activity observed. The nature of the largest transcript obtained with AcCAT is unknown.

The poly(A)-addition sites in the p10 transcripts of the recombinant viruses AcCAT and AcCAT{Delta}poly(A)2 were determined by 3' RACE analysis. A 3' RACE product of approximately 750 bp was detected on an ethidium bromide-stained gel upon analysis of RNA from AcCAT-infected cells (Fig. 4A, lane 2), indicating that the AATAAA motif at +163 was used for polyadenylation, the same motif that was used to polyadenylate the 750 nt transcripts in AcMNPV wild-type infections (lane 1). The 3' RACE product obtained with RNA from cells infected with AcCAT{Delta}poly(A)2 was slightly larger (lane 3) than the product obtained with AcCAT. Sequence analysis of two 3' cDNA clones derived from the infection with AcCAT{Delta}poly(A)2 revealed that the p10 transcripts were polyadenylated several nucleotides further downstream [Fig. 4B; indicated by `cDNAI {Delta}poly(A)2' and `cDNAII {Delta}poly(A)2'] as compared with AcMNPV wild-type (see Fig. 2 B). This indicates that, after inactivation of the original signal by changing AATAAA into AAGGTA (Fig. 4B), an alternative signal is recognized and used for polyadenylation of p10 transcripts. The sequence motif ATTAAA located at position +183 might be this alternative signal.

The effect of a heterologous 3' UTR on gene expression levels
When the p10 promoter is exploited for foreign gene expression, the 3' UTR of the foreign gene is often used. Alternatively, an SV40-derived early terminator sequence functions as the 3' flanking sequence. However, these heterologous 3' UTRs might affect the level of p10 promoter-driven gene expression. In the p10 promoter-based vector system developed by Vlak et al. (1990) , the 3' UTR is derived from the SV40 early terminator sequence, which is part of a gene cassette that enables easy visual screening of p10-recombinant plaques by virtue of their ß-galactosidase expression. In this vector system, the SV40 terminator has a bidirectional function, to polyadenylate not only p10 transcripts, but also transcripts that originate from the hsp70 promoter–lacZ sequence and that run in opposite direction (see Fig. 1B). To test whether the presence of this gene cassette affected foreign gene expression, the transfer vector pAcCAT-SV40-lacZ was constructed (Fig. 1A) and used to generate a recombinant virus. An approximately 60% reduction in CAT expression was observed with this recombinant (AcCAT-SV40-lacZ) (Fig. 5A; lane 3) when compared with infections with recombinant AcCAT (Fig. 5A, lane 2), which has a wild-type p10 3' UTR. This was found in several independent experiments. RNA analysis showed that CAT-specific RNA levels were considerably higher in infections with AcCAT (Fig. 5B, lane 2) than in infections with AcCAT-SV40-lacZ (lane 3). The experiment was repeated several times, showing a reduction in CAT-specific mRNA levels of 60–80%. The longer CAT-specific transcript found for AcCAT-SV40-lacZ (Fig. 5B; indicated by `3'') is probably processed further downstream in the original p10 3' UTR, in line with the results of Roelvink et al. (1992) . Hybridization of the same RNA blot with a polyhedrin-derived probe showed equal levels of infection and RNA loading (Fig. 5C). Although CAT-specific RNA levels were reduced, the size of a 3' RACE product obtained upon analysis of RNA derived from infections with recombinant AcCAT-SV40-lacZ indicates that the AATAAA motif present within the SV40 terminator is recognized as a polyadenylation signal in insect cells (data not shown), albeit inefficiently (Fig. 5B).



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Fig. 5. The effect of a heterologous 3' UTR on CAT activity and mRNA levels. (A) CAT activity was measured at 48 h p.i. in Sf21 cells that were either mock-infected (lane 1) or infected with the recombinant viruses AcCAT (2), AcCAT-SV40-lacZ (3) or AcCAT-SV40 (4). CAM, chloramphenicol; 1 Ac-CAM and 3 Ac-CAM, the two acetylated forms of chloramphenicol. (B) Northern blot analysis of 5 µg total RNA isolated at 48 h p.i. from mock-infected Sf21 cells (lane 1) or cells infected with AcCAT (2), AcCAT-SV40-lacZ (3) or AcCAT-SV40 (4). The CAT coding sequence was used as a probe to visualize CAT-specific transcripts. Polyhedrin transcripts were used as size markers. The arrows `1' and `2' indicate transcripts obtained with AcCAT-SV40. Arrow `1' points to a transcript with a 3' end in the SV40 terminator, while arrow `2' points to a transcript ending in the original p10 3' UTR. Arrow `3' indicates a longer transcript in AcCAT-SV40-lacZ that is probably generated by polyadenylation in the p10 3' UTR. (C) The AcMNPV HindIII fragment was used to prepare a polyhedrin probe to check for equal infection and loading on the RNA blot shown in (B). Two additional polyhedrin transcripts were visible after a longer exposure, in accordance with Smith et al. (1983) . The three polyhedrin transcripts were used as size markers in (B).

 
To test whether the reduction in CAT expression was due to the SV40 terminator sequence or to the presence of the entire gene cassette, an additional recombinant was constructed (AcCAT-SV40), in which only the SV40 terminator was inserted between the CAT coding sequence and the original 3' flanking region. Upon infection with AcCAT-SV40 (Fig. 5A, lane 4), CAT expression levels were as high as in cells infected with recombinant AcCAT, which has a p10 3' UTR. Northern blot analysis showed a minor transcript (Fig. 5B, lane 4, indicated by `1') of the same size as the transcript found with AcCAT-SV40-lacZ, which apparently originated from processing in the SV40 terminator sequence. In addition, a slightly larger, much more abundant transcript was found (indicated by `2'), which corresponds to polyadenylation at position +163 in the p10 3' UTR. Apparently, the presence of this abundant longer transcript is responsible for the high levels of CAT activity observed in AcCAT-SV40 infections. These results show that polyadenylation at the SV40 early terminator, at least in the orientation tested, is very inefficient in Spodoptera frugiperda cells compared with polyadenylation due to the AATAAA signal at position +163 in the p10 3' UTR.


   Discussion
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Abstract
Introduction
Methods
Results
Discussion
References
 
Polyadenylation of transcripts is a process of coupled cleavage and adenylation, which has been studied in detail in mammalian and yeast systems (for recent reviews see Keller, 1995 ; Manley & Takagaki, 1996 ; Edwalds-Gilbert et al., 1997 ). The consensus sequence AAUAAA is found upstream of the cleavage site in 80% of mammalian transcripts and is essential for the binding of the cleavage and polyadenylation specificity factor. For efficient polyadenylation of many mammalian transcripts, a downstream GU- or U-rich element is necessary, which often resembles the consensus sequence YGUGUUYY (McLauchlan et al., 1985 ). This element is recognized by the cleavage stimulation factor (CstF). In general, cleavage occurs between 11 and 23 nucleotides downstream of the AAUAAA element. The downstream GU- or U-rich element is usually located 10–30 nt from the actual cleavage site (Chen et al., 1995 ).

In AcMNPV p10 mRNA, the upstream AAUAAA motif at +65 (Fig. 6, motif I) is not followed by GU- or U-rich sequences within 50 nt. This appears to explain why this motif is not used efficiently as a polyadenylation signal (Fig. 2A). The second AAUAAA motif (Fig. 2B; Fig. 6, motif II), however, is followed 22–38 nt downstream by a GU-rich sequence that may be essential for binding an insect homologue of CstF. The Drosophila gene suppressor-of-forked [su(f)] might be such a homologue (Mitchelson et al., 1993 ; Manley & Takagaki, 1996 ). This gene encodes a protein with homology to the 77 kDa subunit of human CstF. The mRNA pattern is altered in su(f) mutants, suggesting a differential effect on production or stability of mRNAs.



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Fig. 6. Alignment of the p10 3' flanking regions for AcMNPV, BmNPV, OpMNPV and CfMNPV. The AATAAA and ATTAAA sequence motifs are indicated (dark shading), as is the stop codon of the p74 ORF (boxed) that is located on the other strand. Motif II is the actual polyadenylation signal in AcMNPV and is followed 27 nt downstream by a GT-rich sequence (light shading). The alternative ATTAAA motif, which is used in AcMNPV when motif II is mutated, is also accompanied by a GT-rich sequence (light shading) located 35 nt downstream. OpMNPV has an extra AATAAA motif (dark shading) in the p10 3' flanking region. Sequence data were obtained from Leisy et al. (1986) , Kuzio et al. (1989) , Zhang et al. (1995) and Hill et al. (1993) .

 
In general, point mutations altering the AAUAAA sequence drastically reduce the efficiency of polyadenylation (Sheets et al., 1990 ). Therefore, it is unlikely that the sequence AAGGUA, as present in the AcCAT{Delta}poly(A)2 p10 transcripts, can still be recognized by polyadenylation factors. CAT expression was not affected by this mutation, however (Fig. 3). The alternative hexamer AUUAAA is found in natural RNAs and is used for polyadenylation, as was shown in an in vitro mammalian system (Wilusz et al., 1989 ). In the AcMNPV p10 gene, the AATAAA element at +163 is followed by an ATTAAA motif located 20 nt further downstream. Like the original AATAAA signal, the ATTAAA motif is also followed downstream at an equal distance by a GT-rich element (Fig. 4B). Apparently, the signal ATTAAA can be recognized in insect cells and used as an alternative polyadenylation signal.

The p10 3' UTR sequences of other NPV species were compared to that of AcMNPV to obtain further evidence for the pivotal role of an AATAAA sequence in combination with a GT-rich element in the polyadenylation of p10 transcripts (Fig. 6). Besides encoding the p10 3' UTR, this part of the AcMNPV genome also encodes the carboxy terminus of the virulence factor P74 on the opposite strand. This protein is expressed from a late baculovirus promoter, while p10 and polyhedrin are expressed from very late promoters, and is necessary for the infection of larvae (Kuzio et al., 1989 ; Faulkner et al., 1997 ). The presence of the p74 open reading frame downstream of p10 may be the main determinant for the homology observed in this region among the various baculovirus genomes.

In the p10 gene of Bombyx mori (Bm) NPV (Zhang et al., 1995 ; GenBank accession no. S76783), the downstream polyadenylation motif is changed to AACAAA, which is not recognized as a polyadenylation signal, at least in mammalian systems (Sheets et al., 1990 ). Therefore, the alternative motif ATTAAA, located further downstream, might be used in BmNPV, as described here for the AcCAT{Delta}poly(A)2 mutant. In Orgyia pseudotsugata (Op) MNPV (Leisy et al., 1986 ), an additional AATAAA motif is present between the two motifs known from AcMNPV p10, whereas the alternative motif is absent. The motif corresponding to motif II in AcMNPV p10 appears to be used in OpMNPV and is similarly followed by a GT-rich element in OpMNPV (Leisy et al., 1986 ). In Choristoneura fumiferana (Cf) MNPV, the first, inefficient motif is absent but the second and the alternative signal are intact (Hill et al., 1993 ). In Spodoptera exigua MNPV, a longer spacer sequence is found between the open reading frames of p10 and p74 (Zuidema et al., 1993 ) and the sequence is, therefore, not presented in the alignment (Fig. 6). Despite the absence of similarity to AcMNPV in this region, two putative AATAAA polyadenylation signals are again present, of which the more upstream has a GT-rich sequence at a suitable distance. The Spodoptera littoralis (Spli) NPV p10 gene is not flanked by the p74 gene (Faktor et al., 1997 ). In SpliNPV p10, an AATAAA motif is present just upstream of the p10 translational stop codon and this motif is followed by a GT-rich sequence. It is not known whether this motif is used for polyadenylation. In Buzura suppressaria (Busu) NPV, which is a single nucleocapsid (S) NPV, the p10 gene is not followed by p74. Despite the absence of sequence similarity in this region with the NPVs listed above, two putative polyadenylation signals are present (van Oers et al., 1998 ). The first is an ATTAAA motif at the stop codon, which would result in practically no 3' UTR and for which the spacing from a GT-rich element seems to be too small. However, BusuNPV p10 has another ATTAAA motif further downstream, which is followed by a GT-rich domain. In general, all p10 genes have at least one polyadenylation motif, either AATAAA or ATTAAA, in combination with a downstream GT-rich element. The conservation of these elements may indicate that ATTAAA sequences can indeed be used as alternative polyadenylation signals in lepidopteran species and that GT-rich elements are important for efficient polyadenylation of baculovirus genes.

In the AcCAT{Delta}poly(A)2 mutant used in this study, the p74 gene had to be slightly modified, resulting in an amino acid change at position 695 from leucine to threonine (Kuzio et al., 1989 ). Since the CAT expression levels of AcCAT and AcCAT{Delta}poly(A)2 were similar (Fig. 3), we can conclude that this p74 mutation has no negative effect on p10 gene expression in cultured insect cells.

When expressing foreign genes in the baculovirus expression system, heterologous 3' UTRs are often used. However, this might influence the level of foreign protein synthesis. The effect of the heterologous SV40 3' UTR was examined, since this terminator sequence is present in several baculovirus expression vectors. The SV40 early terminator has been tested previously in the opposite orientation in transient expression studies for polyhedrin promoter-driven expression (Westwood et al., 1993 ). It was concluded that the SV40 early polyadenylation signal was used less efficiently than a sequence based on the rabbit ß-globin polyadenylation signal. In the study presented here, the SV40 terminator was tested in the same gene context as in the p10 promoter-based transfer vector pAcAS3 (Vlak et al., 1990 ), where it is part of an hsp70–lacZ–SV40 gene cassette enabling visual selection of recombinant viral plaques. The same gene cassette is also present in the polyhedrin promoter-based vector described by Zuidema et al. (1990) . The data presented in this paper for p10 promoter-based gene expression show that the use of the hsp70–lacZ–SV40 gene cassette results in lower mRNA levels and reduced reporter gene activity (Fig. 5). Various explanations might be given for the observed reduction in mRNA levels in infections with AcCAT-SV40-lacZ, such as inefficient polyadenylation within the SV40 terminator, instability of mRNAs with a heterologous 3' UTR or antisense effects generated by transcripts originating from the hsp70 promoter. To be able to discriminate between these possibilities, the SV40 terminator was tested in the absence of lacZ and hsp70 sequences. In this case, the majority of the transcripts used the original p10 polyadenylation signal at position +163 in the 3' UTR and only a minor fraction had a size corresponding to polyadenylation at the SV40 terminator. This result showed that polyadenylation within the SV40 terminator is very inefficient compared with polyadenylation at the p10 signal at +163, at least in the orientation tested here. The absence of a GT-rich element downstream of the polyadenylation motif in the SV40 sequence is probably responsible for the inefficient polyadenylation, in line with the results described above for the polyadenylation motif at +65 in the p10 3' UTR.

In recombinant AcCAT-SV40, the polyadenylation signal at position +163 in the p10 3' UTR is apparently located close enough to allow efficient polyadenylation of p10 transcripts. In AcCAT-SV40-lacZ, where the spacing between the SV40 and the p10 polyadenylation signal is much larger, only a minor fraction of transcripts appears to be long enough to pass the lacZ and hsp70 sequences and allow polyadenylation at the original p10 signal (Fig. 5B, lane 2, indicated by `3''). However, premature terminations are likely to occur within the gene cassette and may result in non-polyadenylated transcripts of various lengths.

The collective results of this paper show that vectors containing the wild-type p10 3' UTR are to be preferred over vectors containing the hsp70–lacZ–SV40 selection cassette. These data may also have major implications for the use of other heterologous polyadenylation signals in insect cells. Based on all the knowledge gathered thus far, p10-recombinant viruses should preferentially be made by transferring only the open reading frame of the foreign gene, leaving both the 5' and 3' UTRs of the p10 gene intact. In order to achieve this and to be able to select p10-recombinant viruses, the use of the `blue' parental virus AcAS3 is recommended, as performed in this study.


   Acknowledgments
 
The research described in this paper was supported by the Life Science Foundation (SLW) with financial aid from the Netherlands Organization for Scientific Research (NWO). Gert Scheper is acknowledged for providing several plasmid constructs and for helpful discussions. Magda Usmany and Els Klinge-Roode are acknowledged for technical assistance in cell culture.


   References
Top
Abstract
Introduction
Methods
Results
Discussion
References
 
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Received 18 February 1999; accepted 26 April 1999.