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
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Abstract |
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Introduction |
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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.
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Methods |
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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 AcCATpoly(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 TrisHCl, pH 7·6, by three rounds of freezethawing. 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.
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 NcoIBamHI 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 AcCATpoly(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
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.
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Results |
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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
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 24), which has a wild-type 3' UTR, gave the same level of CAT expression as infection with mutant AcCAT
poly(A)2 (lanes 57) at all time-points tested.
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The poly(A)-addition sites in the p10 transcripts of the recombinant viruses AcCAT and AcCATpoly(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
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
poly(A)2 revealed that the p10 transcripts were polyadenylated several nucleotides further downstream [Fig. 4B
; indicated by `cDNAI
poly(A)2' and `cDNAII
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 promoterlacZ 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 6080%. 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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Discussion |
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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 2238 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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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
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 AcCATpoly(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
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 hsp70lacZSV40 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 hsp70lacZSV40 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 hsp70lacZSV40 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.
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Acknowledgments |
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References |
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Received 18 February 1999;
accepted 26 April 1999.