Department of Microbiology, Oregon State University, Corvallis, OR 97331-3804, USA1
Author for correspondence: George F. Rohrmann. Fax +1 541 737 0496. e-mail rohrmanng{at}orst.edu
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Abstract |
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Introduction |
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Baculoviruses have evolved at least two categories of envelope-associated proteins. These include the GP64 (Blissard & Rohrmann, 1989 ) and the LD130 groups (Pearson et al., 2000
), which are both low-pH-induced envelope fusion proteins that appear to be critical for the spread of the infection within host insects. Whereas members of the LD130 group lack homologues of gp64, the gp64-containing viruses encode a homologue of ld130. No fusion function has been demonstrated for the ld130 homologues encoded by gp64-containing viruses (Pearson et al., 2001
). NPVs that encode GP64 appear to be found only in the Group I category of baculoviruses, whereas those employing LD130 homologues as their envelope fusion protein are in Group II (Pearson et al., 2000
).
A feature of all ld130 and gp64 genes found in NPVs is the presence of both early and late promoter elements (Fig. 1). Whereas single RNA polymerase II promoter elements are evident in both AcMNPV and OpMNPV gp64 genes, they both have four late promoter elements upstream of their translational initiation codon (Fig. 1A
). In addition, one to two late promoter elements along with a single TATAA/CAGT motif are found upstream of the translation initiation codons in the ld130 homologues. In AcMNPV and OpMNPV gp64 genes, the distal two late promoters appear to account for most of the late expression from these genes (Blissard & Rohrmann, 1989
; Garrity et al., 1997
) and elimination of both these elements appears to abolish late gene expression. Early gp64 transcription involves single copies of both TATAA and CAGT mRNA start site elements and elimination of either of these motifs causes a marked reduction in gene expression (Blissard et al., 1992
).
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Methods |
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RNA preparation and Northern blot analysis.
Ld652Y cells were infected with LdMNPV or OpMNPV at an m.o.i. of 10. After 1 h of incubation with the virus, the medium was removed and replaced with fresh medium. This point was defined as time zero. Cells were collected at various times thereafter, and total cellular RNA was isolated using TRIzol reagent (Life Technologies) according to the manufacturers instructions.
Riboprobes to ld130 and op21 were synthesized using [-32P]ATP andT7 RNA polymerase (Fermentas) and T3 RNA polymerase (Promega), respectively, according to the manufacturers instructions. pLd130HA was derived from pLd130FL (Pearson et al., 2000
) by inserting a 40 nt sequence encoding a nine amino acid HA epitope in-frame into the NcoI site (nt 128239) located downstream of the transmembrane domain. The HA insert, which contains a stop codon at its 3' end, abolished the NcoI site. pLd130HA was linearized with NcoI, and cRNA to ld130 was generated using T7 RNA polymerase. In order to synthesize cRNA to op21, the entire op21 gene was cloned into pKSie1 (Pearson et al., 2001
). The resulting clone was linearized with HindIII and cRNA was synthesized using T3 RNA polymerase.
Northern blot analysis was carried out as described by Ahrens et al. (1995) with several modifications. Briefly, total RNA from infected cells was electrophoresed on a 1% agarose2·2% formaldehyde gel in 20 mM MOPS buffer and transferred to a Gene Screen Plus membrane. The RNA was cross-linked to the membrane using a UV cross-linker (UV Stratalinker 1800, Stratagene) and the membrane was then baked for 2 h at 80 °C. Prehybridization of the membrane was carried out for at least 2 h at 65 °C in 50% formamide, 5xSSC, 0·1% SDS, 50 mM sodium phosphate, pH 6·8, 0·1% sodium pyrophosphate/5xDenhardts solution, 50 µg/ml salmon sperm DNA (McCaughern-Carucci, 1997
) and hybridization was carried out overnight at 65 °C in the same buffer. Membranes were washed as described by Ahrens et al. (1995)
, followed by a 1 h wash in 0·1xSSC, 0·1% SDS at 65 °C.
5' and 3' rapid amplification of cDNA ends (RACE) and transcriptional mapping.
5' and 3' RACE were carried out using a GeneRacer Kit (Invitrogen) following the manufacturers instructions. PCR on the cDNA was performed using Taq DNA polymerase (Promega) with the GeneRacer 5' and 5' nested primers and internal gene specific primers (GSP). For ld130 the GSPs were BAE4 (5' AACTTGGCGATGTTCACCAG 3') and BAE6 (5' CGATACAGCTCGTCGAGCTC 3') and for op21 they were HdH-F1 (5' GTGCGGCAGCACTCTGACGCGG 3') and HdH-F2 (5' CTGACCAGCTTCACCAGCGG 3'). For 3' RACE the GeneRacer 3' primer was used with GSP B201 (5' CTGCTGCACTACGAGAACTC 3') for ld130 and GSP HdI-10 (5' CACGTGAACACCTCGCTG 3') for op21. The PCR products were cloned into pCR2.1-TOPO vectors (Invitrogen), following the manufacturers instructions. Selected clones were purified (Qiagen) and sequenced as previously described (Kuzio et al., 1999 ).
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Results |
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Identification of a spliced transcript
A 503 bp cDNA band that was less intense than the major op21 cDNA band was observed at 48 and 72 h p.i. (Fig. 3D, lanes 3, 4) One clone each derived from this band at 48 and 72 h p.i. were sequenced and found to be derived from spliced transcripts which initiated at a late promoter element (nt 13795) upstream of OpMNPV orf18. Another spliced transcript was identified in a 36 h p.i. clone which mapped to a late promoter element (nt 14044) within orf18. All these transcripts were spliced just downstream of the orf18 TAG termination codon to a sequence within op21 (Fig. 3C
, Fig. 4E
). This results in an intron with the following coordinates: 14164 GT...AG 17005. The nucleotides, GT...AG, are invariant at the termini of introns involved in nuclear splicing (Lewin, 1997
). Since these RNAs are spliced immediately downstream of the termination codon, they probably would not encode a spliced protein but would only code for orf18 or a small orf within orf18. The two longer spliced transcripts would be about 2300 nt long whereas the other would be somewhat shorter. However, they all would fall within the size range for the major band observed on the Northern blot (Fig. 2B
, lanes 710). Whether these spliced transcripts are an artifact of baculovirus late transcription produced by the presence of the nuclear splicing machinery or actually serve a function remains to be determined.
Transcription termination sites
The transcript stop points for the mRNAs of both ld130 and op21 mapped close to the termination codon of the orf. Two ld130 cDNA clones from the 72 h p.i. timepoint were sequenced and found to terminate 68 nt downstream of the termination codon (Fig. 4B). This site is about 25 nt downstream of a possible 3' processing signal (AATTAAA) in a very AT-rich region. This is somewhat different from conventional 3' regions of eukaryotic genes and also lacks the run of T residues that have been implicated in termination by the baculovirus late RNA polymerase (Jin & Guarino, 2000
). For op21, 24 and 72 h p.i. clones were sequenced and found to terminate 111112 nt downstream of the stop codon (Fig. 4D
). This site was located 12 nt downstream of a consensus 3' processing signal AATAAA and also just downstream of a GTTG sequence.
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Discussion |
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Although early and late promoters were used by both ld130 and op21, they show a different pattern of expression. The relative differences in the 5' mRNA start sites that are used are likely reflected by the 5' RACE amplified cDNA populations because for each population of mRNAs the same primers were employed and the early and late templates were identical except that the late mRNAs were somewhat longer at the 5' end. For ld130, our analyses suggested that there is a shift from almost exclusive use of the early promoter at 24 h p.i. to the exclusive use of the late promoter element later in infection. The kinetics of late ld130 expression are similar to late expression from the LdMNPV 25k fp gene (Bischoff & Slavicek, 1996 ) which also showed maximal levels of late mRNA at 48 and 72 h p.i. In contrast, op21 appeared to be expressed predominantly from the early promoter at 24 through 72 h p.i. Expression from the op21 late promoter was observed (Fig. 3
), but appears to be at a low level. Another cDNA species present at later times was analysed and found to be a spliced transcript that initiates in the orf18 region and is fused to a sequence within op21. Although a scanning model of late transcription initiation has been proposed, evidence suggests that it is not used with AcMNPV gp64 late promoter elements (Garrity et al., 1997
). Therefore, the op21 late promoter element may be in a poor context resulting in low levels of expression, rather than being affected by expression from upstream late promoters. An expression pattern similar to op21 has been observed for the OpMNPV ie-1 gene. It appears to be activated at late times of infection similar to op21 and the late transcripts map to an early transcriptional start site (Theilmann & Stewart, 1991
).
The two envelope fusion protein genes, gp64 and ld130, appear to have similar patterns of gene expression and employ both early and late promoters. Two theories have been advanced to explain the requirement for this pattern of expression for gp64. In one, it has been suggested that the early expression of this protein allows time for its glycosylation, transport and integration into the cell membrane in preparation for the budding of the newly assembled nucleocapsids later in infection (Blissard & Rohrmann, 1989 ). Late expression would sustain this production and allow for replenishing the membrane as the envelope proteins are depleted by nucleocapsid budding. Another theory suggests that early gp64 expression prepares cell membranes for virus budding, thereby permitting some nucleocapsids from the primary infection to travel through gut cells without undergoing replication. Such unreplicated virions might bud directly out through the basal membrane into other tissues, thereby facilitating the early establishment of a systemic infection and avoiding host defence mechanisms such as sloughing of the gut lining (Volkman, 1997
). In this instance, late expression of gp64 would be coordinated with the late expression of the nucleocapsid proteins. Clearly, in both these cases, the early expression of envelope fusion proteins genes could contribute to the acceleration of the establishment of an infection. Whatever the rationale for the expression strategy of gp64, it may be of considerable importance to the success of the virus, as this pattern of expression appears to have evolved independently for both the gp64 and ld130 groups of genes that encode envelope fusion proteins.
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Acknowledgments |
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References |
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Received 27 September 2001;
accepted 11 December 2001.