Boyce Thompson Institute at Cornell University, Tower Road, Ithaca, NY 14853-1801, USA1
Author for correspondence: Gary Blissard. Fax +1 607 254 1366. e-mail gwb1{at}cornell.edu
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Although the lef-11 open reading frame (ORF) was identified by sequence analysis and its expression was implied by complementation experiments in transient late transcription assays, the lef-11 gene transcripts were not previously mapped nor was the presence of the protein documented or localized within infected cells. The lef-11 ORF is somewhat unusual in that it is small and overlaps both the orf38 and the pp31 ORFs. Thus, if expressed as a discrete unit of transcription, the lef-11 promoter may be located within the upstream orf38 gene. In the current study, we examined lef-11 transcription and expression during the infection cycle. To study the expression of the lef-11 gene in the context of an AcMNPV infection, we used 5' and 3' RACE analysis as well as Northern blot analysis to examine RNAs transcribed from the region containing the lef-11 gene.
To map the 5' ends of RNAs transcribed from the lef-11 ORF, two gene-specific primers were used for nested PCR to amplify and clone the 5' ends of the lef-11 transcripts by 5' RACE (Fig. 1A, B
). Using total RNA isolated from AcMNPV-infected Sf9 cells at 12 h post-infection (p.i.) and lef-11-specific primers, two products were detected after 5' RACE analysis: an abundant product of approximately 200 bp and a less abundant product of approximately 520 bp. Cloning and sequencing of the 200 bp 5' RACE product revealed the 5' end of an early transcript that initiates within the lef-11 ORF and corresponds to a previously mapped early transcript encoding the downstream pp31 ORF. The pp31 ORF overlaps the 3' end of the lef-11 ORF by 4 bp (Figs 1A
and 2
). Sequence analysis of the 520 bp 5' RACE product revealed a single transcription initiation site located 196 nt upstream of the lef-11 ORF and 25 nt downstream of a putative TATA box (Figs 1A
, B
and 2
) at nt 30597 on the AcMNPV C6 genome sequence (Ayres et al., 1994
). This transcription start site is within the orf38 ORF and, thus, the lef-11 promoter is probably within the orf38 ORF. Interestingly, a Met codon is found on the relatively long (196 nt) 5' leader sequence of this lef-11 RNA and this Met codon is 139 nt upstream of the lef-11 ORF and in the same frame as the orf38 ORF (Fig. 2
, underlined sequence). Translation initiation from this upstream Met codon would result in the translation of a 174 nt ORF encoding 58 amino acids. This small upstream ORF does not terminate prior to the initiation of the lef-11 ORF, but overlaps it by 35 nt and is in a different reading frame. We resequenced this region and confirmed that the nucleotide sequence of this region of the AcMNPV genome (GenBank accession no. L22858.1 GI:510708) was correct. If initiation occurs at the upstream Met codon, it is possible that this might result in a substantial downregulation of LEF-11 translation. Minicistrons (very small ORFs) in the 5' leader sequences of several baculovirus genes have been reported previously (Blissard & Rohrmann, 1989
; Guarino & Smith, 1990
; Lu & Carstens, 1992
) and translation of a minicistron was shown to downregulate translation of the downstream ORF on gp64 late mRNAs (Chang & Blissard, 1997
). Because previous studies using transient late transcription assays demonstrated that the lef-11 ORF was functional (Rapp et al., 1998
), it is likely that the lef-11 ORF (nt 3040130063) is discretely translated. Initiation at the lef-11 Met codon could occur via a leaky-scanning mechanism in which the upstream Met codon is not used, or is inefficiently used, as a translation initiator.
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To examine the temporal nature of lef-11 transcription, we used Northern blot analysis. A 278 nt strand-specific cRNA probe was labelled with [33P]ATP (Fig. 1A) and hybridized to Northern blots containing total RNA isolated from AcMNPV-infected Sf9 cells at various times p.i. (Fig. 1D
). Four RNAs were detected by hybridization with the lef-11 probe (Fig. 1D
). Two RNAs of 1·5 and 1·1 kb correspond to mapped transcripts from the lef-11 and the pp31 genes, respectively. Two additional bands observed on these blots appear to be artefacts, as they were not identified by either 5' or 3' RACE analysis of this region. Attempts to use a shorter cRNA probe specific for the lef-11 gene alone were unsuccessful, probably due to limitations on the size of these probes and the relatively low abundance of the lef-11 transcripts. Because the lef-11 ORF overlaps the pp31 ORF and the transcription start site of the pp31 gene is within the lef-11 ORF, the cRNA probe used in this study (Fig. 1A
, Northern Probe) detected both the lef-11 and the pp31 transcripts. Mapping the 5' and 3' ends of the lef-11 RNA by RACE analysis identified a 1·376 kb RNA species, consistent with the identification of a 1·5 kb RNA by Northern blot analysis using a lef-11 cRNA probe (Fig. 1D
). The 1·5 kb lef-11 mRNA was detected as early as 4 h p.i. and was most abundant between 12 and 24 h p.i. The steady-state levels of the 1·5 kb lef-11 mRNA appear to be much lower than those of the 1·1 kb pp31 mRNA, since the 278 nt probe was completely homologous to the 1·5 kb mRNA, yet the intensity of the 1·5 kb band was dramatically lower than that of the 1·1 kb band. Only 115 nt (nt 3008430198) of the 278 nt probe were homologous to the 1·1 kb pp31 mRNA (Fig. 1A
, D
). The 1·1 kb pp31 RNA was the most abundant transcript detected. The size and quantity of this transcript are consistent with 5' and 3' end mapping of the pp31 gene in this and previous studies (Guarino & Smith, 1990
, 1992
). The 1·1 kb pp31 mRNA was detected from 4 to 72 h p.i. and was most abundant between 24 and 36 h p.i. (Fig. 1D
). Detection of the pp31 transcripts during the late phase was expected, since a pp31 late promoter characterized previously is located immediately upstream of the pp31 gene early transcription start site (Guarino & Smith, 1992
).
The lef-11 ORF encodes a predicted protein of 112 aa with a molecular mass of approximately 13·1 kDa. The predicted LEF-11 protein contains a single putative zinc finger motif (CysX3CysX9PheX6LeuX1HisX9His) near the N terminus and a basic-charged region near the C terminus. To examine LEF-11 expression in infected cells, an anti-LEF-11 antiserum against a six His-tagged LEF-11 protein expressed in Escherichia coli was generated in rabbits. Anti-LEF-11 antibodies were affinity purified and used for Western blot analysis of extracts from AcMNPV-infected Sf9 cells. Using anti-LEF-11 antibodies, a protein with a molecular mass of approximately 16 kDa was detected in AcMNPV-infected Sf9 cells from 4 to 72 h p.i. (Fig. 3A). Maximal levels of LEF-11 were detected at 12 h p.i., which coincides with times at which maximal levels of the lef-11 RNAs were detected (Fig. 1D
). Although lef-11 mRNA levels declined and were not readily detectable after 36 h p.i., LEF-11 was detected up to 72 h p.i., suggesting that LEF-11 may be relatively stable during the late phase of infection. Although the LEF-11 band appears to be lower in intensity at 36 h p.i., it is not clear how this can be explained. The low intensity of the LEF-11 band at 36 h p.i. may represent either low levels of LEF-11 or perhaps an experimental artefact. To examine the cellular localization of LEF-11, we also used affinity purified anti-LEF-11 antibodies for immunofluorescence microscopy of AcMNPV-infected Sf9 cells (Fig. 3B
). Anti-LEF-11 antibodies were prepared by preadsorption with Sf9 cell extracts and then affinity purified by binding to and eluting from purified LEF-11 protein (Monsma & Wolfner, 1988
). At 18 h p.i., LEF-11 was detected within large and dense nuclear regions, commonly observed in AcMNPV-infected Sf9 cells. A low intensity fluorescence signal in infected cells appeared to localize to the expanded nuclei of infected cells (Fig. 3B
).
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Acknowledgments |
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References |
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Received 2 February 2001;
accepted 4 June 2001.