JOURNAL OF VIROLOGY, Feb. 1992, p. 655-663

Vol. 66, No. 2

0022-538X/92/020655-09$02.00/0 Copyright © 1992, American Society for Microbiology

Transcription Analysis of the EcoRI D Region of the Baculovirus Autographa californica Nuclear Polyhedrosis Virus Identifies an Early 4-Kilobase RNA Encoding the Essential p143 Gene ALBERT LU AND ERIC B. CARSTENS* Department of Microbiology and Immunology, Queen's University, Kingston, Ontario K7L 3N6, Canada

Received 26 August 1991/Accepted 24 October 1991

We have investigated the transcriptional activity of the 60.1- to 68.3-map-unit region of the baculovirus Autographa californica nuclear polyhedrosis virus (AcMNPV). Twelve transcripts mapping to this region were expressed at various times during infection. An early 4.0-kb transcript, potentially coding for a 143-kDa peptide essential for viral DNA replication, was maximally abundant at 6 h postinfection (p.i.). Transcripts of 0.5, 1.1, 1.4, 2.1, and 3.1 kb were most abundant at 12 h p.i., while two large transcripts of 5.2 and 6.8 kb were expressed maximally at 24 h p.i. In the presence of cycloheximide, and in ts8-infected cells at the nonpermissive temperature, only the 4.0-kb RNA was expressed. Northern (RNA) blot analysis using DNA subfragments from the EcoRI D fragment as probes suggested that many of the transcripts overlapped. Strand-specific cRNA probes revealed that the majority of the RNAs were transcribed in the counterclockwise direction. Si nuclease and primer extension analysis were used to map the 5' ends of transcripts coded within the 60.1- to 64.8-map-unit region. Mapping of the 3' ends of the 1.1-, 4.0-, 5.2-, and 6.8-kb transcripts suggested that these RNAs were all coterminal at their 3' ends. A minicistron was found between the early 4.0-kb transcription start site and the predicted ATG start codon of the p143 gene. Several similar sequence motifs were identified in the promoter regions of the p143 gene and the AcMNPV DNA polymerase gene. appears that the regulation of viral DNA replication may also play an important role in regulating late events of the virus infectious cycle. AcMNPV DNA replication begins at 6 to 8 h postinfection (h p.i.) (11) and is possibly mediated by a virus-encoded DNA polymerase (32). The genome location of this polymerase has been identified and sequenced (41). We have identified another gene in AcMNPV, p143, which is essential to viral DNA synthesis (26). The predicted amino acid sequence of p143 contains regions which exhibit amino acid sequence similarities to a group of replicative proteins with putative helicase activities, and therefore p143 may function to unwind duplex DNA at the replication fork and/or at the origin of replication. This gene was identified by sequence analysis of the 60.1- to 65.5-map-unit (m.u.) region of AcMNPV and by marker rescue of the ts8 mutation. In this study, we have investigated the transcriptional organization in the region of the p143 gene. Twelve transcripts were identified in the 60.1- to 68.3-m.u. region (EcoRI-D). Some of these transcripts were mapped to open reading frames derived from the primary DNA sequence (26). S1 mapping and primer extension analysis were used to identify an early 4.0-kb transcript encoding the p143 gene. Analysis of the promoter region of the p143 gene revealed sequence similarities with the AcMNPV DNA polymerase

Autographa californica nuclear polyhedrosis virus (AcMNPV), a member of the family Baculoviridae, contains a double-stranded, covalently closed, circular DNA genome of 128 kb. The developmental program of gene expression in AcMNPV involves the timed expression of four groups of genes; immediate early, delayed early, late, and very late. The expression of these genes is coordinately regulated and sequentially ordered in a cascade fashion (for reviews, see references 10, 13, and 15). The transition from early to late gene expression requires the products of early genes (13). Although the functions of these early proteins are for the most part unknown, studies on four early genes, IE-1 (19), IE-N (4), ETL (7), and PE-38 (24), have indicated that the gene products function in regulating expression of later classes of genes. Late and very late genes are very likely regulated by other viral proteins as well (21, 36). Transcriptional mapping of the viral genome has revealed that overlapping families of transcripts with coterminal 5' or 3' ends are a common occurrence in baculoviruses (14, 15, 20, 28, 29, 35, 37). RNA splicing has been reported only for an immediate-early gene of AcMNPV (6); consequently, promoter occlusion (14) and antisense mRNA inhibition of translation (15, 21) have been proposed as alternate mechanisms of baculovirus transcriptional regulation. The process of DNA replication separates the early and late temporal classes of genes. DNA replication has been weakly implicated in the regulation of late gene expression from observations that the level of late gene products is reduced when viral DNA replication is inhibited by cytosine arabinoside (9) or aphidicolin (38). A temperature-sensitive mutant of AcMNPV, ts8, is unable to replicate its DNA at 33°C. This mutant is also unable to synthesize late proteins and to shut off host protein synthesis (17). Therefore, it *

gene

promoter.

MATERIALS AND METHODS

Cells and virus. Spodopterafrugiperda cells were grown in monolayer cultures of Graces cell culture medium (GIBCO Laboratories) supplemented with 10% heat-inactivated fetal calf serum (Flow Laboratories). AcMNPV was propagated and purified as previously described (26). In all experiments, time

Corresponding author. 655

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added to the cells, corresponding to the beginning of the adsorption period. Preparation of single-strand-specific RNA probes. The 1.28-kb SstI-I fragment from the AcMNPV EcoRI D fragment was cloned into the SstI site of pUC18 by standard methodology (30), giving rise to the chimeric plasmid pAcSstI-I. The EcoRI D fragment, a KpnI-EcoRI 1.75-kb fragment, and a 619-bp EcoRI-HindIII fragment from pAcSstI-I were subcloned into the pGEM3 plasmid vector (Promega Biotec Co.), resulting in the constructs pGEMEco-D, pGEMKE-1.75, and pAcSstl-I/619a, respectively. Uncapped radiolabelled mRNA was prepared in vitro by using either T7 or SP6 RNA polymerase (Promega Biotec) to generate runoff transcripts from these linearized templates. The reactions were carried out according to the manufacturer's protocols. The resulting radiolabelled RNAs were used directly as probes on Northern (RNA) blots. RNA isolation and Northern blot analysis. Total intracellular RNA was isolated by extraction with guanidinium isothiocyanate followed by centrifugation through a CsCl cushion (5). In experiments in which cycloheximide was used, drug (100 Fig/ml) was added to the cells 1 h prior to infection and maintained at that concentration for the duration of the experiment. RNA for Northern blot analysis was denatured by glyoxalation for 1 h at 50°C (31). Different size classes of RNA were separated by electrophoresis through agarose gels in 10 mM Na2HPO4 (pH 7.0), transferred to nitrocellulose filters (Schleicher & Schuell), and hybridized with either radioactive DNA or strand-specific RNA probes. Poly(A)+ RNA was selected by oligodeoxythymidylate-cellulose chromatography (2). Si nuclease mapping. DNA probes for Si nuclease mapping were prepared by 5' end labelling with T4 polynucleotide kinase and [^y-32P]ATP (>3,000 Ci/mmol; Dupont NEN) or 3' end labelling with the Klenow fragment of DNA polymerase (GIBCO-BRL) and [a-32P]dATP (>800 Ci/ mmol; Dupont NEN) (30). Fifty micrograms of total infected intracellular RNA and 2.5 x 104 Cerenkov cpm of uniquely end-labelled probe were hybridized and treated with S1 nuclease (8). The Si nuclease-resistant fragments were sized by electrophoresis through 6% denaturing sequencing gels. Primer extension analysis. The locations of the 5' ends of specific mRNAs were also identified by a modified primer extension protocol as described previously (1). Labelled oligonucleotides (0.15 pmol) were annealed with total infected cell RNA (30 ,ug) and extended with 20 U of reverse transcriptase. Extension products were sized on 6% denaturing sequencing gels, using an M13 sequencing ladder as size markers.

RESULTS Identification of transcripts by Northern analysis. To study the temporal expression of AcMNPV transcripts, total intracellular RNA isolated from infected S. frugiperda cells was analyzed by Northern blot electrophoresis. Probing these blots with the 10.4-kb EcoRI D fragment (60.1 to 68.3 m.u.) identified seven major transcripts mapping to this region throughout the course of infection (Fig. 1). The earliestappearing transcript was a 4.0-kb RNA detectable at 3 h p.i. This 4.0-kb transcript was maximally abundant at early times and decreased in levels by 24 h p.i. The transcript was expressed in the presence of cycloheximide and was also the only RNA observed from this region in ts8-infected cells at the nonpermissive temperature (33°C), suggesting the 4.0-kb

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0.5FIG. 1. Temporal expression of transcripts in the 60.1- to 68.3m.u. region of AcMNPV. Total RNA (30 ,ug) isolated from AcMNPV-infected cells (wt) was glyoxalated, fractionated on 1.4% agarose gels, transferred to nitrocellulose, and hybridized to 32p_ labelled EcoRI-D DNA. RNA was also isolated from AcMNPVinfected cells in the presence of cycloheximide (wt + CX) or from ts8-infected cells (ts8) at the nonpermissive temperature at the times after infection indicated (hpi). ts8 is a DNA replication-defective mutant of AcMNPV (17). The approximate sizes of transcripts detected by the probe are presented on the left.

RNA belonged to the early class of baculovirus transcripts. Increased amounts of the 4.0-kb RNA in these cells at 12 and 24 h p.i. also suggested that this RNA may normally be regulated by virus-specific proteins expressed later during infection. Other transcripts mapping to the 60.1- to 68.3-m.u. region included an abundantly synthesized transcript of 0.5 kb previously identified to encode a basic arginine-rich DNAbinding protein (42) as well as transcripts of 1.1, 2.1, 3.1, 5.2, and 6.8 kb. Similar to the 0.5-kb RNA, the 1.1-, 2.1-, and 3.1-kb RNAs were first observed at 9 h p.i. but were most abundant at 12 h p.i. By 24 h p.i., these transcripts were still detectable. The 5.2- and 6.8-kb RNAs were initially detected at 12 h p.i. but were maximally expressed by 24 h p.i. The initial appearance of these transcripts in infected cells indicated that all temporal classes of RNA, i.e., an early 4.0-kb RNA, late 0.5-, 1.1-, 2.1-, and 3.1-kb RNAs, and very late 5.2- and 6.8-kb RNAs, were expressed by this region of the AcMNPV genome. Transcriptional map of the EcoRI D fragment. The approximate locations of transcripts were mapped by using restriction fragments within the EcoRI D fragment as probes (Fig. 2). Probes 2a to 2g represent small restriction fragments proceeding progressively from 60.1 to 68.3 m.u. (see Fig. 8 for the exact identities and locations of these probes). Probe 2a and adjacent probe 2b detected RNAs of 1.1, 4.0, 5.2, and 6.8. Probes 2c and 2d hybridized to transcripts of 4.0, 5.2, and 6.8 kb. Probe 2e hybridized to the 0.5-, 1.1-, 2.1-, 3.1-, 5.2-, and 6.8-kb transcripts. Transcripts of 1.1 kb were detected by small probes (2b and 2e) separated by a distance of 3 kb. The 1.1-kb transcripts identified with each probe had different temporal expression patterns, indicating that these probes were identifying two different transcripts of similar size. Probe 2f hybridized to 2.1-, 3.1-, and 5.2-kb RNAs, while probe 2g hybridized to a 2.1-kb and a 6.8-kb transcript. To determine whether the transcripts hybridizing to the EcoRI D fragment overlapped adjacent regions, the EcoRI W (59.7 to 60.1 m.u.) and the EcoRI Q (68.3 to 69.5 m.u.) fragments were also used as probes on similar Northern

MAPPING OF AcMNPV TRANSCRIPTS

VOL. 66, 1992

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FIG. 2. Mapping of the transcripts in the 60.1- to 68.3-m.u. region. Northern blots of total RNA isolated from AcMNPV-infected cells at 3 (lane 0), 6 (lane 1), 9 (lane 2), 12 (lane 3), and 24 (lane 4) h p.i. were hybridized to DNA probes representing the whole EcoRI D fragment as well as subfragments from the region. Identities of the 32P-labelled probes used: (a) EcoRI-ScaI (nt 0 to 1395); (b) ScaI-ScaI (nt 1395 to 1695); (c) ScaI-HindIII (nt 1695 to 2754); (d) HindIlI-SalI (nt 3532 to 4417); (e) SalI-KpnI (nt 4794 to 6476); (f) KpnI-KpnI (nt 6845 to 8876); (g) HindIII-EcoRI (nt 10679 to 11120). The sizes (kilobases) of the transcripts hybridizing with each probe are shown on the right. The nucleotide numbers were estimated from sequence data (26) and restriction fragment mapping.

blots. The EcoRI W fragment did not hybridize with any detectable transcripts, whereas the EcoRI Q fragment detected 2.1- and 4.7-kb RNAs (12, 27). Therefore, it appears that the majority of transcripts detected by the EcoRI D fragment mapped entirely within the 60.1- to 68.3-m.u. region. Since some of the probes seemed to hybridize nonspecifically to the blots in the region of 2.1 kb, Northern blots of poly(A)-selected RNA were hybridized with probe 2e and probe 2f to determine whether the hybridization represented a 2.1-kb virus-specific RNA or nonspecific hybridization to cellular rRNA. Probe 2e (Fig. 3A) and probe 2f (Fig. 3B) detected similar transcripts in both total and poly(A)-selected RNAs, indicating that the 2.1-kb transcript was virus specific. In addition, a 1.4-kb transcript not observed in total RNA expressed at late times postinfection was detected with probes 2e and 2f in poly(A)-selected RNA. The 5.2- and 6.8-kb RNAs seen in total RNA were not seen in poly(A)+ RNA. Determination of the direction of transcription. Strandspecific RNA probes were used to determine the direction of transcription within the EcoRI-D region (Fig. 4). Transcripts of 0.5, 1.1, 2.1, 3.1, 4.0, 5.2, and 6.8 kb hybridized to an EcoRI-D cRNA probe generated in the clockwise direction (probe 4a), while no abundant transcripts were detected by the cRNA probe transcribed in the counterclockwise direction (probe 4b). A cRNA probe of the HindIII-SstI 623-bp fragment (62.0 to 62.6 m.u.) transcribed in the clockwise

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FIG. 3. Northern blot analysis of total and poly(A)-selected RNAs. Poly(A) RNA was used to determine whether the 2.1-kb transcripts detected by the SalI-KpnI fragment (nt 4794 to 6476) and the KpnI-KpnI fragment (nt 6845 to 8876) were virus-specific and not nonspecific hybridizations to rRNA. Total and poly(A)-seiected RNAs from 12 (lane 1) and 24 (lane 2) h p.i. were probed with 32P-labelled DNA probes representing the SalI-KpnI fragment (A) or the KpnI-KpnI fragment (B). Sizes (kilobases) of transcripts detected in total RNA samples are shown at the left of each panel; positions of additional transcripts detected in poly(A) RNA samples are indicated at the right.

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LU AND CARSTENS

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FIG. 4. Determination of the direction of transcription. Northern blots of total infected cell RNA harvested at 6, 9, 12, and 24 h p.i. were probed with 32P-labelled strand-specific RNA probes generated from the whole EcoRI D fragment (a and b), a 623-bp HindIII-SstI fragment (c and d), and a 1,650-bp KpnI-EcoRI fragment (e and f). Sizes (kilobases) of transcripts hybridizing to probes detecting RNA transcribed in a counterclockwise direction (a, c, and e) with respect to the conventional map are indicated at the left of the panels; sizes of RNAs hybridizing to probes detecting clockwise transcription (b, d, and f) are indicated at the right.

direction hybridized to the 4.0-, 5.2-, and 6.8-kb RNAs (probe 4c). Probe 4d weakly hybridized to RNAs of 2.1 and 5.2 kb transcribed in the opposite direction. Using cRNA probes derived from a KpnI-EcoRI 1.75-kb fragment at the right end of the EcoRI D fragment, we observed counterclockwise transcripts of 3.1, 4.7, and 6.8 kb (probe 4e) and a clockwise 2.1-kb RNA (probe 4f). These results indicated that the predominant direction of transcription within the 60.1- to 68.3-m.u. region was in the counterclockwise direction on the standard AcMNPV genome map. A 0.5-kb transcript has been previously shown to be transcribed in this direction (42). Mapping of the 4.0-kb RNA start site and 3' end. The early 4.0-kb RNA potentially overlapped the p143 gene open reading frame (ORF). To determine the position of the transcription initiation site of this early RNA with respect to the p143 ORF (26), Si nuclease analysis using a PstI-SalI 665-bp restriction fragment uniquely 5' end labelled at the PstI site was performed (Fig. 5). Following hybridization, three major Si nuclease-resistant fragments of 387, 200, and 198 nucleotides (nt) were observed in infected cell RNA (Fig. 5). The 387-nt fragment was seen exclusively in early RNA (3, 6, and 9 h p.i.) and in RNA synthesized in the presence of cycloheximide. At 12 and 24 h p.i., protected fragments of 200 and 198 nt were seen. Fragments corresponding to the size of the probe were also present at these late times, indicating that larger transcripts overlapped this region. The other band in the probe lane (Fig. 5A, lane P) corresponded to a plasmid DNA fragment that copurified with the PstI-SalI fragment during isolation of the labelled DNA. Since the probe was present in excess amounts relative to the quantity of RNA, the intensity of the protected fragments reflected the relative amount of complementary RNA present at that time point. As shown in Fig. 5, the 387-nt protected fragment was most abundant at 6 h p.i. during normal infection, indicating that the 4.0-kb transcript was maximally present at this time. The amount of this protected fragment was greater in RNA synthesized in the presence of cycloheximide, as expected from the results of the Northern analysis (Fig. 1). The start sites for the 4.0-kb transcript, mapped by Si nuclease, were precisely mapped by primer extension analysis using a 20-nt oligonucleotide primer corresponding to the region -1 to -20 nt relative to the AUG of the p143 gene

(26). An extension product of 154 nt was observed during the course of infection which increased in abundance from 3 to 6 h p.i. and declined from 9 to 24 h p.i. The 5' end of the 4.0-kb RNA mapped at these times by primer extension corresponded to the site mapped by Si nuclease analysis (Fig. 6A). At 12 and 24 h p.i., an additional extension product of 72 nt was produced. In cycloheximide-treated cells and ts8-infected cells at the nonpermissive temperature, only the 154-nt fragment was observed. Thus, the major start site for the 4.0-kb transcript was mapped by both Si and primer extension to a position 154 nt upstream of the predicted translation start codon for the p143 gene, within the sequence 5'GCGTGC-3' (nt 2336 [26]). At 12 and 24 h p.i., another transcriptional start site initiating within the sequence 5'GTAAG-3' was identified (nt 2415). No primer extension product corresponding to a late RNA start site within the sequence 5'ACGTGC-3' (nt 2540) identified by Si nuclease analysis was observed with use of a primer complementary to nt 2580 to 2610 (27). The EcoRI-ScaI 1.3-kb restriction fragment (probe 2b; Fig. 2) (but not the EcoRI W fragment) hybridized to the 1.1-, 4.0-, 5.2- and 6.8-kb RNAs, suggesting that the 3' ends of these transcripts were located in this region. This DNA contains two consensus polyadenylation signals 3' to the translational stop codon for the p143 gene (26). To precisely map the 3' ends of these RNAs, a uniquely 3'-end-labelled AccI-AccI 642-nt fragment was used. This fragment contains asymmetric 5' overhangs which could be preferentially end labelled depending on the radiolabelled deoxynucleotide selected. A single protected fragment of about 445 nt was observed with RNA from all time points (Fig. 5), suggesting that these transcripts were 3' coterminal with ends located approximately 45 nt downstream of the poly(A) signal overlapping the p143 gene stop codon. These data demonstrate that the early 4.0-kb RNA was transcribed from the region of AcMNPV carrying the p143 ORF. Mapping the 5' ends of the 1.1-kb transcripts. A 1.1-kb transcript was mapped by probe 2e (Fig. 2) to an ORF potentially encoding a 38-kDa protein (ORF2) (26). The 5' end of the transcript was mapped by primer extension using a primer complementary to a portion of ORF2 (nt +77 to +107; Fig. 6B). Two extension products of 137 and 196 nt were identified at 12 and 24 h p.i., mapping the start sites to 5'-ATAAG-3' and 5'-TTAAG-3' motifs located 30 nt (nt 834)

VOL. 66, 1992

MAPPING OF AcMNPV TRANSCRIPTS

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J. VIROL.

LU AND CARSTENS

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