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J. gen. Virol. (1989), 70, 2449-2459. Printedin Great Britain 2449 Key words: AcMNPV/transcriptionalmapping/codon usage bias Sequence, Transcription and Translation of a Late Gene of the Autographa californica Nuclear Polyhedrosis Virus Encoding a 34.8K Polypeptide By J I A N G U O W U AND L O I S K. M I L L E R * Departments of Entomology and Genetics, The University of Georgia, Athens, Georgia 30602, U.S.A. (Accepted 4 May 1989) SUMMARY A 1"4 kb region downstream of the D N A polymerase gene of Autographa californica nuclear polyhedrosis virus was sequenced. Two open reading frames (ORFs) were identified of 927 and 474 bases in length. The 927 base ORF encodes a 34.8K protein as determined by in vitro translation of both hybrid-selected R N A and R N A synthesized in vitro from a 927 base ORF template. The predicted amino acid sequence of the 34.8K polypeptide (p34.8) reveals a hydrophobic N terminus, two potential N-glycosylation sites, and potential sites for phosphorylation by casein kinase I and protein kinase C. The p34.8 gene has a strong codon usage bias which is strikingly different from that of the polyhedrin gene. The two 5' ends of the 927 base ORF transcripts initiate from an A T A A G sequence and a G T A A G sequence 11 and 87 bases upstream of the A T G codon respectively. A short upstream reading frame is present in the leader sequence of the longer RNA. The transcripts have multiple 3' ends; the most proximal endpoint correlates with a polyadenylation signal overlapping the translational termination codon of the 927 base ORF. Transcripts of the latter were not observed early in the infection cycle but appeared 6 h after infection and were maximally expressed at 12 to 24 h post-infection. The late nature of these transcripts was confirmed by their sensitivity to aphidicolin and cycloheximide, inhibitors of D N A replication and protein synthesis respectively. Attempts to construct viral mutants carrying a deletion of the p34.8 gene and fusion with the fl-galactosidase gene suggest that the former gene is essential for viral replication. INTRODUCTION Autographa californica nuclear polyhedrosis virus (AcMNPV) serves as a model system for molecular biological studies of baculoviruses (Doerfler & B6hm, 1986) which have been used as both insect pest control agents (Granados & Federici, 1986) and vectors for the high level expression of foreign genes (reviewed in Miller, 1988). Approximately 20 of the genes encoded by the 128 kb circular dsDNA genome of AcMNPV have been sequenced and transcriptionally characterized to date. The genes can be divided into three basic transcriptional classes (Friesen & Miller, 1986): early genes which are expressed before viral D N A replication, late genes which are dependent on D N A replication and are maximally expressed between 12 and 30 h postinfection (p.i.) and very late genes which are maximally expressed after 30 h p.i. Members of all three classes of genes appear to be dispersed throughout the AcMNPV genome without any obvious organizational basis. Transcripts of late nucleocapsid genes (Wilson et al., 1987; Thiem & Miller, 1989) and very late (Rohrmann, 1986) genes are initiated within a (G/A)TAAG sequence which also serves as the major determinant in high level expression of the polyhedrin gene (Rankin et al., 1988). A novel virus-induced ct-amanitin-resistant R N A polymerase activity appears to be responsible for late and very late gene expression (Fuchs et al., 1983 ; Grula et al., 1981). To gain further insight into baculovirus gene organization, we have defined a region downstream of the D N A polymerase gene (dnapol) of AcMNPV. We have found a late gene 0000-8847 © 1989 SGM Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Mon, 19 Jun 2017 01:49:01 2450 J. WU AND L. K. MILLER encoding a 34.8K protein (p34.8) that appears to be essential for viral replication in cell culture and have determined the map location and regulation of transcripts of this gene. The first of two initiation signals for late p34.8 transcription is located 50 nucleotides downstream of the transcriptional polyadenylation signal ofdnapoltranscripts. A short open reading frame (ORF), 18 codons long, lies within the leader sequence of the larger, 927 base ORF transcripts. METHODS Cells and viruses. AcMNPV L1 (Lee & Miller, 1978; Miller et al., 1983) was propagated in the Spodoptera frugiperda IPLB-SF-21 (SF) cell line (Vaughn et al., 1977). The cells were grown at 27 °C in supplemented TC100 medium as described previously (Lee & Miller, 1978; Miller et al., 1986). For RNA and protein studies the cells were inoculated at a multiplicity of 20 p.f.u, per cell. Viruses were allowed to adsorb to the cells for 1 h at room temperature. Inocula were replaced with fresh medium and the infected cells were incubated at 27 °C. Time zero p.i. corresponds to the start of incubation at 27 °C. In experiments requiring the inhibition of protein synthesis, cycloheximide (final concentration of 100 gg/ml) was included in the medium 1 h before inoculation, in the viral inocula, and also in the subsequent incubation medium. In experiments requiring inhibition of DNA synthesis, aphidicolin (final concentration of 5 gg/ml) was included in the medium which was added following the viral adsorption period. Nudeotide sequencing. The PstI H fragment of AcMNPV DNA from 37-8 to 42.0 map units (m.u.) was cloned into a Bluescript phagemid vector [pBSKS(- ), Stratagene] using standard procedures (Maniatis et al., 1982). The restriction fragment resulting from cleavage at the PstI site at 37.8 m.u. and at the NruI site at 40.9 m.u. was cloned into the same vector in the opposite orientation. A series of unidirectional deletions through the inserts of both plasmids were constructed using the exonuclease IIl/mung bean nuclease deletion method described by Stratagene and based on the original method of Henikoff (1984). Selected inserts in the recombinant phage ssDNAs were sequenced by the dideoxyribonucleoside chain termination method (Sanger et al., 1977) using the T3 primer (Stratagene), [~-35S]dATP (500 Ci/mmol; New England Nuclear, NEN) and a sequencing kit (Sequenase). Sequences were compiled and analysed using programs of Pustell & Kafatos (1984; International Biotechnologies). R N A preparation and nuelease protection assay. Total cell RNA was isolated by guanidinium isothiocyanate and CsCI cushion methods (Chirgwin et al., 1979; Davis et al., 1986) from mock- or AcMNPV-infected monolayers (10 T cells per 100 mm plate) at designated times p.i. The 5' and 3' ends of the gene were mapped by a nuclease protection assay using a formamide-based hybridization buffer and S1 nuclease digestion of non-hybrid ssDNA (Friesen & Miller, 1985). The 5' end probe was made by digestion of plasmid pBSMVI/BS with NotI, dephosphorylation with calf intestinal phosphatase (Boehringer-Mannheim) and radiolabelling with T4 polynucleotide kinase (Bethesda Research Laboratories, BRL) and [y-3zp]ATP (3000 Ci/mmol, NEN) at the 5' end. The DNA was further cleaved at the HindlII site in the multicloning site of the vector. The 1556 bp fragment was electrophoretically isolated and used as a probe. The 3' end probe was generated by digestion of the same plasmid with JfbaI and radiolabelling the 3' end with T4 DNA polymerase (BRL), [g-32p]dCTP (3000 Ci/mmol; NEN) and unlabelled dGTP, dTTP and dATP. The labelled DNA was further cleaved at the Sphl site in the insert. This 674 bp fragment was electrophoretically isolated and used as a 3"-terminal probe. D N A - R N A hybridization mixtures contained approx. 100 to 500 ng of labelled DNA and about 200 gg of total cell RNA. Protected fragments were denatured and electrophoresed on 7 ~ polyacrylamide-8 M-urea-Tris-boric acid-EDTA wedge sequencing gets. MspI-digested pUC19 DNA was radiolabelled and used as size markers. In vitro translation. For isolation of RNA by hybrid selection, 30 gg of a single-stranded plasmid DNA (pBSPAF-NX) containing antisense viral DNA between the XbaI and NotI sites (39.2 and 39.55 m.u. respectively) was heated at 100 °C for 2 rain, snap-cooled and absorbed onto a 1 cm 2 section of a nitrocellulose filter (Schleicher & Schuell). The filter was washed with 6 x SSC and then baked for 2 h at 80 °C in a vacuum (Esche & Siegmann, 1982; Friesen & Miller, 1987). Hybridization was conducted for 12 h at 42 °C in 50~ formamide, 10 raM-PIPES pH 6.4, 0-4 M-NaCI, 1 mM-EDTA with 3 mg of total RNA isolated from cells at 12 h after infection. The filters with bound RNA were washed 20 times with 1 x SSC, 0.1 ~ SDS, 2 mM-EDTA at 58 °C. The RNA was released from the filters by boiling for 2 min in 1 mM-EDTA and 10 lag of calf liver tRNA (Boehringer-Mannheim). RNA for translation was also prepared by in vitro transcription of a fragment of AcMNPV DNA from the PstI site at 37.8 m.u. to the SnaBI site at 40.0 m.u. ( - 171 from the ATG of the 927 base ORF) that was subcloned into pBSKS(-). The plasmid template was linearized with BglII which cleaves within the vector 'downstream' of the insert. The solution of linear DNA was treated with 200 lag/ml proteinase K (Boehringer-Mannheim) extracted twice with phenol-chloroform (1 : 1). The DNA was precipitated with ethanol before the transcription reaction. Transcription was carried out in a 25 p.1 reaction volume consisting of 5 gl of x 5 transcription buffer (200 mMTris-HCl pH 8.0, 40 m~l-MgC12, 250 mM-NaC1, l0 mM-spermidine), 1 lag of the linear DNA template, 10 mM each Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Mon, 19 Jun 2017 01:49:01 The p34.8 gene o f A c M N P V 2451 of rATP, rCTP, rGTP and rUTP, 1 ~tl of 0-75 M-dithiothreitol, 25 units RNasin (Promega Biotec) and 10 units T7 RNA polymerase (BRL). The reaction was incubated at 37 °C for 30 rain, diluted 10-fold with 225 p.1 of 40 mMTris-HCl pH 7.5, 6 mM-MgCI2 and 10 mM-NaC1, treated with 1 unit RNase-free DNase I (BRL) and extracted with phenol-chloroform (1:1). The RNA was purified in a Sephadex G50-80 spun column and then precipitated with ethanol (Stratagene method). In vitro translation of hybrid-selected or in vitro synthesized RNA was carried in a reaction containing 35 ~tl nuclease-treated rabbit reticulocyte lysate (Promega Biotec), 1 rtl of an amino acid mixture (containing 1 mM of each amino acid except methionine; Promega Biotec), 6 txl RNA template in H20 (approx. 1 p.g), 5.5 ~tl [35S]methionine (1129 Ci/mmol; NEN) at 10 mCi/ml, 2.5 ~tl RNasin (23000 units/ml; Promega Biotec). The reaction was incubated at 30 °C for 60 rain. Samples were frozen in liquid nitrogen and stored at - 7 0 °C until use. Viral proteins were also labelled in vivo by pulse-labelling with [35S]methionine. Cells (106 per 35 mm plate) were infected with 20 p.f.u, per cell. At designated times p.i., the medium was replaced with methionine-deficient medium lacking supplements and ceils were incubated for 1.5 h. This medium was then replaced with 0.5 ml of methionine-deficient medium containing 50 ~tCi [35S]methionine (see above) and the ceils were incubated for 1 h to radiolabel the proteins. The cells were then collected, washed with phosphate-buffered saline (Lee & Miller, 1978), and lysed in 1~o Nonidet P-40, 50 mM-Tris-HCl pH 8-0 and 150 mM-NaC1. In vitro translation products and intracellular proteins were subjected to SDS-PA G E (Laemmli, 1970) followed by autoradiography using a Kodak XAR5 film. fl-Galactosidase gene insertion into the 927 base ORF. The Escherichia coil fl-galactosidase gene (lacZ) was obtained from plasmid pSKSI06 (Casadaban, 1983) by digestion with Sail, BamHI and then PstI. The cohesive ends were removed with mung bean nuclease. The largest fragment, containing lacZ, was purified by agarose gel electrophoresis and inserted between blunt-ended SstI and Xbal restriction sites in a Bluescript minus-sense plasmid containing AcMNPV DNA from the PstI site at 37-8 m.u. to the Nrul site at 40.9 m.u. to construct the transplacement plasmid pBSKS106. Ten ~tg of pBSKS106 DNA and 1 ~tg of AcMNPV L1 DNA were cotransfected into SF cells (Miller et al., 1986). The recombinant viruses were selected as blue plaques on SF monolayers in the presence of X-gal (Pennock et al., 1984). Purified DNAs from the putative recombinant viruses were compared with that of wild-type virus by restriction endonuclease digestion (Pstl and SstI) and Southern blot analysis. RESULTS ORFs downstream of the AcMNP V DNA polymerase gene Both strands of A c M N P V D N A extending from the SmaI site at 40.3 m.u. (see Fig. 1, centre) to a SphI site (38.6 m.u. approximately 1.4 kb downstream of the carboxy terminus of dnapol were sequenced using the strategy shown in Fig. 1 (top section). A major ORF, 927 bases in length, was observed on the same strand downstream from but in a different reading frame than that of dnapol (see Fig. 1, bottom). A second smaller ORF, 474 bases in length, was observed on the opposite strand, downstream of the 927 base ORF (Fig. 1, bottom section). The D N A sequence of this region and the predicted amino acid sequence of the 34.8K polypeptide product (p34.8) of the 927 base O RF are both presented in Fig. 2. The 12 N-terminal amino acids of p34.8 are hydrophobic and two potential glycosylation sites (Asn-X-Ser) are present at amino acid residues 195 and 295. There is a potential protein kinase C phosphorylation site (Arg-Lys-Ser) at amino acid 292 and a potential casein kinase I phosphorylation site (Glu-Glu-Ser) at amino acid 285. There are three potential tyrosine kinase sites (Glu-Xz-Tyr) but as the X residues are not acidic, it is unlikely that these sites are utilized. The polypeptide p34.8 is rich in basic and aromatic amino acids. The D N A sequence and the two polypeptide sequences were screened for nucleotide or amino acid sequence homology against GenBank release 55.0 using the IBI/Pustell 'Cyborg' software which employs a modification of the FASTP algorithm of Lipman & Pearson (1985). N o significant homology was observed for either ORF. Mapping of 927 base ORF transcripts The 3' and 5' ends of transcripts of the 927 base ORF were mapped using two probes derived from plasmid pBSMVI/BS which is schematically presented in Fig. 3 (a). A 674 bp probe (probe A) was labelled at the 3' end at the XbaI site at 39.2 m.u. R N A isolated from infected cells at 6, 12 and 24 h p.i. in the absence of inhibitors protected D N A fragments of approximately 187, 337 and 674 bases (Fig. 3 b). These fragments were not protected by R N A from mock-infected cells Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Mon, 19 Jun 2017 01:49:01 J. WU AND L. K. MILLER 2452 D 4 4 BclI XbaI Sphl ~;1(386) II If llIII II|I II Ill II I IIIllgIlll~ 474 base ORF II I i gill II II I II i 1[ SnaBI NotI SstI 11(39-6 I (39-2) I PvulI I I l I II I fl gill ii II1111 II II 1 0 I [ II g i l II llI II II illll I! Ill 11 II ill I I 1 lift III II li II 1 I 927 base ORF ! III g Sinai t(403t: II I 1 II 12 II 1 3 --dnapol-- / III II I II [ [ /llllllI I II lilt I n 1' 2' 3' Fig. 1. Key restriction sites, sequencing strategy and distribution of ORFs for the AcMNPV region between 38-6 and 40-3 m.u. In the centre, a restriction map of a 2106 bp region from 38.3 to 40.3 m.u. on the AcMNPV physical map is schematically presented. Numbers in parentheses below selected restriction endonucleases indicate the m.u. scale. At the top, the strategy employed for sequencing both strands of DNA is presented. At the bottom of the figure, the distribution of ORFs on the top strand (frames 1, 2 and 3) and on the bottom strand (1', 2' and 3') are presented. The dnapol ORF and an ORF of 927 bases are read from right to left (counterclockwise with respect to the circular AcMNPV map) whereas a 474 base ORF is read from left to right. nor by R N A isolated 2 or 4 h p.i. (Fig. 3b). Significant levels of protection of the 674 bp probe were observed with RNA from 6, 12 and 24 h p.i. but not with R N A from mock-infected cells or infected cells at other times p.i. indicating that the appearance of this fragment is due to R N A protection (i.e. some transcripts cross the entire length of this 674 bp fragment). Aphidicolin, an inhibitor of both cellular and viral D N A polymerases, inhibited the synthesis of transcripts through this region indicating that the transcripts are 'late' in the sense that they depend on D N A replication for expression. Cycloheximide, an inhibitor of protein synthesis, also inhibited synthesis of these RNAs. Thus protein synthesis during virus infection is required for transcription; this suggests that one or more viral proteins are required for the activation of the 927 base ORF transcription. The position of the ends of the protected 187 and 337 base fragments are shown in Fig. 2 and are located just downstream of polyadenylation signals ( A z U A 3 ) , strongly suggesting that these ends represent polyadenylation sites. Fig. 2. Nucleotide sequence of the region encompassing the 927 base ORF and the amino acid sequence of its predicted polypeptide product. The nucleotide sequence of the region encompassing the SnaBI to SphI region (Fig. l) is presented; the positions of the SnaBI and SphI sites are underlined and identified. The NotI site (GCGGCCGC) between +217 and +224 and the XbaI site (CTCTAG) at 39.2 m.u. are underlined once and twice respectively. The end of the dnapol gene is indicated under the top line of sequence (dnapol/) where the slash mark notes the TAA termination codon of the dnapol ORF. The (G/A)TAAG boxes at the 5' ends of transcripts through the 927 ORF are indicated by addition signs ( + ) under the boxes. Polyadenylation signals (A2TA3) near the 3' ends of transcripts are indicated by carets (A) below the nucleotides. The exclamation marks below two nucleotides indicate the approximate position of the polyadenylation or 3' ends of the RNAs as mapped by nuclease protection experiments. Two potential sites for N-glycosylation (Asn-X-Ser) are noted in bold print. Amino acids located at a potential casein kinase I site and a protein kinase C phosphorylation site are singly and doubly underlined respectively. The nucleotides are numbered (along the right side) according to + 1, + 2 and -I-3 in reference to the ATG of the 927 base ORF. Amino acids of the 927 base ORF product are numbered in parentheses below the nucleotide numbers. The asterisks are above every tenth nucleotide. Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Mon, 19 Jun 2017 01:49:01 2453 The p34.8 gene of AcMNPV AAA TGA CAC T C A ATG TGC TAA CAA TAC GTA TAA AAT GAA CTG TAA + +++ GGT + GTT TCC GAT TAG TGT -.19 GCG Ala ATT CAC Ile His GCG Ala +42 (14) ATT TTG TTT GTA TAA TTA SnaBl * TTT ATA ATT ATA TAC GAT ACT ATG TTT TAT TGT ATG ATG TTT CAA CAT AAC GTT TTT GAC CAA GAT TAC GAC AGC GGT TAT TAT AAG AGT ++ TTG ATA ATC ATA ATG Met ATT GCA Ile Ala TTA TTA Leu L e u ATT Ile CGC CAC GGC TAT TCC CCG ACG Pro T h r GCC Ala GAT ATT TCT ATA +++ CCG Pro GCA GTG Ala Val A r g S e r H i s G l y T y r L e u Ser V a l AAA GAT AAC GGA TCT TTT TAT TGG CCC TTG GAC AAT GTG GGC GCA TTG Ala Leu AAC L y s ASp G l y Asn Phe T y r T r p P r o Asp A s n G l y Asp Asn I l e AAT ASh GCT Ala GCT TCC Ala Ser TAC AAA TCA GTC TAT TAT AAA TAT GCT CTC ACA GCG CAA TAC ATG TTT CAA AAT TAT GAC GAT TTT GAC CTA ATC A s h T y r Asp Asp Phe Asp L e u I l e AAA GGT CAG TAT TTT AAC GAT CGC AAT TCT GTG CAA ATG GAA AGG GTT GTG Lys Gin Arg Val Val GGT TGG AGG CCA AAT ACG CTA TAT TTA AAT TAC GCG GAT AAA AGC CGC TAT CAA --79 AAA Lys TGT TTT Cys P h e +102 (34) GCC GCG TGC +162 (54) CCC G A C TTA --139 CAA TAT Gln Tyr CGC P r o Asp A ! a A l a Cys A r g T y r Met G l u T y r A l a G l y S e r Asn Asp A r g Asn S e r V a l Phe G l y Asp L y s S e t AAC Ash GAT CGT Arg ATT GAA TCC GGG GCG GCC T y r L y s S e r V a l T y r T y r L y s T y r A r g A l a L e u Asp L e u G l u S e t G l y A l a A l a T h r A l a G l n T y r Met P h e G i n G i n TCA CGA TTT GCT Phe Ala AAA ATA dnapol/ A l a Val Ala GGT GIy CCT Pro . +282 (94) GCT GTG GCC +222 (74) CCG Pro CAC A C A His Thr CTT Leu TGC Cys GGT Gly GCC Ala -~342 (114) GGA ATG GAA CCT TTT AAC +402 (134) GAC G l y Met Asp G l u P r o Phe A s n , CCG GTT TAT CAG ATG T r p A r g P r o Asn T h r L e u T y r L e u A s n A r g T y r G l n F r o V a ! T y r G l n M e t . AAT GTT ASh Val +462 (154) CAT TTT H i s Phe TGT Cys CCA Pro ACA Thr GCC Ala A T A CAC G A G Ile H i s G l u CCC Pro AGT TAT Set T y r TTT Phe GAA Glu GTG Val TTT Phe ATT Ile ACT Thr AAA TCA Lys S e r , ~522 (174) AAC Asn GAT CGT CGC AAT CCA ATA ACT TGG AAC TTA T r p A s p A r g A r g ASh P r o Iie T h r T r p A s n GI,] L e u G A A TAC glu Tyr ATA ile GGT Gly GGT AAC GAT GIy Asn Asp . +582 (194) GAT Asp TAT TCT ATA T y r Ser Ile ~642 {214) TGG TCA AAT Set Asn TCA Set GAA TTA TGT GAC AAC L e u Cys Asp A s n AGT Ser CTA GTC Leu Val ATG Met TAT Tyr GTG Val CGT Arg TGG Trp CAG Gln CGC Arg ATA ile +702 (234) CTG GTG TTT Phe GAA Glu ACT Thr CTA GAC Leu A~p GAC Asp , +762 (254) TTA Leu ATT Ile CCA AAT Pro A s h CCG Pro GGC GI¥ CCC GTA GTA Pro Val Val ATA Ile CCG Pro TAC Tyr AGG Arg TCA AAT Set A s h CAA TTT GTA G l n Phe V a l GAT GGT GAA GGA TTT TAT TGC CCC GTG AAT GCC GAT Asp P r o V a l G l y G l u G l y P h e T y r Asn Cys A l a Asp L e u V a l GAA TGC AGA TAC GCT CAA ATG GCT AAA GTG GTC G l u Cys A r g T y r A l a G l n Met A i a L y s V a l V a l GAT GCA CGC ATT GAT CAT AAT GAC GAA GAA TCT AGA Arg AGC Ser CAA TTA CAA Gln Leu Gln AAG Lys CAT His CTT Leu * +822 (274) TGT TGG CGT AAA TCA AAT TAT Ser Ash Tyr +882 (294) AAT TAC ATA TTA TAT ! ~942 ATT TCA AAC ATG TCT TCA +1002 GCA CGC Asp A l a A r g I l e Asp H i s Asn Asp G l u G l u Ser Cys T r p A r g A i a A r g L y s TCG TCA TTT Set Ser Phe TTT Phe AAT Asn TTA GCA AGT AAT AAC AAT TCG TAG TCT GCT CTG TCA TAA TTA TCA TCG TCA AAA AGA CCA TTT TTG CAG CCA Pro GGA TTT G l y Phe TAA TAA AAC AAA CAA AAT TTT CTA CAA TCA ATA CGA ACA AAA TAT AAA Lys ...... AG~ TCT ATG CTA AAA TTG TTA ATT AAA TCT TCC ATA TTT TCA CAC +1062 TGT AAT AAA AAT AGT TTA AAG CCA TCT TGA TCT CGT TTG GAT ATT TCG +1122 AGA TAT TGC AAA GCA AAC TGT ACT TCT TTG GCG TAA GGA TTT AAC AGT +1182 ACT CTG TCT ACG AGC GTA TTG AGA CTT TCC GCA GTT AAA GTG TTG +1242 TCA CAA AAC CGC TTG ATT TCA TCG GCT ACT TGA +1302 TAT TTT GCT TCT CCG TGC AGC CCA +1362 ATG TTG CTC TAA AAT TAA TAA AAC ACT +1422 ACG CAA CTG ATT GCC GGT TTC AGA CGA GTT TTT TCA AAA TCC ATT TGA GAC ATT AAA TAT TTG TTC AAG TGA AGA TAG ATC ATA AAT TTA GAT TGC ATG CAA TCA AAT G TCG TAC TAG SphI Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Mon, 19 Jun 2017 01:49:01 2454 J. WU AND L. K. MILLER (a) ATAAG-"-Iv U G T A A G 3'(~ v -4--I5' RNA ~-304"~ 674 ~] ~-337"-t 927 base ORF ) PstI I Hss,i (40.9)/N (39.6) XbaI (39.2) NotI SphI (37-8) ] HindlII 674 bases 1556 bases [ Probe A SphI (3') XbaI Probe B (5') HindlII NotI (b) (c) M 0 2 4 6 6A 12 A C 24 M 2 6 12 A C 24 48 P 1556~ ,674 501N_ 489--404 m "337 331~ 3091, 242 2331" 190~ 4187 147~ Fig. 3. Nuclease protection mapping of transcripts of the 927 base ORF. (a) A schematic diagram of the plasmid pBSMVI/BS from which DNA probes were prepared in order to map transcripts of the 927 base ORF. Vector sequences are denoted by shaded blocks ([]) labelled 'BS-'. AcMNPV sequences between the PstI site at 37.8 m.u. and the NruI site at 40.9 m.u. are represented by the open blocks and the position of the 927 base ORF is represented by the open arrow immediately above the region. Key restriction sites and m.u. are noted immediately below the region. The NruI site was removed by fusing this site and the EcoRV site of the vector; both sites are noted by a shaded block (~1). Below the restriction sites are representations of the radiolabelled DNA probes (A and B) used for nuclease protection studies. Probe A was 674 bases in length and was labelled exclusively at the 3' end of the Xba I site as noted by the asterisk. Probe B was 1556 bases long and labelled exclusively at the 5' end of the NotI site as indicated by the asterisk. A summary of the 5' and 3' ends determined from nuclease protection experiments are shown at the very top of the figure. The thin open arrow shows the position Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Mon, 19 Jun 2017 01:49:01 ) The p34.8 gene of A c M N P V 2455 The 5' ends of the 927 base O R F transcripts were analysed using a 1556 bp probe which was 5' end-labelled at the NotI site within the O R F (Fig. 3, probe B). R N A from 6, 12, 24 and 48 h p.i. protected two fragments of approximately 233 and 309 nucleotides in size (Fig. 3b). The R N A from mock-infected cells or infected cells at 2 h p.i. did not protect the probe (Fig. 3 b). Similarly R N A from infected cells that were inhibited by aphidicolin did not protect the probe, indicating that these are late transcripts. Cycloheximide also inhibited the synthesis of these transcripts. The end of the protected 233 nucleotide fragment maps to an A T A A G sequence at - 11 relative to the A T G of p34.8 and the protected 309 nucleotide fragment maps to a G T A A G sequence at - 8 7 (Fig. 2). Translation of the 927 base ORF transcripts To determine the nature of the product of the transcripts through this ORF, R N A homologous to the antisense strand of the O R F was hybrid-selected using a plasmid containing the region spanning the XbaI to NotI sites (see Fig. 3). The selected R N A was translated in vitro using a rabbit reticulocyte in vitro translation system including radiolabelled methionine. A polypeptide product of 34K to 35K was observed by autoradiography (34.8K, Fig. 4, lane H); controls with no added exogenous R N A or vector DNA-selected R N A showed that this 34.8K protein was a specific product of the 927 base O R F selected R N A (control data not presented). The same size product was observed when R N A synthesized in vitro from a template containing the entire 927 O R F beginning at - 171 from the A T G (Fig. 4, lane S). The 34-8K polypeptide migrated in SDS-polyacrylamide gels just above the polyhedrin protein (Fig. 4, 30 h lane). Attempts to construct p34.8-~-galactosidase fusions It has been possible to isolate mutant viruses containing deletions in genes which are nonessential for virus replication in cell culture by fusing the N-terminal portion of the O R F to the E. coli /~-galactosidase gene and selecting recombinant virus plaques using a chromogenic indicator of/~-galactosidase (Pennock et al., 1984; Crawford & Miller, 1988; Vlak et al., 1988). To determine whether p34.8 is non-essential in cell culture, a transplacement plasmid was constructed based on pBSMHVI. The transplacement plasmid contained the E. coli ~galactosidase gene fused in frame with the p34.8 gene at the SstI site within the 927 base O R F (39.6 m.u. ; Fig. 3). The ~-galactosidase gene replaced 927 base O R F sequences from the SstI site down to the XbaI site at 39.2 m.u. and was flanked by approximately 1-5 kb of viral sequences on both sides to permit allelic replacement. Cotransfection of the plasmid with wild-type viral D N A resulted in virus stocks which contained a low proportion of blue plaques. Eight representative blue plaques were picked and plaque-purified at least four times. Stable stocks of pure blue plaque viruses could not be isolated; instead, clear (non-blue) plaques continued to arise at a high frequency. Restriction endonuclease characterization of the viral D N A from stocks of four of these isolates suggested that the plasmid had recombined by a single rather than and direction of transcripts. The closed arrowheads show the positions of the two 5' ends whereas the open arrowheads show the positions of the mapped 3' ends or polyadenylation sites. The dashed lines and arrowhead indicate that some of these transcripts extend through the SphI site used in the probe construction. The numbers between the bracket below the transcripts indicate the lengths (in bases) of the protected fragments shown in (b) and (c). (b) An autoradiogram of DNA fragments protected from S 1 nuclease digestion by RNA isolated from mock-infected cells (M) or from AcMNPV-infected cells at time zero (0) and at 2, 4, 6, 12 and 24 h p.i. in the absence of inhibitors or in the presence of aphidicolin at 6 and 12 h p.i. (6A and A respectively) or cycloheximide at 12 h p.i. (C). Mr markers are shown at the far left. The sizes of the protected fragments are shown at the right of (b). (c) An autoradiogram of DNA protected by RNA isolated from mock-infected cells (M) or AcMNPV-infected cells at 2, 6, 12, 24 and 48 h p.i. in the absence of inhibitors or at 12 h p.i. in the presence of aphidicolin (A) or cycloheximide (C). Lane P contains the probe alone. Molecular size (bases) markers are on the far right. The positions of the protected fragments of 233 and 309 bases and of the 1556 base probe are indicated by arrows at the left of (c). Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Mon, 19 Jun 2017 01:49:01 2456 M J. WU AND L. K. MILLER 3 6 12 30 S H --Endogenous Fig. 4. SDS-PAGE of products from in vitro translation of both hybrid-selected RNA and in vitro synthesized RNA of the 927 base ORF, also of in vivo labelled proteins from infected cells and their analysis by SDS-PAGE. RNA selected by hybridization to antisense DNA of the 927 base ORF was translated (H) and the products were compared by p34.8 SDS-PAGE to those obtained from translation of - - Polyhedrin RNA synthesized in vitro from the 927 ORF (S) by SDS-PAGE. For additional comparison, proteins were pulse-labelled in vivo at 3, 6, 12 and 30 h p.i. and in mock-infected cells (M). The position of the major background protein produced from RNA endogenous to the rabbit reticulocyte lysate is noted (endogenous) on the right. The solid arrowhead on the right indicates the major 34.8K product of translation of both the hybrid-selected RNA and in vitro synthesized RNA. The position of polyhedrin from AcMNPV-infected cells (note 30 h p.i. lane) is also indicated on the right. a double crossover event (data not shown). Thus, wild-type viruses were generated upon passage with the loss of the fl-galactosidase gene. These results strongly suggest, but do not prove because of their negative nature, that the p34.8 gene is essential for virus replication. DISCUSSION We have found that a late gene, encoding a 34.8K polypeptide is located downstream of the D N A polymerase gene of A c M N P V . Like dnapol, the p34-8 gene is transcribed in the counterclockwise direction but the regulation of the two genes is distinctly different. Transcription of dnapol is maximal at 6 h p.i. (Tomalski et aL, 1988) and occurs in the presence of cycloheximide, indicating that it is an early gene. Normally, transcripts of dnapol are switched off by 12 h p.i. but their presence is extended past 12 h p.i. in the presence of cycloheximide. In contrast, the p34.8 gene transcripts are initiated around 6 h p.i., the beginning of viral D N A replication, and are present maximally from 12 h to 48 h p.i. Transcription of the p34-8 gene does not occur in the presence of cycloheximide suggesting that a viral gene product is required for transcription. Transcription of the p34.8 gene also appears to be dependent on D N A replication since transcription of the 927 base O R F is not observed in the presence of aphidicolin. These properties place the p34.8 transcripts in the late class which persists through the very late (occlusion) phase of viral infection. The organization of genes around p34.8 most closely resembles the gene organization around the p 10 gene (Rankin et al., 1986; Kuzio et al., 1984). It remains to be determined whether such gene organization has regulatory significance. Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Mon, 19 Jun 2017 01:49:01 The p34.8 gene of A c M N P V 2457 Similar to all other late and very late transcripts of AcMNPV that have been characterized to date, p34.8 RNAs initiate within an (G/A)TAAG sequence which forms the nucleus of the major determinant for high level expression from the polyhedrin promoter (Rankin et al., 1988). The p34.8 RNAs initiate from two different (G/A)TAAG sequences, an ATAAG which is located only 11 bases upstream of the ATG of p34.8 and a G T A A G which is 87 bases upstream. Utilization of multiple (G/A)TAAG sites has been observed previously for transcripts of the major capsid protein (Thiem & Miller, 1989). Three (G/A)TAAG sequences are utilized for capsid gene transcriptional initiation; one is a GTAAG site located 330 bases upstream of the ATG codon of the capsid ORF. Linker scan mutations of the polyhedrin promoter which convert the ATAAG to GTAAG decrease expression fourfold (Rankin et al., 1988) suggesting that the GTAAG start is less well utilized as an initiation site than ATAAG. Consistent with this observation, the level of p34-8 transcripts initiating at the G T A A G site appears to be approximately one-third of that of transcripts initiating at the A T A G G site (Fig. 3). An interesting feature of the GTAAG-initiated p34.8 transcript is that it contains a short ORF which would encode an 18 amino acid peptide terminating at a UAG encoded upstream of the ATAAG initiation site. Whether this peptide is synthesized and functional or whether this ORF represents some type of translational regulation remains to be determined. The codon usage of this upstream ORF is similar to that of the 927 base ORF. The GTAAG-initiated transcript of the major capsid gene also has an upstream ORF that would encode a very short peptide (six amino acids). The dnapol gene has two ATGs in the leader sequence of its RNAs (Tomalski et al., 1988) but unlike the late RNA leader ATGs of the capsid and p34.80RFs, translation products beginning at these 'upstream' ATGs in the polymerase RNA would terminate downstream of the ATG of the polymerase ORF. Such an arrangement could dramatically decrease levels of DNA polymerase expression. In the case of the capsid and p34.8 ORFs, reinitiation at the ATG of the major ORF could occur. The positions of two 3' ends or polyadenylation sites for p34-8 transcripts have been identified and map just downstream of consensus polyadenylation signals (AEUA3). One of these signals overlaps with the double (tandem) UAA termination codons of p34.8. Such a tight signal organization appears to be a common feature in AcMNPV. The polyadenylation sites, however, do not appear to be utilized efficiently as transcripts crossing both sites and terminating further downstream appear to be abundant, particularly late in infection (Fig. 3a, 674 base probe protection at 12 and 24 h p.i.). The lack of efficient polyadenylation of late transcripts at consensus polyadenylation sites seems to be a common characteristic of late gene transcription; it has been observed for pl0, polyhedrin and 6.9K core protein gene transcription (Friesen & Miller, 1985; Wilson et al., 1987) as well as the series of late transcripts in the EcoRI H and S region (Friesen & Miller, 1987; Oellig et al., 1987). Transcripts continuing through the polyadenylation site within the 927 base ORF could regulate expression of the downstream 474 base ORF because they would be antisense. It is possible that the p34.8 protein is modified post-translationally. It contains signals for both N-glycosylation and phosphorylation. The highly hydrophobic N terminus might serve as a signal sequence for membrane transport or anchoring; however, the region lacks basic residues at the N terminus which are usually found in signals for endoplasmic reticulum transport. Glycosylated proteins of 34K and 37K (Stiles & Wood, 1983) and a prominent phosphorylated protein of 34K have been observed in AcMNPV-infected cells (Maruniak & Summers, 1981 ; O'ReiUy & Miller, 1988). A 34K 'calyx' protein which is phosphorylated was recently identified as a component of the outer envelope of occlusion bodies (Whitt & Manning, 1988) but the gene encoding this calyx protein is reported to be at 83 m.u. (Gombart et al., 1989) and would be expected to be non-essential. A 35K early protein was previously identified (Friesen & Miller, 1986). If p34.8 is modified by either glycosylation or phosphorylation, its migration with respect to in vivo labelled proteins might be altered. Thus, there is some difficulty in ascribing a particular infected cell-specific protein to p34.8. The nature and function of p34.8 will need to be further pursued using specific antibodies raised to p34.8 epitopes. The failure of our attempts to delete this gene strongly suggests that p34.8 plays an essential role in viral replication. Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Mon, 19 Jun 2017 01:49:01 2458 I. W U AND L. K. M I L L E R A rather unusual feature of the p34.8 gene is the codon bias utilized. Usually, strong codon usage bias is correlated with high level gene expression. The p34.8 gene has a strong codon usage bias which differs from that of the abundantly expressed very late polyhedrin gene. For example, all 16 phenylalanines of p34-8 are encoded by TTT codons whereas 13 of the 14 phenylalanines of polyhedrin are encoded by the other codon for phenylalanine, TTC. Strong and opposite codon biases between polyhedrin and p34-8 are also observed for tyrosine, lysine and isoleucine. Some codon bias is also found in other highly expressed baculovirus genes. The pl0 gene tends to utilize codons in a fashion similar to the polyhedrin gene. The major capsid gene, however, has a codon usage bias that shares similarities with those of both polyhedrin and p34-8. With the exception of the valine codon, the p34.8 codon usage favours A and T residues at the wobble position. Codon bias may reflect specific aspects of gene evolution to regulate expression or the acquisition of these genes from different sources during baculovirus evolution. The emerging picture for the gene organization of the AcMNPV genome is one in which early and late genes are intermixed; transcriptional, post-transcriptional and translational regulatory signals are brief and closely spaced. 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