Biomedical subjects

M D Summers

Publications and source records attributed to M D Summers.

At least 55 records · Page 3Linked to original sources

PubMed1989

Location and nucleotide sequence of the 25K protein missing from baculovirus few polyhedra (FP) mutants.

Wild-type and few polyhedra (FP) mutants of the baculovirus Autographa californica nuclear polyhedrosis virus (AcMNPV) were studied to identify and sequence the gene encoding the 25-kDa (25K) protein normally present in AcMNPV-infected Spodoptera frugiperda cells but which is often missing from FP mutant-infected cells. Our previous study had mapped two overlapping late transcripts to the insertion site of host cell DNA within the HindIII-I fragment (33.8 to 37.7 map units) of wild-type AcMNPV. An FP mutant, AcFP875-2, had a 1.6-kbp insertion of S. frugiperda DNA near the 5' end of these transcripts which by S1 analysis were shown to initiate within the host cell sequence. Primer extension analysis revealed that the transcription start for this gene in wild-type virus occurred within a conserved 12-base sequence found near the transcription start sites of several baculovirus late and hyper-expressed genes. A similar 12-base sequence was found at the transcription start site within this 1.6-kbp pair host cell DNA sequence in AcFP875-2. mRNAs from wild-type virus-infected cells were hybridization-selected using a 542-bp SalI subfragment of the 3.2-kbp EcoRI-HindIII fragment (35.0 to 37.7 map units). These mRNAs directed the synthesis of a 25K protein which in size was identical to the 25K protein in wild-type virus-infected cells and the translation product of a 1.15-kb cRNA transcribed from a RsaI fragment (36.4 to 37.4 map units). Comparison of gel band patterns following partial proteolysis of the translation product of the 1.15 cRNA and the 25K protein from wild-type virus-infected cells revealed that the two proteins were closely related if not identical. Nucleotide sequence analysis within this EcoRI-HindIII fragment revealed an open reading frame which encodes a 25K protein. Insertion of the Escherichia coli lacZ gene encoding the beta-galactosidase enzyme into the transcribed portion of this EcoRI-HindIII fragment yielded a recombinant virus which lacked a 25K protein and exhibited an altered (FP) plaque phenotype.

Animals↗

PubMed1989

High level expression of nonfused foreign genes with Autographa californica nuclear polyhedrosis virus expression vectors.

High levels of nonfused chloramphenicol acetyltransferase, beta-galactosidase, and beta-glucuronidase expressed under the control of new vector constructs of the polyhedrin promoter in Spodoptera frugiperda cells infected with Autographa californica nuclear polyhedrosis virus were investigated by SDS-PAGE and RNA dot blot analysis of total cytoplasmic RNA. When the polyhedrin ATG start codon was converted to ATT by site-directed mutagenesis, translation initiated at downstream ATG codons resulting in high yields of nonfused foreign proteins. When a stop codon was inserted downstream from and in phase with the polyhedrin ATG codon but upstream from the ATG of a foreign gene, nonfused proteins were also produced, but at lower levels. The level of steady-state polyhedrin gene-promoted mRNA was not affected by the mutation from ATG to ATT or the insertion of in phase stop codons downstream from the polyhedrin ATG.

Animals↗

PubMed1989

Insertion of the SfMNPV polyhedrin gene into an AcMNPV polyhedrin deletion mutant during viral infection.

The Autographa californica nuclear polyhedrosis virus (AcMNPV) polyhedrin deletion mutant, d10A, was cotransfected with the recombinant plasmid SfMNPV.HindIII-L, which contains the Spodoptera frugiperda nuclear polyhedrosis virus (SfMNPV) polyhedrin gene. An occlusion-positive hybrid virus was obtained which contained a DNA insertion at approximately 13 map units on the AcMNPV.d10A genome. A fine structure restriction map of cloned viral DNA fragments from this region revealed that most of the 1.7-kb AcMNPV.PstI-O fragment (13.4-14.7 map units) had been replaced with a 3.7-kb fragment, containing the SfMNPV polyhedrin gene with flanking sequences and the entire pUC8 plasmid. Subclones of this region were sequenced and the exact site of insertion was determined. Flanking the insert were 43 and 60 bp of unknown origin at the 5' and 3' ends, respectively. Transcription was examined in the region of the insertion in both wild-type AcMNPV and the AcMNPV/SfMNPV (Ac/Sf) hybrid. In the AcMNPV, a nested set of seven overlapping transcripts ranging from 2.2 to 5.3 kb was found, each with coterminal 3' ends. Only the 2.2-kb transcript was found to be expressed early and throughout infection. The SfMNPV insertion interrupted this transcriptional unit and produced a more complex pattern of transcription. Alterations included a nested set of three overlapping transcripts with coterminal 5' ends, including the SfMNPV polyhedrin mRNA, transcripts originating in AcMNPV and terminating in either SfMNPV or pUC8 sequences, and other minor transcripts. The SfMNPV polyhedrin gene was sequenced and the locations of the 5' and 3' ends of polyhedrin mRNA were mapped. An analysis of SfMNPV polyhedrin protein expression showed that the SfMNPV polyhedrin gene in the hybrid virus was expressed at approximately one-fourth the level of the wild-type AcMNPV polyhedrin gene. Expression of a beta-galactosidase gene under the control of the SfMNPV polyhedrin promoter in the AcMNPV.d10A mutant was also investigated.

Amino Acid Sequence↗

PubMed1989

Cell-surface expression and purification of human CD4 produced in baculovirus-infected insect cells.

CD4 is an integral membrane glycoprotein that acts as the cellular receptor for human immunodeficiency virus (HIV). A cDNA encoding full-length CD4 was inserted into the genome of Autographa californica nuclear polyhedrosis virus under transcriptional regulation of the viral polyhedrin gene promoter. The recombinant virus was used to infect insect cells, which resulted in the abundant expression of CD4 as evaluated by flow cytometry and immunoblot analysis. Recombinant CD4 expressed on the surface of infected insect cells was immunologically indistinguishable from human CD4 when using 11 different anti-CD4 monoclonal antibodies. The extraction of infected cells by phase-transition separation with Triton X-114 followed by immunoaffinity chromatography yielded a single protein detected by NaDodSO4/PAGE using silver staining. N-terminal sequence analysis of the purified recombinant protein showed that CD4 produced in Sf9 cells is efficiently cleaved from the precursor protein. Immunoblot analysis under nondenaturing conditions showed that the purified protein reacted with the anti-CD4 monoclonal antibody Leu-3a. The potential use of the recombinant membrane-associated CD4 in anti-HIV therapy is discussed.

Amino Acid Sequence↗

PubMed1989

Expression of Sindbis virus 26S cDNA in Spodoptera frugiperda (Sf9) cells, using a baculovirus expression vector.

To study protein processing in an insect Spodoptera frugiperda (fall armyworm; Sf9) cell line, a 26S cDNA encoding the sequence of Sindbis virus structural proteins (capsid protein, of 30 kilodaltons [kDa]; p62 [the precursor of E3 and E2], of 62 kDa; a 6-kDa peptide; and the E1 protein, of 56 kDa) was inserted into the genome of Autographa californica nuclear polyhedrosis virus (AcNPV) adjacent to the polyhedrin promoter. By immunoblot analysis with antisera directed against whole Sindbis virus and the individual structural proteins (capsid, E2, and E1), we have shown that polypeptides similar in size and antigenicity to those synthesized in Sindbis virus-infected BHK cells are expressed in Sf9 cells infected with the recombinant baculovirus Ac373-SV26. By pulse-chase labeling in the presence or absence of tunicamycin, by endo-beta-N-acetylglucosaminidase H (endo-H) treatment of the recombinant glycoproteins, and by N-terminal sequence analysis of the E1 envelope glycoprotein, we have further shown that the 26S transcription translation unit of Sindbis virus, although normally encoded by nonnuclear RNA, is expressed and proteolytically cleaved similarly, if not identically, in Sf9 cells as compared with BHK cells when a baculovirus expression vector is used.

Animals↗

PubMed1989

Expression and characterization of hepatitis B virus surface antigen polypeptides in insect cells with a baculovirus expression system.

The baculovirus Autographa californica nuclear polyhedrosis virus was used as an expression vector to produce hepatitis B virus surface antigen with and without the pre-S domain. The S gene product was expressed as both fusion and nonfusion polypeptides. No difference was observed in the posttranslational modification of the fusion and nonfusion polypeptides. The S proteins were not secreted into the medium but were inserted into the endoplasmic reticulum, glycosylated, and partially extruded into the lumen of the endoplasmic reticulum as 22-nm lipoprotein particles. The oligosaccharide chains on the insect cell-derived S protein were of the N-linked high-mannose form, in contrast to the complex-type oligosaccharides detected on plasma-derived hepatitis B virus surface antigen. The pre-S-S polypeptides were inserted into the endoplasmic reticulum, glycosylated, and modified by fatty acid acylation with myristic acid. A procedure was developed to purify the S protein from cellular membranes by using detergent extraction and immunoaffinity chromatography. The purified S protein was in the form of protein-detergent micelles and was highly antigenic and immunogenic.

Animals↗

PubMed1989

Glycosylation and secretion of human tissue plasminogen activator in recombinant baculovirus-infected insect cells.

Cell lines established from the lepidopteran insect Spodoptera frugiperda (fall armyworm; Sf9) are used routinely as hosts for the expression of foreign proteins by recombinant baculovirus vectors. We have examined the pathway of protein glycosylation and secretion in these cells, using human tissue plasminogen activator (t-PA) as a model. t-PA expressed in Sf9 cells was both N glycosylated and secreted. At least a subset of the N-linked oligosaccharides in extracellular t-PA was resistant to endo-beta-N-acetyl-D-glucosaminidase H, which removes immature, high-mannose-type oligosaccharides. This refutes the general conclusion from previous studies that Sf9 cells cannot process immature N-linked oligosaccharides to an endo-beta-N-acetyl-D-glucosaminidase H-resistant form. A nonglycosylated t-PA precursor was not detected in Sf9 cells, even with very short pulse-labeling times. This suggests that the mammalian signal sequence of t-PA is efficiently recognized in Sf9 cells and that it can mediate rapid translocation across the membrane of the rough endoplasmic reticulum, where cotranslational N glycosylation takes place. However, t-PA was secreted rather slowly, with a half-time of about 1.6 h. Thus, a rate-limiting step(s) in secretion occurs subsequent to translocation and N glycosylation of the t-PA polypeptide. Treatment of Sf9 cells with tunicamycin, but not with inhibitors of oligosaccharide processing, prevented the appearance of t-PA in the extracellular medium. This suggests that N glycosylation per se, but not processing of the N-linked oligosaccharides, is required directly or indirectly in baculovirus-infected Sf9 cells for the secretion of t-PA. Finally, the relative efficiency of secretion decreased dramatically with time of infection, suggesting that the Sf9 host cell secretory pathway is compromised during the later stages of baculovirus infection.

Animals↗

PubMed1989

Insect cells as substrates for biologicals.

Various methods for the growth of insect cells and the production of recombinant human beta-interferon (rHu beta-IFN) by insects cells, Spodoptera frugiperda, IPL-Sf-21AE after infection with recombinant AcNPV are described. Using a suspension perfusion Biospin filter culture system, an IPL-Sf-21AE cell line was propagated in a semi-controlled environment. To obtain and maintain high cell densities and viabilities in this system, the culture medium for suspension methods was modified by supplementing with ZnSO4, ALCl3, methyl cellulose and Darvan #2. Using these improved conditions, multiple harvests of rHu beta-IFN were collected in the cell culture supernatant for downstream processing.

Animals↗

PubMed1988

Characterization of the lipid acyl hydrolase activity of the major potato (Solanum tuberosum) tuber protein, patatin, by cloning and abundant expression in a baculovirus vector.

Patatin is a family of glycoproteins that accounts for 30-40% of the total soluble protein in potato (Solanum tuberosum) tubers. This protein has been reported not only to serve as a storage protein, but also to exhibit enzymic activity. By using a baculovirus system to express protein from the patatin cDNA clone pGM01, it was unambiguously shown that the patatin coded by this DNA has lipid acyl hydrolase and acyltransferase activities. The enzyme is active with phospholipids, monoacylglycerols and p-nitrophenyl esters, moderately active with galactolipids, but is apparently inactive with di- and tri-acylglycerols.

Acyltransferases↗

PubMed1988

Signals important for high-level expression of foreign genes in Autographa californica nuclear polyhedrosis virus expression vectors.

The transcriptional and translational signals required for efficient expression of the chloramphenicol acetyltransferase, beta-galactosidase, and tissue plasminogen activator genes, under the control of the polyhedrin promoter in Spodoptera frugiperda cells infected with Autographa californica nuclear polyhedrosis virus, were investigated by SDS-PAGE and RNA dot blot analysis. The recombinant baculoviruses all contained alterations in the leader sequence or 5' proximal coding region of the polyhedrin gene. Highest levels of foreign proteins and polyhedrin-linked mRNAs were observed when portions of the coding sequence of the polyhedrin gene were fused in phase with the foreign gene. Recombinant viruses in which the foreign gene was inserted upstream from the polyhedrin ATG start codon expressed nonfused products but at lower levels than contructs which produced fusion proteins. A corresponding decrease in the levels of mRNAs produced by such constructs was also observed. Some constructs in which the foreign gene was inserted out of phase downstream from the polyhedrin start codon expressed nonfused protein products at low levels but produced polyhedrin-linked mRNA at levels comparable to vectors which produced protein fusions. These data suggest that reinitiation of translation can take place at AUG start codons a short distance downstream from the primary polyhedrin start codon. These results indicate that sequences immediately upstream from the polyhedrin start codon are important for regulation of transcription and that additional sequences near the AUG start codon can have a dramatic influence on the levels of translation observed.

Animals↗

PubMed1988

Comparisons of host cell DNA insertions and altered transcription at the site of insertions in few polyhedra bacilovirus mutants.

Few polyhedgra (FP) mutants of Autographa californica nuclear polyhedrosis virus (AcNPV) and the closely related strain Galleria mellonella (Gm)NPV have been reported which contain Trichoplusia ni host cell DNA sequences inserted into the viral genome between map units 35.0 and 37.7. New FP mutants are described with alterations of the HindIII-I restriction enzyme fragment (33.8 to 37.7 map units) of AcNPV, either deletions of viral DNA sequences or insertions of spodoptera frugiperda host cell DNA sequences. S. frugiperda DNA insertions from FP mutants were compared to T. ni host DNA insertions from FP mutants previously isolated (M.J. Fraser, G.E. Smith, and M.D. Summers (1983) J. Virol. 47, 287-300). Two host cell DNA sequences isolated from FP mutants, one of T. ni origin and one from S. frugiperda DNA, were transcribed in infected cells. Deletions of viral DNA sequences and insertions of host DNA sequences produce altered transcripts at the site of mutation as determined by both Northern and S1 nuclease analysis. Cell-free translation of cRNAs transcribed from wild-type viral DNA revealed an open reading frame coding for a 25-kDa protein at the site where host cell DNA insertions have been mapped. This was the same size as an infected-cell protein missing from most FP mutants examined.

DNA Transposable Elements↗

PubMed1988

Functional mapping of an AcNPV immediately early gene which augments expression of the IE-1 trans-activated 39K gene.

An early gene which augments the expression of the delayed early/late 39K gene of the baculovirus Autographa californica nuclear polyhedrosis virus (AcNPV) was identified by functional mapping. Transient expression of the plasmid p39CAT, containing the bacterial chloramphenicol acetyltransferase coding sequences under the control of the promoter of the 39K protein, was observed in cells cotransfected with AcNPV DNA digested with several restriction endonucleases. However, when p39CAT was cotransfected with viral DNA digested with Bg/II restriction endonuclease, no CAT activity could be detected. To map the location of the Bg/II-sensitive sequences required for efficient expression of 39CAT, p39CAT and Bg/II-digested viral DNA were cotransfected with a PstI library of AcNPV DNA. The PstI-N fragment restored 39CAT activity. A major early 1.3-kb transcript from this fragment was mapped by S1 nuclease analysis. Transient assay experiments indicated that this major transcript of the PstI-N fragment was produced by an immediate early gene, named IE-N. The PstI-N fragment alone did not activate expression of p39CAT but was required when IE-1 was present in limiting quantities.

Chromosome Mapping↗

PubMed1988

Functional mapping of Autographa california nuclear polyhedrosis virus genes required for late gene expression.

A plasmid containing the bacterial chloramphenicol acetyltransferase (CAT) gene under the control of an Autographa california nuclear polyhedrosis virus (AcNPV) late gene promoter was constructed. This plasmid (pL2cat) also contained the AcNPV hr5 enhancer element. Transient-expression assay experiments indicated that the late promoter was active in Spodoptera frugiperda cells cotransfected with pL2cat and AcNPV DNA but not when pL2cat was transfected alone. Low levels of CAT activity were observed in cells cotransfected with pL2cat and pIE-1 DNAs. However, CAT activity was not induced in a similar plasmid which lacked the cis-linked enhancer element, indicating that the enhancer was required for expression of the late gene. Cotransfection mapping of pPstI clones of AcNPV DNA indicated that the pPstI-G clone of viral DNA contained a factor which further stimulated late gene expression 3- to 10-fold. Transient-expression assay analysis of subclones of pPstI-G localized the trans-active factor to a 3.0-kilobase XbaI fragment. The nucleotide sequence of this fragment was determined and found to contain three potential open reading frames. A computer-assisted search of a protein database revealed no closely related proteins. One of the predicted amino acid sequences contained potential metal-binding domains similar to those found in nucleic acid-binding proteins. Subcloning and subsequent CAT assay indicated that two of the open reading frames were required for the activation of pL2cat. Nuclease S1 mapping of infected and transfected RNAs indicated that the two open reading frames were transcribed as delayed-early genes. Quantitative nuclease S1 analysis and differential DNA digestion of recovered plasmids indicated that the activation of pL2cat was not due to an increase in steady-state levels of mRNA replication of the viral DNA.

Amino Acid Sequence↗

PubMed1988

Identification and comparison of Campoletis sonorensis virus transcripts expressed from four genomic segments in the insect hosts Campoletis sonorensis and Heliothis virescens.

The Campoletis sonorensis virus (CsV; Polydnaviridae) genome consists of at least 28 closed circular superhelical (SH) DNAs. In this study we used complete clones of four SH DNAs to analyze viral transcription both in the adult parasitic wasp host Campoletis sonorensis (Ichneumonidae) and in the lepidopteran host, Heliothis virescens (Noctuidae). CsV genes are expressed in parasitized H. virescens, but no viral transcripts had been characterized from C. sonorensis until this study. The clones of the SH DNAs B, H, M, and O1 were used to probe Northern blots of poly(A)+ RNA isolated from C. sonorensis reproductive tissue and from parasitized H. virescens larvae. All four SH DNAs hybridized to viral transcripts. SH-H,-M, and -O1 hybridized to messages expressed in both hosts. SH-B and -M hybridized to transcripts that were detected only in either C. sonorensis reproductive tissue or parasitized H. virescens larvae. These results suggest that some CsV genes are expressed in a host-specific manner. In a previous study we identified a family of imperfectly conserved tandemly repeated 540-bp repeat elements on SH-B,-H and -O1 (D. A. Theilmann and M. D. Summers, 1987 J. Virol. 61; 2589-2598). Hybridization of the 540-bp repeat regions to Northern blots showed that they were all homologous to viral transcripts. A cDNA clone of a mRNA that is transcribed from the 540-bp repeat region of SH-B was isolated from a lambda gt 10 library and completely sequenced. The sequence data revealed that the 540-bp repeat element was contained within the open reading frame of this gene. These results indicate that transcribed sequences homologous to the 540-bp repeat elements represent a second gene family to be identified within the CsV genome.

Amino Acid Sequence↗

PubMed1987

Two related viral genes are located on a single superhelical DNA segment of the multipartite Campoletis sonorensis virus genome.

Campoletis sonorensis virus (CsV) (Polydnaviridae) is a large eucaryotic DNA virus with a structurally complex genome consisting of 28 or more superhelical (SH) DNA segments. Little is known of the relationship between different SH DNAs, but some SH DNAs do cross hybridize, indicating a relatedness between certain SH DNAs. In a previous study of viral expression in parasitized Heliothis virescens larvae, several partially homologous viral mRNAs were also identified (G. W. Blissard, S. B. Vinson, and M. D. Summers, 1986, J. Virol. 57, 318-327). To study the organization of the viral genome and the relationship between two partially homologous viral mRNAs, we analyzed cDNA clones and a cloned SH DNA segment of the CsV genome. Of the two CsV mRNAs examined (1.6 and 1.0 kb), both mRNAs were abundant at 48 hr after parasitization and the 1.6-kb mRNA was detected as early as 2 hr after parasitization. Nucleotide sequence analyses of cDNA clones representing the two partially homologous CsV mRNAs (1.6 and 1.0 kb) show that the two CsV mRNAs share five regions of imperfect homology (68 to 88%) which include a large part of each mRNA. These data indicate that the two mRNAs are transcribed from two separate but closely related CsV genes. Comparison of predicted amino acid sequences shows that the two related viral genes encode proteins with divergent amino acid sequences. Northern and Southern hybridization analyses using cloned cDNAs as probes showed that one CsV mRNA (1.6 kb) is homologous to CsV SH DNAs W, R, and M, while the other mRNA (1.0 kb) shows strong homology only to SH DNA W. By cloning and Southern hybridization mapping of the 15.8-kbp SH DNA W, we demonstrate that the genes for the related 1.6- and 1.0-kb mRNAs are located on different regions of this single 15.8-kbp SH DNA. Alignment of nucleotide sequences from a cloned viral genomic DNA and a cDNA demonstrates that the CsV gene encoding the 1.6-kb mRNA is a spliced gene containing at least two introns. Conservation of splice junctions between the two mRNAs suggests that the 1.0-kb mRNA is also spliced. These data represent the detailed analysis of two closely related CsV mRNAs abundantly expressed in parasitized H. virescens larvae, the first observation of related viral genes in a eucaryotic DNA virus, the demonstration of splicing in the Polydnaviridae, and the cloning and mapping of one of the largest SH DNA segments of the CsV genome.

Base Sequence↗

PubMed1987

Two of the three influenza viral polymerase proteins expressed by using baculovirus vectors form a complex in insect cells.

Each of the influenza virus polymerase (P) genes PB1, PB2, and PA was inserted into a baculovirus vector under the control of the polyhedrin promoter. In insect (Spodoptera frugiperda) cells infected by each baculovirus recombinant containing a P gene insert, a large amount of the encoded P protein was synthesized. Gel electrophoretic analysis of the total proteins in infected cells revealed the presence of a new protein band corresponding to the encoded P protein that was abundant enough to be stained with Coomassie blue. In cells infected simultaneously with both the PB1 and PB2 baculovirus recombinants, a PB1-PB2 complex was formed that was immunoprecipitated with an antiserum specific for either PB1 or PB2. In cells infected simultaneously with all three P baculovirus recombinants, a PB1-PB2 complex lacking the PA protein was formed. Formation of this PB1-PB2 complex partially mimics events that occur in influenza virus-infected cells, where all three P proteins form a complex with each other (B. M. Detjen, C. St. Angelo, M. G. Katze, and R. M. Krug, J. Virol. 61:16-22, 1987). These results indicate that the ability of PB1 and PB2 to form a complex is an intrinsic property of these two proteins that does not require the participation of other influenza viral gene products. Possible reasons for the absence of the PA protein from the immunoprecipitable P protein complex in insect cells infected by the three P baculovirus recombinants are discussed.

Animals↗

PubMed1987

Synthesis and immunogenicity of the rotavirus major capsid antigen using a baculovirus expression system.

Rotaviruses are the major pathogens that cause life-threatening diarrhea in young children and animals. We inserted a simian rotavirus SA11 gene 6 cDNA into the genome of the baculovirus Autographa californica nuclear polyhedrosis virus adjacent to the strong polyhedrin promoter. The major capsid antigen (VP6) was expressed in high yields (20 to 150 micrograms/10(6) cells) when Spodoptera frugiperda cells were infected with baculovirus recombinants containing SA11 gene 6 inserts. Reactivity with monospecific polyclonal and monoclonal antibodies suggested that VP6, expressed intracellularly or found in the media, maintained native antigenic determinants. VP6 purified from the media from infected cells also possessed a native oligomeric structure, was immunogenic in guinea pigs, and was able to spontaneously assemble into morphologic subunits. Antisera from immunized guinea pigs failed to neutralize virus in plaque reduction assays, but detected homologous and heterologous rotavirus strains when tested by immunofluorescence, immunoprecipitation, and enzyme-linked immunosorbent assays.

Antibodies, Viral↗

PubMed1987

Nucleotide sequence and temporal expression of a baculovirus regulatory gene.

The nucleotide sequence of a trans-activating regulatory gene (IE-1) of the baculovirus Autographa californica nuclear polyhedrosis virus has been determined. This gene encodes a protein of 581 amino acids with a predicted molecular weight of 66,856. A DNA fragment containing the entire coding sequence of IE-1 was inserted downstream of an RNA promoter. Subsequent cell-free transcription and translation directed the synthesis of a single peptide with an apparent molecular weight of 70,000. Quantitative S1 nuclease analysis indicated that IE-1 was maximally synthesized during a 1-h virus adsorption period and that steady-state levels of IE-1 message were maintained during the first 24 h of infection. Northern blot hybridization indicated that several late transcripts which overlap the IE-1 gene were transcribed from both strands. The precise locations of the 5' and 3' ends of these overlapping transcripts were mapped using S1 nuclease. The overlapping transcripts were grouped in two transcriptional units. One unit was composed of IE-1 and overlapping gamma transcripts which initiated upstream of IE-1 and terminated downstream of IE-1. The other unit, transcribed from the opposite strand, consisted of gamma transcripts with coterminal 5' ends and extended 3' ends. The shorter, more abundant transcripts in this unit overlapped 30 to 40 bases of IE-1 at the 3' end, while the longer transcripts overlapped the entire IE-1 gene. Transcription of several early A. californica nuclear polyhedrosis virus genes, in addition to 39K, was shown to be trans-activated by IE-1, indicating that IE-1 may have a central role in the regulation of beta-gene expression.

Journal Article↗