DNA SEQUENCE CODING FOR A POLYPEPTIDE WHICH ENHANCES VIRUS INFECTION OF HOST INSECTS

Abstract: This disclosure relates to an isolated and cloned DNA from a granulovirus virus which comprises an amino acid sequence of the viral gene encoding a polypeptide isolated from occlusion bodies of certain baculoviruses and which polypeptide possesses the biological activity of enhancing baculovirus infectivity. Such proteins termed herein as "enhancins" are found within the viral occlusion body, have a disruptive effect on the insect peritrophic membrane (PM) proteins, and/or interact with the midgut epithelium in such a manner as to permit the increased adsorption, penetration and uptake of virus particles by midgut cells with a concomitant increase in host mortality. Disclosed herein is a recombinant DNA sequence which codes for the enhancin protein of the Helicoverpa armigera granulovirus virus. The DNA sequence is shown in SEQ. ID. NO. :1 and the open reading frame is shown in SEQ. ID. NO.: 1: base pairs 271-2976. The amino acid sequence of the enhancin protein is shown in SEQ. ID. NO.: 2.

Description: DNA SEQUENCE CODING FOR A POLYPEPTIDE WHICH ENHANCES VIRUS INFECTION OF HOST INSECTS REFERENCE TO RELATED APPLICATIONS This is a continuation-in-part patent application of a copending parent provisional patent application serial number 60/002,743, filed August 24, 1995. FIELD OF THE INVENTION The invention relates to the cloning and sequencing of novel viral genes from certain baculoviruses for insect control. More particularly, the invention relates to an isolated and cloned DNA from a virus which encodes an amino acid sequence of a polypeptide isolated from occlusion bodies of certain baculoviruses and which enhance baculovirus infectivity, termed herein as "enhancins". BACKGROUND OF THE INVENTION The Baculoviridae, comprised of the nucleopolyhedroviruses (NPV) and granuloviruses (GV), are a family of viruses that primarily infect insects. Autographa californica multiple NPV (AcMNPV) is the best studied insect virus. Well developed in vitro systems have permitted extensive molecular characterization. In addition, it is utilized as a tool for foreign gene expression and an alternative to chemical pesticides In 1959 a protein, the synergistic factor (SF), was identified that increased the susceptibility of Pseudaletia unipuncta larvae to a dual infection by both PsunGV-H and a PsunNPV (Tanada, 1959). The SF was estimated to be present at levels up to 5% of the granulin protein (Tanada, 1985). The granules of the cabbage looper granulovirus, Trichoplusia ni, TnGV, also contain a protein, the enhancin (Derksen & Granados, 1988; Corsaro et al. , 1993) that enhances infections of AcMNPV and other viral species like the T. ni single enveloped nucleopolyhedrovirus TnSNPV (Greenspan Gallo et al., 1991). The exact mechanisms of enhancement are unknown, but two different modes of action have been proposed. The PsunGV-H enhancin was reported to interact with viral particles and increase the binding of the latter to the insect midgut microvilli (Tanada, 1985). Studies on the mode of action of the VEF isolated from Trichoplusia ni (cabbage looper) granulovirus virus (TnGV) showed that the VEF caused rapid degradation of the peritrophic membrane which lines the midgut lumen of lepidopteran larvae. Larval bioassays suggested that this alteration made the peritrophic membrane more permeable to invading baculoviruses resulting in at least a 25-fold increase in larval mortality. Closely related to, or identical with, the VEF protein is a lipoprotein, originally isolated in crude form from a Hawaiian strain of Pseudaletia unipuncta granulovirus virus (PuGV-H), but not cloned or sequenced. It is described by Tanada and co-workers as the "synergistic factor" (SF) and as having a calculated molecular weight between 90K and 160K. The SF was released from the capsule upon dissolution in the midgut, and was the localized to the microvillar surface of the midgut cell membrane where it caused an apparent increase in the uptake of enveloped nucleocapsids. The binding of SF to the midgut membrane was found to be specific with a calculated equilibrium constant of 1.57X10 sup-9 M. It was postulated that the two proteins (VEF and SF) are closely related and have similar dual modes-of-action: peritrophic membrane disruption and increased virus uptake. Evidence to support this relationship comes from southern hybridization's of PuGV-H genomic DNA with the VEF gene and western blots of dissolved PuGV-H occlusion bodies with an anti- VEF polyclonal antiserum. Tanada determined that this SF in the capsule of PuGV-H increased the larval susceptibility to P. unipuncta nuclear polyhedrosis virus (PuNPV). This was confirmed by the sequence similarity shown in Pat. No. 5,475,050. These proteins are referred to herein generally as "enhancins", but are also referred to as virus enhancing factors (VEF) and/or as synergistic factors (SF). Genes encoding enhancins (VEF and SF) and pest control compositions comprising this factor and nuclear polyhedrosis viruses are the subject matter of U.S. Pat. Nos. 5,475,090, 4,973,667, and 5,011,685. Since viral enhancing proteins are important at early stages of host infection, it is important to identify and locate the genes for other similar proteins within the viral genome. A need, therefore, exists to clone and sequence the genes of other related proteins. It is an object of this invention to satisfy such a need. SUMMARY OF THE INVENTION The invention relates to the cloning and sequencing of novel viral genes from certain baculoviruses for insect control. More particularly, the invention relates to an isolated and cloned DNA from a granulovirus virus which comprises an amino acid sequence of the viral gene encoding a polypeptide isolated from occlusion bodies of certain baculoviruses and which polypeptide possesses the biological activity of enhancing baculovirus infectivity. This invention also relates to isolated and purified baculovirus proteins which are characterized by enhancing the infectivity of baculoviruses. Such proteins termed herein as "enhancins" are found within the viral occlusion body, have a disruptive effect on the insect peritrophic membrane (PM) proteins, and/or interact with the midgut epithelium in such a manner as to permit the increased adsorption, penetration and uptake of virus particles by midgut cells with a concomitant increase in host mortality. The invention relates to the cloning and sequencing of a novel viral gene. The present invention includes a recombinant DNA sequence which codes for the enhancin protein of the Helicoverpa armigera granulovirus virus. The DNA sequence is shown in SEQ. ID. NO.: 1 and the open reading frame is shown in SEQ. ID. NO.: 1: base pairs 271- 2976. The amino acid sequence of the enhancin protein is shown in SEQ. ID. NO.: 2: base pairs 1-901, which has two domains one stretching from residues 1-550, and the other from 551-901. A recombinant protein can be produced from this newly isolated DNA sequence with an expression vector comprising the DNA sequence and a suitable promoter which, when put into a suitable host will be capable of resulting in the expression of an amino acid sequence having the physical, chemical, and/or biological properties of the enhancin protein of the Helicoverpa armigera granulovirus virus. The expression vector can be either a recombinant plasmid adapted for transformation of a microbial host or recombinant baculovirus. A recombinant plasmid which includes a recombinant DNA sequence which codes for the enhancin protein of the Helicoverpa armigera granulovirus virus was used to transform E. coli and deposited at the Agricultural Research Service Culture Collection (NRRL), Northern Regional Research Center, Agricultural Research Service, U.S. Department of Agriculture, 1815 North University Street, Peoria, Illinois 61604 and assigned accession number: NRRL B-21614. The recombinant enhancin protein, and/or the recombinant baculovirus for expressing the enhancin protein can be used in a composition toxic to insects along with a compound toxic to insects. The activity of the composition is partially through the action of the enhancin, the enhancin having a disruptive effect on the insect peritrophic membrane proteins such that it interacts with the midgut epithelium in such a manner as to enhance the increased absorption, penetration, and/or uptake of virus by midgut cells with a corresponding increase in host mortality. A more complete appreciation of the invention and the advantages thereof will be apparent as the same becomes better understood by reference to the following details of description when considered in connection with the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWING Figures la and lb show a comparison of the amino acid sequences of the Pseudaletia unipuncta, Trichoplusia ni and Helicoverpa armigera enhancins. DESCRIPTION OF THE PREFERRED EMBODIMENT One of the discoveries of the present invention is the cloning and sequencing of the enhancin gene found in the Helicoverpa (Heliothis) armigera granulovirus (HearGV). While, the enhancin gene found in the PsunGV-H virus is virtually identical to the previously characterized Trichoplusia ni GV (TnGV) enhancin gene, a comparison of the predicted amino-acid (aa) sequences of TnGV enhancin (901 aa) with the HearGV enhancin (902 aa) revealed herein demonstrated an overall identity of only 80%, with greater conservation (88%), and hence higher homology, from amino-acids 1-550. The addition of the newly discovered HearGV enhancin to an AcMNPV inoculom increased the infectivity of baculoviruses in insect larvae. This discovery will aid in the effort to control certain pests without the use of pesticides. Moreover, the host range of the Helicoverpa (Heliothis) armigera granulovirus is significantly broader than that of other known granulovirus viruses, such as TnGV or PuGV, which suggests that the HearGV enhancin disclosed herein may have broader biological activity, increasing its potential effectiveness in the control of insects (John J. Hamm, 1982, incorporated herein by reference). A pathogen, here a insect baculovirus, that is an effective microbial control against several species of economically damaging insects, as is HearGV due to its broad specificity, is much more useful against a complex of insects attacking a crop and therefore more economically viable as a commercially useful insect pathogen than a similar enhancin with a narrower insect target range. I. Identification of Enhancins in Granulovirus Species Proteins from eight different granuloviruses that infected 4 different Lepidopteran families were analyzed by SDS-PAGE and Western blotting in an attempt to identify novel enhancin proteins. The granulovirus (GV) species used in this study were: CpGV, Estigmene acrea (EsacGV), Heliocoverpa armigera (HearGV), PlinGV, Pieris rapae (PiraGV), PsunGV-H, Scotogramma trifolii (SctrGV) and TnGV. Viral stocks were maintained in the lab as granule suspensions and/or infectious hemolymph isolated from previously infected larvae. Larval infections, purification of granules and enhancin was as previously described (Derksen & Granados, 1988; Greenspan & Gallo et al., 1991, both incorporated herein by reference). For the SDS-PAGE and Western blot analysis granules were dissolved in 100 mM NaHCO3 (pH 10.5) and incubated at room temperature for 15 minutes. The protein concentration was determined using the Bradford assay kit (Promega Corp., Madison, WI). Approximately 10 jug of each GV solution was analyzed on SDS-PAGE. Gels were silver stained. A duplicate gel was transferred onto PVDF membrane (NEN Research Products, Dupont, Boston, MA) using a protocol provided by the manufacturer (Biorad, Melville, NY). Western blots were analyzed using a rabbit anti-VEF-TrpE polyclonal antibody at a dilution of 1:5000. Cross-reactive bands were visualized using goat anti-rabbit alkaline phosphatase conjugated secondary antibody, this was done at a dilution of 1:3000 (Sigma, St. Louis, MO). Five different GV species, four infecting the Noctuidae family and one infecting the Pieridae family, were found to contain proteins that did cross-react with the anti-enhancin polyclonal antibodies already mentioned. However, PlinGV, which infects indian meal moth larvae (Noctuidae), CpGV, that infects codling moth larvae (Tortricidae) and EsacGV, that infects saltmarsh caterpillar larvae (Arctiidae), were not seen to have crossreacting proteins. Two granulovirus species, C. pomonella GV, and E. acrea GV, were previously identified as having an enhancin, and were used as positive controls in the research herein disclosed. We also used standard neonate bioassays performed with both EaGV and CpGV, in attempt to demonstrate enhancin activity therein, however, neither displayed any ability to enhance the infectivity of baculovirus. The enhancins can be subdivided into of three different groups based on their respective molecular weights, and migration patterns: 104 kD for the TnGV, PsunGV-H and PiraGV enhancins, 108-110 kD for HearGV enhancin and 120 kD for SctrGV enhancin. In standard T. ni neonate bioassays HearGV, PsunGV-H and SctrGV demonstrated an ability to "enhance" the level AcMNPV infectivity, confirming the presence of an enhancin. PsunGV-H, was the first granulovirus for which the presence of an enhancin in its occlusion body was described and documented. A. Cloning and Sequencing Viral genomic DNA was isolated from granules as described by Smith and Summers (1982), incorporated herein by reference. All of the restriction endonucleases and modifying enzymes were purchased from Promega Corp. (Madison, WI). Restriction endonucleases were routinely used in DNA digests using a universal enzyme restriction endonuclease buffer (lOx = 0.33 M Tris/ Acetate pH 7.85, 0.65 M Potassium acetate, 0.1 M Magnesium acetate, 0.04 M Spermidine Tri-Chloride, 5 mM Dithiothreitol). DNA gel electrophoresis, Southern blotting and hybridization were performed as described (Hashimoto et al., 1991). Viral genomic DNA was cloned into either pUC18/19 (Yanisch- Perron et al., 1985) or Bluescript SK (Stratagene, La Jolla, CA). The complete HearGV enhancin gene was identified through hybridization of a 1.75 Kb Kpnl fragment of the HearGV gene, that crosshybridized to a TnGV internal enhancin fragment on a blot, where the HearGV genomic DNA had been digested with BamHI, EcoRI, Hindlll and Kpnl. A cross-hybridizing BamHI fragment with an estimated size of 5.2 Kb was cloned into the pUC18 expression vector and a DNA restriction map was thereafter generated. The Kpnl fragment already mentioned, as well as the 1.45 Kb Sstll- BamHI fragment, and deletion fragments derived thereof, were sequenced on both strands. HearGV genomic clones were sequenced using cesium chloride purified DNA and a commercially available sequencing kit (United States Biochemical Corp., Cleveland, OH). Deletion clones were generated by Bal31 digestion as described, the Erase-a-Base ExoIII/Sl digestion kit (Promega Corp., Madison, WI) or an ExoIII/Mung bean nuclease kit (Stratagene). Sequence products were analyzed on gels prepared with 6% Sequagel Rapid Sequencing Solution (National Diagnostics, Atlanta, GA). Sequence information from the generated genomic clones was developed through the use of the Sequence Analysis Software Package of the Genetics Computer Group (Madison, WI, Versions 7.2 and 7.3). The enhancin gene from HearGV was cloned and sequenced as described above. Every nucleotide on both strands of the enhancin gene sequence was sequenced a minimum of 2 times. The DNA sequence and deduced amino acid sequence of the HearGV gene is shown in SEQ. ID. NO: 1 and SEQ. ID. NO: 2, respectively. Sequence data analysis of the HearGV DNA revealed an open reading frame (ORF) of 2706 nucleotides that encodes a protein containing 902 amino acids and a mass of 104.6 kD. Similarly, PsunGV-H clones have an ORF of 2703 bp that encodes a protein of 901 amino acids with a mass of 104.2 kD. (Please note that the DNA and amino acid sequence for PsunGV-H and TnGV enhancins are presented in the Sequence Listing of Patent No. 5,475,090). B. Isolation of RNA and Primer Extension Analysis Total RNA was isolated from HearGV infected T. ni larvae at daily intervals from 0-8 days post infection (p. ), using guanidine isothiocyanate (GIT) as described (Sambrook et al., 1989 inc. by ref.). Four to eight larvae were collected for each timepoint, frozen in liquid nitrogen and then ground in a glass Potter tube in the presence of 4 ml of GIT. The resulting larval suspension was layered onto a CsCl gradient and spun for 16 hours at 35K. The RNA pellet was then collected, precipitated, washed, dried and quantified. For primer extension analysis of the enhancin promoter, a primer (HAZR2) 5' CAC GGC GGC AGC ACG G 3' complementary to nucleotides 43-28 downstream of the AUG initiator codon of the enhancin gene was used. Approximately 100 ng of the primer was labeled using 100 mCi of g-ATP (Dupont Company, Boston, MA) and T4 polynucleotide kinase (Promega Corp., Madison. WI). Five nanograms of the primer were incubated with 50 μg of total RNA isolated from HearGV infected T. ni larvae isolated at 1, 4, 5, 6, 7 and 8 days post infection. The primer extension reaction was according to Ausubel et al., (1989 inc. by ref.) with two modifications: the AMV reverse transcriptase (RVT) (Promega, Madison, WI) was incubated at 50°C and actinomycin D was added at a final concentration of 75 μg/ml to inhibit the DNA-dependent DNA polymerase activity of the RVT. Reaction products were analyzed on a 6% polyacrylamide (PAA)-gel and compared to a sequencing ladder of clone HABAM, that contains a 5.2 Kb BamHI fragment from the HearGV genome and has the complete enhancin coding sequence also sequenced with the same primer. C. Generating the Plasmid For the analysis of the 5' end of the HearGV enhancin message, a 800 bp Ncol fragment from clone HABAM was subcloned in vector pSL1180 (Pharmacia, Piscataway, NJ) to yield pHANCO. A 470 bp Munl-BamHI fragment (425 bp HearGV sequences and 45 bp pSL1180 multilinker) was subcloned into Bluescript KS+ digested with EcoRI and BamHI to yield pHAMB. To generate a probe complementary to the 5' end of the HearGV enhancin gene, plasmid pHAMB was linearized at the Hindlll multilinker site, which lies upstream of the Munl/EcoRI fusion site. Transcription with T7-RNA polymerase (Gibco BRL, Gaithersburg, MD) was according to the manufacturer's protocol in the presence of 20 μCi UTP. After transcription, the template was digested for 15 minutes at 37°C by adding 2 μl RNase free DNase (10,000 U/ml; Boehringer Mannheim, Indianapolis, IN ). Following digestion, 100 μl TSE (TE plus 0.5% SDS) was added, followed by extraction with PCI (phenol/chloroform/isoamyl alcohol, 25:24: 1). The probe was further purified by elution from a Nick G-50 gravity flow column (Pharmacia, Piscataway, NJ). Two microliters of the resulting purified probe were analyzed on a 6% polyacrylamide-gel. The presence of several premature stops further necessitated purification on a 6% PAA gel. The full length probe (530 nucleotides) was isolated from this gel. For analysis of the 3' end of the enhancin message, clone HASB, which has a 1.45 Kbp Sstll-BamHI fragment of the HearGV enhancin gene cloned into Bluescript SK-, was cut with M . An antisense T7 RNA probe was synthesized and purified as described above. Upon gel analysis the probe was found to be > 95 % full length (372 nucleotides) and used without further purification. The HearGV gene inserted into the plasmid vector was transfected into E. coli and deposited under the Budapest Treaty on August 22, 1996 at the Agricultural Research Service Culture Collection (NRRL), Northern Regional Research Center, Agricultural Research Service, U.S. Department of Agriculture, 1815 North University Street, Peoria, Illinois 61604 and assigned accession number: NRRL B-21614. The applicant agrees to be bound by the terms of the Budapest Treaty regarding availability. D. RNAse Protection Analysis For RNase protection analysis, 20 μg of total RNA were mixed with 105 cpm of antisense RNA probe, precipitated and resuspended in 30 μl of hybridization buffer (80% formamide, 40 mM PIPES, 400 mM NaCl, 1 mM EDTA). After heating for 10 minutes at 80°C, the mixture was left to hybridize overnight at 50°C. Following hybridization, unprotected RNA was digested by adding 350 μl RNase mix (10 mM Tris pH 7.5, 5 M EDTA, 0.3 M NaCl, 2 μl 10 mg/ml RNase A, 5 μl 100,000 U/ml RNase Tl) and incubated at 30oC for 60 minutes. The digestion was stopped by adding SDS to a final concentration of 0.5 % . Samples were further purified by digestion with Proteinase K (3 μl of a 15 mg/ml premix; Boehringer Mannheim) for 30 minutes at 37°C. The samples were extracted twice with 400 μl PCI and precipitated at room temperature (RT) for 30 minutes by addition of 1 ml of 96% ethanol. Samples were spun at room temperature, washed twice with 70% ethanol at room temperature, dried briefly and resuspended in 20 μl loading buffer (50% formamide, BFB and XCFF 0.05%). Reaction products were analyzed on 6% polyacrylamide sequencing (3 1 analysis) or slab gels (5* analysis). Product sizes were estimated by comparison to a sequencing ladder of clone HABAM sequenced with primer HAZR2 and comparison to a 123bp ladder (GIBCO BRL). Gels were exposed to XAR 5 film in cassettes with intensifying screens (Kodak, Rochester, NY). The RNase protection analysis of the 3 'end of the HearGV enhancin offers some evidence both for transcription termination and transcriptional read through into the second ORF. No canonical polyadenylation signal AAUAAA, was present upstream of the transcription termination signal. An alternative poly (A) sequence closely resembling the poly(A) sequence, AACAAA, was present between nucleotides 3003-3008. It has been reported that the presence of a similar sequence downstream of the β-thalassaemia gene or other alternative poly(A) like sequences results in elongated and unstable transcripts and changes to the steady state levels of mRNA. This may explain why, apart from the two major protected RNA species during RNase protection, several minor protected species appeared to be protected. Alternatively, it is possible that the HearGV enhancin gene transcript does not have a poly(A) tail. This would not be unique for baculoviruses. Recently, a convincing case has been made for the absence of a poly(A) tail on Spodoptera exigua NPV polyhedrin mRNA. The 3' RNase protection assay also indicated that the enhancin ORF and the downstream ORF maybe on one message. The occurrence of bi-cistronic messengers is not uncommon for baculoviruses. Analysis of the Hindlll-M region of Orgyia pseudotsugata NPV for instance, has shown that bi,- and multi-cistronic messages originated from baculovirus late promoter motifs that had different 5' ends but the same 3' end. E. Transcriptional Analysis of the Enhancin Genes Transcriptional analysis of the HearGV gene reveals several more interesting features of enhancin genes. Of the three baculovirus late promoter motifs conforming to the consensus NTAAG sequence, present in the region upstream of the enhancin ORF the one that is predominantly used, TTAAG, is positioned only 3 nucleotides from the translational AUG start codon. Transcriptional analysis of the TnGV enhancin gene has also shown that the ATAAG motif present at -8 to -4 relative to the AUG initiator codon serves as the only transcriptional initiation point. From our work it has become clear that the AUG closest to the promoter is the translation initiation codon. That this codon is the translation initiator codon is based on several observations seen infra. First, the context of the initiator codon -3 AUCAUGC +4 is similar to the Kozak consensus sequence for translation initiation, -3 A/GYYAUGG +4. The pyrimidine C present at position +4 in the HearGV gene, and in all enhancin genes sequenced so far, is the only notable exception to the Kozak consensus. It has been found through mutation studies that replacement of the consensus G in position +4 acts to downregulate eukaryotic translation. Second, the mass of the protein observed in protein gels corresponds well with the first AUG in the ORF acting as the translational initiator codon. Third, the next possible AUG lies 240 nucleotides downstream in the coding region and does not confer to Kozak rules at all. The observation that a baculovirus late promoter in the HearGV gene is present so proximal to the start codon that upon transcription it gives rise to a 5' leader with a maximum length of 7 nucleotides, is unique to the enhancin genes, and has only been previously reported for the TnGV enhancin gene. Leaders of eukaryotic messages are seldom shorter than 7-10 nucleotides and usually average between 25 and 50 nucleotides. Deletion studies of the 5' non-coding region on the translational efficiency of phosphoglycerate kinase mRNA in yeast, for instance, have shown that even if the leader length is decreased to 7 nucleotides, translation at 50% of the optimal rate still occurs. The observation that a shorter leader impairs the fidelity of initiation by eukaryotic ribosomes, has been confirmed using an in vitro transcription and translation system (Kozak, 1991, inc. by ref.). This negative effect on fidelity of initiation, however, can be almost completely eliminated by the presence, or through the introduction of, secondary structure with a DG of -19 kcal/mol in the mRNA at an optimal distance of 14 nucleotides from the AUG initiator codon (see, Kozak, 1990, 1991). Transcription of the HearGV enhancin gene also results in a short 5' leader sequence with considerable secondary structure (DG -16.7 kcal/mol: GCG MFold analysis) within the first 100 nucleotides of the mRNA. The total amount of enhancin present in granules has been estimated to be 5% of total protein. This suggests that enhancin gene promoters are rather strong. The combination of promoter proximity, suboptimal AUG context and secondary structure downstream of the AUG initiator codon, may very well result in a complex mechanism of transcription regulation, mRNA stability, and translation efficiency unique to baculoviruses π. Comparisons of Homology After sequencing the HearGV enhancin gene, as laid out above, it was determined that it shared significant sequence homology with both the TnGV and PsunGV-H enhancin genes (see Table 1 below). Analysis of this similarity in sequence between the PsunGV-H and HearGV enhancin genes at the DNA and deduced amino acid sequence level, and thereafter comparison with the TnGV enhancin gene sequence revealed several interesting characteristics of the genes, and their respective homology. The TnGV and PsunGV-H enhancin genes are virtually identical from 325 nucleotides upstream of the enhancin ORF and throughout the partial ORF identified downstream of the enhancin gene. Since in Hindlll digests of TnGV and PsunGV-H genomic DNA, 19 of the 26 visible fragments comigrate the observed conservation of homology suggests that both granuloviruses are strongly related and may have only recently evolved divergently. TABLE 1- Comparison of the TnGV, PsunGV-H and HearGV DNA and amino acid sequences ND: Not Determined ID: Identity SIM: Similarity n: nucleotide A comparison of the PsunGV-H and TnGV enhancin genes (Figure 1 & Table 1), showed that they are virtually identical at both the DNA and the amino acid sequence level over almost their entire length. The amino acid sequences differ in only fifteen amino acids. Seven changes are caused by a 21 nucleotide reciprocal frameshift (reflected by the dissimilarity in amino acids 652-658, Fig. 4). The remaining eight amino acid changes are caused by point mutations in the PsunGV-H enhancin gene. The DNA homology from - 450 through -325 nucleotides upstream of the translational initiator codon is 35 % . This increases to 94% from -325 to -1 and 98% for the remaining DNA. There is a 98% identity for the peptide sequences. When the HearGV and PsunGV-H enhancin genes are compared, virtual identity is not present (Figure 1 & Table 1). For 270 nucleotides upstream of the translational start codon the homology is 40%. This increases to an overall homology of 77% within the ORF, decreases to 46% for the intergenic region and increases again to 69% for the putative downstream ORF. Comparison of the amino acid sequences of the two enhancins revealed that the overall identity and similarity are 81 % and 90% respectively. Amino acids 1-550 show an identity and similarity of 89% and 95%. This similarity decreases to 69% and 84%, when amino acids 551-902, respectively, are compared. While amino acid sequences 1-550 score significantly above the similarity average, the amino acids 551-901 scored significantly below it, when the GCG PlotSim software program is used to generate a similarity comparison. A. Genetic Analysis The above analysis demonstrates that enhancins have two distinct domains. This may be a reflection of the fact that two activities have been found to be associated with enhancins, namely the breakdown of peritrophic membranes through a proteolytic activity and the enhancement of viral binding to receptors present in the midgut microvilli. The HearGV gene has a consensus baculovirus late promoter motif shared with the PsunGV-H gene (PsunGV-H: ATAAG; HearGV: TTAAG,) at positions -8 through -4 relative to the translational start codon. Two other late motifs, GTAAG and ATAAG, were present in the HearGV sequence within 270 basepairs upstream of the ATG. The PsunGV-H sequence has no other late promoter motifs within 400 nucleotides upstream of the AUG. A second late promoter motif, ATAAG was present at 36-32 nucleotides upstream of the enhancin translational stop codon (positions 2991-2993) with the start codon of a putative ORF (PsunGV-H: 3057-3059; HearGV: 3051-3053) downstream of it. No canonical polyadenylation consensus sequences were identifiable in the short intergenic region between the translational stop codon of the enhancin gene and the translational start codon of the downstream ORF. It was noted however, that the HearGV DNA sequence here is relatively AT rich, and the PsunGV-H enhancin gene has two sequential stop codons, TAATAA. Either of these DNA sequences may meet the requirements for polyadenylation sites. B. Genetic Divergence HearGV Enhancin Gene Sequence The HearGV enhancin gene has several baculovirus late promoters upstream of the enhancin ORF and encodes a significantly different protein from previously described enhancins, one which differs in both mass and deduced amino acid sequence from the enhancin of TnGV and PsunGV-H. Therefore it is a separate and novel gene product from previously described genes. While the HearGV enhancin has 902 amino acids and a mass of 104.6 D, it migrates at a higher than expected mass of 108-110 kD. We hypothesize that this variant migration behavior may be caused by the presence of a sequential stretch of proline residues at the C-terminal end of this sequence (positions 880-883), that is lacking from the PsunGV-H sequence. Comparison of the partial amino acid sequence of the downstream ORF (HearGV: AUG=3051, PsunGV-H: AUG=3057) shows an identity and similarity of 82 and 89%, respectively (Fig. 5; Table 1). Analysis of the amino acid sequences with a GCG program that recognizes signal sequences with a probability of 95% and an accuracy of 75 % at a score level of 3.5, revealed that the first twenty amino acids of both the HearGV and the PsunGV-H downstream ORF, most likely encode a similar signal sequence (the signal cleave scores are 8.8 and 7.3 respectively). The PsunGV-H downstream ORF has been shown to be open for at least 170 amino acids. The organization, transcription and translation of these enhancin genes hold several special features that can be modulated to serve as useful alternatives to the application of chemical pesticides for insect management. As Table 2, below, demonstrates that the use of enhancin proteins, specifically the HearGV enhancin protein, dramatically affects the mortality of host larvae (see Table 2 below). Also as seen in Table 2, the amounts of the enhancins needed to substantially increase insect host mortality are very small. SUBSΠTLΠΈ SHEET (RULE 26) TABLE 2- The Use of Various Enhancin Proteins to Increase the Infectivity of Baculoviruses, and Thereby the Mortality in Treated Insect Larvae Percent mortality in T.Ni neonate larvae following ingestion of one AcMNPV OB per larva at two different concentrations of enhancin derived from four different granulovirus viruses. AcMNPV control at 1 OB/larva was 50% and 47% for 2 replications; Tn= Trichoplusia ni GV; Ha= Heliothis armigera GV; St= Scotogramma trifolii GV; Pu= Pseudaletia unipuncta GV. Accordingly, it is to be understood that the embodiments of the invention herein described are merely illustrative of the application of the principles of the invention. Reference herein to details of the illustrated embodiments are not intended to limit the scope of the claims, which themselves recite those features regarded as essential to the invention. SEQUENCE LISTING (1) GENERAL INFORMATION: (i) APPLICANT: Granados, Robert R. (ii) TITLE OF INVENTION: DNA SEQUENCE CODING FOR A POLYPEPTIDE WHICH ENHANCES VIRUS INFECTION OF HOST INSECTS (iii) NUMBER OF SEQUENCES: 2 (iv) CORRESPONDENCE ADDRESS: (A) ADDRESSEE: Barnard, Brown _ Michaels (B) STREET: 306 E. State St., Suite 220 (C) CITY: Ithaca, (D) STATE: NY. (E) COUNTRY: USA (F) ZIP: 14850 (v) COMPUTER READABLE FORM: (A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS (D) SOFTWARE: Patentln Release #1,0, Version #1.30 (vi) CURRENT APPLICATION DATA: (A) APPLICATION NUMBER: (B) FILING DATE: 23-AUG-1996 (C) CLASSIFICATION: (vii) PRIOR APPLICATION DATA: (A) APPLICATION NUMBER: 60/002,743 (B) FILING DATE: 24-AUG-1995 (viii) ATTORNEY/AGENT INFORMATION: (A) NAME: Michaels, Christopher A. (B) REGISTRATION NUMBER: 34,390 (C) REFERENCE/DOCKET NUMBER: BTI-32 (ix) TELECOMMUNICATION INFORMATION: (A) TELEPHONE: (607)273-1711 (B) TELEFAX: (607)273-2609 (2) INFORMATION FOR SEQ ID NO:l; (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 3186 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: circular (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: Helicoverpa armigera granulovirus virus (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION: 271..2976 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:l: CTATATGTGG TAAGATTGAT TGAATGTTCG TTTGCTCTCG CGTTGTTTAA AAAACGCAAC 60 AAACACCGGT ATTAGCGGAG CGGTCTCCTG TATCCGGCAG CGGACTGTCG CGTTAGCGTG 120 CACCACTACG CCGGCCGGAA CGCGACCGTG TTTGAGCGCC AACCAATTGT CCTGTGGCCT 180 CAAATAAGGC GGCACTGCTA AAGATGGTAC CGGTAGAATA ATGGAAGTTT TGTTCTTTCA 240 CACAACATTA CGCTGTCTTA AATTAAGATT ATG TCG TAC AAC GTA ATT GTG CCT 294 Met Ser Tyr Asn Val lie Val Pro 1 5 ACT ACC GTG CTG CCG CCG TGG CTG AGG ATC GGT CAA AAT TGG ATA TTC 342 Thr Thr Val Leu Pro Pro Trp Leu Arg lie Gly Gin Asn Trp lie Phe 10 15 20 GCT AGA CAC AGA CGC ACC GAA GTC GGT GTG GTG TTA CCT GCA AAC ACA 390 Ala Arg His Arg Arg Thr Glu Val Gly Val Val Leu Pro Ala Asn Thr 25 30 35 40 AAG TTT CGG GTT CGA GCC GAT TTC GCT AAA TGG GGC ATC ACG AGG CCC 438 Lys Phe Arg Val Arg Ala Asp Phe Ala Lys Trp Gly lie Thr Arg Pro 45 50 55 GTG ATC GTG CGC CTC TTG AAC AAC AAC CGT AAC ACC GAG CGC GAG ATA 486 Val lie Val Arg Leu Leu Asn Asn Asn Arg Asn Thr Glu Arg Glu lie 60 65 70 AAT TTA ACC AAC GAC CAA TGG ATA GAG ATG GAG CAC GAG CAC GAG TGT 534 Asn Leu Thr Asn Asp Gin Trp lie Glu Met Glu His Glu His Glu Cys 75 80 85 GTG CCG TTC GTC GAC TGG CCG GTG GGT GAA AAA AAC ACC ATG GCC GAG 582 Val Pro Phe Val Asp Trp Pro Val Gly Glu Lys Asn Thr Met Ala Glu 90 95 100 GTA CAC TTT GAA ATC GAC GGA CCA CAC ATA CCG CTT CCC GTG TAC GTG 630 Val His Phe Glu lie Asp Gly Pro His lie Pro Leu Pro Val Tyr Val 105 110 115 120 TTC AAC ACG AGA CCC GTG GAA AAC TTT AAG AGC GAG TAC CGC CAG AGT 678 Phe Asn Thr Arg Pro Val Glu Asn Phe Lys Ser Glu Tyr Arg Gin Ser 125 130 135 TCG TCG GGC TAC TGC TTC CTG TAT TTG GAC CTG GTG TGT ATT TTG GTG 726 Ser Ser Gly Tyr Cys Phe Leu Tyr Leu Asp Leu Val Cys lie Leu Val 140 145 150 CCG CCG GCT AGT AAA AAC GTG TTA CTA GAC ACG GAC CTG TTT GAG CTC 774 Pro Pro Ala Ser Lys Asn Val Leu Leu Asp Thr Asp Leu Phe Glu Leu 155 160 165 CAT CAA TTT TAT AAC GAA ATT ATT AAT TAC TAT GAC GAT TTG TGC GGT 822 His Gin Phe Tyr Asn Glu lie lie Asn Tyr Tyr Asp Asp Leu Cys Gly 170 175 180 TTG GTC GAG GAC CCG TAC GCA GAC ACT GTG GAT TCA AAC CTA CCC AAC 870 Leu Val Glu Asp Pro Tyr Ala Asp Thr Val Asp Ser Asn Leu Pro Asn 185 190 195 200 AAG GCG GCA TTC GTG AAA GCC GAC GGT GGT GGT CCC GGC GGT GCT TAT 918 Lys Ala Ala Phe Val Lys Ala Asp Gly Gly Gly Pro Gly Gly Ala Tyr 205 210 215 TAC GGG GCA TTC TGG ACG GCT CCC GCC AGC ACA AAT CTA GGC GAA TAT 966 Tyr Gly Ala Phe Trp Thr Ala Pro Ala Ser Thr Asn Leu Gly Glu Tyr 220 225 230 CTC CGG GTG TCG CCC ACC AAT TGG ATG GTT ATT CAC GAG CTG GGT CAC 1014 Leu Arg Val Ser Pro Thr Asn Trp Met Val lie His Glu Leu Gly His 235 240 245 GCG TAC GAT TTC GTG TTT ACT GTG AAC ACT CGC CTT ATA GAA ATC TGG 1062 Ala Tyr Asp Phe Val Phe Thr Val Asn Thr Arg Leu lie Glu He Trp 250 255 260 AAC AAC TCG TTC TGC GAT CGG ATA CAA TAC ACG TGG ATG AAC AAA ACC 1110 Asn Asn Ser Phe Cys Asp Arg He Gin Tyr Thr Trp Met Asn Lys Thr 265 270 275 280 AAG CGA CAG CAA CTG GCT CGC ATT TAC GAG AAC CAA CGA CCC CAG AAG 1158 Lys Arg Gin Gin Leu Ala Arg He Tyr Glu Asn Gin Arg Pro Gin Lys 285 290 295 GAG GCG GCT ATT CAA GCG CTA ATC GAC AAC AAT GTA CCG TTT GAT AAT 1206 Glu Ala Ala He Gin Ala Leu He Asp Asn Asn Val Pro Phe Asp Asn 300 305 310 TGG GAT TTT TTT GAG AAA CTC AGC ATT TTT GCA TGG CTG TAC AAT CCG 1254 Trp Asp Phe Phe Glu Lys Leu Ser He Phe Ala Trp Leu Tyr Asn Pro 315 320 325 CAA AGG GGA CTG GAC ACT TTG CGT AAT ATC AAT CAT TCG TAC AGG TTG 1302 Gin Arg Gly Leu Asp Thr Leu Arg Asn He Asn His Ser Tyr Arg Leu 330 335 340 CAC GCT GCC CGC AAT CCA GTT ACG CCA TAC CCG CAA ATT TGG GCA TGG 1350 His Ala Ala Arg Asn Pro Val Thr Pro Tyr Pro Gin He Trp Ala Trp 345 350 355 360 TTG ATG AGT TGT GGT TAC GAC AAC TTT TGG TTG TAC TTT AAT CGA ATA 1398 Leu Met Ser Cys Gly Tyr Asp Asn Phe Trp Leu Tyr Phe Asn Arg He 365 370 375 GGT TTG TAC CCT GCC GAT TTT TAC ATT AAC GAA CAC AAT AAA GTC GTG 1446 Gly Leu Tyr Pro Ala Asp Phe Tyr He Asn Glu His Asn Lys Val Val 380 385 390 CAT TTC AAT CTG CAC ATG CGC GCC TTA GCG CTG GGA CAG AGT GTG CGT 1494 His Phe Asn Leu His Met Arg Ala Leu Ala Leu Gly Gin Ser Val Arg 395 400 405 TAC CCT ATC AAA TAT ATT ATT ACC GAC TTT GAT TTA TTG CAA AAG AAC 1542 Tyr Pro He Lys Tyr He He Thr Asp Phe Asp Leu Leu Gin Lys Asn 410 415 420 TAC GAC ATA AAG CAA TAT TTA GAG AGT AAC TTT GAT CTT GTA ATA CCG 1590 Tyr Asp He Lys Gin Tyr Leu Glu Ser Asn Phe Asp Leu Val He Pro 425 430 435 440 GAA GAG TTG AGA CAG ACC GAT CTG GTT GCG GAC GTA CGA GTG GTG TGC 1638 Glu Glu Leu Arg Gin Thr Asp Leu Val Ala Asp Val Arg Val Val Cys 445 450 455 GTC ATC GAC GAC CCA TCA CAA ATT ATA GGT GAA CCG TTT AGT TTG TAC 1686 Val He Asp Asp Pro Ser Gin He He Gly Glu Pro Phe Ser Leu Tyr 460 465 470 GAC GGT AAC GAA CGA GTT TTT GAG AGC ACA GTA GCC ACG GAT GGT AAC 1734 Asp Gly Asn Glu Arg Val Phe Glu Ser Thr Val Ala Thr Asp Gly Asn 475 480 485 ATG TAT TTA GTT GGC GTG GGT CCG GGA GTG TAC ACT CTA CGC GCG CCC 1782 Met Tyr Leu Val Gly Val Gly Pro Gly Val Tyr Thr Leu Arg Ala Pro 490 495 500 CGC GGC AAA GAC AAA CGC TAC AAA CTC CAC TTG GCA CAC TCG CCC AAC 1830 Arg Gly Lys Asp Lys Arg Tyr Lys Leu His Leu Ala His Ser Pro Asn 505 510 515 520 GAG CCG GTT CAT CCG GCT AAC GAT CAC ATG TAT CTA CTC GTG ACA TAT 1878 Glu Pro Val His Pro Ala Asn Asp His Met Tyr Leu Leu Val Thr Tyr 525 530 535 CCA TAT TAC AAT CAA ACG TTA ACC TAC ACA CGA TAT ATA ACT TCG GAC 1926 Pro Tyr Tyr Asn Gin Thr Leu Thr Tyr Thr Arg Tyr He Thr Ser Asp 540 545 550 CTT GCA ATA GAC GCG GCC CAC TTA TTC GGT ACC GAC CGC TTG TAT GTG 1974 Leu Ala He Asp Ala Ala His Leu Phe Gly Thr Asp Arg Leu Tyr Val 555 560 565 GCC ACG ATA TAT TTC GAC GCA TTA CAG CAG ACT GTG ACC GTG TAT CTG 2022 Ala Thr He Tyr Phe Asp Ala Leu Gin Gin Thr Val Thr Val Tyr Leu 570 575 580 AAC AAT ATT CGC ACT GGC AGG GAA AAC AAC ACC ACC TTG TAT TTT GAA 2070 Asn Asn He Arg Thr Gly Arg Glu Asn Asn Thr Thr Leu Tyr Phe Glu 585 590 595 600 ATG GAA ATA CAT AAT CCG TTT ATT GGC ACT TCT TCG AAA TTT ACT TTG 2118 Met Glu He His Asn Pro Phe He Gly Thr Ser Ser Lys Phe Thr Leu 605 610 615 TTA GAG GAT AAC GTC ACG ATG CGC CAG GGA TAT TAT AAA TTT CCG GCG 2166 Leu Glu Asp Asn Val Thr Met Arg Gin Gly Tyr Tyr Lys Phe Pro Ala 620 625 630 GTC ACC TTT AGC TCG ATT CGT TTA CAC ATA AGA GAT GAC AAC AGA CTA 2214 Val Thr Phe Ser Ser He Arg Leu His He Arg Asp Asp Asn Arg Leu 635 640 645 ATG CTG GTA GAT AAA TAT TTA CCA GCG GGC GAC ACG TTG CTG TTC ATG 2262 Met Leu Val Asp Lys Tyr Leu Pro Ala Gly Asp Thr Leu Leu Phe Met 650 655 660 TTT CCC AAT CAA ATC GTT GAC AAT AAT ATA TTT CCC GAT GGG TCA ATA 2310 Phe Pro Asn Gin He Val Asp Asn Asn He Phe Pro Asp Gly Ser He 665 670 675 680 TTG ACC AGC ACA TAC AAC CGT ATA AAA GAA CAA GCT GCT TTC ATC GAA 2358 Leu Thr Ser Thr Tyr Asn Arg He Lys Glu Gin Ala Ala Phe He Glu 685 690 695 AAC CAT AAA CAG CTG TTG TAC ATT GAA AAC GAA TTA CGC GAC AGC ATA 2406 Asn His Lys Gin Leu Leu Tyr He Glu Asn Glu Leu Arg Asp Ser He 700 705 710 TAC TTG GCG TCA CAA TTT GTG AAT AGT GAT TCC AAC GAA TTT TTA AAG 2454 Tyr Leu Ala Ser Gin Phe Val Asn Ser Asp Ser Asn Glu Phe Leu Lys 715 720 725 TAT TTT CCT GAT TAT TTT AGA GAC CCT CAT ACG TTC TCA TAC CTG TTT 2502 Tyr Phe Pro Asp Tyr Phe Arg Asp Pro His Thr Phe Ser Tyr Leu Phe 730 735 740 CGG TTC AGA GGC TTG GGT GAT TTC ATG TTG CTA GAA TTA CAA ATT GTG 2550 Arg Phe Arg Gly Leu Gly Asp Phe Met Leu Leu Glu Leu Gin He Val 745 750 755 760 CCT ATA CTA AAT TTG GCT TCG GTA CGT GTA GGT AAC CAT CAC AAC GGG 2598 Pro He Leu Asn Leu Ala Ser Val Arg Val Gly Asn His His Asn Gly 765 770 775 CCC CAC TCG TAT TTC AAT ACA ACG TAT CTA TCG GTG GAA GTG CGC GAC 2646 Pro His Ser Tyr Phe Asn Thr Thr Tyr Leu Ser Val Glu Val Arg Asp 780 785 790 ACA AGC GGT GGT GTT GTG TTT TCG TAT TCA CGC CTC GGT AAC GAA CCG 2694 Thr Ser Gly Gly Val Val Phe Ser Tyr Ser Arg Leu Gly Asn Glu Pro 795 800 805 ATG ACA CAC GAA CAT CAC AAA TTC GAA GTG TTC AAA GAT TAT ACA ATA 2742 Met Thr His Glu His His Lys Phe Glu Val Phe Lys Asp Tyr Thr He 810 815 820 CAC TTG TTC ATA CAA GAA CCT GGC CAA AGG TTA CAA TTA ATA GTC AAC 2790 His Leu Phe He Gin Glu Pro Gly Gin Arg Leu Gin Leu He Val Asn 825 830 835 840 AAA ACA CTC GAC ACG GCG CTG CCC AAC TCT CAA AAC ATT TAC GCT CGC 2838 Lys Thr Leu Asp Thr Ala Leu Pro Asn Ser Gin Asn He Tyr Ala Arg 845 850 855 CTC ACG GCC ACG CAA TTA GTA GTG GGA GAA CAG AGC ATT ATC ATT AGC 2886 Leu Thr Ala Thr Gin Leu Val Val Gly Glu Gin Ser He He He Ser 860 865 870 GAC GAT AAC GAC TTT GTA CCG CCA CCA CCA CGC GTT AAT TGT GGC GAC 2934 Asp Asp Asn Asp Phe Val Pro Pro Pro Pro Arg Val Asn Cys Gly Asp 875 880 885 CAG CAG ATA AGA GTA GTG GAA ACT TTA AAA ATG ATA GCG TTC 2976 Gin Gin He Arg Val Val Glu Thr Leu Lys Met He Ala Phe 890 895 900 TAGAAATTTT TTAACAAAAC ACAAAGTGAA TTGCAGTCGC TTGTTATCTT TGGCCACGGT 3036 ATGGCGCGCG CTTCGTATTA TGTGCTACTA CTATCCTTGG TGGTGTTATC GGTTAATGGA 3096 TATTCGTTTT ATTCGTCCAT CGAAGCCCTA CTTTTGAACG ATCGCACACA AATCTGCATT 3156 GGCGATTGTT ACGAGCGCAA CGGTCAACAT 3186 (2) INFORMATION FOR SEQ ID NO:2: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 902 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: Met Ser Tyr Asn Val He Val Pro Thr Thr Val Leu Pro Pro Trp Leu 1 5 10 15 Arg He Gly Gin Asn Trp He Phe Ala Arg His Arg Arg Thr Glu Val 20 25 30 Gly Val Val Leu Pro Ala Asn Thr Lys Phe Arg Val Arg Ala Asp Phe 35 40 45 Ala Lys Trp Gly He Thr Arg Pro Val He Val Arg Leu Leu Asn Asn 50 55 60 Asn Arg Asn Thr Glu Arg Glu He Asn Leu Thr Asn Asp Gin Trp He 65 70 75 80 Glu Met Glu His Glu His Glu Cys Val Pro Phe Val Asp Trp Pro Val 85 90 95 Gly Glu Lys Asn Thr Met Ala Glu Val His Phe Glu He Asp Gly Pro 100 105 110 His He Pro Leu Pro Val Tyr Val Phe Asn Thr Arg Pro Val Glu Asn 115 120 125 Phe Lys Ser Glu Tyr Arg Gin Ser Ser Ser Gly Tyr Cys Phe Leu Tyr 130 135 140 Leu Asp Leu Val Cys He Leu Val Pro Pro Ala Ser Lys Asn Val Leu 145 150 155 160 Leu Asp Thr Asp Leu Phe Glu Leu His Gin Phe Tyr Asn Glu He He 165 170 175 Asn Tyr Tyr Asp Asp Leu Cys Gly Leu Val Glu Asp Pro Tyr Ala Asp 180 185 190 Thr Val Asp Ser Asn Leu Pro Asn Lys Ala Ala Phe Val Lys Ala Asp 195 200 205 Gly Gly Gly Pro Gly Gly Ala Tyr Tyr Gly Ala Phe Trp Thr Ala Pro 210 215 220 Ala Ser Thr Asn Leu Gly Glu Tyr Leu Arg Val Ser Pro Thr Asn Trp 225 230 235 240 Met Val He His Glu Leu Gly His Ala Tyr Asp Phe Val Phe Thr Val 245 250 255 Asn Thr Arg Leu He Glu He Trp Asn Asn Ser Phe Cys Asp Arg He 260 265 270 Gin Tyr Thr Trp Met Asn Lys Thr Lys Arg Gin Gin Leu Ala Arg He 275 280 285 Tyr Glu Asn Gin Arg Pro Gin Lys Glu Ala Ala He Gin Ala Leu He 290 295 300 Asp Asn Asn Val Pro Phe Asp Asn Trp Asp Phe Phe Glu Lys Leu Ser 305 310 315 320 He Phe Ala Trp Leu Tyr Asn Pro Gin Arg Gly Leu Asp Thr Leu Arg 325 330 335 Asn He Asn His Ser Tyr Arg Leu His Ala Ala Arg Asn Pro Val Thr 340 345 350 Pro Tyr Pro Gin He Trp Ala Trp Leu Met Ser Cys Gly Tyr Asp Asn 355 360 365 Phe Trp Leu Tyr Phe Asn Arg He Gly Leu Tyr Pro Ala Asp Phe Tyr 370 375 380 He Asn Glu His Asn Lys Val Val His Phe Asn Leu His Met Arg Ala 385 390 395 400 Leu Ala Leu Gly Gin Ser Val Arg Tyr Pro He Lys Tyr He He Thr 405 410 415 Asp Phe Asp Leu Leu Gin Lys Asn Tyr Asp He Lys Gin Tyr Leu Glu 420 425 430 Ser Asn Phe Asp Leu Val He Pro Glu Glu Leu Arg Gin Thr Asp Leu 435 440 445 Val Ala Asp Val Arg Val Val Cys Val He Asp Asp Pro Ser Gin He 450 455 460 He Gly Glu Pro Phe Ser Leu Tyr Asp Gly Asn Glu Arg Val Phe Glu 465 470 475 480 Ser Thr Val Ala Thr Asp Gly Asn Met Tyr Leu Val Gly Val Gly Pro 485 490 495 Gly Val Tyr Thr Leu Arg Ala Pro Arg Gly Lys Asp Lys Arg Tyr Lys 500 505 510 Leu His Leu Ala His Ser Pro Asn Glu Pro Val His Pro Ala Asn Asp 515 520 525 His Met Tyr Leu Leu Val Thr Tyr Pro Tyr Tyr Asn Gin Thr Leu Thr 530 535 540 Tyr Thr Arg Tyr He Thr Ser Asp Leu Ala He Asp Ala Ala His Leu 545 550 555 560 Phe Gly Thr Asp Arg Leu Tyr Val Ala Thr He Tyr Phe Asp Ala Leu 565 570 575 Gin Gin Thr Val Thr Val Tyr Leu Asn Asn He Arg Thr Gly Arg Glu 580 585 590 Asn Asn Thr Thr Leu Tyr Phe Glu Met Glu He His Asn Pro Phe He 595 600 605 Gly Thr Ser Ser Lys Phe Thr Leu Leu Glu Asp Asn Val Thr Met Arg 610 615 620 Gin Gly Tyr Tyr Lys Phe Pro Ala Val Thr Phe Ser Ser He Arg Leu 625 630 635 640 His He Arg Asp Asp Asn Arg Leu Met Leu Val Asp Lys Tyr Leu Pro 645 650 655 Ala Gly Asp Thr Leu Leu Phe Met Phe Pro Asn Gin He Val Asp Asn 660 665 670 Asn He Phe Pro Asp Gly Ser He Leu Thr Ser Thr Tyr Asn Arg He 675 680 685 Lys Glu Gin Ala Ala Phe He Glu Asn His Lys Gin Leu Leu Tyr He 690 695 700 Glu Asn Glu Leu Arg Asp Ser He Tyr Leu Ala Ser Gin Phe Val Asn 705 710 715 720 Ser Asp Ser Asn Glu Phe Leu Lys Tyr Phe Pro Asp Tyr Phe Arg Asp 725 730 735 Pro His Thr Phe Ser Tyr Leu Phe Arg Phe Arg Gly Leu Gly Asp Phe 740 745 750 Met Leu Leu Glu Leu Gin He Val Pro He Leu Asn Leu Ala Ser Val 755 760 765 Arg Val Gly Asn His His Asn Gly Pro His Ser Tyr Phe Asn Thr Thr 770 775 780 Tyr Leu Ser Val Glu Val Arg Asp Thr Ser Gly Gly Val Val Phe Ser 785 790 795 800 Tyr Ser Arg Leu Gly Asn Glu Pro Met Thr His Glu His His Lys Phe 805 810 815 Glu Val Phe Lys Asp Tyr Thr He His Leu Phe He Gin Glu Pro Gly 820 825 830 Gin Arg Leu Gin Leu He Val Asn Lys Thr Leu Asp Thr Ala Leu Pro 835 840 845 Asn Ser Gin Asn He Tyr Ala Arg Leu Thr Ala Thr Gin Leu Val Val 850 855 860 Gly Glu Gin Ser He He He Ser Asp Asp Asn Asp Phe Val Pro Pro 865 870 875 880 Pro Pro Arg Val Asn Cys Gly Asp Gin Gin He Arg Val Val Glu Thr 885 890 895 Leu Lys Met He Ala Phe 900 INDICATIONS RELATING TO A DEPOSITED MICROORGANISM (PCT Ru\e \ 3bis) A. The indication . made below relate lo the microorganism referred lo in the descriptii on page 4 . line 10 B. IDENTIFICATION OF DEPOSIT Further deposits are identified on an additional shed P~j Name of depositary institution AGRICULTURAL RESEARCH SERVICE CULTURE COLLECTION (NRRL) Address of depositan institution (including postal code and country) Northern Regional Research Center Agricultural Research Service, U.S. Department of Agriculture 1815 North University Street Peoria, Illinois 61604 U.S.A. Date of deposit Accession Number August 22, 1996 NRRL B-21614 C. ADDITIONAL INDICATIONS tleave Manic if not applicable) This information is continued on an additional sheet Q D. DESIGNATED STATES FOR WHICH INDICATIONS ARE MADE (if the indications' arc not fm all designated Slate*) E. SEPARATE FURNISHING OF INDICATIONS (leave blank if not applicable) 1 he indications listed hclov. w ill be siihmilled to the International Bureau later (specif,- the general nature n) the indications c , e . ". ic t riiion Λ umber n/ 1 >epo<aι "1 for recci . mg Office use onl \ For international Bureau use onl \ [ I T his sheet w s recci \ cd w ith the internalional application | ) T ins sheet w as received In the International Bureau < Authorized officer Authorized officer Form PCT/RO'l 34 (JtιK 1992)

Claims: What is claimed is: 1. The DNA sequence selected from the group consisting of: a) A DNA sequence which codes for an amino acid sequence having the sequence of SEQ. ID. NO.2; b) A DNA sequence which codes for the enhancin protein of the Helicoverpa armigera granulovirus virus; c) A DNA sequence having the sequence of SEQ. ID. NO.: 1; d) A DNA sequence having the sequence of SEQ. ID. NO. : 1: base pairs 271- 2976; e) A DNA sequence encoding an amino acid sequence having the SEQ. ID. NO.: 2: residues 1-550; and f) A DNA sequence encoding an amino acid sequence having the SEQ. ID. NO.: 2: residues 551-901. 2. An expression vector comprising a DNA sequence of claim 1 and a suitable promoter which, when put into a suitable host will be capable of resulting in the expression of a amino acid sequence having the physical, chemical, and/or biological properties of the enhancin protein of the Helicoverpa armigera granulovirus virus. 3. A recombinant protein produced using a DNA sequence of claim 1. 4. A recombinant DNA sequence which codes for the enhancin protein of the Helicoverpa armigera granulovirus virus. 5. A recombinant protein which retains the physical, chemical and biological properties of the enhancin of the Helicoverpa armigera granulovirus virus enhancin, said enhancin having a disruptive effect on the insect peritrophic membrane proteins such that it interacts with the midgut epithelium in such a manner so as to enhance the increased absorption, penetration, and/or uptake of virus by midgut cells with a corresponding increase in host mortality 6. A recombinant plasmid adapted for transformation of a microbial host, said plasmid comprising a plasmid vector into which a DNA segment which codes for the Helicoverpa armigera granulovirus virus enhancin gene, or the variant DNA sequences thereof which, when translated into a protein, would retain the physical, chemical and biological properties of said enhancin. 7. The plasmid of claim 6, wherein said plasmid has all of the identifying characteristics of the plasmid deposited at the Agricultural Research Service Culture Collection (NRRL), Northern Regional Research Center, Agricultural Research Service, U.S. Department of Agriculture, 1815 North University Street, Peoria, Illinois 61604 and assigned accession number: NRRL B-21614. 8. The recombinant protein as produced by the plasmid in claim 6. 9. A composition toxic to insects comprising an enhancin produced using a DNA sequence of claim 1 and a compound toxic to insects, the activity of such composition is partially through the action of said enhancin, said enhancin having a disruptive effect on the insect peritrophic membrane proteins such that it interacts with the midgut epithelium in such a manner as to enhance the increased absorption, penetration, and/or uptake of virus by midgut cells with a corresponding increase in host mortality. 10. A recombinant baculovirus adapted for transformation of a microbial host, said recombinant baculovirus comprising a DNA segment which codes for the Helicoverpa armigera granulovirus virus enhancin gene, or the variant DNA sequences thereof which, when translated into a protein, would retain the physical, chemical and biological properties of said enhancin. 11. The baculovirus of claim 10 further comprising a compound toxic to insects, the activity of such composition is partially through the action of said baculovirus when ingested by insects. 12. The recombinant baculovirus vector of claim 10, wherein said recombinant baculovirus is an Autographa calif ornica baculovirus. 13. The recombinant protein as produced by said recombinant baculovirus expression vector in claim 10. 14. A composition toxic to insects comprising an enhancin produced using a DNA sequence of claim 1 and a compound toxic to insects, the activity of such composition is partially through the action of said enhancin, said enhancin having a disruptive effect on the insect peritrophic membrane proteins such that it interacts with the midgut epithelium in such a manner as to enhance the increased absorption, penetration, and/or uptake of virus by midgut cells with a corresponding increase in host mortality.

Inventors: GRANADOS ROBERT R, GRANADOS, ROBERT, R.
Applicants: THOMPSON BOYCE PLANT RES, BOYCE THOMPSON INSTITUTE FOR PLANT RESEARCH, INC.
Application year: 1996
Main classification: C07K14
IPC codes: A01N63, C07K14