[go: up one dir, main page]

WO1996001320A2 - Sequence genomique complete du virus autographa californica de la polyhedrose nucleaire - Google Patents

Sequence genomique complete du virus autographa californica de la polyhedrose nucleaire Download PDF

Info

Publication number
WO1996001320A2
WO1996001320A2 PCT/IB1995/000578 IB9500578W WO9601320A2 WO 1996001320 A2 WO1996001320 A2 WO 1996001320A2 IB 9500578 W IB9500578 W IB 9500578W WO 9601320 A2 WO9601320 A2 WO 9601320A2
Authority
WO
WIPO (PCT)
Prior art keywords
sequence
acnpv
orf
virus
gene
Prior art date
Application number
PCT/IB1995/000578
Other languages
English (en)
Other versions
WO1996001320A3 (fr
Inventor
David Bishop
Robert Possee
Martin Ayres
Original Assignee
Natural Environment Research Council
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Natural Environment Research Council filed Critical Natural Environment Research Council
Priority to AU28972/95A priority Critical patent/AU2897295A/en
Publication of WO1996001320A2 publication Critical patent/WO1996001320A2/fr
Publication of WO1996001320A3 publication Critical patent/WO1996001320A3/fr

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/14011—Baculoviridae
    • C12N2710/14111—Nucleopolyhedrovirus, e.g. autographa californica nucleopolyhedrovirus
    • C12N2710/14141—Use of virus, viral particle or viral elements as a vector
    • C12N2710/14143—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector

Definitions

  • This invention relates to Autographa califomica nuclear polyhedrosis virus DNA sequences and particularly to the DNA sequence of the complete virus genome.
  • Autographa califomica nuclear polyhedrosis virus (AcNPV) is a widely studied baculovirus which has been used to form the basis of a polypeptide expression systems (see e.g. US-P-4,745,051 and EP 0 327 626). Modified baculoviruses have also been proposed for use as viral insecticides.
  • Baculoviruses are invertebrate-specific viruses with large, circular, covalently closed, double-stranded DNA genomes (Francki et al., 1991).
  • early genes and early gene promoters are identified in the AcNPV C6 sequence, genes that have not been reported hitherto and which via substitution of the downstream gene, or by promoter duplication, alone, or in concert with other promoters of AcNPV, or other baculoviruses will allow the expression of foreign genes, or operons, or duplicated AcNPV genes at defined times in the baculovirus infection process.
  • the genome data provides information on which specific restriction enzymes do not cut the AcNPV C6 genome.
  • the data also identifies restriction enzymes that cut the sequence only once, or twice, or thrice, etc, and the location of all such sites.
  • the latter sites can now be removed by deletion (for non-essential, including coding sequences), or by site directed mutage ⁇ esis (for essential, including coding sequences).
  • AcNPV derivatives can be constructed that only cut the genome at defined locations (new sites) by these specific enzymes. This will allow the linearisation of the virus DNA at defined locations in order to facilitate the introduction of foreign genes.
  • the new sites may be located within a reporter gene sequence for the efficient identification of recombinant expression vectors by the loss of the reporter gene function.
  • Additional sequences representing these restriction sites may also be placed in flanking sequences of essential genes to improve the efficient recovery of recombinants using transfer vectors that provide both the foreign gene and the unmodified essential flanking sequences. Further, the use of a number of such enzyme sites strategically located in the virus genome, will allow the preparation of genetically stable, multiple gene expression vectors.
  • the genome sequence allows for the identification of essential and non- essential genes in relation to the infection course of the virus in different types of cultured cells and host insects.
  • genes that will be proven to be essential to the infection course of the virus in cultured cells and insect hosts and other genes that are non-essential to one or other or both substrates can now be specifically removed from the AcNPV genome without affecting the expression of essential, including flanking, genes, or the replication of the virus in certain cultured cells. Removal of such genes and corresponding reduction in the AcNPV genome size and hence cost to the overall transcription, translation and other processes induced by the virus, or certain other processes and structures naturally operative in the host cell, will provide a preferred expression vector system and improved virus replication.
  • the modifications will allow the time when foreign gene products are made to be regulated and improvements to the amounts and quality of such products.
  • removal of such genes will be to the benefit of commercial manufacturing processes and environmental safety.
  • the removal of natural AcNPV genes that facilitate the persistence of AcNPV in the environment and, or that provide for the productive infection of insect larvae and, or that facilitate the transmission of infectious virus in the environment by affecting characters such as determinants of host range, cell death and larval degradation will be suitable candidate genes to remove.
  • the loss of any or all such functions and the derivation of disabled virus expression vectors will prohibit the occurrence of any adverse consequences of virus escape from laboratories or manufacturing establishments, by eliminating any potential effect on natural insect populations in the environment, or the likelihood of re-acquisition of such genes and functions from natural sources.
  • both ⁇ uclease and protease genes deleterious to the transcription, expression and product accumulation of foreign genes expressed by baculovirus vectors have been identified. Removal of such genes will also provide for improved expression vectors.
  • sequence information allows new sites to be identified for the insertion of single or multiple gene expression cassettes composed of viral promoters, foreign gene(s) of choice, including new polyadenylation sites and transcription terminators.
  • cassettes can now be positioned so that they do not affect resident genes, their promoters, terminators, polyadenylation sites, or give mRNA species that act as antisense sequences to required viral genes.
  • the sites may be contiguous. Additionally, or alternatively, the sites may be non-contiguous thereby facilitating expression of foreign genes without incurring deleterious positional effects on mRNA transcription.
  • the genome sequence allows genetically engineered virus insecticides to be produced by exploiting the advantages described above with regard to tailored genome size, genetic stability, multiple foreign gene expression, and by the exploitation of gene dose.
  • the ability to introduce genes into proscribed sites in the AcNPV genome and derivatives without affecting resident genes thereof includes the ability to transfer from other baculoviruses and other origins individual genes, cassettes of genes, and other DNA sequences that will affect the virus host range, its transmission and stability in the environment.
  • the benefits will include effects on the LD50 (lethal doses required to kill 50% of target species) and LT50 (lethal time in 50% of members of an infected host species) and other biological properties of the natural virus.
  • Such sequences will include, for example, genes representing baculoviruses with alternative host ranges, including genes from viruses that have proved impossible to grow, or to clone in cultured cells.
  • the AcNPV genome contains genes and sequences that alone or in concert with host factors regulate the expression of viral genes generally in a temporally controlled fashion.
  • the genome sequence allows the identification of all such regulatory viral genes and sequences.
  • sequence information contained in SEQ LD NO. 1 may be used in the manufacture of a range of novel polynucleotides which may be used industrially.
  • the invention according to one aspect thereof provides the use of sequence information derivable from the complete genomic sequence of AcNPV in the manufacture of a polynucleotide for use in an industrially applicable process.
  • the invention further provides the use of sequence information derivable from the complete genomic sequence of AcNPV in the manufacture of a polynucleotide capable of acting as a control sequence in the expression of a foreign gene in an insect or insect cell.
  • sequence information is derivable solely and/or primarily from said complete genomic sequence.
  • the information may be derived from sequence data present in said complete genomic sequence, but essentially absent from or present in incomplete form in previously available sequence data.
  • sequence analysis of the complete genomic sequence contained in SEQ ID NO. 1 has revealed the presence of 154 open reading frames of which 91 have not hitherto been described.
  • These novel open reading frames are identified in Table 1 as ORF 13, 22-26, 28-30, 32, 38, 41-46, 50-60, 62-63, 66, 68-79, 81-87, 91-92, 96-98, 101-103, 106-126, 129-130, 140-146, 148-150, 152 and 154.
  • the present invention thus includes isolated polynucleotides containing a nucleotide sequence which corresponds to one of the aforementioned ORFs.
  • corresponding to as used herein is meant a nucleotide sequence which is identical to the disclosed sequence or which has sufficient homology to hybridize to the aforementioned sequence under hybridization conditions corresponding to TM -19 to TM -25.
  • the corresponding sequences may be at least 80%, preferably at least 90% and most preferably at least 95% homologous to the stated sequence. Desirably the degree of homology is not less than 98%),
  • the invention also includes polypeptides obtainable by expressing polynucleotides corresponding to the aforementioned ORFs. Such expression may be achieved by incorporating an insert having a sequence corresponding to one of the aforementioned polynucleotides into a suitable expression vector in association with and under the control of appropriate expression control sequences.
  • Information derived from the SEQ ID NO. 1 may be used to optimize polypeptide expression in expression systems based upon baculoviruses by selecting appropriate control sequences.
  • the present invention further provides a method of synthesizing a polypeptide by expressing the polypeptide in an insect or cultured insect cell which has been transformed by an expression vector derived from AcNPV, the expression vector containing a coding sequence coding for the polypeptide and control sequences responsible for control of replication of the expression vector and/or transcription of the coding sequence, characterized in that the control sequences are selected on the basis of sequence information derived from SEQ ID NO. 1.
  • the information derived from SEQ ID NO. 1 additionally enables the efficiency of polypeptide expression to be increased by modifying the nucleotide sequence being expressed so as to take advantage of the preferred codon usage which is characteristic of the ORFs which have been identified in SEQ ID NO. 1.
  • the invention provides a method of synthesizing a polypeptide by expressing the polypeptide in an insect or cultured insect cell which has been transformed by an expression vector derived from AcNPV, the expression vector containing a coding sequence coding for the polypeptide and control sequences responsible for control of replication of the expression vector and transcription of the coding sequence, characterized in that the coding sequence is adapted by selecting codons in accordance with the preferred codon usage of AcNPV.
  • Preferred codon usage differs between species and expression of foreign polypeptides can often .be hampered if codons contained in the coding sequence to be expressed correspond to less preferred codons in the expression host.
  • Knowledge of the preferred condo ⁇ usage for AcNPV allows the DNA sequence of the insert being expressed to be modified so as to increase the proportion of codons which are preferred for AcNPV.
  • the coding sequence should be modified (if necessary) so as to ensure that one or more (and preferably at least ten, most preferably at least 15) of the amino acids indicated below are encoded by the indicated codons:
  • Val GTG A person of ordinary skill in this art could therefore employ the preferred codons for the different amino acids as described herein in order to the optimize expression of a variety of different heterologous proteins using the claimed expression vector and the claimed methods.
  • the genes encoding a desired heterologous protein could be modified to include the more preferred codons (see list above) and to exclude the less preferred codons (see list below for codons to avoid).
  • DNA sequences encoding different enzymes, hormones, toxins, antibodies and receptors may be modified as described herein to enhance production.
  • proteins useful in agriculture proteins are modified to alter insect behavior in a desirable way), clinical therapy, and or diagnosing disease could be modified.
  • these different proteins include, but are not limited to the following: hepatitis B virus core antigen, hepatitis B virus surface antigen, bovine Herpesvirus-1 glycoprotein glV, Human immunodeficiency virus type 1 (HIV-l) envelope protein gp 120, HTV-l envelope protein gp 160, HTV-l Gag protein, HTV-l Gag-pol fusion protein, HTV-l Integration protein, HTV-l Major core p24, HTV-l Nef protein, HTV-l Pol protein, HTV-l protease, HTV-l Rev protein, Human immunodeficiency virus type 2 Gag precursor protein, Human T-cell lymphotxophic virus type 1 (HTLV-1) p20E protein, HTLV-1 gp46 protein, HTLV-1 040* protein, Bacillus thuringiensis subspecies kurstaki HD-73 delta endotoxin, Bacillus thuringiensis subspecies aizawai 7.21 crystal
  • the coding sequence should be modified so that the following codons are avoided (these being less preferred codons for the indicated amino acids): Amino Acid Codon(s) to be avoided
  • Chaperon sequences shall be defined as a sequence encoding a protein which contains a nucleotriphosphate and which is capable of leading, escorting or "chaperoning" a different protein into the nucleus from the cytoplasm.
  • open reading frame shall refer to a specific length of DNA with a methioni ⁇ e start codon and terminated by a translation stop codon.
  • Predicted sequences describes a sequence of putative protein as derived from the DNA sequence in the open reading frame. Using the genetic code one of ordinary skill in this art could readily define a protein sequence corresponding to each of the 154 open reading frames presented in Table 1. Putative is defined as "assumed to exist” e.g. “encodes a putative alkaline exonuclease” (infra under the Heading "Gene functions", last para.).
  • data is used to define nucleotide sequences based on computer predictions; particularly when assuming the function of putative gene product.
  • a consensus sequence is defined as a sequence specific for a biological function or characteristic as determined by computer sequence analysis. Consensus sequences may also be used to define a sequence (and corresponding characteristic or function for this sequence) which is shared or found to be homologous among different species.
  • DNA wobble is a term used to explain how the third nucleotide of a codon can vary or "wobble" and still encode the same amino acid.
  • TTT and TTC both encode the amino acid phenylalanine and that ATT, ATC, and ATA all encode for the amino acid isoleucine.
  • Protease sequence defines those amino acid sequences found on certain proteins which are known or presumed (because of a consensus sequence) to play a role in the enzymatic digestion of other proteins.
  • Ligase sequences refers to an amino acid sequence that is capable of joining or ligating the ends of RNA molecules or joining the ends of DNA molecules.
  • T4 DNA ligase is used to join or ligate compatible "sticky” or “blunt” ends of DNA derived after restriction enzyme digestion.
  • "Sticky” and “blunt” are terms in the art to define how the ends of DNA molecules appear after restriction enzyme digestion.
  • Helicase sequences are protein sequences in enzymes associated with the unfolding of DNA molecules.
  • polymerase sequences refer to either RNA or DNA polymerases. These enzymes are responsible for synthesizing RNA or DNA from the appropriate template.
  • Deleterious sequences refer to a sequence that can have a deleterious effect on the production or efficiency of certain proteins being produced in the host cells.
  • a protease sequence might be deleterious if this portease specifically breaks down the foreign recombinant protein synthesized in the insect cell via a baculovirus expression vector.
  • Enhancer sequences are DNA sequences which increase the transcription of a virus gene. For example, dot matrix analysis of the AcNPV sequence against itself and its complement revealed eight regions of direct and inverted repetitive DNA sequences (hr ⁇ -hr5). The hrs are involved in enhancing early mRNA transcription and act as origins of DNA replication (infra, first two sentences under the heading "AcNPV genomic organization and repetitive DNA”.
  • disrupted, interrupted, mutated, and deleted are sometimes used interchangeably in reference to specific ORFs. It is intended that these terms refer to a condition where the encoded protein is no longer functional due to a disruption, interruption, mutation, or some other interference that prevents, shuts down, nullifies or inhibits the otherwise named function.
  • SEQ ID NO. 1 was derived from the C6 clone of AcNPV
  • sequence information provided according to the invention may be used to optimize expression in other baculovirus expression systems.
  • published partial sequence data, restriction enzyme and hybridization analysis can be used to identify other clones and baculovirus isolates from insects which may be strains, variants or varieties of AcNPV.
  • isolates include viruses obtained from Autograph califomica, Autographa gamm, Galleria mellonella, Plutella xylostella, Rachiplusia ou, Spodoptera exempta, Spodoptera litura and Trichoplusio ni.
  • Such viruses are likely to possess DNA sequences, genes, origins and replication, transcriptional promoters, terminators and regulatory factors in common with those of AcNPV C6 and such entities are likely to be involved in directing the course of infection, multiplication and morphogenesis of these viruses as well as their interactions with hosts, host cells and components thereof. Accordingly, the information provided according to the invention of SEQ ID No. 1 may be used in the development of expression systems utilizing these alternative viruses and virus strains.
  • the complete nucleotide sequence of the genome of clone 6 of the baculovirus Autographa califomica nuclear polyhedrosis virus (AcNPV) has been determined.
  • the molecule comprises 133,894 base-pairs and has an overall A + T content of 59%.
  • Our analysis suggests that the virus enclodes some 154 methionine-initiated, and potentially expressed, open reading frames (ORFs) of 150 nucleotides or greater. These ORFs are distributed evenly throughout the virus genome on either strand.
  • the ORFs are arranged as adjacent, non-overlapping reading frames, separated by short intergenic regions.
  • Figure 1 A physical map and summary of coding strategy of the
  • Figure 2. A dot matrix analysis of AcNPV genomic DNA.
  • Figure 3. A circular map of the AcNPV genome.
  • Figures 4 - 14. A construct for modififying the following respective genes to identify which genes are dispensable (non-essential) and which genes are indispensable (essential) for viral replication in cell culture or insect larvae.
  • Figure 15 Single restriction enzyme site within the AcNPV EGT gene.
  • AcNPV genomic DNA was prepared as described by Possee (1986).
  • the DNA was digested with an appropriate restriction endonuclease (Bam ⁇ I, Bg ⁇ i, EcoBl, HindTH, Pstl, Sstl, Sst ⁇ ).
  • the derived DNA fragments were inserted into pUC18/19, pUC118/119 or pT7T318/19 vectors using standard protocols (Sambrook et al., 1989).
  • plasmids containing larger regions of virus DNA were digested with a restriction enzyme to release the insert, the virus DNA purified using agarose gel electrophoresis and then digested with another restriction enzyme. These smaller DNA fragments were inserted into plasmid vectors to provide materials more convenient for DNA sequencing.
  • Reaction mixtures contained the dGTP analogue, 7-deaza dGTP, in lieu of dGTP in order to reduce sequence compressions.
  • dITP was substituted for dGTP in the sequencing reactions.
  • the M13 primer (5' GTAAAACGACGGCCAGT) was used to sequence the ends of each virus DNA fragment.
  • Oligonucleotide primers prepared using an Applied Biosystems Instruments synthesizer (ABI, Model 380B, Warrington, UK), were employed to obtain the internal sequences of the viral fragments. Where appropriate, double-stranded DNA templates were used to complete regions of the AcNPV sequence not analysed as single-stranded DNA.
  • An ABI automated sequencer (model 370A) was also used on occasion. Using the established nomenclature for describing (by rank of size) the AcNPV restriction endonuclease fragments (e.g., A, B, C, etc.,), the following cloned virus DNA fragments were completely sequenced: BamHl-D, -E and -G; BgHl-G', HinaH-C to -K, -O to -S, -U, -W and -X; PsrI-J to -M, Sstl-F to -H.
  • the AcNPV restriction endonuclease fragments e.g., A, B, C, etc.
  • Partially sequenced fragments included: BamHI-E; BgU -E and -H; HindHl-L; Pstl-B and -C; Sstl-O and Ssf ⁇ -I. All the DNA sequences between adjoining virus DNA fragments were determined using appropriate subclones spanning the respective junctions.
  • the DNA sequences of the AcNPV C6 homologous region (hr) 1, .EcoBI-I and -R fragments have been reported (Possee et al., 1991).
  • the remaining sequence of this AcNPV clone was determined from a data set comprising approximately 106 nucleotides.
  • the complete AcNPV genomic sequence has been determined to consist of 133,894 base-pairs (bp) and has an A+T content of 59%.
  • the distributions of purines and A+T nucleotides for the plus strand (+ strand; see convention established by Vlak and Smith, 1982) throughout a linearized representation of the circular AcNPV genome is shown in Fig. 1, using a moving window of 250 nucleotides.
  • FIG. 1 A physical map of the genome was derived from the sequence data and is also illustrated in Fig. 1. This shows the arrangement of some of the common restriction enzyme sites frequently used to map the virus DNA (Ec ⁇ Rl, HindUL, Pstl, Sstl, BgiU, Xhol). Although circular, the map is presented with the first JEcoRI site of ⁇ rl as the left end of the genome.
  • the virus DNA fragments shown in Fig. 1 are labelled alphabetically, in decreasing order of size (Vlak and Smith, 1982).
  • a small fragment of 38 nucleotides is present between the HindUI-L and -M fragments and a 12 nucleotide fragment between the Hind ⁇ I-C and -W fragments (see Lu and Carstens, 1991 for the data on the clone HR3).
  • the only exceptions to labelling fragments uniquely according to their size are the HindUl-Al (15,293 bp) and -A2 (7,576 bp) fragments. These are designated Al and A2 in Fig. 1 solely for convenience of comparison with previously published data.
  • the Sstl map is modified to interchange the SsrI-A and -B fragments and the BgUL map is modified to interchange the B ⁇ ZII-G and -H fragments.
  • the Ar4c represents an imperfect copy of the typical AcNPV 30 bp palindrome since there is a base change that mutates to AAATTC the characteristic JBCORI site (GAATTC) found in the centre of all other AcNPV hr palindromes (Table 2).
  • Fig. 1 shows the positions (black boxes) of 337 open reading frames (ORFs) that are initiated with a methionine codon (vertical bars) and which could encode polypeptides of at least 50 amino acids.
  • ORFs open reading frames
  • This strategy of analysis does not identify gene products that may be smaller than 50 amino acids, or products that are generated by removal of introns from primary mRNA transcripts representing larger regions of the genome.
  • ORFs open reading frames
  • ORF 1 encodes a virus protein tyrosine/serine phosphatase (FTP) previously identified by Kim and Weaver (1993).
  • FTP virus protein tyrosine/serine phosphatase
  • Table 1 provides a more detailed summary of the information concerning the selected ORFs.
  • the left end of each ORF identified in Table 1 (column Left) represents the site of either the translation initiation or termination codon, as determined by the orientation of the ORF.
  • the right end of each ORF (Table 1, column Right) indicates the respective translation termination or initiation codon.
  • the .direction of transcription (Table 1, column D), relative to that of the polyhedrin gene, is indicated by an arrow.
  • the predicted number of amino acids (Table 1, column aa) per methionine initiated polypeptide derived from the ORF, and the M r of that polypeptide are also given.
  • ORF128, Fig. 1 the large ORF encoded entirely within the region of gp67 (ORF128, Fig. 1), but on the opposite strand, was excluded from our final dataset.
  • ORF100 which encodes the basic DNA binding protein, p6.9, of AcNPV (Wilson, et al., 1987), was included in our final dataset. As a consequence the two similar sized ORFs that overlap ORFIOO were not. Further analyses of the selected and non-selected ORFs will determine whether these assumptions are correct.
  • ORF6 (lef-2) starts within the 3' region of ORF5.
  • ORF14 (lef-1) overlaps the start of ORF13.
  • ORF25 in Table 1 was recorded as 2 smaller ORFs by the same authors. In the vicinity of residue 7,497 there are 4 extra nucleotides compared to the previous published AcNPV C6 sequence data (Possee et al., 1991). This causes a frameshift in the coding region and results in an extension of a predicted protein, PKl (ORF10), from 196 to 272 amino acids.
  • PKl predicted protein
  • hrl Dot matrix analysis of the AcNPV sequence against itself and its complement revealed 8 regions of direct and inverted repetitive DNA (Fig. 2, identified as hrl, Aria, hrl, hrZ, Ar4a, hr4b, ⁇ r4c, hr ⁇ ).
  • the hr regions are involved in enhancing early mRNA transcription and as origins of DNA replication (Pearson et ⁇ l., 1992; .Leisy.and Rohrmann, 1993; Kool et ⁇ l., 1993a,b).
  • Other regions of DNA sequence were identified that have direct or inverted repetitive DNA that meet the minimal 21/24 bp matching criteria. The significance of these sequences is unknown.
  • Table 2 is listed a number of the larger, non- ⁇ r inverted repeats that could in single-stranded forms produce hairpin structures. These may be relevant to the secondary structure of mRNA species and affect the transcriptional or translational efficiencies of a particular ORF. In this regard, it is noted that most of these sequences occur within ORFs, rather than in intergenic sequences (Table 2). Their presence may be solely a consequence of the encoded amino acid sequence and the codons used. However, of particular note is the palindromic sequence found within the 25K gene (FP-protein; ORF61) and its similarity to the hr palindromic sequences (see Table 2).
  • RNA polymerase II RNA polymerase II
  • AGT first potential translation start codon
  • TATA boxes shown in Table 3 represents a sampling of several of the core DNA elements that are recognised to bind transcription factors (TFIID and TFUD-like proteins) (Ghosh, 1992).
  • TFIID transcription factors
  • TFIID transcription factors
  • One general, loosely-defined consensus for the TFIID binding site is TATA(A/T)A(A/T) (Nikolov et al., 1992).
  • the patterns that were employed were selected to limit the number of matches obtained when only TATA was used as the search motif. In the TATA motif search it was observed that the two patterns that favoured the A residue at position 6 were preferred over the third pattern (TA AAT, see Table 3).
  • the TATAAA motif occurs in 46% of the cases, the TATATA motif in 34%, and the TATAAT motif in 19%.
  • the CAGT motif is not always found at the start site of AcNPV early mRNA species. It should also be noted that in identifying possible RNA pol II promoter sites, we only considered the relative positions of the TATA box and CAGT motif (i.e., a TATA box 5' to a CAGT motif within the 5' leader sequence that was analysed, see above). Generally, however, in eukaryotes the TATA box motif is within 20 to 40 nucleotides of the mRNA cap site (Roeder, 1991; Zawel and Reinberg, 1992).
  • AcNPV late genes are transcribed from a consensus late promoter transcription start signal (TAAG; Blissard and Rohrmann, 1990).
  • the TAAG motif shows a dramatic difference in occurrence within the leader sequences of the selected ORFs (71 ORFs, 46%, Tables 1, 3) compared to the non- selected ORFs (11 ORFs, 6%; Tables 1, 3).
  • A- T rich regions flank AcNPV ORFs (Kuzio et al., 1984). While the nucleotide composition of the genome is 59% A+T, A+T rich regions are not uniformly ( randomly) distributed.
  • Fig. 1 shows several regions of A+T composition th approaches 85% when measured with a 250 nucleotide moving window.
  • Althoug A+T rich regions often flank AcNPV genes this characteristic is not absolute
  • the region 5' to the viral DNA polymerase (ORF65) is not especiall A+T rich.
  • the TAAG motif occurs less frequently than would b expected for a random sequence.
  • GAAT a sequence of similar composition, GAAT, occurs 574 times on the strand and 595 times on the — strand.
  • th expected frequency of a sequence conforming to the composition (A2TG) in 133,894 bp genome of the base composition of AcNPV and involving randoml distributed bases is 705 occurrences per strand.
  • a frequency distribution profile of the nucleotides surrounding the start codon of the 154 selected ORFs is shown in Table 4.
  • the dominance of an A residue at the -3 and perhaps -2 positions relative to the A of the ATG translation start sites in the corresponding DNA is the only significant characteristic of the selected ORFs. G at -3 is not favoured in the selected ORFs.
  • ORFs in AcNPV initiate translation at an ATG downstream of an in-frame ATG in the transcribed mRNA (Table 1, column K, identified as "2"). These are gp67 (ORF128) and PCNA (ORF49) (O'Reilly et al., 1989; Whitford et al, 1989).
  • gp67 ORF1278
  • PCNA ORF49
  • the amino acids and predicted M r of the selected ORFs are based on the calculations for the largest potential ORF initiated with a methionine. This assumption over-estimates the size of the primary translation products for gp67 and PCNA, and for any other product for which translation is initiated at a downstream in-frame ATG.
  • mini-cistrons There are 15 short ORFs (mini-cistrons) that are located immediately upstream (within 80 nucleotides) of the translation start site of the selected ORFs. All these mini-cistrons have ATG flanking sequences that conform to Kozak's rules. These are identified as "! in Table 1, column K. For mini-cistrons that are out-of-frame with respect to the larger ORF, a termination codon occurs either upstream of the selected ORF, or within a short distance into its coding region. Mini-cistrons have been reported in the 5' leaders of other baculovirus genes (Tomalski et a/., 1988; Blissard and Rohrmann, 1989) and may have regulatory roles in the translation of mRNA species.
  • codons that are used (Table 5), for example AGG and CGG (arginine), GGG (glycine), CTA, CTC, CTT (leucine) which are each used at less than half of the frequencies that may be expected if all the possible codons were utilized equally. While some codons appear to be discriminated against in the selected ORFs, others appear to be favoured (Table 5), for example CAA (glutamine), GAA (glutamic), GGC (glycine), ATT (isoleucine), TTG (leucine), and AAA (lysine). To what extent codon bias affects the expression level of AcNPV genes, or foreign genes expressed from AcNPV- derived expression vectors, remains to be determined.
  • the predominant translation termination codon utilized by the selected ORFs is TAA. It terminates 117 of the 154 ORFs (76%, Table 5).
  • CpGV LAP Cydi ⁇ pomonell ⁇ granulosis virus
  • AcNPV encodes a gene with identity to the acidic and basic fibroblast growth factors (FGFs), also known as heparin binding growth factors (HBGF, reviewed by Burgess and Maciag, 1989; Klagsbrun and D'Armore, 1991).
  • FGFs acidic and basic fibroblast growth factors
  • HBGF heparin binding growth factors
  • the AcNPV FGF- like gene product shows c ⁇ . 35% identity (75% similarity) with known members of the FGF superfamily.
  • GTAs Global transactivators
  • D. mel ⁇ nog ⁇ ster brahma gene is encoded by a 1638 codon ORF (Tamkun et al., 1992) while the yeast SNF2 gene contains an ORF of 1703 codons (Laurent era/., 1991).
  • PNK/PNL encodes a protein that may have multiple functions.
  • the amino terminal portion is strongly related to T4 RNA ligase (31% identity, 72% similarity) while the carboxy terminal half of this protein is related to T4 polynucleotide kinase (26% identity, 66% similarity).
  • AcNPV encodes a chitinase (ORF126) that resembles those of other organisms, most notably Serr ⁇ ti ⁇ m ⁇ rcescens (57% identity; 88% similarity; Jones et ⁇ l., 1986). Analyses of the function of the viral chitinase indicates that it has a role in the liquefaction of infected larvae (R. Hawtin and R.D. Possee, manuscript in preparation).
  • AcNPV also encodes a putative alkaline exonuclease (ORF133).
  • ORF133 has 53% identity with its Orgyi ⁇ pseudotsug ⁇ t ⁇ NPV (OpNPV) homologue (Gombart etal., 1989).
  • RNA binding motifs As part of our search for potential virus-encoded RNA polymerase subunits, we searched for DNA binding motifs. A sample of the motifs used for the searches are shown in Table 3. They include zinc fingers (Table 1, Dom column, “Z”), leucine zippers (Table 1, Dom column, “L”), nucleoside triphosphate binding domains (Table 1, Dom column, “NTP”) and nuclear translocation signals (Table 1, Dom column, “NTS”).
  • Zinc fingers were found in two potential apoptosis inhibitory proteins IAPl (ORF27) and IAP2 (ORF71) (Table 1). Zinc fingers were also found in the early genes IE-1 (ORF147), ME53 (ORF139) and PE38 (ORF153). The zinc finger suggested to be in cg30 was not identified by our analysis. However, the leucine zipper in the cg30 protein (ORF88) was identified. Leucine zippers were found in 7 other potential polypeptides, including the calyx protein, pp34 (Table 1).
  • NTP binding motif was identified in 4 ORFs, 3 of which are known as late enhancing factors (lefs, Table 1).
  • the fourth protein was PNK/PNL (ORF86).
  • searches with a simplified motif for the ATP-binding site in protein kinases would not have found matches in either PKl (ORF10), or PK2 (ORF123), both of which have extensive overall identity with known protein kinases.
  • PKl lacks a consensus ATP-binding motif, having IxGxxG at the ATP-binding site, while PK2 completely lacks this N- terminal domain.
  • NTS motifs were found in 12 of the selected ORFs.
  • Known nuclear localising proteins that have an NTS include 39K, DNA polymerase, and p6.9.
  • No NTS was found for the plO protein, which is the component of fibrous bodies present in the nuclei of AcNPV infected cells. It is possible that this and other viral proteins enter the nucleus using an alternative pathway, or are chaperoned by a protein containing an NTS. None of the AcNPV proteins that are known to be solely cytoplasmic had a predicted NTS.
  • the cDNA sequence information for A. califomica can be used to design a vector which is capable of optimally expressing a desired protein product (called a "designer vector").
  • a design vector An investigator of ordinary skill in this art would analyze a variety of different factors prior to deciding on which genetic elements should be included in a specific designer vector. For example, an investigator might study the following factors before designing a vector; the protein to be synthetically produced, the host cells to be used, desired temporal timing for protein production, available insertion sites for the non-natural promoters, any known deleterious sequences or proteases that could reduce the amount of protein being produced, etc.
  • the designer vector can include a single promoter, multiple promoters, tandem promoters, combinations of synthetically constructed promoters, natural promoters and derivatives thereof.
  • the choice of promoters depends on several factors and is usually performed on a vector to vector basis (case by case basis). Additionally, many different genetic elements can be included in the designer vector and deciding which to include or exclude depends on the desired protein to be recombina ⁇ tly produced in the baculovirus expression vector system.
  • a vector can be designed and constructed to optimize the isolation and recovery of the desired protein.
  • the vector can be designed to include specifically identified secretion sequences determined from the cDNA sequence data.
  • califomica cDNA sequence information locations of transcription and translation signal sequences can be determined. Additionally, specific flanking sequences near the ATG sequence of the open reading frame can be identified and then used in order to optimally transcribe the ORF.
  • the A. califomica cDNA sequence information can be used to identify new genes. Once these new genes are identified, their promoters (early, late, immediate early or immediate late) may then be obtained and used in vectors. The new promoters from these new late genes may then be used to drive the expression of desired genes more efficiently and effectively when compared to the polyhedrin.
  • essential and non-essential gene regions can be identified.
  • essential and non-essential genes refer to the virus replication in cell culture (e.g. Spodoptera frugiperda cells).
  • ORFs 126 chitinase
  • 127 cathespin
  • ORFs 126 and 127 have been shown to be non-essential genes.
  • these two gene could be eliminated from the A. califo ica sequence and not affect the nature of the sequence. Elimination of these two non-essential genes could be performed by standard protocols known to those skilled in the art.
  • the rationale for identifying non-essential genes is to reduce the genome size to smaller and more functional pieces in order to create a more effective, and environmentally acceptable pesticide or in order to create a more effective vector.
  • regions that are essential for enabling a virus to live in an insect cell can be identified. Once these essential regions are identified, the essential sequence can be used to produce a virus that will not propagate in live insects. One use of such an environmentally safe virus would be used as a selective pesticide.
  • the A califomica cDNA sequences claimed in this invention can be used to design a plasmid vector capable of optimizing expression of the desired protein.
  • One way in which this plasmid vector can be tailored to more effectively and efficiently produce the desired protein of choice is to optimize it for the particular host.
  • SF9 cells are the optimal cells of choice for production of desired proteins in the baculoviral expression vector system.
  • a designer vector as in Example 11 above can be constructed for optimal expression of the desired protein in the SF9 cells by deleting selected deleterious sequences and/or providing enhancer sequences.
  • the A. califomica cDNA sequences claimed in this invention can be used to design a complete virus which is specifically constructed to contain specific and unique elements which will enhance the infectivity of this virus in a particular insect cell.
  • a viral particle can -be designed to infect and kill the insect at an early stage.
  • the claimed sequence can also be used to produce a virus capable of infecting larvae and not adult insects.
  • An additional embodiment of this invention is to use the claimed A. califomica cDNA sequence to tailor or design a virus which is capable of infecting only specific insects, thereby constructing a very host specific virus.
  • a self destructive mechanism may be included in the viral particle. This mechanism can be designed such that once the viral particle has killed the host specific insect, the virus destroys itself via a time, chemical, or enzymatic attack. This self destructive mechanism will effectively eliminate any residual virus and therefore produce a more environmentally acceptable pesticide.
  • a sequence known to trigger lysis may be inserted adjacent to a late or early promoter.
  • the availability of the complete AcNPV sequence and subsequent experimental data will allow the identification of those virus genes with roles in determining those insect species which can be infected with the virus.
  • the virus could be modified to limit infection to the target pest species, while leaving other species unaffected.
  • baculoviruses may be engineered to expand their host range to include several pest species.
  • AcNPV has a wide host range in comparison to other baculoviruses and therefore may be a source of "host range genes" which can be added to these other baculoviruses.
  • Certain proteins are naturally expressed in A. califomica (for example, heparin binding factor).
  • the cDNA sequence information of the claimed invention can be used to enhance or increase production of the proteins that are naturally expressed in califomica, for example, by inserting additional promoter sequences and/or by deleting certain sequences deleterious to the production of the desired protein.
  • the deletion of the annihilator gene (ORF 135) from the virus results in a phenotype in which virus-infected cells die through a process of apoptosis or early cell death. In effect, the cell commits suicide to prevent replication of the virus.
  • ORF 135 The deletion of the annihilator gene (ORF 135) from the virus results in a phenotype in which virus-infected cells die through a process of apoptosis or early cell death. In effect, the cell commits suicide to prevent replication of the virus.
  • ORF 135 The deletion of the annihilator gene (ORF 135) from the virus results in a phenotype in which virus-infected cells die through a process of apoptosis or early cell death. In effect, the cell commits suicide to prevent replication of the virus.
  • other genes could be identified with a similar function to the annihilator gene, i.e. preventing the cell from undergoing an apoptotic response. These genes would
  • Fig. 1 a linear representation of the map is shown. Since the virus genome is circular, a more conventional map for the AcNPV genome is given in Fig. 3. In this map the identified genes (hatched arrows), and unassigned selected ORFs (open arrows) are shown as well as their orientations. Also indicated in Fig. 3 are the sites of Ar sequences and insertion (IS) and retroposon sequences (RP). This circular map includes the revised Eco ⁇ l (outer ring) and HindUL (inner ring) fragment lengths of AcNPV C6.
  • ORFs were identified within the virus genome that could potentially encode proteins of greater than 50 amino acids. This selection allowed inclusion of the 55 amino acid, arginine-rich p6.9 protein (basic protein, Wilson et al., 1987). It disregards smaller ORFs, some of which may encode proteins or peptides that are made during the virus infection process.
  • the 154 ORFs were selected on the basis of their possession of a methionine codon and the absence of a larger, overlapping ORF. Again these assumptions may prove to be incorrect in some cases (e.g., where a spliced mRNA is involved).
  • the number of gene products encoded by the AcNPV genome may be larger or smaller than 154, depending on the extent that the assumptions made in these analyses prove to be correct.
  • other strains of the virus may include additional sequences (insertions, or ORFs), or lack sequences by comparison to those in the C6 virus. Since it is valuable to have a reference point for comparison purposes, it is suggested that the AcNPV C6 ORF numbering nomenclature is adopted pro temporis and until virus gene functions are described for the particular ORFs.
  • the complete AcNPV sequence was analysed using a neutral-net ORF identification programme, GRATL (Uberbacher and Mural, 1991), in order to predict potential protein coding regions.
  • GRAIL was originally designed as a programme for identifying coding exons in human and other DNA sequences.
  • the GRATL coding recognition module incorporates seven sensor algorithms. Each component of the module provides an indication of the coding potential of the DNA sequence. The various sensor outputs are integrated using a neutral network which also predicts the locations of the coding regions.
  • the system has been demonstrated to be effective in the identification of 90% of exons over 100 bases long in human DNA (Uberbacher and Mural, 1991). In part this success rate depends on the G+C content of the DNA.
  • Coding regions are recognised less easily in DNA sequences with a lower G+C than A+T content.
  • the G+C content of the AcNPV genome is only 41%, so the coding regions predicted from the GRATL an lysis must be treated with some caution.
  • the candidate ORFs that were identified by GRATL were rated as excellent, good, marginal or null (Table 1).
  • Most of the AcNPV genes which have been assigned functions gave excellent or good ratings using this method. The most notable exceptions were the protein tyrosine phosphatase (Kim and Weaver, 1993), p6.9 (Wilson et al., 1987) and conotoxin (Eldridge et al., 1992).
  • GRAIL provided a complementary analysis of the likely coding potential of the AcNPV genome. The value is confirmed by the fact that GRAIL predicted 84% of the 154 selected ORFs (Table 1), whereas only 4% of the 183 non-selected ORFs were identified by GRATL as having potential protein coding capacity.
  • TATA boxes TFITD binding sites
  • CAGT possible mRNA transcription start sites
  • the CAGT motif is associated with many baculovirus early gene promoters and is probably a good indicator of whether or not a virus gene is transcribed in the early phase of the replication cycle.
  • the TATA boxes are more problematic, in part due to the high A+T content of the AcNPV genome and its intergenic regions. More than one TATA box was present upstream of many of the ORFs.
  • Table 3 we located TATA boxes upstream of 40% of the selected ORFs. Of the 3 TATA box patterns utilised to identify possible TFIID-type binding motifs (Table 3),. the TATAAA motif, which is the preferred TFIID binding site, was the most frequent in the selected ORFs identified to be early genes.
  • RNA pol II promoter within 160 nucleotides of the ATG codon of the respective ORF.
  • the known AcNPV early genes identified by this procedure include: ME53 (ORF139), IE-1 (ORF147), IE-N (ORF151), and PE38 (ORF153).
  • the presence of a TATA motif does not prove that it is used in early transcription by RNA pol EL This can only be determined by experimentation.
  • lef-3 has consensus TATA and CAGT motifs in its 5' leader, but no evidence has been reported that these are utilised in early mRNA synthesis (Li et al., 1993).
  • the polyhedrin gene has an RNA pol II motif within its promoter region.
  • CGTGC motif Alternative transcription start sites, initiating from a CGTGC motif, have been identified in some AcNPV early gene promoters.
  • the CGTGC motif is utilised as early start sites for pl43 (Lu and Carstens, 1991), DNA polymerase (Tomalski et al., 1988) and p47 (Carstens et al., 1993).
  • pl43 Long and Carstens, 1991
  • DNA polymerase Tomalski et al., 1988
  • p47 Carstens et al., 1993.
  • this motif is involved in the expression of the AcNPV delayed-early genes and may be a site of recognition by virus-encoded, trans-activating proteins.
  • the CGTGC motif is broadly similar to sequences found in AcNPV Ar regions, i.e., TYC(A/T)(A/T)A(AT)CGXGTRA (where Y is a pyrimidine, R a purine and X any nucleotide).
  • the CGTGC motif is evenly distributed between the selected ORFs and the non-selected ORFs, suggesting that the definition of this motif is not refined enough toie of predictive value. If it is important, its placement may not be confined to the immediate 5' leader sequence of a neighbouring gene.
  • the late and very late transcription start sites involve a TAAG motif (Blissard and Rohrmann, 1990).
  • TAAG rather than the canonical ATAAG or RTAAG sequences to search for ORFs that might be transcribed late in infection in an endeavour to maximise the chance of finding matches.
  • the 46% of the selected ORFs that are identified as probable late/very late genes may under ⁇ estimate such genes.
  • cg30 ORF88
  • initiates from the sequence ATTAG Wang and Miller 1989.
  • the late gene p74 (ORF138) initiates transcription at the sequence TATTG (Kuzio et al, 1989) and p47 (ORF40) has a late transcription start site GTAAAAC (Carstens et al., 1993).
  • a search for similar matches to the start site used in p47 revealed a good match at nucleotide 66,740 in the coding region for gp41 (ORF80).
  • an ATAAG motif is present 145 nucleotides upstream of this site.
  • codon usage table for the 154 selected ORFs presented in this study (Table 5). There appears to be some codon bias.
  • the codon usage bias shown by the AcNPV ORFs may reflect some state of the tRNAs available to the virus during the infection process. However although the sample base is low, so far we have not been able to detect a differential codon bias between early and late expressed genes.
  • C ⁇ s-acting elements (hrs) involved in the origins of AcNPV DNA replication have been shown to be A+T rich.
  • OpNPV appears to have at least one origin that is slightly G+C rich, but with a neutral purine composition, i.e., different from the hrs of AcNPV, or the transcription enhancer regions found within OpNPV (Pearson et al, 1993).
  • the region in AcNPV homologous to the OpNPV origin of replication lies within ORF13 and ORF14 (lef-1).
  • Choristoneura fumiferana NPV CfNPV
  • Bombyx mori NPV BmNPV
  • the Ar sites of baculoviruses may be active in inter- or intra-molecular recombination. If recombination was involved in the one or other inversion, how this occurred is not certain since there is no obvious relationship between the left and right arms of the second inverted region in
  • OpNPV the corresponding regions of AcNPV are A+T rich. This suggests that an intramolecular inversion may have taken place in OpNPV. However, a detailed analysis of this region in that virus has yet to be undertaken.
  • the Ar regions of AcNPV have been implicated in replication of the AcNPV genomic DNA and may act as origins of replication (Pearson et al., 1992; Leisy and Rohrmann, 1993; Kool et al., 1993a,b). Furthermore, recent studies have identified regions of the AcNPV genome that encode products that act on the origins of replication (Kool et al., 1994).
  • the AcNPV + strand G-rich sequence at position 78,300 of AcNPV shows no overall bias in A+T content but has a pronounced spike with respect to total purine composition (ca. 78%, Fig. 1).
  • Purine-rich tracts can potentially form an intrastrand triple-helix and tetrads.
  • Triple helical DNA has been implicated as an origin of replication for some plasmids, as well as having other potential regulatory functions (Caddie et al., 1990).
  • the only other region of elevated purine composition in AcNPV occurs within the coding region of ORF66 (ca. 68% purines).
  • the purine rich region within ORF66 is also A+T rich, thus A residues- contribute highly to the purine composition of the + strand.
  • the CpGV IAP gene provides the 3 ⁇ k gene function in AcNPV 35k-negative mutants, thereby preventing the annihilator phenotype of the mutant (Crook et al., 1993).
  • the CpGV IAP provides the 3 ⁇ k gene function in AcNPV 35k-negative mutants, thereby preventing the annihilator phenotype of the mutant (Crook et al., 1993).
  • there is no sequence identity between AcNPV 35k and CpGV IAP In view of the structural homologies between the CpGV IAP and the AcNPV IAPl and IAP2 genes, the roles and functions of these AcNPV genes warrant further investigation. It has been shown that the 35k-negative AcNPV mutant, while unable to replicate efficiently in S. frugiperda cells in culture, or in whole larvae, can be propagated in T. ni cells, or insects.
  • the AcNPV IAPl and IAP2 genes may prevent apoptosis in AcNPV infections of other cell types or larval species. Further, it has been shown that over-expression of a human inhibitor of apoptosis (BCL2) in S. frugiperda cells (Alnemri et at., 1992), using an AcNPV expression vector, results in the protection of the cells against apoptosis. These recombinant virus infected cells have an extended survival time and do no . show the degradation of host cell DNA that is evident in cells infected with wild- type AcNPV. It is not known if over-expression of the AcNPV IAP genes results in extended survival of virus-infected cells. The AcNPV LAP genes do not share any structural similarity with BCL2, or any other known IAP gene. However, the viral LAP genes are similar to certain DNA binding proteins by the possession of 3 copies of a zinc finger motif.
  • AcNPV encodes a gene with identity to the FGFs and HBGF family of growth factors.
  • Two conserved cysteines have been identified in all the human FGFs sequenced to-date. These are Cys31 and Cys98 (relative to human acidic FGF). These cysteines have been implicated in intramolecular disulfide bond formation (Burgess and Maciag, 1989). The N-terminal cysteine is lacking from the putative AcNPV FGF.
  • site-directed mutagenesis of cDNA clones has implicated Lysl33 in heparin binding (Burgess and Maciag, 1989).
  • the AcNPV FGF has an arginine at this position.
  • Hbg3 This substitution of one basic residue for another also occurs in the int-2 proto-oncogene precursor Hbg3.
  • Free heparin is known to inhibit the growth of herpes simplex viruses (Nahmias and Kilbrick, 1964). More recently, it has been shown that heparin binds to HSV-1 virions via the glycoprotein gC (WuDunn and Spear, 1989; Herold et al., 1991) and prevents their adsorption to heparin sulphate moieties resident on cell surface proteogl cans. Heparin similarly inhibits plaque formation of pseudorabies virus by binding to glycoprotein gELI (Mettenleiter et al., 1990).
  • heparin binding factor by the baculovirus could be a method to complex free heparin (or heparin-related compounds) thereby facilitating virus spread within the host.
  • the virus FGF has a signal peptide sequence at its amino terminal sequence which may facilitate secretion from virus-infected cells.
  • the GTAs are non-DNA binding proteins thought to have a role in the regulation of homeotic genes (Tamkun et al., 1992).
  • Homeotic genes are involved in the expression of a large group of other genes that have been implicated in directed development and growth of an organism (McGinnis et al., 1984a,b; Scott and Weiner, 1984; Levine and Hoey, 1988; Hayashi and Scott, 1990).
  • the AcNPV ORF42 has homology with the GTAs of D. mel ⁇ nog ⁇ ster (Tamkun etal., 1992) and yeast (Laurent et al., 1991).
  • the AcNPV GTA-like protein does not have either the early CAGT, or late TAAG transcription initiation sites, so it is difficult to predict when it may be expressed in virus-infected cells. Transcriptional analysis is required to determine if and when it is synthesized.
  • a viral GTA might be involved in regulating a number of genes involved in viral processes, such as late gene transactivation. It is also conceivable that the AcNPV GTA-like gene acts as a repressor to inhibit host gene expression.
  • RNA polymerase has at least 8 subunits with apparent sizes of 95, 76, 50, 47.5, 40, . 33.5, 27.5, and 26 kDa (Yang et ⁇ l., 1991). These subunits are believed to be distinct from host encoded RNA polymerase subunits.
  • the level of processing of viral RNA polymerase subunits i.e., cleavage of primary products phosphorylation
  • ORF144 encodes a 33.5 kDa peptide that has similarity to the yeast MSS18 protein (Seraphin et al., 1988). MSS18 is known to be involved with yeast mitochondrial RNA splicing. Also, ORF124 encodes a 28.5 kDa peptide with similarity to a plasmid copy number protein from Clostridium perfringens (Gamier and Cole, 1988).
  • lefs late enhancing factors
  • This Example identifies a new AcMNPV gene which is dispensable for virus replication in cell culture or insect larvae. This gene is located at ORF 27: 22600 > 23458 and named "Inhibitor of Apoptosis-Like Gene 1" (IAPl).
  • This AcMNPV gene can be deleted from the baculovirus genome to: (a) provide additional sites for inserting single or multiple copies of foreign genes and (b) to reduce the size of the virus genome.
  • Each gene is ascribed a number corresponding with the order of open reading frames (ORFs) within the AcMNPV genome. This is followed by the precise coordinates of the left and right ends of the coding region. Genes which are on the same strand as the polyhedrin gene are indicated by " > " between the left and right coordinates. Genes which are antisense to the polyhedrin are indicated by " ⁇ " between the left and right coordinates.
  • the translation initiation codon (ATG) for polyhedrin-sense genes is located at the left coordinate while the translation initiation codon for antisense genes is located at the right coordinate. All coordinates in this description are relative to the AcMNPV genomic sequence, even after subcloning of virus DNA fragments into plasmid vectors.
  • the cassette was chosen to derive an in-frame fusion between the IAPl and beta-galactosidase coding regions.
  • the plasmid was designated pUC118.IAPl.lacZ. This was used to cotransfect Spodoprera frugiperda cells with infectious AcMNPV C6 DNA to produce recombinant virus with a copy of the beta- galactosidase gene in frame with the IAPl, this disrupting IAPl function.
  • the results from this Example demonstrated that the recombinant virus (AcIAPl.lacZ) replicated normally in S. frugiperda cells and Trichoplusia ni insect larvae.
  • ORF 30 24315 ⁇ 25704: Haemolysin Secretory Protein (HSP)
  • This Example identifies a new AcMNPV gene which is dispensable for virus replication in cell culture or insect larvae. This gene is located at ORF 30: 24315 ⁇ 25704 and named "Haemolysin Secretory Protein” (HSP).
  • This AcMNPV gene can be deleted from the baculovirus genome to: (a) provide additional sites for inserting single or multiple copies of foreign genes and (b) to reduce the size of the virus genome.
  • Each gene is ascribed a number corresponding with the order of open reading frames (ORFs) within the AcMNPV genome. This is followed by the precise coordinates of the left and right ends of the coding region. Genes which are on the same strand as the polyhedrin gene are indicated by " > " between the left and right coordinates. Genes which are antisense to the polyhedrin are indicated by " ⁇ " between the left and right coordinates.
  • the translation initiation codon (ATG) for polyhedrin-sense genes is located at the left coordinate while the translation initiation codon for antisense genes is located at the right coordinate. All coordinates in this description are relative to the AcMNPV genomic sequence, even after subcloning of virus DNA fragments into plasmid vectors.
  • ORF 32 27041 ⁇ 27584: Fibroblast Growth Factor (FGF)
  • This Example identifies a new AcMNPV gene which is dispensable for virus replication in cell culture or insect larvae. This gene is located at ORF 32: 27041 ⁇ 27584 and named "Fibroblast Growth Factor: (FGF).
  • FGF Fibroblast Growth Factor
  • This AcMNPV gene can be deleted from the baculovirus genome to: (a) provide additional sites for inserting single or multiple copies of foreign genes and (b) to reduce the size of the virus genome.
  • Each gene is ascribed a number corresponding with the order of open reading frames (ORFs) within the AcMNPV genome. This is followed by the precise coordinates of the left and right ends of the coding region. Genes which are on the same strand as the polyhedrin gene are indicated by " > " between the left and right coordinates. Genes which are antisense to the polyhedrin are indicated by " ⁇ " between the left and right coordinates.
  • the translation initiation codon (ATG) for polyhedrin-sense genes is located at the left coordinate while the translation initiation codon for antisense genes is located at the right coordinate. All coordinates in this description are relative to the AcMNPV genomic sequence, even after subcloning of virus DNA fragments into plasmid vectors.
  • ORF 71 61016 > 61763: Inhibitor of Apoptosis-Like Gene 2 (IAP2)
  • This Example identifies a new AcMNPV gene which is dispensable for virus replication in cell culture or insect larvae. This gene is located at ORF 71: 61016 > 61763 and named "Inhibitor of Apoptosis-Like Gene 2" (IAP2).
  • This AcMNPV gene can be deleted from the baculovirus genome to: (a) provide additional sites for inserting single or multiple copies of foreign genes and (b) to reduce the size of the virus genome.
  • Each gene is ascribed a number corresponding with the order of open reading frames (ORFs) within the AcMNPV genome. This is followed by the precise coordinates of the left and right ends of the coding region. Genes which are on the same strand as the polyhedrin gene are indicated by " > " between the left and right coordinates. Genes which are antisense to the polyhedrin are indicated by " ⁇ " between the left and right coordinates.
  • the translation initiation codon (ATG) for polyhedrin-sense genes is located at the left coordinate while the translation initiation codon for antisense genes is located at the right coordinate. All coordinates in this description are relative to the AcMNPV genomic sequence, even after subcloning of virus DNA fragments into plasmid vectors.
  • an AcMNPV DNA fragment coordinates 60448 (Sad Site) to 63194 (Sad site) was subcloned into pUC118 digested with SacI and treated with CEP to derive pUC118.IAP2 (See Figure 7, Panels a-e).
  • This Example identifies a new AcMNPV gene which is dispensable for virus replication in cell culture or insect larvae. This gene is located at ORF 86: 72131 ⁇ 74213 and named "Polynucleotide Kinase/Polynucleotide Ligase" (PNK/PNL).
  • This AcMNPV gene can be deleted from the baculovirus genome to: (a) provide additional sites for inserting single or multiple copies of foreign genes and (b) to reduce the size of the virus genome.
  • Each gene is ascribed a number corresponding with the order of open reading frames (ORFs) within the AcMNPV genome. This is followed by the precise coordinates of the left and right ends of the coding region. Genes which are on the same strand as the polyhedrin gene are indicated by " > " between the left and right coordinates. Genes which are antisense to the polyhedrin are indicated by " ⁇ " between the left and right coordinates.
  • the translation initiation codon (ATG) for polyhedrin-sense genes is located at the left coordinate while the translation initiation codon for antisense genes is located at the right coordinate. All coordinates in this description are relative to the AcMNPV genomic sequence, even after subcloning of virus DNA fragments into plasmid vectors.
  • an AcMNPV DNA fragment coordinates 71417 (Hindm site) to 83121 (Hin ⁇ T ⁇ site) was subcloned into pAT153 digested with HindEQ and treated with CEP to derive PAT153.PNK/PNL (See Figure 8, Panels a-e).
  • the plasmid was designated pUCll ⁇ .PNK/PNL.lacZ. This was used to cotransfect S. frugiperda cells with infectious ACMNPV C6 DNA to produce recombinant virus with a copy of the beta-galactosidase gene in frame with the PNK/PNL, thus disrupting PNK/PNL function. The results showed that the recombinant virus (AcPNK/PNL.lacZ) replicated normally in S. frugiperda cells. EXAMPLE 26
  • ORF 123 102964 ⁇ 103609: Protein Kinase 2 (PK2)
  • This Example identifies a new AcMNPV gene which is dispensable for virus replication in cell culture or insect larvae. This gene is located at ORF 123: 102964 ⁇ 103609 and named "Protein Kinase 2" (PK2).
  • PK2 Protein Kinase 2
  • This AcMNPV gene can be deleted from the baculovirus genome to: (a) provide additional sites for inserting single or multiple copies of foreign genes and (b) to reduce the size of the virus genome.
  • Each gene is ascribed a number corresponding with the order of open reading frames (ORFs) within the AcMNPV genome. This is followed by the precise coordinates of the left and right ends of the coding region. Genes which are on the same strand as the polyhedrin gene are indicated by " > " between the left and right coordinates. Genes which are antisense to the polyhedrin are indicated by " ⁇ " between the left and right coordinates.
  • the translation initiation codon (ATG) for polyhedrin-sense genes is located at the left coordinate while the translation initiation codon for antisense genes is located at the right coordinate. All coordinates in this description are relative to the AcMNPV genomic sequence, even after subcloning of virus DNA fragments into plasmid vectors.
  • an AcMNPV DNA fragment coordinates 102148 (Pstl site) to 105164 (Pstl site), was subcloned into pUCll ⁇ digested with Pstl and treated with CEP to derive pUC118.PK2 (See Figure 9, Panels a-e).
  • Apal-BglH adaptor AGATCTGGCC
  • coli lacZ coding region to provide an in-frame fusion between the virus and bacterial genes.
  • the plasmid was designated pUC118.PK2.lacZ. This was used to cotransfect S. frugiperda cells with infectious AcMNPV C6 DNA to produce recombinant virus with a copy of the beta-galactosidase gene in frame with the PK2, thus disrupting PK2 function. The results demonstrated that the recombinant virus (AcPK2.1acZ) replicated normally in S. frugiperda cells. 56 EXAMPLE 27
  • ORF 126 105282 ⁇ 106935: Chitinase (CHID
  • This Example identifies a new AcMNPV gene which is dispensable for virus replication in cell culture or insect larvae. This gene is located at ORF 126: 105282 ⁇ 106935 and named "Chitinase” (CHIT.
  • This AcMNPV gene can be deleted from the baculovirus genome to: (a) provide additional sites for inserting single or multiple copies of foreign genes and (b) to reduce the size of the virus genome.
  • Each gene is ascribed a number corresponding with the order of open reading frames (ORFs) within the AcMNPV genome. This is followed by the precise coordinates of the left and right ends of the coding region. Genes which are on the same strand as the polyhedrin gene are indicated by " > " between the left and right coordinates. Genes which are antisense to the polyhedrin are indicated by " ⁇ " between the left and right coordinates.
  • the translation initiation codon (ATG) for polyhedrin-sense genes is located at the left coordinate while the translation initiation codon for antisense genes is located at the right coordinate. All coordinates in this description are relative to the AcMNPV genomic sequence, even after subcloning of virus DNA fragments into plasmid vectors.
  • an AcMNPV DNA fragment coordinates 105164 (Pstl site) to 107943 (Pstl site), was subcloned into pUC118 (lacking a Hind HI site) digested with Pstl and treated with CIP to derive pUC118.CHIT (See Figure 10, Panels a-e). This was digested with Hind (106337), treated with CIP and ligated with a Hindm-BamHl adaptor (AGCTGGATCC) to insert a BamHl site within the CHIT gene to derive pUC118.CHIT-BamHl. This was digested with BamHl, treated with CEP and ligated with a DNA cassette containing the E.
  • the plasmid was designated pUC118.CHIT.lacZ. This was used to cotransfect S. frugiperda cells with infectious ACMNPV C6 DNA to produce recombinant virus with a copy of the beta- galactosidase gene in frame with the chitinase, thus disrupting chitinase function.
  • the recombinant virus (AcCHIT.lacZ) replicated normally in S. frugiperda cells. In T. ni insect larvae, the virus replicated but failed to induce liquefaction of the host.
  • This Example identifies a new AcMNPV gene which is dispensable for virus replication in cell culture or insect larvae. This gene is located at ORF 127: 106983 > 107952 and is named "Cathepsin” (CATH).
  • This AcMNPV gene can be deleted from the baculovirus genome to: (a) provide additional sites for inserting single or multiple copies of foreign genes and (b) to reduce the size of the virus genome.
  • Each gene is ascribed a number corresponding with the order of open reading frames (ORFs) within the AcMNPV genome. This is followed by the precise coordinates of the left and right ends of the coding region. Genes which are on the same strand as the polyhedrin gene are indicated by " > " between the left and right coordinates. Genes which are antisense to the polyhedrin are indicated by " ⁇ " between the left and right coordinates.
  • the translation initiation codon (ATG) for polyhedrin-sense genes is located at the left coordinate while the translation initiation codon for antisense genes is located at the right coordinate. All coordinates in this description are relative to the AcMNPV genomic sequence, even after subcloning of virus DNA fragments into plasmid vectors.
  • This mutated plasmid was designated pUC119.M.CHTT- /CATH-. It was used to cotransfect S. frugiperda cells with infectious virus DNA, purified from the AcCHTT.lacZ, which had been digested with Bsu361 to enhance the recovery of recombinant viruses.
  • the recombinant virus, AcCH_T-/CATH- replicated normally in S. frugiperda cells. In T. ni insect larvae, the virus replicated but failed to induce liquefaction of the host.
  • ORF 42 34010 > 33924: Global Transactivator (GTA)
  • This Example identifies a new AcMNPV gene which is indispensable for virus replication in cell culture or insect larvae. This gene is located at ORF 42: 34010 > 33924 and is named "Global Transactivator" (GTA).
  • GTA Global Transactivator
  • This AcMNPV gene was modified in a similar manner as Examples 21-28 (described above), but the modification did not result in the production of an infectious virus stock. This information is a strong indication that this virus gene is indispensable for replication and cannot be removed from the virus genome.
  • an AcMNPV DNA fragment coordinates 33403 (EcoRI site) to 37088 (Asp718 site) was inserted into pUC118 digested with EcoRI and Asp718 and treated with CEP, to derive pUCll ⁇ .GTA (See Figure 12, Panels a-e).
  • This plasmid was designated pUC118.GTA.lacZ. This was used to cotransfect S. frugiperda cells with infectious AcMNPV C6 DNA to produce recombinant virus. Although some blue plaques were derived, these could not be titrated to genetic homogeneity and it was concluded that the GTA gene is essential for virus replication in cell culture.
  • This Example identifies a new AcMNPV gene which is indispensable for virus replication in cell culture or insect larvae. This gene is located at ORF 124: 103793 > 104534 and named "Plasmid Copy Number Protein” (PCNP).
  • This AcMNPV gene was modified in a similar manner as Examples 21-28 (described above), but the modification did not result in the production of an infectious virus stock. This information is a strong indication that this virus gene is indispensable for replication and cannot be removed from the virus genome.
  • an AcMNPV DNA fragment coordinates 102148 (Pstl site) to 105164 (Pstl site) was inserted into pUC118, digested with Pstl and treated with CEP, to derive pUC118.PCNP (See Figure 13, Panels a-e).
  • This plasmid was designated pUC118.PCNP.lacZ. This was used to cotransfect Spodoptera frugiperda cells with infectious AcMNPV C6 DNA to produce recombinant virus. Although some blue plaques were derived, these could not be titrated to genetic homogeneity and it was concluded that the PCNP gene is essential for virus replication in cell culture.
  • ORF 132 112560 > 113817: Alkaline Exonuclease (ALK-EXO)
  • This Example identifies a new AcMNPV gene which is indispensable for virus replication in cell culture or insect larvae. This gene is located at ORF 132: 112560 > 113817 and named "Alkaline Exonuclease" (ALK-EXO).
  • This AcMNPV gene was modified in a similar manner as Examples 21-28 (described above), but the modification did not result in the production of an infectious virus stock. This information is a strong indication that this virus gene is indispensable for replication and cannot be removed from the virus genome.
  • an AcMNPV DNA fragment coordinates 112044 (Smal site) to 113913 (HindELT site) was subcloned into pUC118 digested with HindHT and Smal and treated with CEP (See Figure 14, Panels a-e).
  • This combination of enzymes served to remove an intervening BamHl site within the polylinker of the plasmid.
  • the plasmid was designated pUCl 18.
  • ALK-EXO This was digested with BamHl (113033), treated with CEP and Ugated with a DNA cassette containing the E. coli lacZ coding region to provide an in-frame fusion between the virus and bacterial genes.
  • the plasmid was designated pUC118.ALK-EXO.lacZ. This was used to cotransfect S. frugiperda cells with infectious AcMNPV C6 DNA to produce recombinant virus. Although some blue plaques were derived, these could not be titrated to genetic homogeneity and it was concluded that the ALK-EXO gene is essential for virus replication in cell culture.
  • This Example identifies three restriction enzymes which do not have recognition sites within the AcNPV genome. These three enzymes are:
  • Bsu36I sites were inserted within the ORF 9 (immediately downstream of the polyhedrin gene in AcNPV) and ORF 7 (immediately upstream of the polyhedrin gene).
  • the polyhedrin gene was replaced with the beta-galactosidase coding region which also contains a Bsu36I site.
  • Srfl and Sse8387I could be utilized in a similar manner in other regions of the virus genome. For example, they could be used to alter the AcNPV genome to incorporate these sites and facilitate genomic DNA linearization.
  • This Example identifies two restriction sites which only digest AcNPV DNA once: Avril (See Figure 15) and Fsel (See Figure 16).
  • AvriT digests within the non-essential EGT (ecdysteroid UDP-glucosyltransferase) gene and Fsel digests within the essential GTA (global transactivator) gene.
  • EGT ecdysteroid UDP-glucosyltransferase
  • GTA global transactivator
  • Information derived from the entire AcMNPV genomic sequence could afford development of novel baculovirus transfer vectors that encode baculoviruses with favorable agronomic properties. Identification of genes encoding proteins that modify viral host range would lead to generation of recombinant NPVs wherein said recombinant viruses would be capable of infecting and therefore neutralizing a wider spectrum of important agronomic pests. Alternatively, genetic manipulation could lead to changes in viral properties that render the virus capable of infecting only a very narrow spectrum of insect pests, thus affording precise control of targeted insect species while sparing beneficial insect populations.
  • genes involved in viral replication could be identified. Manipulation of these genes could afford recombinant baculoviruses that multiply more rapidly within infected insect cells, thus leading to more rapid neutralization of the infected insect.
  • the Global Transactivator Gene ORF 42; see Example 29
  • ORF 42 the Global Transactivator Gene
  • Other genes influencing viral infectivity could also be identified and modified in order to raise the efficiency of the infectious process. This would also afford more rapid neutralization of targeted populations, and thus approach the rapidity of insect neutralization commonly associated with appUcation of traditional chemical insect control agents.
  • genes could be identified that qu ⁇ ditatively control viral repUcation outside of a permissive propagation system.
  • viral mutants deficient in a protein or proteins required for in vivo infectivity could be propagated in an insect cell culture system that is permissive to viral replication. While efficient viral repUcation in cell culture takes place, as well as initial infection of target insects, further viral replication in vivo is curtailed, and environmental impact of appUcation of recombinant baculoviruses is minimized.
  • ALTSCHUL S.F., GISH, W., MILLER, W prisms, E.W. and LTPMAN, D.J.
  • COCHRAN M.A.
  • CARSTENS E.B.
  • EATON B.T.
  • FAULKNER FAULKNER
  • Viral transcription during Autographa califo ica nuclear polyhedrosis virus infection a novel
  • baculovirus polyhedral envelope-associated protein genetic location nucleotide sequence, and immunocytochemical characterization.
  • Glycoprotein C of herpes simplex virus type 1 plays a principal role in the adsorption of virus to cells and in infectivity. J. Virol.65, 1090-1098. HODGMAN, T.C (1988a). A new superfamily of replicative proteins. Nature 333,
  • Baculovirus gene ME53 which contains a putative zinc finger motif, is one of the major early-transcribed genes. J. Virol. 67, 753-758. KOGAN, P.H. and BLISSARD, G.W. (1994). A baculovirus gp64 early promoter is activated by host transcription factor binding to CACGTG and GATA elements. J. Virol. 68, 813-822. KOOL, M. and VLAK, J.M. (1993). The structural and functional organization of the Autographa califomica nuclear polyhedrosis virus genome. Arch. Virol.
  • Point mutations define a sequence flanking the AUG initiator codon that modulates translation by eukaryotic ribosomes.
  • RAWLINGS N.D., PEARL, L.H. and BUTTLE, D.J. (1992).
  • the baculovirus Autographa califomica nuclear polyhedrosis virus genome includes a papain- like sequence. Biol. Chem. Hoppe-Seyler 373, 1211-1215. ROEDER, R.G. (1991).
  • the complexities of eukaryotic transcription initiation regulation of preinitiation complex assembly. TIBS 16, 402-408.
  • Brahma a regulator of Drosophila homeotic genes structurally related to the yeast transcriptional activator SNF2/SW12. Cell 68, 561-572. THIEM, S.M. and MELLER, L.K. (1989a). Identification, sequence, and transcriptional mapping of the major capsid protein gene of the baculovirus
  • FIG. 1 Physical map and summary of coding strategy of the AcNPV genome.
  • the upper part of each panel represents a map of the sites in the virus genome for the commonly used restriction endonucleases (see text). Also shown are the hrs within the EcoRI map.
  • the middle part of each panel summarizes the coding potential of all six reading frames of the virus DNA (1,2,3, ,2',3').
  • ORFs are identified as black boxes starting at methionine codons (vertical Unes).
  • the selected ORFs (see text) are numbered 1-154, with appropriate designations for the genes which have been characterized previously (see Table 1).
  • Non-selected ORFs represent potential genes which overlap with other coding regions (see text).
  • the lower section in each panel summarizes the percent purine or A+T composition for the + strand of the virus genome, using a sliding 250 nucleotide window. Units at the bottom are in base-pairs.
  • FIG. 2 Dot matrix analysis of AcNPV genomic DNA. The genomic sequence of AcNPV
  • + strand was compared to itself (left panel), or to its complementary - strand (right panel), using a 24 nucleotide moving window.
  • the direction of sequence (strandedness) relative to the standard map in each comparison is indicated by the arrows on the x and y axes. Dots represent sites where there is 21 out of 24 or greater nucleotide sequence match (88% identity).
  • matches in the left panel indicate sites of positional identity (diagonal Une mnning lower left to upper right), or direct DNA repeats (dots off the diagonal Une).
  • Matches in the right panel indicate regions of inverted repetitive DNA.
  • Dots close to the position where a diagonal Une should be in the right panel represent potential stem-and-loop (hairpin) structures.
  • the columns and rows of dots marking the positions of the repetitive DNA associated with hrs are labelled across the top and on the right-side y-axis. Scales on the x and y axes are in kilobase-pairs.
  • FIG. 3 Circular map of the AcNPV genome. The sites for the EcoRI (outer ring) and
  • HindUL (inner ring) restriction enzymes are presented. The positions of the 154 ORFs described PCMB95/00578
  • Fig. 1 Fig. 1
  • arrows representing the direction of transcription for these putative genes. Shaded arrows indicate that the gene is known to be expressed, or has a weU characterized homologue in the protein sequence databases.
  • Insertion sites and names of well characterized insertion sequences (IS) and retroposons (RP) are indicated, as are the positions of the hr sequences.
  • the scale on the inner circle is in 100 map units.
  • Fig. 10 Modification of the AcNPV CHITINASE gene.
  • Panel (a) Pstl restriction maps for AcNPV (linearized form).
  • Panel (b) Exploded view of genome coordinates 105164-107943 within pUC118.CHTE.
  • Panel (c) pUC118.CHrr-Bgi ⁇ .
  • Panel (d) pUC118.CHTr.LacZ.
  • Fig. 12. Modification of the AcNPV GTA gene.
  • Fig. 13 Modification of the AcNPV PCNP gene.
  • Fig. 14 Modification of the AcNPV ALK-EXO gene.
  • Panel (a) Hindm/Smal restriction map for AcNPV (linearized form).
  • Fig. 15 Single restriction enzyme site (AvrEQ within the AcNPV EGT gene. Panel (a):
  • Avrll restriction enzyme map for AcNPV (linearized form).
  • Fig. 16 Single restriction enzyme site (Fsel) within the AcNPV GTA gene.
  • Fsel restriction enzyme map for AcNPV (linearized form).
  • the selected ORF's are numbered sequentially, in their order of appearance in the + strand of the genome (see text and Fig. 1).
  • the left (column Left) and right (column Right) columns define the ends of the ORF irrespective of its encoding strand.
  • the direction of the transcripts (column D) that could express the ORF is indicated by arrows.
  • the number of amino acids encoded by the ORF (column aa) and the predicted molecular mass of the primary translation product (column M r ) from the first ATG are Usted (see text).
  • the transcription column (Trans) indicates if at least one early (e/E), or TATA-like (t/T), or cap (c/Q motif is present in the 160 nucleotides upstream of an ORF (see text and Table 3). where a TATA-box is positioned 5' to a CAGT in a poUL- like promoter orientation, this is indicated by "TC".
  • TC late promoter motif
  • TAAG L
  • ORFs that have an initiation methionine that conforms to Kozak-rules (column K) for higher eukaryotes are indicated (k).
  • ORFs representing potential mini-cistrons initiating upstream of one of the selected ORFs and with an ATG condon that conforms to Kozak-rules are indicated (*, see text).
  • ORFs that initiate at an ATG codon downstream of the first ATG or an ORF and producing a translation product that is smaller than the computer predicted product are marked (*, see text).
  • Representative motifs in putative translation products (Table 3) are indicated in the domains column (Dom).
  • the motifs included signal peptide (S), zinc finger (Z), leucine zipper (L), nuclear translation signal (N), and NTP binding domain (P).
  • S signal peptide
  • Z zinc finger
  • L leucine zipper
  • N nuclear translation signal
  • P NTP binding domain
  • the comments column includes differences in genomic organization pubUshed for other strains of AcNPV, functional properties of predicted peptide products, or other relevant features. References are Usted as a guide to the Uterature regarding previously pubUshed sequences or studies defining AcNPV gene functions.
  • TATA box TATAAA; TATATA; TATAAT " 61/154 40/183
  • Late promoter TAAG 71/154 1 1/183 oza consensus AxxATG(A/G); GxxATGG 91/154 52/183
  • Zinc finger C/H X 2 -5 C/H X1 L13 C/H X2/5 C/H 31/154
  • the motifs, their patterns and the number of the selected and non-selected ORFs with at least one copy of the indicated motifs are presented.
  • the searches for motifs representing putative early transcription sites involved analyses of DNA sequences 160 nucleotides upstream of the first ATG codon (i.e., CGTGC, TATA box, Cap site and Pol II promoter motifs).
  • the •search involved 80 nucleotides upstream of the ATG codon.
  • Only the selected ORFs were analysed for motifs in the putative gene products (see text).
  • Val GTA 508 18 lie ATA 822 30 Val GTC 4S2 18 lie ATC 590 22 Val GTG 1083 39 lie ATT 1286 48 Val GTT 678 25 SEQUENCE LISTING
  • MOLECULE TYPE DNA (genomic)

Landscapes

  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biomedical Technology (AREA)
  • Organic Chemistry (AREA)
  • Biotechnology (AREA)
  • General Engineering & Computer Science (AREA)
  • Chemical & Material Sciences (AREA)
  • Wood Science & Technology (AREA)
  • Microbiology (AREA)
  • Physics & Mathematics (AREA)
  • Plant Pathology (AREA)
  • Virology (AREA)
  • Molecular Biology (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Biophysics (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Peptides Or Proteins (AREA)

Abstract

La présente invention concerne la séquence nucléotidique complète du génome du clone 6 du baculovirus Autographa californica du virus de la polyhédrose nucléaire (AcNPV). La molécule comporte 133 894 paires de bases et une teneur globale en A+T de 59 %. Des analyses effectuées, il semble découler que le virus est capable de coder environ 154 cadres de lecture ouverts (ORF) d'au moins 150 nucléotides à médiation méthioninique, et potentiellement exprimés. Ces ORF sont régulièrement répartis sur chacun des brins du génome de tout le virus. Ces ORF sont disposés en cadres de lecture adjacents et ne se chevauchant pas, séparés grâce à de courtes régions intergéniques. D'après la séquence nucléotidique primaire, il semble possible de déterminer les fonctions de certains gènes, les sites initiaux de réplication de l'ADN viral, les modalités de régulation des transcriptions géniques précoce et tardive, et des facteurs susceptibles d'affecter l'efficacité translationnelle du gène de l'AcNPV. Les données concernant la séquence génomique confirment, à quelques différences mineures près, les informations obtenues dans le cas des autres clones d'AcNPV. Le clone C6 semble donc proposable comme archétype de l'AcNPV pour les modalités comparatives.
PCT/IB1995/000578 1994-07-04 1995-06-30 Sequence genomique complete du virus autographa californica de la polyhedrose nucleaire WO1996001320A2 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU28972/95A AU2897295A (en) 1994-07-04 1995-06-30 Autographa californica complete genome sequence

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
GB9413420A GB9413420D0 (en) 1994-07-04 1994-07-04 Autographa californica nuclear polyhedrosis virus dna sequences
GB9413420.2 1994-07-04

Publications (2)

Publication Number Publication Date
WO1996001320A2 true WO1996001320A2 (fr) 1996-01-18
WO1996001320A3 WO1996001320A3 (fr) 1996-07-25

Family

ID=10757768

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/IB1995/000578 WO1996001320A2 (fr) 1994-07-04 1995-06-30 Sequence genomique complete du virus autographa californica de la polyhedrose nucleaire

Country Status (3)

Country Link
AU (1) AU2897295A (fr)
GB (1) GB9413420D0 (fr)
WO (1) WO1996001320A2 (fr)

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000005391A1 (fr) * 1998-07-21 2000-02-03 Dow Agrosciences Llc Regulation negative de proteines vegetales a mediation par anticorps
US6635748B2 (en) * 1997-12-31 2003-10-21 Chiron Corporation Metastatic breast and colon cancer regulated genes
CN114058598A (zh) * 2021-11-04 2022-02-18 中国科学院精密测量科学与技术创新研究院 新的重组杆状病毒基因组插入位点及其应用
CN114317608A (zh) * 2020-12-28 2022-04-12 陕西杆粒生物科技有限公司 一种基因敲除型杆状病毒表达载体
CN118086400A (zh) * 2024-04-17 2024-05-28 和元生物技术（上海）股份有限公司 核酸分子、包含其的重组杆状病毒及其应用

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AUPN570295A0 (en) * 1995-09-29 1995-10-26 Commonwealth Scientific And Industrial Research Organisation Biologically active proteins of viral origin

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
ARCH. VIROL., vol.130, pages 1 - 16 M. KOOL AND J.M. VLAK; 'The structural and functional organization of the autographa californica nuclear polyhedrosis virus genome' cited in the application *
VIROLOGY, vol.185, 19 October 0 pages 229 - 241 R.D. POSSEE ET AL.; 'Nucleotide sequence of the Autographa californica nuclear polyhedrosis 9.4 kbp Eco RI-I and -R (polyhedrin gene) region' cited in the application *
VIROLOGY, vol.191, pages 1003 - 1008 S.C. BRAUNAGEL ET AL.; 'Sequence, genomic organization of the EcoRI-A fragment of Autographica californica nuclear polyhedrosis virus, and identification of a viral-encoded protein resembling the outer capsid protein VP8 of Rotavirus' cited in the application *
VIROLOGY, vol.202, pages 586 - 605 M.D. AYRES ET AL.; 'The complete DNA sequence of Autographa californica nuclear polyhedrosis virus' *

Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6635748B2 (en) * 1997-12-31 2003-10-21 Chiron Corporation Metastatic breast and colon cancer regulated genes
US7279307B2 (en) 1997-12-31 2007-10-09 Chiron Corporation Metastatic breast and colon cancer regulated genes
US7795407B2 (en) 1997-12-31 2010-09-14 Novartis Vaccines And Diagnostics, Inc. Metastatic breast and colon cancer regulated genes
WO2000005391A1 (fr) * 1998-07-21 2000-02-03 Dow Agrosciences Llc Regulation negative de proteines vegetales a mediation par anticorps
CN114317608A (zh) * 2020-12-28 2022-04-12 陕西杆粒生物科技有限公司 一种基因敲除型杆状病毒表达载体
CN114317608B (zh) * 2020-12-28 2023-08-22 陕西杆粒生物科技有限公司 一种基因敲除型杆状病毒表达载体
CN114058598A (zh) * 2021-11-04 2022-02-18 中国科学院精密测量科学与技术创新研究院 新的重组杆状病毒基因组插入位点及其应用
CN114058598B (zh) * 2021-11-04 2023-04-28 中国科学院精密测量科学与技术创新研究院 新的重组杆状病毒基因组插入位点及其应用
CN118086400A (zh) * 2024-04-17 2024-05-28 和元生物技术（上海）股份有限公司 核酸分子、包含其的重组杆状病毒及其应用

Also Published As

Publication number Publication date
AU2897295A (en) 1996-01-25
WO1996001320A3 (fr) 1996-07-25
GB9413420D0 (en) 1994-08-24

Similar Documents

Publication Publication Date Title
DK2467489T3 (en) Baculovirus-based production of biopharmaceuticals free of contaminating baculoviral virions
Cummings et al. The complete DNA sequence of the mitochondrial genome of Podospora anserina
KR102147007B1 (ko) Fad3 성능 유전자좌 및 표적화 파단을 유도할 수 있는 상응하는 표적 부위 특이적 결합 단백질
AU2013312198B2 (en) Fluorescence activated cell sorting (FACS) enrichment to generate plants
CN111163803B (zh) 表达car t细胞靶物的溶瘤病毒及其用途
AU2017353868C1 (en) Synthetic chimeric poxviruses
CN112543806B (zh) 合成嵌合痘苗病毒
KR102080055B1 (ko) 식물 조절 요소 및 그의 용도
CN113215109B (zh) 非洲猪瘟多基因联合缺失减毒株的构建及作为疫苗的应用
KR20220165731A (ko) Sars-cov-2 바이러스에 대한 재조합 폭스바이러스 기반 백신
KR20230113832A (ko) 키메라 폭스바이러스 조성물 및 이의 용도
Van Oers et al. The baculovirus 10-kDa protein
CN112899290B (zh) 一种天然免疫抑制基因缺失的减毒非洲猪瘟病毒株及应用
CN113025629A (zh) 一种基因缺失的减毒非洲猪瘟病毒株及应用
KR20220148823A (ko) 천연 또는 합성 dna에 의해 생산된 폭스바이러스-기반 벡터 및 그의 용도
CN116670153A (zh) 允许在稳定细胞系中高效生长的非洲猪瘟疫苗的基因组缺失
WO1996001320A2 (fr) Sequence genomique complete du virus autographa californica de la polyhedrose nucleaire
CN112261951A (zh) 包含合成嵌合痘苗病毒的干细胞及其使用方法
US20040185565A1 (en) High throughput system for producing recombinant viruses using site-specific recombination
EP0975787A1 (fr) Vecteur d'administration de gene a base d'entomopoxvirus pour vertebres
US6180098B1 (en) Recombinant helicoverpa baculoviruses expressing heterologous DNA
HK40078138A (en) Poxvirus-based vectors produced by natural or synthetic dna and uses thereof
KR100270928B1 (ko) 누에 핵다각체병 바이러스 p10 유전자를 이용한 전이 벡터 및 제조방법
KR100325394B1 (ko) 신규한 진핵세포용 유전자 클로닝 전달운반체와 재조합 바이러스 운반체 및 이들을 이용한 재조합 단백질 제조방법
HK40042130A (en) Stem cells comprising synthetic chimeric vaccinia virus and methods of using them

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A2

Designated state(s): AM AT AU BB BG BR BY CA CH CN CZ DE DK EE ES FI GB GE HU IS JP KE KG KP KR KZ LK LR LT LU LV MD MG MN MW MX NO NZ PL PT RO RU SD SE SG SI SK TJ TM TT UA UG US UZ VN

AL Designated countries for regional patents

Kind code of ref document: A2

Designated state(s): KE MW SD SZ UG AT BE CH DE DK ES FR GB GR IE IT LU MC NL PT SE BF BJ CF CG CI CM GA GN ML MR NE SN TD TG

DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
121 Ep: the epo has been informed by wipo that ep was designated in this application
REG Reference to national code

Ref country code: DE

Ref legal event code: 8642

122 Ep: pct application non-entry in european phase
NENP Non-entry into the national phase in:

Ref country code: CA