Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
  • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
  • C07K16/081—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from DNA viruses
  • C07K16/085—Herpetoviridae, e.g. pseudorabies virus, Epstein-Barr virus
  • C07K16/087—Herpes simplex virus
• • 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
  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/14—Hydrolases (3)
  • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
  • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
  • C12N9/503—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from viruses

Definitions

• the present invention relates to the identification of a new enzyme in the area of herpes simplex virology. More particularly, it relates to the identification of a protease associated with herpes simplex virus type-2 (HSV- 2) protease, to nucleic acid sequences encoding such a protease, and to the expression of the protease by a host cell.
• HSV- 2 herpes simplex virus type-2
• the present invention also relates to the use of the proteolytic activity as a target for anti-viral therapy.
• HSV herpes simplex virus
• HSV falls into two distinct serotypes, designated type 1 and type 2 (HSV-1 and HSV-2, respectively).
• the HSV-1 genome specifies an abundant capsid protein (Gibson etal.,J. Virol. 13: 155-165 (1974)) and a set of genetically and immunologically related viral capsid proteins have been identified and designated infected-cell proteins 35 (ICP35) (Braun et al. J. Virol.49: 142-153 (1984)).
• HSN-2 a member of the alphaherpesviridae subfamily of the herpesviridae family, has a variable host range, a short reproductive cycle, and the ability to establish a latent infection in the sacral ganglia (Roizman et al. Virology, 2 ⁇ ⁇ . New York: Raven Press 65: 1795-1841 (1990).
• the HSV- 2 virion is composed of a nucleoprotein core surrounded by a capsid, tegument, and a lipid membrane. These structural features are characteristic of all herpes viruses.
• the HSN-2 genome is approximately 150 kilobases in size. It consists of two components, the unique long (UL), and unique short (Us) regions (Braun et al.
• a major drawback in developing effective therapy against HSV-1 and HSV-2 has been the failure to discover viral-specific replicative mechanisms which can be blocked by antiviral agents.
• the ideal antiviral agent should interrupt viral replication at an essential step of the life cycle without significantly altering host-cell metabolism. While some progress has been made in controlling HSV infections through the use of drugs such as 5- fluorodeoxyuridine and acyclovir, no satisfactory treatment for HSV infections has been found. Consequently, improvement for antiviral therapy is needed in this area.
• Maturation of herpes virus particles is believed to occur through the formation of a procapsid structure, which acquires DNA and an envelope to become an infectious virion (Whitley, supra, 1990; Roizman, supra, 1990).
• Proteases appear to be essential to the development of the capsid of the virus. Consequently, inhibiting protease action will lead to disruption of the lytic cycle of the virus. Thus, inhibitors of protease action are desirable targets for antiviral therapy.
• the present invention provides a HSN type 2 protease.
• the amino acid sequence of the protease and the D ⁇ A sequence, or degenerate equivalents thereof, which encodes the protease is shown in Figure 1.
• the present invention further provides expression vectors capable of expressing HSN type 2 protease in a host cell.
• a D ⁇ A segment encoding a HSN type 2 protease or a portion thereof is operably linked to suitable regulatory regions in a vector, whereby the vector is replicated and carried by the host cell.
• Recombinant host cells carrying said vectors are additionally provided.
• Figure 1 shows the nucleotide and predicted amino acid sequences of the HSN-2 genomic region comprising the coding sequences of the protease gene and ICP35 protein.
• the HSN-2 gene comprises a D ⁇ A sequence beginning at nucleotide 211 and extending through nucleotides 951-1120; this range is flexible and is meant to denote all portions of the sequence which encode for protease activity.
• the open reading frame for the ICP35, the substrate for the protease comprises the amino acid sequence presented in Figure 1 between nucleotides 1124 and 2119.
• FIG. 2 is a schematic representation of the HSN gene showing the expression cassettes used in the examples.
• Figure 3 shows the SDS-PAGE for the expression and self-processing of HSN-2 protease-CKS fusion protein in E. coli.
• Figure 4 shows the SDS-PAGE for the expression and self-processing of HSN-2 protease translationally coupled to CKS in E. coli.
• Figure 5 is a Western Blot of HSN-2 protease expression and self- processing in E. coli.
• Figure 6 is a schematic illustration of the construction of plasmid pSSPIl as described in Example 13.
• Figure 7 shows the SDS-PAGE for the expression and self-processing of HSN-2 protease/ICP35 translationally coupled to CKS in E. coli.
• Figure 8 is a Western Blot of HSN-2 protease expression and self- processing in S. cerevisiae.
• Figure 9 shows the SDS-PAGE for the expression and self-processing of HSN-2 protease in Sf9 cells.
• Figure 10 is a Western Blot of HSN-2 protease expression and self- processing in Sf9 cells.
• Figure 11 shows the SDS-PAGE for the expression and self-processing of HSN-2 protease in Baculovirus-infected Trichoplusia ni larvae.
• Figure 12 is a Western Blot of HSN-2 protease expression and self- processing in Baculovirus-infected Trichoplusia ni larvae.
• CKS refers to CTP:CMP-3-deoxy-D- ann ⁇ 3-octulosonate cytidylyl transferase, also known in the art as CMP-KDO synthetase or CKS, an enzyme derived from Escherictiia coli (E. coli), according to methods known in the art.
• D ⁇ A expression vector is any autonomous element capable of replicating in a host independently of the host's chromosome, after additional sequences of D ⁇ A have been incorporated into the autonomous element's genome.
• Gene is a segment of D ⁇ A, a portion of which codes for a specific polypeptide or R ⁇ A molecule.
• ICP35 refers to a set of genetically and immunologically related viral capsid proteins identified with the HSN-1 and -2 genomes.
• Promoter is a D ⁇ A sequence generally described as the 5' region of a gene, located proximal to the start codon. At the promoter region, transcription or expression of an adjacent gene is initiated. This is referred to as the transcription initiation site. At the promoter region may be a sequence of nucleotides that interacts as a control over the expression of any operably linked structural gene or genes. "Operably linked” is a term for the control exerted by the promoter over the initiation of expression of the polypeptide encoded by a structural gene.
• Open reading frame (ORF) is a D ⁇ A sequence containing a series of triplets coding for amino acids without any termination codons. Sequences of this type are potentially translatable into a protein.
• "Protease” refers to a proteolytic activity and the corresponding encoding nucleic acid sequences which are capable of cleaving a herpes virus assembly protein precursor.
• the HSN-2 protease gene of the present invention comprises a D ⁇ A sequence beginning at nucleotide 211 and extending through nucleotides 951-1120; this range is flexible and is meant to denote all portions of the sequence which encode for protease activity.
• Transcription initiation site is a D ⁇ A sequence of a promoter to which R ⁇ A polymerase binds, thereby initiating transcription of succeeding codons in a 5' to 3' direction.
• Transcription terminator is a D ⁇ A sequence at the end of the transcript that causes R ⁇ A polymerase to terminate transcription.
• UL26 refers to that part of the HSN-2 genome believed to encode the protease and the adjoining ICP35 gene.
• the present invention relates to the discovery of a new viral protease encoded by HSN type 2.
• the genome of HSN-2 is approximately 150 kilobases in size. Unlike HSN-1, much of the genomic D ⁇ A of HSN-2 has not been sequenced. And, to date, only a few analogous genes related to HSN-1 have been identified within the HS V-2 genome. The proteins expressed by these genes which have been identified to date include thymidine kinase, glycoproteins C & D, D ⁇ A polymerase and alkaline exonuclease.
• HSN-2 D ⁇ A was isolated from Nero cells infected with HSV-2 strain G using the procedure of Straus et al. J. Virol. 40: 516-525 (1981). Approximately 10 ⁇ g of D ⁇ A was digested with -B ⁇ mHI enzyme and separated on a 1% TBE gel.
• the gel was stained with ethidium bromide, photographed, and placed on a southern blotting apparatus for transfer to nitrocellulose.
• a nick-translated 32p. ⁇ a beled probe of the HSV-1 protease gene was added to the blot and left to hybridize overnight. After extensive washing, the blot was subjected to autoradiography for 30 minutes. A single band corresponding to approximately 4.0 kilobases in size was visualized. This segment of DNA contained the putative HSV-2 protease coding sequence.
• a Bam ⁇ I digest of the DNA demonstrated that each clone contained the 4.0 kilobases DNA fragment encoding the HSV-2 protease gene.
• the 4.0 kilobases fragment was subjected to several different restriction digests and Southern blot analyses.
• a single 1.5 kilobases BaniHl-Sa ⁇ . fragment containing the protease gene was identified by Southern blot hybridization and subcloned into pUC19. This plasmid has been designated pH2Pro which contains the entire HSV-2 protease coding sequence.
• the - ⁇ mHI-S ⁇ -ZI fragment was cloned into M13mpl8 and M13mpl9 phage RF vectors for single-stranded D ⁇ A sequence analysis.
• Several phage clones from transformed mpl8 and mpl9 plates were picked and sequenced through the first 100 bases to determine the correct orientation for sequence analysis.
• One mpl9 clone was correctly oriented and became the focus of the D ⁇ A sequencing analysis.
• Approximately 1900 bases of the protease gene have been sequenced from the Ban ⁇ r ⁇ l-SaU. mpl9 clone.
• the complete coding sequence for the HSV-2 protease is shown in Figure 1 between nucleotides 211 -951.
• the promoter region for the HSN-2 protease has also been isolated and maps between positions 1-210 as shown in Figure 1.
• the coding sequence for ICP35 is between nucleotides 1126-2119.
• the sequence can be derived from plasmid pH2proA (which contains the entire HSN-2 protease coding sequence along with flanking promoter/regulatory sequences and partial ICP35 coding sequences) or pH2proB (which contains the remaining coding sequence for the assembly protein ICP35). Plasmids pH2proA and pH2proB were deposited on April 26, 1993 at the Agricultural Research Service Patent Culture Collection, Peoria, Illinois and have the accession numbers NRRL B-21185 and NRRLB-21186, respectively.
• the nucleic acid sequences of the present invention may include a sequence or portion thereof as illustrated in Figure 1. For example, the sequence may be either smaller or larger than those illustrated in Figure 1 as long as the nucleic acid segment encodes a functional equivalent of the protease.
• the HSV-2 protease coding region encodes a polypeptide of approximately 247 amino acids which has the amino acid sequence presented in Figure 1 between nucleotide 211 and 951. It is understood that the size of the protease may be smaller or larger than this range as long as the protein fragment retains its biological or functional activity.
• any herpes protease containing at least 70% homology, and preferably 90% homology, to any contiguous stretch of ten or more amino acids presented herein which is isolated from a HSV-2 source is also intended to be within the scope of the present invention. This homology is determined by any of the available sequence analysis software packages, such as that available from DNAstar, Intelligenetics, the Genetics Computer Group of the University of Wisconsin, and the like.
• the production of a recombinant form of HSN-2 typically involves (a) isolating a D ⁇ A that encodes the mature enzyme; (b) placing the recovered coding sequence in operable linkage with suitable control sequences in a replicable expression system; (c) transforming a suitable host with the vector; and (d) culturing the transformed host under conditions to effect the production of the recombinant HSN-2 protease.
• control sequences, expression systems, and transformation methods are dependent on the type of host cell used to express the gene.
• Prokaryotes are most frequently represented by various strains of Escherichia coli. However, other microbial strains may also be used, for example, Bacillus subtilis, various strains of Pseudomonas, or other bacterial strains. In such prokaryotic systems, plasmid vectors that contain replication sites and control sequences derived from a species compatible with the host are used. For example, E. coli is typically transformed with derivatives of pBR322, a plasmid derived from an E. coli species described by Bolivar, et al, Gene 2: 95 (1977).
• Infection of insect larvae with baculovirus can be achieved by injection of the virus into the larval hemolymph (Medin, et al., 1990, supra), or by oral ingestion (Price, et al., 1989, supra). It has been reported that infection of larvae by the oral route with recombinant viruses lacking the polyhedrin protein can be improved by coinfection with wild- type nuclear polyhedrosis virus (Price, et al., 1989, supra.). In some of the examples described below, infection of cabbage looper larvae was achieved using recombinant virus alone as well as with a mixture of recombinant and wild type baculoviruses.
• the present invention also provides suitable vectors for the expression of the HSN-2 protease.
• suitable vectors containing the desired coding and control sequences employs standard ligation and restriction techniques that are well understood in the art.
• Site-specific D ⁇ A cleavage is performed by treating the D ⁇ A with the suitable restriction enzyme under conditions that are generally well-understood in the art.
• any selectable marker may be used which is functional in E. coli or other selected host and allows cells transformed with a vector of the present invention to be distinguished from cells not so transformed.
• a gene that provides a dominant selectable marker for antibiotic resistance in E. coli is such a selectable marker.
• the gene for ampicillin resistance is especially preferred.
• Other D ⁇ A segments which confer resistance to other antibiotics including apramycin, tylosin, picromycin, oleandomycin, viomycin, neomycin, tetracycline, chloramphenicol, hygromycin and the like, can be used either as replacements of, or in addition to, the drug resistance segment described herein.
• a transforming D ⁇ A according to the present invention may include elements for its selection and replication in bacteria, especially E. coli, whereby production of large quantities of D ⁇ A by replication in bacteria will be facilitated.
• a preferred D ⁇ A of the present invention is a plasmid which includes a segment comprising the origin of replication and ampicillin resistance gene or fragment thereof of plasmid pBR322.
• Yeast offer an attractive alternative host system to E. coli.
• a typical yeast expression vector will comprise (i) a yeast selective marker, (ii) a yeast origin of replication and (iii) yeast promoter and terminator sequences positioned relative to a unique restriction site in such a way that expression of HSN-2 protease may be obtained.
• SDH sorbitol dehydrogenase
• Both cassettes may be inserted into a 30 copy yeast plasmid containing the yeast TRP1 gene as a selectable marker and 2 micron origin of replication. Any yeast replication origin known in the art may be used to construct the vector.
• the replication region of the natural yeast plasmid 2 micron can be employed. This plasmid is cryptic in that it confers no readily detectable phenotype and is present in about 100 copies per cell. For example, S.
• HSV-2 protease of the present invention is placed under the control of transcriptional and translation initiation and termination regulatory sequences of the alcohol dehydrogenase I gene (as described in the Example section) and used to express HS V-2 in any yeast cell capable of transformation, including, but not limited to, yeast mutants that alter regulation, and the like.
• yeast nitrogen base a chemically defined medium which contains a number of trace elements, vitamins, trace amounts of amino acids to stimulate growth, and the principal minerals potassium phosphate, magnesium sulfate, sodium chloride, and calcium chloride.
• the nitrogen source is ammonium sulfate.
• the desired carbon source is added at a concentration of from between about 0.5% and between about 3%.
• the pH range of the medium is usually from between about pH 3.0 and about pH 8.0, preferably from between about pH 4.5 and about pH 6.5.
• segments of the HSV-2 viral genome including all or portions of the UL26 gene, believed to encode the protease and the adjoining ICP35 gene, are cloned into a series of vectors designed to give efficient expression in E. coli, S. cerevisiae or insect cells.
• the segments to be cloned are amplified from the viral genome employing the polymerase chain reaction (PCR) as described in US. Patents 4,883,195 and 4, 883,202, the entire disclosures of which are incorporated herein by reference.
• the segments of UL26 amplified are sequenced after cloning into the respective vectors to determine that they match the sequence obtained from pH2proA and pH2proB, which carry the unamplified UL26 gene derived from genomic HSN-2 D ⁇ A.
• Segments of UL26 cloned include the first 247 amino acids, from the ⁇ -terminus to the site believed to be cleaved by the HSN-2 protease or the first 306 amino acids, from the ⁇ -terminus to the methionine residue which is believed to correspond to the start of the ICP35 gene.
• protease as part of an operon wherein the protease gene is downstream of a highly expressed gene is also described.
• expression of the entire UL26 gene, comprising the protease and adjacent ICP35 segments in yeast is described.
• the protease of the present invention is useful in a screening method for identifying potential herpes viral protease inhibitor compounds, also known as “candidate antiviral inhibitor compounds". It is contemplated that this screening technique will prove useful in the general identification of any compounds that will serve the purpose of inhibiting HSN-2 protease. It is further contemplated that useful compounds in this regard will not be limited to proteinaceous or peptidyl compounds but may include synthetic organic compounds which are non-peptidyl in nature and which will be recognized and bound by the protease, and serve to inhibit the enzyme through a tight binding or other chemical interaction. The use of such inhibitors to block the action of the protease will serve to treat or alleviate an HSN-2 infection. Inhibitors of HS V-2 protease will be useful by themselves or in conjunction with other herpes therapies.
• the present invention is directed to a method for determining the ability of a candidate compound to inhibit HS V-2 protease, the method comprising: obtaining a composition comprising HS V-2 protease that is capable of cleaving an appropriate substrate in a reaction mixture; mixing a candidate compound with the protease and suitable substrate; and determining whether the candidate compound inhibited the protease from cleaving the substrate.
• An important aspect of the candidate compound screening assay is the ability to prepare a protease composition in a relative purified form. This is an important aspect of the candidate compound screening assay in that without at least a relatively purified preparation, one will not be able to assay specifically for HS V-2 protease inhibition, as opposed to inhibition by extraneous substances in the assay. In any event, the successful expression of the recombinant HS V-2 protease now allows for the first time the ability to identify new compounds which can be used for inhibiting this herpes-related protein.
• Plasmid DNAs used for co-transfection of insect cell cultures were prepared by equilibrium centrifugation in cesium chloride gradients according to Maniatis et. al., supra. DNA fragments were recovered from low melting temperature agarose (SeaPlaque Agarose, FMC, Rockland, ME.). Plasmid preparations were done using the Magic DNA preparation systems by Promega. Insertion of DNA fragments into pUC18 or its derivatives, pKB130, pJO201, etc. often employed the use of X-gal as a color reagent to screen for the presence of inserts.
• the standard PCR mixture contains the following components: 50 ng HSV-2 genomic DNA, 20 mM Tris-HCl pH 8.3, 1.5 mM MgCl2, 50 mM KC1, 0.2 mM dATP, dCTP, dTTP and dGTP, 0.4 pmol/ml each primer, 10% formamide, 2% glycerol and 1 U Taq DNA polymerase in a 50 ⁇ l reaction.
• the standard PCR conditions can be varied to include: 50 ng HSV-2 genomic DNA, 20 mM Tris-HCl, pH 8.8 10 mM KC1, 10 mM ammonium sulfate, 6 mM magnesium sulfate, and 0.1% Triton X-100, 0.2 mM dATP, dCTP, dTTP and dGTP, 0.4 pmol ml each primer.
• the variation can also include 0.1 mg ml acetylated BSA, 10% formamide, and 2% glycerol.
• thermostable polymerase such as Taq DNA polymerase ⁇ Thermus aquaticus), Vent DNA polymerase, or Tth DNA polymerase (Thermus thermophilus) must also be added. Cycling temperatures and times are described for each application. Growth of E. coli in L broth and transformation of plasmid DNA into E. coli is done as described by Maniatis et al., supra. General methods used in the manipulation of yeast are described by
• Minimal medium contain 0.67% yeast nitrogen base and 2% glucose. Amino acids are added according to Sherman et al., supra. Transformation of yeast is described by Percival et al., Anal. Biochem. 163:39 (1987). Transformants containing plasmids derived from pVTIOO-U and it's derivatives were grown selectively at 30°C for 48 hrs in minimal liquid medium containing 2% glucose as the carbon source.
• soluble protein extracts of Sf9 insect cells 2 x 10 ⁇ cells were plated in 25 cm ⁇ flasks and infected with 1 ml of culture fluid containing recombinant virus plus 4 ml of fresh media. Infections were allowed to proceed for three days, after which time cells were harvested by low speed centrifugation and washed once with phosphate buffered saline. Cells were then resuspended in 100 ml of hypotonic lysis buffer (10 M Tris pH 7.4, 10 mM NaCl, 1.5 mM MgCl2) and incubated on ice with occasional vortexing for 20 minutes. The extract was then pelleted in a microfuge for 2 minutes to remove any insoluble material and used for SDS-PAGE.
• hypotonic lysis buffer 10 M Tris pH 7.4, 10 mM NaCl, 1.5 mM MgCl2
• Cabbage looper moths and larvae were reared according to the method of Guy, R., Leppla, N., Rye, J., Green, C, Barrette, S. & Hollien, K. (1985) in "Handbook of Insect Rearing, Vol. II", edited by P. Singh and R. Moore, Elsevier, Amsterdam.
• Adults were maintained in environmental growth chambers at 28 °C, 80% relative humidity, with a 14 hour photophase and fed a 10% sucrose solution. Oviposition occurred on paper toweling which was wrapped around the wire mesh cages. The egg laden toweling was surface sterilized with dilute formalin and rinsed thoroughly with water.
• Vero cells were grown in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% Fetal Calf Serum.
• DMEM Dulbecco's Modified Eagle Medium
• HSV-2 strain G was obtained from the American Type Culture Collection (ACT VR-734). Viral stocks were grown and titered on Vero cells.
• E. coli strain XLl-Blue and DH5 ⁇ competent cells for transformation were purchased from Stratagene and BRL, respectively.
• Saccharomyces cerevisiae strain YJO ura3-52 leu2-3,l 12 gal4 ⁇ gal80 ⁇
• Yeast vector pVTIOO-U which contains the yeast alcohol dehydrogenase 1 [ADH1] expression cassette, a yeast URA3 selectable marker and the yeast 2 ⁇ origin of replication; Gene 52, 225-233, 1987
• Dr. D. Thomas Biotechnology Research Institute, National Research Council of Canada, Montreal, Que., Canada.
• the Baculovirus expression system including Baculogold insect virus, expression vector pVL1392, Sf9 insect cells, and TMN-FH serum- supplemented culture media were obtained from Pharmingen, San Diego, California. Handling of tissue culture cells and propagation of recombinant virus were performed according to the suppliers specifications as instructed in the accompanying manual by Gruenwald et. al., Baculovirus Expression Vector System: Procedures and Methods Manual, 2nd Ed. Sf9 insect cell cultures were maintained in 75 cm ⁇ tissue culture flasks at 27 °C. Cells were fed with fresh TMN-FH media every 2 to 3 days and split 1:5 once per week. Cells were split into fresh cultures 1 or 2 days prior to their use in transfections or infections to insure healthy log phase growth. All viral stocks were stored at 4 °C. and protected from exposure to light.
• HSV-2 DNA was isolated from Vero cells infected with HSV-2 (G) at a multiplicity of 0.001 plaque-forming units per cell using the procedure of Straus et al. supra (1981). Cells were collected, spun down at low speed (3000 rpm) and resuspended in IX lysis buffer [0.5% NP-40, 3.6 mM CaCl2, 5 mM magnesium acetate, 125 mM KC1, 0.5 mM EDTA (pH 7.5), 6 mM ⁇ - mercaptoethanol, 0.5% deoxycholate]. Cell lysate was extracted one time with freon by shaking for 1 minute and centrifuging at 1000 rpm for 10 minutes at 4°C.
• IX lysis buffer 0.5% NP-40, 3.6 mM CaCl2, 5 mM magnesium acetate, 125 mM KC1, 0.5 mM EDTA (pH 7.5), 6 mM ⁇ - mercaptoethanol, 0.5% deoxychol
• the aqueous phase was removed and layered onto a discontinuous gradient of 5% and 40% glycerol in IX lysis buffer and spun at 33,000 rpm for 45 minutes. After spinning, cell pellets were taken up in 2X STE [0.1M Tris- HCl pH 7.5), 20 mM EDTA, 2% SDS], proteinase K was added to a final concentration of 200 mg ml and incubated at 50°C for 30 minutes. Viral DNA was gently extracted by washing one time with phenol, one time with a phenol- chloroform mixture, and one time with a chloroform/isoamyl alcohol mixture. The upper aqueous phase containing HS V-2 DNA was carefully removed with a wide-bore pipette and precipitated in three volumes of ethanol at -20°C.
• Southern blotting and hybridization was done essentially as described by Maniatis et al. supra (1982). Approximately 10 ⁇ g of HSV-2 DNA was digested with the restriction enzyme BamHI and separated by gel electrophoresis on a 1% TBE agarose gel for 3 hours at 100V. After electrophoresis, the gel was stained with ethidium bromide, photographed, and placed in a Southern blotting unit for transfer to nitrocellulose. The DNA was depurinated for 5 minutes in a solution of 0.25N HC1, denatured in a solution of 1.5M NaCl and 0.5M NaOH, and neutralized. A nick-translated probe derived from the HSV-1 protease sequence was used for the hybridization.
• HS V-2 DNA was subjected to -B ⁇ mHI, Sal I, and -B ⁇ mHI- Sal I digests, and the resulting digested DNA was cloned into the pUC19 vector enzymatic restriction as described in Straus et al. supra (1981).
• the ligated DNA was used to transform competent JM109 cells and colonies were picked to generate an HS V-2 derived genomic library.
• colony hybridization was used to identify HS V-2-protease specific clones in pUC19. Colonies derived from the genomic library were replica-plated onto nitrocellulose. The bacterial colonies were lysed and prepared as described in Maniatis et al, supra, (1982). After neutralization, the colony blot was transferred onto 3 MM paper and baked at 80°C for 2 hours. Colony blots were prehybridized for 4 hours, then a nick-translated probe derived from HSV-1 protease strain F was denatured and added to the prehybridization solution and left to hybridize overnight. After washing, the blot was dried and autoradiographed. Positive colonies were picked, grown, and subjected to restriction digest analysis.
• HSV-2 DNA was isolated from Vero cells infected with HS V-2 strain G as described in Example 1, above. Approximately 10 ⁇ g of DNA was digested with BamHL enzyme and separated on a 1% TBE gel. For the hybridization and Southern blot analysis, the gel was stained with ethidium bromide, photographed, and placed on a southern blotting apparatus for transfer to nitrocellulose. A nick-translated 32p-i beled probe of the HSV- 1(F) protease gene was added to the blot and left to hybridize overnight. After extensive washing, the blot was subjected to autoradiography for 30 minutes. A single band corresponding to approximately 4.0 kilobases in size was visualized. This segment of DNA contained the putative HS V-2 protease gene.
• the protease gene was cloned from the HS V-2 genome employing a shotgun cloning procedure utilizing the - ⁇ /nHI-digested HS V-2 genome randomly cloned into the plasmid vector pUC19. Transformed bacterial colonies from the ligated pUC19 vector were screened by DNA-DNA colony hybridization. Five HS V-2-specific DNA clones containing the protease gene were identified by hybridizing the nick-translated HSV-1 protease DNA probe to nitrocellulose. For verification of the HS V-2 DNA sequence contained within pUC19, positive bacterial colonies were picked from a replica plate, grown, and plasmid DNA extracted from them.
• a -S ⁇ mHI digest of the DNA demonstrated that each clone contained the 4.0 kilobases DNA fragment encoding the HS V-2 protease gene.
• the 4.0 kilobases fragment was subjected to several different restriction digests and Southern blot analyses.
• a single 1.5 kilobases BamHI- Sal I fragment containing the protease gene was identified by Southern blot hybridization and subcloned into pUC19 .
• Further restriction analyses also revealed a pUC19-derived clone containing a 1.9 kilobases Sal I fragment with additional coding sequences.
• This plasmid has been designated pH2ProA which contains the entire HS V-2 protease coding sequence along with flanking promoter/regulatory sequences and partial ICP35 coding sequences.
• the BamHL-Sal I fragment was cloned into M13mpl8 and M13mpl9 phage RF vectors for single-stranded DNA sequence analysis.
• Several phage clones from transformed mpl8 and mpl9 plates were picked and sequenced through the first 100 bases to determine the correct orientation for sequence analysis.
• One mpl9 clone was correctly oriented and became the focus of the DNA sequencing analysis.
• both the 1.9 kilobases SalL fragment derived from pH2ProA and the BamHL fragment derived from pH2ProB were cloned into M13mpl9 for sequencing.
• a cassette system was utilized to clone portions of the UL26 gene for expression of the protease gene in E. coli as shown in Figure 2.
• the cassettes were PCR amplified from HS V-2 genomic DNA utilizing primers with non- homologous tails and appropriate restriction sites for cloning.
• Part A contains the 5' MET ATG of protease up to a unique AccL site at nucleotide 177.
• Part B contains the AccL site at nucleotides 177 through 741 which encodes the protease gene up to the cleavage site at amino acid 247.
• Part C contains nucleotides 725 to nucleotides 921 which encodes the 3' end of the protease from a unique AflLTL site through to the start of the ICP-35 portion of the molecule.
• Plasmid pAlB6 was acheived by PCR amplification of parts A and B and cloning into the expression vector pJO201 resulting in the expression of C-terminally fused HSV-2 protease(l-247).
• PCR was accomplished using a mixture containing 50 ng HSV-2 genomic DNA, 20 mM Tris-HCl pH 8.8, 2 mM MgSO4, 10 mM KC1, 10 mM (NH4)2SO4, 0.2 mM dATP, 0.2 mM dCTP, 0.2 mM dTTP, 0.2 mM dGTP, 0.4 ⁇ M each oligonucleotide primer, 10% formamide, 2% glycerol and 1U Vent DNA polymerase in a 50 ⁇ l reaction. Vent was added after heating the mixture at 95 °C for 1 minute.
• the 5' (upstream) PCR primer for part A was: 5-TAGATGAATIC ⁇ TAGAAGGT ⁇ 3T ⁇ T ⁇ TrAG GGaXXnC-n-XXX-L ⁇ CGGG-S'. It has an EcoRI site for cloning and a Factor Xa cleavage site at the CKS/HS V-2 junction. The codons for the first 10 amino acids were optimized in the third position; the following 21 nucleotides are completely homologous for PCR priming.
• the 3' primer for It has an Xb ⁇ L site for cloning and 28 nucleotides of homologous sequence ending in the AccL site.
• the PCR product was 213 nucleotides.
• the 5' primer for part B was: 5-AGTAGTCTAGAGTAGAOCA ⁇ X-ra- ⁇ It has an Xb ⁇ l site for cloning and 22 nucleotides of homologous sequence starting from the ⁇ ccl site.
• the 3' primer for part B was: 5 AGATCOGCAGTTAa-KXriGAAGG CCnGlGT ⁇ It has a PstL site for cloning, a TAA stop codon and 27 nucleotides of homologous sequence.
• the PCR product was 591 nucleotides.
• the expression vector was pJO201 which carries the ampicillin resistance gene and ⁇ kdsB, the gene which codes for CKS, driven by a modified lac promoter.
• CKS CMP-KDO Synthetase
• the enzyme has been overexpressed in E. coli using a modified lac promoter and has been used as a fusion partner to overexpress a number of genes (BioTechniquesNol.8, No. 5 [1990]).
• a multiple cloning site was engineered at the 3' end of kdsB; the cassettes were cloned as EcoRL/Pstl restriction fragments.
• Parts A and B were cloned in pJO201 (4.0 kilobases) to give plasmid pAlB6 of 4.8 kilobases.
• the plasmid was transformed in E. coli strain XLl-Blue to give strain SSHP1.
• the strain was grown in L Broth to an OD600 of 0.5-1.0, induced with 1 mM IPTG and grown for an additional 4-16 hours.
• CKS/HSV-2 protease was evaluated by SDS-PAGE; a band corresponding to the predicted molecular weight for HS V-2 protease fused to CKS (55 kilodaltons) was obtained as shown in Figure 3.
• the fusion protein was cleaved by digestion with factor Xa at an enzyme to substrate ratio of 1:200 (w/w) for 12 hours.
• Cleavage was evaluated by SDS-PAGE and Western Blots using an antibody against the CKS protein; production of bands corresponding to the predicted molecular weight for HSV-2 protease (26.6 kDa) and for CKS (28.8 kDa) were obtained.
• Strain SSHP1 containing plasmid pAlB6 (prepared as desribed above) encoding CKS/HSV-2 protease( 1-247) was grown for expression as described in Example 5.
• Preparative SDS-PAGE was done on aliquots of whole cell lysates. Proteins were visualized with 0.25 M KCl/1 mM dithiothreitol and then destained with 1 mM dithiothreitol.
• the protein was suspended in Freund complete adjuvant to approximately 0.1 mg/ml and was used to inject the animals.
• the cassette system described in Example 5 and Figure 2 was used to construct a CKS fusion to HSV-2 Protease (1-307).
• Part C was a new PCR product, parts A and B were identical to that described in Example 5. PCR conditions were as described in Example 5.
• the 5' primer for part C was: 5-lAGATCTAGAATOCXXXX-L ⁇ CACA ⁇ It contains an XbaL site for cloning, the AflLLL site and 26 nucleotides of homologous sequence.
• the 3' primer was:
• the PCR product was 223 nucleotides.
• Parts A, B and C were cloned in pJO201 to give the 5.0 kilobases plasmid pAlB6C3 which was transformed in E. coli strain XLl-Blue to give strain SSHP2.
• the strain was grown and evaluated for expression as described in Example 1. As shown in Figure 4, a band corresponding to the predicted molecular weight for the fusion protein (61 kilodaltons) was observed on SDS- PAGE. An additional band corresponding to the molecular weight of self- processed CKS/HS V-2 protease at amino acid 247 cleavage site (55kDa) was also observed.
• Part Cmut was a new PCR product, parts A and B were identical to Example 1.
• the mutation was incorporated into the 5' PCR primer for part Cmut-
• the 5' primer for part Cmut was:
• 5"-TAGATCTAGAAT03 ⁇ GGA ⁇ - ⁇ It contains an Xb ⁇ L site for cloning, the AflLLL site, the Ala-Gly mutation and 33 nucleotides of homologous sequence.
• the 3' primer was identical to the 3' primer used for part C described in Example 7. PCR was conducted as described in Example 5 to give a 223 nucleotides PCR product.
• Parts A, B and Cmut were cloned in pJO201 to give the 5.0 kilobases plasmid pAlB6C ut which was transformed in XLl-Blue to give strain SSHP3.
• the strain was grown and evaluated for expression as described in Example 5.
• a band corresponding to the predicted molecular weight for the fusion protein (61 kDa) was observed on SDS-PAGE as shown in Fgure 3.
• a 55 kDa band which would correspond to self-processed CKS/HSV-2 protease was not observed.
• HSV-2 protease(l-247) was expressed translationally coupled to CKS.
• a ribosome binding site was inserted between a 40 nucleotides 5' fragment of kdsB (described in Example 5) and the gene for HSV-2 protease in order to produce non-fused HS V-2 protease.
• the cassette system was as described in
• Example 5 part A2 was a new PCR product, part B was identical to Example
• the 5' primer for part A2 was:
• the expression vector was pJO201.
• a unique SalL site at 40 nucleotides downstream of the kdsB ATG start codon and the PstL site in the multi-cloning site were used to clone the PCR products. Translational coupling yielded a 19 aa product from kdsB and HSV-2 protease (1-247). Parts A2 and B were cloned in pJO201 to give plasmid pA2B6 of 4.1 kilobases. The plasmid was transformed in E. coli strain XLl-Blue to give strain SSHP4. The strain was grown and evaluated for expression as described in Example 5. A band corresponding to the predicted molecular weight of HS V-2 protease (27 kDa) was obtained on SDS-PAGE ( Figure 4) and Western Blot (Figure 5).
• HSV-2 protease(l-307) was expressed translationally coupled to CKS.
• the cassette system described in Example 5 and Figure 2 was used again in this example; part A2 was described in Example 9, parts B and C were described in Examples 5 and 7. Parts A2, B and C were cloned in pJO201to give the 4.3 kilobases plasmid pA2B6C3.
• the plasmid was transformed in E. coli strain XLl-Blue to give strain SSHP5.
• the strain was grown and evaluated for expression as described in Example 5.
• a band corresponding to the predicted molecular weight for HSV-2 protease(l- 307) was observed (33 kDa).
• a band corresponding to the molecular weight for self-processed HS V-2 protease 27 kDa was also observed.
• Example 8 The mutation at the amino acid 247 protease self-processing site described in Example 8 was incorporated into the non-fused translationally coupled protease.
• the cassette system in Example 5 was again used: part A2 was as described in Example 9 part B described in Example 9 and part Cmut described in Example 8 were cloned in pJO201 to give the 4.3kb plasmid pA2B6Cmut.
• the plasmid was transformed in E. coli strain XLl-Blue to give strain SSHP6.
• the strain was grown and evaluated for expression as described in Example 5. As shown in Figure 3, a band corresponding to the predicted molecular weight for HSN-2 protease( 1-307) was observed (33 kDa). There was no band corresponding to the molecular weight for self-processed HSV-2 protease (27 kDa).
• a subclone was constructed of pH2ProB (prepared as described in Example 3) which contained only the 0.7 kilobases BamHL fragment encoding the remaining sequence of the assembly protein ICP35.
• the 0.7 kilobases - ⁇ HI fragment from pH2ProB was subcloned in pUC18. This construct was needed for subsequent construction of full length UL26 clones containing HSV-2 protease and ICP35.
• Example 13 Construction of pSSPH-11 and Expression of HSV-2 Protease/ICP35
• a CKS fusion was constructed to the entire UL26 encoding HS V-2 protease and ICP35 as outlined in Figure 6.
• Plasmid pAlB6C, described in Example 7 was digested with Nc ⁇ L/PsiL and, in a triple ligation, was ligated to a 557 bp NcoL/SalL fragment of pH2ProA and a 81bp SalL/PstL fragment of pHPB-1, described in Example 12.
• the resulting intermediate clone pITL-6 was then digested with RsrLL/Hin ⁇ LLL and ligated with a 610 bp RsrLL/Hin ⁇ LLL fragment of pHPB-1 to give the final plasmid pSSPIl-11 of 5.9 kilobases.
• the plasmid was transformed in E. coli strain XLl-Blue to give strain SSHP7. The strain was grown and evaluated for expression as described in Example 5. A band corresponding to the predicted molecular weight for CKS/HS V-2 protease/ICP-35 fusion was observed (95 kDa).
• the histidine residue at position 148 of HS V-2 protease was mutagenized to an alanine residue.
• the Altered Sites Mutagenesis Kit (supplied by Promega) was used following the manufacturer's protocols.
• a KpnL/SphL fragment of pSSPIl-11 was subcloned in the pALTER-1 mutagenesis vector provided in the kit.
• Mutagenesis at nucleotides 442-443 of HS V-2 protease was performed: CAC to GCC converting His- 148 to Ala and creating an EagL site for analysis of mutants.
• pALTHM-4 contained the desired mutation which was confirmed by DNA sequencing.
• the UL26 gene encoding HS V-2 Protease and ICP35 was translationally coupled to CKS.
• the plasmid pA2B6C was the parent vector for this construct.
• a 1441bp NcoL/Hin ⁇ LLL fragment from pSSPIl-11 containing the 3' portion of HSV-2 Protease and the ICP35 gene was cloned in pA2B6C to give the 5.2 kilobases plasmid pSSPI2.
• the plasmid was transformed in E. coli strain XLl-Blue to give strain SSHP8. The strain was grown and evaluated for expression as described in Example 5.
• the UL26 gene encoding HS V-2 protease with the amino acid 247 deletion, described in Example 8, and ICP35 was translationally coupled to CKS.
• the plasmid pA2B6Cmut was the parent vector for this construct.
• a 1441 bp NcoL/Hin ⁇ LLL fragment from pSSPIl-11 containing the 3' portion of HSV-2 protease and the ICP35 gene was cloned in pA2B6Cmut to give plasmid pSSPI2M.
• the plasmid was transformed in E. coli strain XLl-Blue to give strain SSHP9. The strain was grown and evaluated for expression as described in Example 5.
• the plasmid pSSPIl-11 prepared as described in Example 13, was digested with EcoRI and the sticky ends were blunt ended by large fragment of DNA polymerase according to manufacturer's instructions. The DNA was then digested with Xbal and electrophoresed on 1% low melt agarose gel. A 2.3 kilobases fragment containing the UL26 gene was excised from the gel and then ligated to pVTIOO-U at the Pvul l/Xbal sites. The ligation mix was transformed into E. coli DH5 ⁇ . The plasmid pVTIOO-U UL26, containing the UL26 gene was identified by digestion with Hindi 11/ Xbal which released a 2.3 kilobases fragment.
• Yeast strain YJO was transformed with pVTIOO-U or pVTIOO-U UL26 employing selection for uracil prototrophy. Transformants were grown at 30 °C in liquid media lacking uracil and containing 2% glucose and examined for expression as described in Example 5. An immunoreactive band of 27 kDa was detected ( Figure 8, lanes 5, 6) indicating that the UL26 gene product (HS V-2 protease) is active in S. cerevisiae.
• the gene was cloned into the transfer vector pVL1392, placing it under the control of the strong Polyhedrin promoter of the Baculovirus, Autographa californica
• the gene was then integrated into the linearized Baculogold virus in the insect cell host by homologous recombination. Recombinant viruses were then used to infect insect cells in tissue culture to produce the protease.
• a DNA fragment coding for the HSV-2 protease UL26 gene was excised from plasmid pSSPIl-11 (described in Example 13) in the following manner: the plasmid was first digested with Xba I, then treated with the Klenow fragment of DNA Polymerase to generate a blunt end, followed by digestion with Eco Rl.
• a resulting DNA fragment of approximately 2 kilobases was purified through an agarose gel and ligated to the transfer vector pVL1392 which was previously digested with Eco Rl and Sm ⁇ L, to create the plasmid pAcUL26.
• the plasmid pALTHM-4 (described in Example 13) containing the HSV-2 U 26 gene with a mutation at the active site His- 148 was digested with Hwdi ⁇ , made blunt with Klenow polymerase, cut with Eco Rl, and the 2 kilobases DNA fragment generated was gel purified and ligated to Eco Rl and Sma I cut pVL1392, to generate the plasmid pAc ⁇ 148.
• Recombinant viruses were derived by co-transfection of 5 ⁇ g of either pAcUL26 or pAcH148 with 0.5 ⁇ g of linearized Baculogold DNA using the reagents for calcium phosphate precipitation supplied in the kit.
• the Baculogold DNA contains a lethal deletion of ORFl 629 and is not viable unless rescued by recombination with the transfer vector containing ORFl 629 and the gene to be expressed, thus providing a selection for recombinant virus.
• the Sf9 cells were incubated in 4 ml TMN-FH media for 5 days and monitored for signs of infection, ie, enlargement of cells, loss of cell viability, or lysis.
• FIG. 9 shows the results of SDS-PAGE analysis of 20 ⁇ l aliqouts of soluble extracts of uninfected cells (lane 3), wild-type Baculovirus infected lysate (lane 4), VACUL26 infected lysate (lane5), and vAcH148 infected lysate (lane 6), after staining with Coomasie blue.
• the polyprotein from the UL26 gene made in vAcUL26-infected cells undergoes autoproteolysis to produce the 27 kDa protease and the 40 kDa ICP-35 protein, indicating that the HS V-2 protease is active in these cells.
• the mutation in the active site of the protease greatly reduces autoproteolysis and the 67 kd polyprotein is the predominant form observed. This shows that processing of the polyprotein is not due to an endogenous protease of viral or cellular origin but an intrinsic function of the active HS V-2 protease, itself.
• the Western blot, shown in Figure 10 confirms that the proteins produced in virus infected cells are the pro- and mature forms of HSV-2 protease.
• Example 19 Preparation of antibody to the 236-247 region of HSV-2 protease
• a peptide of the sequence Gln-Ala-Gly-Ile-Ala-Gly-His-Thr-Tyr-Leu- Gln-Ala was synthesized commercially using standard techniques. This sequence corresponds to the residues 236-247 of the UL26 gene product and the carboxy terminal 12 amino acids of the mature form of HSV-2 protease.
• the peptide was conjugated to keyhole limpet hemocyanin using a commercially available kit (Pierce). The conjugated peptide was then emulsified with Freund's adjuvant and injected into several subcutaneous dorsal sites of New Zealand white rabbits (approx. 0.5 mg peptide per immunization). Immunization boosts were also administered two and six weeks later.
• the animals were bled prior to primary immunization and also at four, eight and ten weeks after primary immunization.
• the serum was collected from clotted blood by centrifugation, heated to 56°C for 30 minutes, and then stored at -20 °C.
• Example 20 Expression of the U ⁇ _26 gene in insect larvae using a recombinant baculovirus vector, Cabbage looper larvae were grown on the semisynthetic diet at 27 °C and 50% relative humidity until the fourth instar stage. Shortly after molting, the insects were removed from their diets, housed individually in plastic Petri dishes, and starved for 18 hours.
• insect cell culture fluid (see Example 18) which was infected with either 1) wild-type nuclear polyhedrosis virus (Autograp ⁇ ha californica ), 2) recombinant baculovirus with the UL26 gene for HS V-2 ( VACUL26), 3) recombinant baculovirus and wild type nuclear polyhedrosis virus in a 10:1 ratio, or 4) no baculovirus.
• Each of the cell culture fluids contained 10% sucrose to stimulate drinking. The larvae were observed until they drank part of their culture fluid, and then they were left in contact with their respective culture fluid for an additional three hours.
• the Coomasie stained gel shown in Figure 11, shows the presence of a 27 kDa band not present in larvae infected with only wild-type baculovirus or with no virus .
• These data suggest that the recombinant VACUL26 baculovirus infected larvae expressed a protein of about 27 kDa.
• the Western blot analysis ( Figure 12) confirms this interpretation, showing that the primary antibody to the 236-247 peptide fragment of HSV-2 protease reacts positively only with the 27 kDa band in VACUL26 treated larvae and some minor degradation products.
• MOLECULE TYPE DNA (genomic)
• HYPOTHETICAL NO
• ATC AAC GTA GAC CAC CGC GCT CGG TGC GAG GTG GGC CGG GTG CTC GCC 426 lie Asn Val Asp His Arg Ala Arg Cys Glu Val Gly Arg Val Leu Ala 60 65 70
• Gly Ala lie Ala Ala Asp Arg Gin Ala Gly Gly Leu Pro Ala Ala Ala 100 105 110
• MOLECULE TYPE cDNA
• HYPOTHETICAL NO
• ORIGINAL SOURCE

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