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WO1997009345A1 - Purification de proteines de virus a partir de virosomes - Google Patents

Purification de proteines de virus a partir de virosomes Download PDF

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Publication number
WO1997009345A1
WO1997009345A1 PCT/US1996/014187 US9614187W WO9709345A1 WO 1997009345 A1 WO1997009345 A1 WO 1997009345A1 US 9614187 W US9614187 W US 9614187W WO 9709345 A1 WO9709345 A1 WO 9709345A1
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Prior art keywords
vap
ser
cleaved
thr
leu
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Allen Portner
Toru Takimoto
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St Jude Childrens Research Hospital
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St Jude Childrens Research Hospital
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    • 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
    • C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
    • C07K16/1027—Paramyxoviridae, e.g. respiratory syncytial virus
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • 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
    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/18011—Paramyxoviridae
    • C12N2760/18111—Avulavirus, e.g. Newcastle disease virus
    • C12N2760/18122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes

Definitions

  • the present invention generally pertains to the field of viral protein crystallization.
  • the present invention specifically pertains to crystallization methods and crystallized viral attachment proteins (VAPs), obtained from virosomes.
  • VAPs crystallized viral attachment proteins
  • the crystallized VAP is biologically active.
  • Crystallized VAP, nucleic acid, vectors and host cells of a strain of paramyxovirus are also provided.
  • viruses contain an inner virion core having nucleic acid and a lipid envelope which holds the transmembrane (hydrophobic) domains of the envelope proteins.
  • Some of the envelope proteins are viral attachment proteins (VAPs) that contain extracellular domains. Viruses infect a target cell by association ofthe virus' VAP with the target cell's viral receptor.
  • VAPs viral attachment proteins
  • the extracellular domain ofthe VAP binds the target cell receptor and the transmembrane domain anchors the VAP to the viral envelope. (White et al, Quant. Rev. Biophys. 16: 151-195 (1983)). After association of the VAP with the cell's viral receptor, the virion core enters the cytoplasm ofthe bound cell and the viral replication process is initiated. In some cases, viruses that are bound to target cell receptors can enter the cells by receptor mediated endocytosis. Infectious Envelope-Containing Viruses
  • infectious envelope-containing viruses include, but are not limited to, togaviruses (yellow fever, RSSE and rubella); retroviruses (leukemia, sarcomas); orthomyxoviruses (influenza A, B, C); paramyxoviruses (mumps, measles, paramfluenza, Newcastle disease); rhabdoviruses (rabies); hepatitis virus; herpes viruses (herpes simplex, varicella zoster, cytomegalovirus, Epstein-Barr); and poxviruses (varcola, vaccinia, Molluscum Contagiosum). See, e.g., Lycke andNorrby, eds. Textbook of Medical Virology, Chs. 1-4 and 7-9, Butterworths, London (1983).
  • Paramyxoviruses are one ofthree genera ofthe family Paramyxoviridae, which includes enveloped, negative-stranded RNA viruses. Paramyxoviruses utilize hemagglutinin neuraminidase (HN) as the target VAP (Fraenkel-Conrat and Wagner, eds., Comprehensive Virology, Vol. 4,
  • paramyxoviruses include mumps, measles, parainfluenza virus (PIV), Sendai virus (SV) and Newcastle disease virus (NDV).
  • Electron micrographs have demonstrated that all paramyxovirus species have the same basic morphology.
  • This morphology includes highly pleomorphic particles that are enclosed by a lipid envelope acquired during maturation. Maturation usually occurs by virus budding from the plasma membrane ofthe host cell.
  • the viral membrane of a paramyxovirus contains two virus-specified glycoproteins, HN and F.
  • HN and F are found in all strains of paramyxoviruses.
  • HN and F are attached to the viral membrane by short N-terminal and C-te ⁇ ninal transmembrane peptide sequences, respectively.
  • the nucleotide sequence of HN genes of several paramyxoviruses has been determined. See, e.g., Gorman etal, Virology 775:211-221 (1990); Merson etal, Virology 167:91-105 (1988)); Blumberg et al., Cell 41:269-218 (1985); Paterson et al, Proc. Natl. Acad. Sci.
  • HN and F glycoproteins from different paramyxovirus strains exhibit the same biological activities.
  • Hemagglutination activity is the capacity of a virus to absorb to erythrocytes and, as a result, cause the erythrocytes to aggregate (agglutinate).
  • a protein projecting from the virus membrane surface (HN) mediates the attachment to a sialic acid glyco- conjugate receptors on the erythrocyte surface.
  • the hemagglutination reaction (HA) is an example of a relatively simple, quick, convenient and semi-quantitative way of detecting, identifying, titrating viruses, detecting viral antibody and studying virus attachment.
  • Cell-binding activity is the capacity ofa virus to attach to a variety of infectible host cells.
  • the HN protein of paramyxoviruses mediates the attachments to host cells, via a sialic acid-containing glyco-conjugate receptor.
  • Neuraminidase activity is the enzyme catalyzed cleavage of the ⁇ -ketosidic linkage between terminal sialic acid and an adjacent sugar residue.
  • the HN protein of paramyxoviruses possess neuraminidase activity. Fusion promoting activity is the capacity of paramyxovirus VAPs (e.g., HN, H) to provide an essential function that allows the fusion (F) protein to directly mediate virus host-cell and cell-to-cell membrane fusion. (Fraenkel-Conrat, supra, pp.
  • HN cell-binding, neuraminidase, and fusion promoting activities, essential for virus infection and spreading, are conserved among all or most strains of paramyxoviruses, as reflected in the high degree of sequence identity among these proteins. Therefore, the determined three-dimensional structure of an HN from a strain of paramyxovirus is useful for rational design of inhibitors to treat infections of many or most paramyxoviruses and may be applicable to other members of Paramyxoviridae family.
  • the crystal structures of neuramimdases, from influenza virus, Salmonella tryphimurium, and Vibrio cholerae show similar three-dimensional structures (Crennell et al, Structure 2:535-544 (1994); Crennell et al, Proc. Natl. Acad. Sci. USA 0:9852-9856 (1993)).
  • the present invention provides methods of purifying and crystallizing a viral attachment protein (VAP) from an envelope containing virus.
  • VAP viral attachment protein
  • the present invention also provides crystallized VAP which is soluble and biologically active.
  • the present invention also provides antibodies specific for the VAP and host cells that produce the antibody.
  • the present invention further provides nucleic acid molecules encoding the VAP, as well as nucleic acid probes specific for portions ofthe nucleic acid molecule. Also provided are vectors and host cells comprising the molecule.
  • the present invention also provides a crystallized HN protein from a strain ofa species of a paramyxovirus.
  • the present invention also provides antibodies specific for the HN and host cells that produce the antibody.
  • the present invention further provides nucleic acid molecules encoding the HN, as well as nucleic acid probes specific for portions ofthe nucleic acid molecule. Also provided are vectors and host cells comprising the nucleic acid.
  • the present invention also provides a crystallized HN protein from the Kansas strain of a species of a paramyxovirus: Newcastle disease virus (NDV).
  • This HN crystallized protein is suitable for x-ray diffraction analysis.
  • the x-ray diffraction patterns obtained by this analysis provide coordinates of moderately high to high resolution. These coordinates are useful for three dimensional modeling of the HN protein.
  • the three dimensional modeling programs use these coordinates and the amino acid sequence to generate secondary, tertiary and quaternary structures ofthe Kansas NDV HN.
  • the present invention also provides antibodies specific for the Kansas NDV HN and antibody expressing host cells.
  • the present invention further provides nucleic acid molecules encoding the Kansas NDV HN, as well as nucleic acid probes specific for portions ofthe nucleic acid molecule. Also provided are vectors and host cells comprising the nucleic acid.
  • FIG. 3 A diffraction pattern is presented from a crystal of Kansas strain NDV cleaved HN using an X-ray source. The resolution was 3.5 A at the edge ofthe pattern.
  • Figure 4 A diffraction pattern is presented from a crystal of cleaved HN using a more powerful X-ray beam than in Figure 3, produced in synchrotron storage rings. The resolution ofthe pattern was 2.6 A at the edge. Data was collected using a crystal frozen at -175 °C.
  • FIG. 1 The nucleotide sequence of the Kansas strain of NDV is presented.
  • Figure 6. The deduced amino acid sequence of an HN ofthe Kansas strain of NDV is presented.
  • the present invention overcomes one or more deficiencies ofthe related background art, by providing methods for crystallizing a viral attachment protein (VAP) from virosomes, where the crystals diffract x-rays with high resolution of 1.5-3.9A, such as 2.4-27A.
  • VAP viral attachment protein
  • the present invention thus includes methods of purifying and crystallizing a VAP from virosomes derived from a virus.
  • the present invention also provides crystallized VAP by these methods which is soluble and biologically active.
  • the present invention in a non-limiting example, provides methods of purifying and crystallizing hemagglutinin neuraminidase (HN) from a strain of a paramyxovirus using virosomes.
  • HN hemagglutinin neuraminidase
  • the present invention also provides crystallized HN by these methods which is soluble and biologically active.
  • the present invention also provides biologically active VAPs.
  • a non-limiting example is an HN from the Kansas strain of a species ofa paramyxovirus, the Newcastle disease virus (NDV).
  • the VAP is also provided as a crystallized protein.
  • a VAP from a virus is isolated in soluble form (e.g., lacking the transmembrane domains) by cleavage employing a protease applied to purified viruses or virosomes, as described herein.
  • the resulting cleaved VAP is in sufficient purity and concentration (e.g., a monomer or dimer) for crystallization.
  • the cleaved VAP is then isolated and assayed for biological activity and for lack of aggregation (which interferes with crystallization).
  • the purified and cleaved VAP preferably runs as a single band under reducing or nonreducing polyacrylamide gel electrophoresis (PAGE) (nonreducing is used to evaluate the presence of cysteine bridges).
  • PAGE polyacrylamide gel electrophoresis
  • the purified cleaved VAP is preferably crystallized using the hanging drop method under varying conditions of at least one ofthe following: pH, buffer type, buffer concentration, salt type, -o-
  • crystallized protein is also tested for neuraminidase or cell binding biological activity and differently sized and shaped crystals are further tested for suitability for X-ray diffraction. Generally, larger crystals provide better crystallography than smaller crystals, and thicker crystals provide better crystallography than thinner crystals.
  • a strain of virus is diluted in a buffer solution at about neutral pH.
  • the diluted virus solution can also be inoculated into the allantoic cavity of embryonated hen eggs for amplification.
  • Tissue culture of a virus strain, or recombinant expression ofthe VAP can alternatively be used according to known method steps.
  • infected, embroynated eggs are incubated for several days and then chilled at about 4°C or less overnight.
  • the allantoic fluids are collected and centrifuged at about 4°C or less to remove red blood cells.
  • the virus in the supernatant is sedimented by ultracentrifugation at about 4°C or less. After the virus pellet is soaked in buffer solution overnight at about 4°C or less, the pellet is resuspended, e.g., by homogenization.
  • the resuspended virus is optionally further purified by centrifugation in a sucrose gradient of about 5-50% at about 4°C or less.
  • the sedimented virus is collected at a suitable sucrose percentage (e.g., in the range of 5-50% sucrose), and sedimented again (after dilution with buffer) by ultracentrifugation at about 4°C or less.
  • the sedimented, purified virus is then suspended in buffer containing suitable preservatives. See, e.g., Portner et al, Virology 755:61-68 (1987); Takimoto et al, J. Virol. 66:1591-1600 (1992).
  • the purified virus can then be used for virosome preparation.
  • the protein be pure, in high concentration, biologically active, and/or have the transmembrane sequence removed. Removal ofthe transmembrane domains is preferred since aggregation ofthe transmembrane or hydrophobic domains can inhibit crystallization. These objectives are alternatively accomplished by foraiing virosomes, when the purified virus itself cannot be suitably cleaved to provide cleaved VAP for crystallization.
  • Virosomes comprise reconstituted viral lipid envelope or liposome, containing surface viral proteins. The surface proteins have lipophilic or hydrophobic portions in the viral envelope or liposome, as well as extra cellular portions projecting from the envelope or liposome.
  • the virosomes used in the present invention comprise a VAP such as hemagglutinin (HA), hemagglutinin neuraminidase (HN) or neuraminidase (NA), or other surface proteins that include, but are not limited to, F protein, sialidase, measles virus H protein, VSV G protein, gpl 20.
  • VAP such as hemagglutinin (HA), hemagglutinin neuraminidase (HN) or neuraminidase (NA), or other surface proteins that include, but are not limited to, F protein, sialidase, measles virus H protein, VSV G protein, gpl 20.
  • purified virus is added to a proportional volume of a saline buffer containing a suitable detergent (e.g. , non-ionic detergent) to solubilize the virus.
  • a suitable detergent e.g. , non-ionic detergent
  • the mixture is then incubated at about room temperature with shaking.
  • the preparation is then ultra-centrifuged at about 4°C or less to sediment the virus nucleocapsid and matrix proteins.
  • the supernatant containing at least one type of VAP is collected and the detergent removed.
  • the solution is then shaken at about room temperature or colder. Withdrawal of the detergent allows the virus membrane lipids and the virus envelope proteins to reform into a virosome as a lipid envelope containing the VAP extracellular portion projecting from the surface ofthe envelope.
  • the solution is collected and the procedure repeated to substantially remove the detergent.
  • the final solution contains the virosomes. See, e.g., Almeida et al, LANCET
  • the purified virosomes are optionally tested for biological activity (such as neuraminidase or sialidase activity) using known assays. See, e.g., Aymard-Henry et al, Bulletin ofthe World Health Organization, ⁇ 5.199-202 (1973); Thompson et al, J. Virol. 62:4653-4660 (1988); Takimoto et al, J. Virol. 66:1591-1600 (1992).
  • biological activity such as neuraminidase or sialidase activity
  • Proteolytic cleavage by a protease is used to remove soluble portions of a VAP, from the transmembrane portion, contained in either the virus or the virosome.
  • a proteolytic enzyme e.g., pronase
  • the preparation is ultra-centrifuged at about 4 °C or less. The cleaved VAP in the supernatant (as soluble protein) is collected and then optionally concentrated by further centrifugation.
  • the cleaved VAP is assayed for neuraminidase activity and for lack of aggregation, indicating that the transmembrane portion ofthe VAP has remained embedded in the virus envelope or virosome and is not part ofthe isolated protein. Removal ofthe hydrophobic membrane spanning region is preferred since aggregation ofthe hydrophobic regions can inhibit crystallization.
  • proteolytic treatment virosomes and cleaved VAP are separated by centrifugation, with the cleaved VAP remaining in the supernatant. For example, a modification of a procedure described previously can be used (Thompson et al, J. Virol. 62:4653-4660 (1988)).
  • the cleaved VAP fraction is preferably further concentrated by centrifugation through a filter, such as using a CENTRICON filter.
  • the results ofthe purification are optionally analyzed by polyacrylamide gel electrophoresis (PAGE) under reducing or non-reducing conditions. A single band is preferably obtained.
  • PAGE polyacrylamide gel electrophoresis
  • the analysis of the cleaved VAP be under non ⁇ reducing conditions to indicate whether the cleaved protein formed disulfide linked dimers.
  • the amino acid sequence can also be determined according to known methods, or otherwise obtained, as this sequence is important in determining the three dimensional structure ofthe cleaved protein (in combination with crystallographic analysis), as described herein, using molecular modeling techmques.
  • biological activity e.g., neuraminidase (or sialidase) activity for HN, or other activity ofthe VAP
  • biological activity is determined using equivalent amounts of both virion- or virosome- associated VAP and purified and cleaved protein. It is preferred that the biological activity exceed the activity of the virion- or virosome-associated protein. The preferred result indicates that the cleaved protein retains its native structure, which is important for determining the three-dimensional crystal structure of the biologically active molecule.
  • the purified and cleaved protein can be sequenced using known techniques. See, e.g. , Murti et al. , Proc. Natl. Acad. Sci. USA P ⁇ :1523-1525 (1993); Takimoto et al., J. Virol. 66:7597-7600 (1992), entirely incorporated herein by reference.
  • the hanging drop method is preferably used to crystallize the cleaved protein. See, e.g., Taylor et al., J. Mol. Biol. 226:1287-1290 (1992); Takimoto et ⁇ /., J. Virol. 66:7597-7600 (1992); CRYSTAL SCREEN, Hampton Research.
  • a mixture ofthe cleaved protein and precipitant can include the following: • pH (e.g., 4-9);
  • buffer type e.g., phosphate, sodium, or cacodylate acetates, imidazole, Tris HCl, sodium hepes
  • buffer concentration e.g., 10-200 mM
  • salt type e.g., calcium chloride, sodium citrate, magnesium chloride, ammonium acetate, ammonium sulfate, potassium phosphate, magnesium acetate, zinc acetate; calcium acetate
  • polymer type and concentration (e.g., polyethylene glycol (PEG) 1-50%, type 200- 10,000);
  • a non-limiting example of such crystalization conditions is the following: • purified cleaved protein (e.g., 5-30 mg/ml);
  • precipitant 2-60% Polyethylene glycol (PEG) 500-5000 buffered with 10-200 mM phosphate or acetate buffer and 50-300 mM of a precipitating salt (e.g., ammonium sulphate)); • at an overall pH of about 3.5-8.5.
  • a precipitating salt e.g., ammonium sulphate
  • the above mixtures are used and screened by varying at least one of pH, buffer type; buffer concentration, precipitating salt type or concentration, PEG type, PEG concentration, and cleaved protein concentration. Crystals ranging in size from 0.2-0.9 mm are formed in 1-14 days.
  • crystals diffract X-rays to at least 3.5 A resolution, such as 1.5 -3.5 A, or any range of value therein, such as 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, or 3.0, with 3.0 ⁇ or less being preferred.
  • Crystals appear after 1-14 days and continue to grow on subsequent days. Some of the crystals are removed, washed, and assayed for biological activity, which activity is preferred for using in further characterizations. Other washed crystals are preferably run on a stained gel and those that migrate in the same position as the purified cleaved VAP are preferably used. From two to one hundred crystals are observed in one drop and crystal forms can occur, such as, but not limited to, bipyramidal, rhomboid, and cubic. Initial X-ray analyses indicate that such crystals diffract at moderately high to high resolution, such as 1.5-3.5 A or 2.2-2.7 A. When fewer crystals are produced in a drop, they can be much larger size, e.g., 0.4-0.9 mm.
  • antibody refers both to monoclonal antibodies which are a substantially homogeneous population and to polyclonal antibodies which are heterogeneous populations. Such antibodies can be of any immunoglobulin class including IgG, IgM, IgE, IgA, IgD and any subclass thereof.
  • antibody is also meant to include both intact molecules as well as fragments thereof, such as Fab and F(ab') 2 , which are capable of binding antigen. Fab and F(ab') 2 fragments lack the Fc fragment of intact antibody, clear more rapidly from the circulation, and/or have less non-specific tissue binding than an intact antibody (Wahl et al, J. Nucl. Med.
  • Such fragments are typically produced by proteolytic cleavage, using enzymes such as papain (to produce Fab fragments) or pepsin (to produce F(ab') fragments).
  • enzymes such as papain (to produce Fab fragments) or pepsin (to produce F(ab') fragments).
  • Antibodies can be generated against VAP produced recombinantly or isolated from cells and tissues where the VAP is present, as in virally infected cells. Antibodies can be generated against the entire VAP or, more preferably, antibodies are generated against peptide subfragments representing functional domains ofthe VAP required for its cell binding activity, e.g., the extracellular portion or a domain thereof. Antibodies for specifically inhibiting a VAP can be generated against peptide fragments unique to that protein. Alternatively, antibodies for generally inhibiting more than one member of a related class of VAPs can be generated against peptide fragments shared by the class of VAPs desired to be inhibited.
  • cDNA is generated from an envelope containing virus's RNA or virus-specific RNA from infected cells, or (in the case of DNA viruses) viral DNA is isolated, both from the virus and host cells containing the virus.
  • a suitable oligonucleotide, or set of oligonucleotides, which is complementary to a sequence encoding a VAP is identified and hybridized to the DNA or cDNA.
  • Single stranded oligonucleotide probes complementary to a unique portion of a VAP encoding sequence can be synthesized and labeled using known method steps. Such a probe can be used by known procedures (or as a basis for synthesizing PCR probes) for amplifying DNA encoding a VAP from an envelope containing virus. Such oligonucleotide probes can be at least about 10 nucleotides in length (such as 10-30, 30-100, 100-500, or any range or value therein), in order to be specific for a target VAP encoding nucleic acid. Such procedures are well-known in the art. See, e.g., Ausubel, infra, Sambrook, infra, and Kaufman, infra.
  • Culturing ofthe host and introduction of corresponding or complementary DNA or RNA into a vector and/or host cell can be performed by known methods. Any of a wide variety of vectors can be employed for this purpose. See, e.g., Ausubel, infra, ⁇ 1.5, 1.10, 7.1, 7.3, 8.1, 9.6, 9.7, 13.4, 16.2, 16.6, and 16.8-16.11.
  • a nucleic acid sequence encoding a VAP ofthe present invention can be recombined with vector DNA in accordance with conventional techniques, e.g. , as disclosed by Ausubel, infra, Kaufman, infra, or Sambrook, infra.
  • the vector is then incorporated into host cells (bacterial, yeast, insect or mammalian cells) using such vectors or viral vectors (e.g., vaccinia, a retrovirus, an adenovirus or a baculovirus), according to known techniques.
  • host cells bacterial, yeast, insect or mammalian cells
  • viral vectors e.g., vaccinia, a retrovirus, an adenovirus or a baculovirus
  • Host cells comprising a nucleic acid which encodes a VAP ofthe present invention can be grown under conditions that provide expression ofthe VAP in recoverable or commercially useful amounts. See, e.g., Ausubel, infra, at ⁇ 1 and 13; Palese, U.S. Patent No. 5,166,057, which are entirely inco ⁇ orated herein by reference.
  • NDV HN cDNA was cloning of NDV HN cDNA by RT-PCR (reverse transcriptase polymerase chain reaction). Briefly, viral mRNA was isolated from virus infected mammalian cells and was then reverse transcribed into cDNA. The cDNA was subjected to PCR amplification using gene-specific (NDV HN specific) primers (corresponding to the DNA sequence presented in Figure 5. The amplified cDNA, wliich encodes NDV HN gene, was ligated into vector plasmid and then the plasmid was introduced into E. coli.
  • RT-PCR reverse transcriptase polymerase chain reaction
  • Virus (NDV) infected mammalian cells BHK cells were washed and suspended in a lysis buffer containing the nonionic detergent (Nonidet P-40). The intact nuclei were removed by a brief microfuge spin, and sodium dodecyl sulfate was added to the cytoplasm supernatant to denature protein. Protein was digested with protease and removed by extractions with phenol chloroform and chloroform. The cytoplasmic RNA which includes viral mRNA was recovered by ethanol precipitation. The isolated viral mRNA was used as a template to synthesize cDNA. First strand synthesis was driven by AMV reverse transcriptase and the oligo dT primer. Reverse transcriptases were derived from retroviruses such as avian myoblastosis virus (AMV) or Molony murine leukemia virus (MMLV), which use them to make DNA copies of their RNA genomes.
  • AMV avian myoblastosis virus
  • Oligonucleotides were used as primers for extension on RNA templates.
  • the DNA synthesized from the RNA template is complementary DNA (cDNA).
  • PCR was used to amplify a segment of the cDNA.
  • Two oligonucleotides were used as primers for a series of synthetic reactions that are catalyzed by a DNA polymerase (e.g., Taq DNA polymerase).
  • a DNA polymerase e.g., Taq DNA polymerase
  • These oligonucleotides are complementary to sequences that (1) lie on opposite strands of the template DNA and (2) flank the segment of DNA that is to be amplified.
  • These primers contain a potential restriction site at their 5' termini to facilitate cloning ofthe amplified double-stranded cDNA into an appropriate vector.
  • the major product of this reaction is a segment of double- stranded DNA whose termini are defined by the 5' ofthe oligonucleotide primers and whose length is defined by the distance between the primers.
  • the PCR product was cleaved with restriction enzyme which recognition sites were involved in the primers designed.
  • the NDV HN cDNA was then ligated into the plasmid vector pTFl (Takahashi et al, Genet. Anal Tech. Appl. 9:91-95 (1992)).
  • the NDV HN cDNA was subcloned into Hindlll and Kpnl sites of the pTFl vector.
  • the ligated DNA was introduced into Escherichia coli (E. coli). E. coli cells were transformed with the pTFl vector containing the NDV
  • HN cDNA using the calcium chloride precipitation method.
  • the transfected cells were grown in nonselective medium to allow synthesis of plasmid-encoded antibiotic resistance protein, then plated on antibiotic-containing medium to allow identification of plasmid containing colonies. Positive transformants were selected using ampicillin containing medium for the ampicillin resistance gene in the pTFl vector. Clones which included the plasmid pTFl with NDV HN cDNA insert were isolated, grown in the ampicillin-containing medium and, after adding glycerol to 50%, stored at -70 °C.
  • HN Hemagglutinin-Neuraminidase
  • Newcastle disease virus (Kansas strain) was diluted to 0.2 hemagglutination units (HA) in lOmM phosphate buffered saline (PBS pH7.4) containing gentamicin (0.5mg/ml: Bio Whittaker). Virus was inoculated into the allantoic cavity of 11 -day-old embryonated hen eggs (0.1 ml/egg). The eggs were incubated at 35 °C for two days and then chilled at 4°C overnight. The allantoic fluids were collected and centrifuged at 2,000 rpm for 30 min at 4°C in IEC CR-6000 centrifuge to remove red blood cells.
  • HA hemagglutination units
  • the virus in the supernatant was sedimented by ultracentrifugation at 30,000 rpm for 1 hr at 4°C. After the virus pellet was soaked in PBS overnight at 4°C, the pellet was resuspended by homogenization in a dounce homogenizer. The resuspended vims was purified by centrifugation in a 30-50% sucrose gradient (PBS) at 27,000 rpm for 2 hrs at 4°C. The vims which sedimented at approximately 40% sucrose was collected and sedimented again, after adding at least 1.5 vol of PBS, by ultracentrifugation at 35,000 rpm for 1 hr at 4°C. The sedimented purified vims was suspended in PBS containing 0.1% sodium azide.
  • PBS sucrose gradient
  • HN To prepare HN for crystallization, it is important that the HN be pure, in high concentration, biologically active, and have the transmembrane sequence removed. By forming virosomes these objectives were unexpectedly accomplished.
  • PBS PBS containing 2% Triton X-100 (Sigma) was added to solubilize the vims. The mixture was then incubated at room temperature for 1 hr with gentle shaking. The preparation was next centrifuged at 35,000 rpm for 2 hrs at 4°C to sediment the vims nucleocapsid and matrix proteins.
  • the supernatant containing HN and F proteins was collected and Bio-Beads (Bio-Rad) (1 gram/5 ml supernatant) added to remove the detergent.
  • the solution was gently shaken at room temperature for 1 hr. Withdrawal of the detergent allows the vims membrane lipids and the vims envelope proteins, HN and F, to reform into an envelope containing HN and F spikes projecting from the surface ofthe envelope.
  • the solution was collected by syringe with a 27G needle. The procedure was repeated twice more to remove the detergent completely.
  • the final solution contained the purified virosomes.
  • Proteolytic cleavage with a protease was used to remove HN from the virosome.
  • virosome solution 1.5mg/ml
  • pronase 0.5mg/ml in PBS
  • CACHEM pronase
  • the preparation was then centrifuged at 35,000 rpm for 1.5 hrs at 4°C.
  • the cleaved HN protein in the supernatant was concentrated by centrifugation through CENTRICON- 100 (AMICON) filter tubes. The concentrated HN was used for crystallization.
  • Figure 1 lane 3 shows the results of the purification, analyzed by polyacrylimide/gel electrophoresis (PAGE) under non-reducing conditions. A single band was obtained. Analysis of the HN under non-reducing conditions indicated that this HN ofthe Kansas strain of NDV did not form disulfide linked dimers. This strain is similar to the LaSota NDV strain which also does not show oligomeric HN under non-reducing PAGE analysis (Mirza et al, J. Biol. Chem. 265:21425- 21431 (1993)). The lack of cysteine in position 123 of the Kansas and LaSota strains (which is thought to be involved in disulfide bond formation) is likely responsible for the monomeric HN seen in the non-reducing gels.
  • PAGE polyacrylimide/gel electrophoresis
  • HN of this strain forms non-disulfide linked oligomers which are unstable under PAGE conditions. Additional characterization ofthe Kansas HN showed a protein migration pattem typical of NDV. We cloned and sequenced the Kansas HN gene (See Example 3) (Fig. 5) which showed a typical NDV HN sequence with up to 99% identity to HN from other NDV strains in the GenBank database. This sequence information is important in determining the three dimensional stmcture of HN from crystallographic analysis.
  • NDV HN Neuraminidase Activity of NDV HN Purified From Virosomes or on Virus Particles
  • HN Purified from HN Purified from of HN Protein ⁇ g
  • Virosomes A 549
  • HN Neuraminidase activity of equivalent amounts of HN, comparing the native viral activity with cleaved and purified HN.
  • the equivalent amounts of HN were incubated for 30 min at 37°C with 2.0 mg of N-acetylneuramin-lactose and then assayed for free sialic acid.
  • HN represents -25% of total virion protein.
  • the hanging drop method was used to crystallize the cleaved HN protein.
  • Crystals ranging in size from 0.2-0.7 mm were formed in 2-7 days (Fig. 2). Some of these crystals were removed, washed, and assayed for neuraminidase activity, which they were found to retain. Other washed crystals were run on a stained gel and found to migrate in the same position as the cleaved HN. As many as 40 crystals were observed in one drop and a number of different crystal forms were noted, including bipyramidal, rhomboid, and cubic. Initial X-ray analyses discussed in the next section indicates that the 0.2-0.25 mm crystal diffracts at moderately high resolution. Fewer crystals were also produced in a drop, but of much larger size, 0.4-0.6 mm.
  • the first crystals produced ( ⁇ 0.2-0.25 mm) were X-ray analyzed on a rotating Cu anode X- ray source operating at 40 kV and 100 mA.
  • Figure 3 shows the diffraction pattem from a single frame of several hundred collected. Crystals were stable for at least 20 hrs. Frozen crystals were used for longer X-ray exposures (48 hrs), the crystals being stable to the X-rays in the frozen state. To collect the maximum number of useful reflections, multiple frames were collected as the crystal was rotated in the X-ray beam for 48 hrs. In this analysis, crystals diffracted to a resolution of 3.5 A (Fig. 4, edge). To increase the resolution further, slightly larger crystals (0.25 mm) were analyzed in a synchrotron high energy X- ray source. Using frozen crystals, X-ray diffraction data was collected every 6 minutes over a 24-hr period. A single frame is shown in Figure 5. The crystals diffracted to a relatively high resolution of 2.6 A.
  • Example 3 Cloning and Sequencing of Nucleic Acid Encoding a Paramyxovirus HN Protein
  • HN gene of NDV was cloned by polymerase chain reaction (PCR) method using RNA extracted from vims infected BHK cells.
  • First strand cDNA was synthesized by Moloney murine leukemia vims reverse transcriptase (Promega) using primer designed from consensus sequence found in NDV strains obtained from NIH GenBank. The synthesized cDNA was amplified by PCR using similarly designed primers.
  • NDV HNcDNA containing full coding region was subcloned into plasmid pTFl (Takahashi et al, 1992, Bousse et al, 1994) at Hindlll and Kpnl sites.
  • ADDRESSEE Sterne, Kessler, Goldstein & Fox, P.L.L.C.
  • GCA AAA AAT ACA TGG CGC TTG ATA TTC CGG ATT GCA ATC TTA CTC TTA 96 Ala Lys Asn Thr Trp Arg Leu Ile Phe Arg Ile Ala Ile Leu Leu Leu 20 25 30

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Abstract

Cette invention se rapporte à des procédés de purification et à une protéine de fixation virale (VAP) cristallisée de virosomes dérivés d'un virus, où (a) la protéine VAP cristallisée peut servir dans des analyses par cristallographie aux rayons X, (B) l'analyse aux rayons X fournit des réseaux de diffraction de résolution suffisante pour déterminer la structure tridimentionnelle de la protéine VAP, (c) la protéine VAP cristallisée est sous forme biologiquement active; ainsi qu'à une protéine VAP cristallisée spécifique, à de l'hémagglutinine-neuraminidase (HN) provenant d'une souche de Paramyxovirus, y compris des acides nucléiques, des vecteurs et des cellules hôtes comportant des séquences nucléoditiques codant l'enzyme HN.
PCT/US1996/014187 1995-09-08 1996-09-06 Purification de proteines de virus a partir de virosomes Ceased WO1997009345A1 (fr)

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002010459A3 (fr) * 2000-07-27 2003-02-27 Biocryst Pharm Inc Structure tridimensionnelle d'hemagglutine-neuraminidases de paramyxovirus et utilisation correspondante
US7153824B2 (en) 2003-04-01 2006-12-26 Applied Research Systems Ars Holding N.V. Inhibitors of phosphodiesterases in infertility
EP2193808A1 (fr) 1999-08-21 2010-06-09 Nycomed GmbH Combinaision synergique
US7772318B2 (en) 2005-04-04 2010-08-10 Rohm And Haas Company Aqueous polymer dispersions

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
ARCH. VIROL., 1987, Vol. 97, WEMERS et al., "The Hemagglutinin-Neuraminidase (HN) Gene of Newcastle Disease Virus Strain Italien (NDV Italien): Comparison With HNs of Other Strains and Expression by a Vaccinia Recombinant", pages 101-113. *
J. VIROL., December 1988, Vol. 62, No. 12, THOMPSON et al., "Isolation of a Biologically Active Soluble Form of the Hemagglutinin-Neuraminidase Protein of Sendai Virus", pages 4653-4660. *
J. VIROL., December 1992, Vol. 66, No. 12, TAKIMOTO et al., "Crystallization of Biologically Active Hemagglutinin-Neuraminidase Glycoprotein Dimers Proteolytically Cleaved From Human Parainfluenza Virus Type 1", pages 7597-7600. *
PROC. NATL. ACAD. SCI. U.S.A., February 1993, Vol. 90, MURTI et al., "Crystals of Hemagglutinin-Neuraminidase of Parainfluenza Virus Contain Triple-Stranded Helices", pages 1523-1525. *
VIROL., 1989, Vol. 169, SAKAGUCHI et al., "Newcastle Disease Virus Evolution: I Multiple Lineages Defined by Sequence Variability of the Hemagglutinin-Neuraminidase Gene", pages 260-272. *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2193808A1 (fr) 1999-08-21 2010-06-09 Nycomed GmbH Combinaision synergique
WO2002010459A3 (fr) * 2000-07-27 2003-02-27 Biocryst Pharm Inc Structure tridimensionnelle d'hemagglutine-neuraminidases de paramyxovirus et utilisation correspondante
US7153824B2 (en) 2003-04-01 2006-12-26 Applied Research Systems Ars Holding N.V. Inhibitors of phosphodiesterases in infertility
US7772318B2 (en) 2005-04-04 2010-08-10 Rohm And Haas Company Aqueous polymer dispersions

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