WO2002000924A2

WO2002000924A2 - Viral phospholipase a2 enzymes, anti-viral agents and methods of use

Info

Publication number: WO2002000924A2
Application number: PCT/CA2001/000932
Authority: WO
Inventors: Peter Tijssen; Zoltan Zadori
Original assignee: Institut National De La Recherche Scientifique
Priority date: 2000-06-28
Filing date: 2001-06-28
Publication date: 2002-01-03
Also published as: AU2001267239A1; WO2002000924A3

Abstract

The present invention provides a novel class of phospholipase A2 enzyme (PLA2) from viruses and a method for identifying, isolating, purifying and characterizing enzymes of this class. The present invention includes viral PLA2 proteins, nucleic acids, and antisense oligonucleotides, and the use of these molecules in screening methods for anti-viral agents, in decreasing the infectivity and/or replication of viruses and as research tools. The present invention further includes treatment or prevention of virus-associated diseases using viral PLA2 inhibitors and the use of the viral PLA2-encoding region to improve virus-based gene therapy vectors.

Description

VIRAL PHOSPHOLIPASE A₂ ENZYMES, ANTI-VIRAL AGENTS

AND METHODS OF USE

FIELD OF THE INVENTION

The present invention pertains to a class of phospholipase A₂ proteins and nucleic acids and their use. More particularly the present invention pertains to viral phospholipase A₂ proteins and nucleic acids and their use.

BACKGROUND OF THE INVENTION

Phospholipase A₂

Phospholipase enzymes catalyze the removal of fatty acid residues from phosphoglycerides. Phospholipases A₂ (PLA₂s) are a superfamily of key enzymes involved in a multitude of (patho)physiological and cellular processes, including lipid membrane metabolism, signal transduction pathways, inflammation, acute hypersensitivity, and degenerative diseases (Balsinde et al, (1999) Annu. Rev. Pharmacol. Toxicol. 39:175-189; Kramer and Sharp (1997) FEBS Letters 410:49-53; Nishizuka (1992) Science 258:607-614; Dennis (1994) J Biol. Chem. 269:13057-13060; Dennis (1997) Trends Biochem. Sci. 22:1-2; Yuan and Tsai (1999) Biochim BiophysActa 1441:215).

PLA₂s hydrolyze the ester bonds at the sn-2 position of the glycerol moiety of membrane phospholipids to yield lysophospholipids and fatty acids, from which secondary messengers may be generated. These secondary messengers may modulate enzyme activities, ion channels, and (post-)transcription factors (Nishizuka (1992) Science 258:607-614; Rao etal, (1993) Oncogenes 8:2759-2764; Yao et al, (1995) Nature 378:307-310).

PLA₂s are found in many living species and form a diverse family of enzymes. The main groups of PLA₂s are the cytosolic PLA₂s, the Ca²⁺-independent PLA₂s, and the secretory PLA₂s (sPLA₂s). A large number of sPLA₂s, from host cells as well as from snake and bee venoms, have been structurally characterized, and show a high degree of sequence homology (See, for example, Chang etal.,(\981) Biochemical Pharmacology 36:2429-2436). Although the known sPLA₂s were originally divided into groups by source organism and their primary amino acid sequences, they are now characterized by a growing list of other attributes, such as the number and position of disulfide bonds. Group I includes pancreatic sPLA₂s from vertebrates, including mammals, and snake venom sPLA₂s. These sPLA₂s are active at pH 6-8 and their activity is dependent on the presence of calcium ion (Ca²⁺). Group II contains a mixture of non-pancreatic, or synovial, sPLA₂s and sPLA₂s from the venom of crotalids and viperids. Both group I and group II members have a similar 3D-structure. Group III contains sPLA₂s from bee venoms. sPLA₂s from all three groups have a 14 kDa molecular mass and are disulfide-rich (Dennis (1994) J Biol. Chem. 269:13057-13060; Dennis (1997) Trends Biochem. Sci. 22:1-2). While all low-molecular weight (-13.5-16.8 kDa), Ca²⁺-dependent PLA₂s possess secretion sequences and have been found extracellularly, some are also cell- associated (Anderson et al. (1994) Prostaglandins, Leukotrienes and Essent Fatty Acids 51 : 19; Fayardet-ϊ/. (l 998) JCellSci 111: 985). Other types of PLA₂s include group V PLA₂ (Chen et al. (1994) J Biol. Chem. 269:2365), and group X PLA₂ isoforms (Valentin et al (1999) J Biol. Chem. 21 A: 31195).

The product of sPLA₂ activity, arachadonic acid, is processed into bioactive lipid mediators or shuttled into pathways for the synthesis of eicosanoids. In mammals eicosanoids, such as prostaglandins, prostacyclins, thromboxanes and leukotrienes, are involved in pain, inflammation and fever. As such, inhibitors of mammalian sPLA₂ have been described for use in the treatment of a variety of inflammatory disorders (for example, see International Patent Application No. WO 96/27604, European Patent Application No. EP 950657 A2 and U.S. Patent No. 5,948,626). In addition, a recent report has shown that sPLA₂ isolated from bee or snake venom inhibits human immunodeficiency virus (HIV) by blocking viral entry into host cells (Fenard et al, (1999) J Clin. Invest. 104:611).

While prokaryotic versions of PLA₂ have been reported in bacteria such as Escherichia coli and Streptomyces violaceoruber, viral PLA₂ has not previously been identified.

Viruses

Viruses are infectious agents that are found in virtually all life forms, including humans, animals, plants, fungi, and bacteria. Viruses often damage or kill the cells that they infect, causing disease in infected organisms. The difficulty in developing anti- viral therapies stems from the large number of variant viruses that can cause the same disease, as well as the inability of drugs to disable a virus without disabling healthy cells. The development of specific anti- viral agents, therefore, is a major focus of current research.

Therapeutic Anti-Viral Treatments

Although a number of methods exist currently for the treatment of viral infections, many of these are complicated by the side effects the anti-viral agent(s) has on the host system.

One example of a method for inhibiting a viral infection is provided by U.S. Patent No. 5,449,608, which describes a method of inhibiting the human parvovirus, B19. The method comprises administering compositions containing a B19 binding domain, thus preventing binding of B19 to host cells. Unfortunately, such an agent competes with similar molecules already present in the host cells, without a clear competitive edge, and is prone to catalytic breakdown.

Similarly, a soluble form of the receptor (CD-4) for the human immunodeficiency virus, HΓV-1, has been shown to be somewhat effective in reducing the infectivity of HIV in tissue culture, but this result could not be replicated in vivo. This is likely due to the large number of receptor binding sites present on a single virus particle, thus making it difficult for a soluble receptor to block all sites and prevent binding of the virus to cells (see Flint et al, Principles of Virology (Washington, DC: ASM Press, 1999) 119).

Clearly, a need remains to provide easily applicable methods and agents that may be used to effectively treat viral infections, particularly in cases where vaccination has failed or where subjects were not vaccinated.

The present invention provides a means of inhibiting virus infection and/or replication in animals, including humans, and of treating persistent infections and associated syndromes. This background information is provided for the purpose of making known information believed by the applicant to be of possible relevance to the present invention. No admission is necessarily intended, nor should be construed, that any of the preceding information constitutes prior art against the present invention.

SUMMARY OF THE INVENTION

An object of the present invention is to provide viral phospholipase A₂ enzymes, antiviral agents and methods of use. In accordance with an aspect of the present invention, there is provided a viral polypeptide that has phospholipase A₂ activity.

hi accordance with another aspect of the present invention, there is provided an isolated polynucleotide encoding a viral polypeptide that has phospholipase A₂ activity, antisense oligonucleotides complementary to the polynucleotides, vectors comprising the polynucleotides, and host cells genetically engineered with the polynucleotides or vectors.

In accordance with another aspect of the present invention there is provided methods of decreasing parvovirus infection and replication, and thereby methods of treating parvovirus- related diseases.

In accordance with another aspect of the present invention viral PLA₂s are used in manufacturing, clinical, and research settings. At present, bee venom is commonly used as a standard in measuring PLA₂ activity. The viral PLA₂ of porcine parvovirus (PPV), however, has a 40 to 100-fold higher specific activity than bee venom. This high activity makes PPV PLA₂ ideal for use as a PLA₂ standard.

In accordance with another aspect of the present invention there is provided methods for screening compounds in order to identify inhibitors of viral PLA₂ activity, comprising adding a candidate inhibitor compound to a solution containing a viral PLA₂ and a phospholipid substrate, and detecting the inhibition of PLA₂ activity. In accordance with another aspect of the present invention there is provided antisense oligonucleotides designed to inhibit expression of viral PLA₂.

In accordance with a further aspect of the present invention is the use of these viral PLA₂ inhibiting agents as anti- viral agents in the treatment of various viral infections.

In accordance with still another aspect of the present invention there is provided the use of viral PLA₂ nucleic acids, or variations thereof, to improve gene-therapy vectors.

Various other objects and advantages of the present invention will become apparent from the detailed description of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 depicts a parvovirus genome.

The right-half of the parvovirus genome contains a large ORF coding for a nested set of capsid proteins (VPs) from alternative in-frame initiation codons so that these VPs differ by their N-terminal extensions. The VPs can be subdivided into 3 domains: common C-terminal domains required for capsid formation (60 copies per capsid); a small hinge-(like) domain; and unique N-terminal extensions. The capsid-forming domain consists of a β-barrel in which β-strands (black boxes: αA and αB are helices) are connected by loops (Ll-4). About 100 amino acids upstream of the start of VP is a conserved domain (cd). The shaded boxes represent the domains (Vlups) cloned in expression vectors (VPlup: PPV-VPlup amino acids 2-174 [SEQ ID NO: 50]; B19-VPlup amino acids 2-240 [SEQ ID NO: 52]; and G DNV-VPlup amino acids 1-378 [SEQ ID NO: 54]).

Figure 2 presents a protein sequence alignment of parvovirus VPlup regions and representatives of sPLA₂ groups. Parvoviral and sPLA₂ sequences are separated by numbers of the common structural numbering for group I/ϋsPLA₂ (Renetseder et al, (1985) Annu. Rev. Pharmacol. Toxicol. 39:175; Kramer and Sharp (1997) FEBSLett. 410:49; Nishizuka (1992) Science 258:607). Black shading indicates 100% identity among sPLA2s. Dark grey indicates 100% identity among vPLA2s. Medium grey shading indicates 100% identity among all PLA₂s. GenBank accession numbers are indicated except for those not yet deposited.

Figure 3 demonstrates the specificity of PLA₂ activity. A. Thin layer chromatography after hydrolysis of phosphatidylcholine substrate (PL) into fatty acid (FA) and lysophosphatidylcholine (LPL) by expressed pvPLA₂ in the mixed micelles assay. PLA₂s used were: lanes 1, 2 and 5 (0.6 ng of PPV PLA₂), lanes 3 and 4 (250 ng of B 19 PLA₂), lane 6 (350 ng of G DNV PLA₂), lane 7 (2,000 ng thioredoxin as negative control) and lane 8 (15 ng bee venom PLA₂). The 3C9 monoclonal antibody, which binds to the C-terminus of PPV- VP 1 up, reduced PPV PLA₂ activity of VP 1 up (lane 2), but not that of B19 (lane 4). Adding EGTA to a final concentration of 5 mM abolished PLA₂ activity (lane 5).

B. Sequences outside the conserved pvPLA₂ domain increased pvPLA₂ activity of the PLA₂ motif of expressed PPV-VPlups. VPlup from Ml to SI 74 was used throughout this work and was assigned the^'relative specific activity of 1.0. Sequences within this expressed peptide but outside the PLA₂ motif (dark-grey) contributed to the activity as shown by the relative specific activities.

C. The impact of Ca²⁺ concentration, pH and different substrates on the activity of pvPLA₂. Since 1 mM Ca²⁺, 50 mM Tris-HCl, pH 8.0, and phosphatidylcholine (PC) were used in standard assays, these were set at 100% relative activity. PE = phosphatidylethanolamine, PI = phosphatidylinositol. Samples were measured in triplicate.

D. PLA₂ activity of untreated 0.2 μg virions (lane 1) and after dissociation (lanes 2 & 3) and heat shock (lanes 4 & 5). Bee venom was included as a positive control (lanes 6 & 7). Lane 8 contains the negative control. Samples in lanes 3, 5 and 7 were treated with anti- VPlup antibody. Figure 4 depicts the predicted 3D-structure of the vPLA₂ domain. The predicted 3D-structure of the vPLA₂ domain, as shown for PPV/B19, showed sequence homology with group III sPLA₂ at the - and C-termini, whereas the centre section resembled group I/II sPLA₂ (sequence of IB pancreatic sPLA₂ shown). The top line shows the position of helices (H) for group I I, which are inferred from the homologous porcine pancreatic PLA₂. The bottom line shows the position of helices (H) for group III PLA₂. The predicted three-dimensional model of B19/PPV PLA₂ was obtained by homology modeling using the program MOE. Both the three-dimensional structure of bee venom PLA₂ (Group III, in magenta) and the three- dimensional structure of the pancreatic porcine PLA₂ (Group I/II, in orange) were used for homology modeling of the vPLA₂ (centre). Note that residues L, V, I as well as residues Y, F, W were considered equivalent residues. The three-dimensional images were generated with the program GRASP. The color code in the sequence matches that in the vPLA₂ structure. Residues coloured green in vPLA₂ indicate residues that are conserved in both group I/II and group III PLA₂s; orange-coloured residues are conserved in group I/II; and magenta-coloured residues are conserved in group III PLA₂s. The amino acids identified in the vPLA₂ stracture were submitted to site-directed mutagenesis.

Figure 5 depicts immunofluorescence of infected cells using wild-type (wt) or mutant virus, with or without NH₄C1 treatment. Immunofluorescence was used to follow the entry and infection of PT cells by the wt or mutant virus. At 0 hours of entry, a strong staining, often patchy, was observed on the cellular membrane. After 4 hours, virus entry led to a cytoplasmic, granular, andperinuclear staining, whereas with NH₄C1 treatment, the virus was still mostly distributed throughout the cytoplasm. After 8 hours, all incoming virus was perinuclear except for wt without NH₄C1, for which some nuclei became positive. At 12 hours, only wt without NH₄C1 yielded significant numbers of positive nuclei. Some positive nuclei appeared at 18 hours for wt with NH₄C1, and for the P21W or P21L mutants. The delivery of the viral genome can be seen to be impaired in the case of the mutant virus, and is further inhibited by treatment with NH₄C1.

Figure 6 shows the binding (A) and entry (B) of ³⁵S-labelled porcine Parvovirus using 4x10⁵ cells and 5x10⁹ wild type (wt) virus particles. Equivalent amounts of mutant viruses were used for the binding studies, and twice as much for the entry studies (experiments in quadruplicate).

C. ³⁵S-labeled PPV particles (wt or mutant) were added to 2x10⁷ cells in Petri dishes and about 20% was recovered in the cells. After cellular fractionation, the distribution of virus over cytosolic and nuclear fractions was measured by liquid scintillation. The relative radioactivity is calculated as follows: [nuclear fraction-bound activity]/[cellular fraction- bound activity]. Standard errors were calculated from three experiments.

D. Co-localization of PPV and LAMP-2 was studied by confocal microscopy. Anti-PPV antibodies were labeled with FITC, giving green fluorescence, and anti-LAMP-2 antibodies with Texas Red, giving red fluorescence. Co-localization of PPV and LAMP-2 resulted in yellow staining.

Figure 7 demonstrates distribution of viral DNA in infected cells. Incoming viral DNA was detected in the cytoplasm by in situ hybridization 4 (A) and 8 (B) hours post-infection (p.i.), whereas replicating DNA in the nucleus was found at 12 hours p.i. (C). At 12 hours p.i., no DNA could be detected in the nucleus in the case of the HD (D) and P21^pvW mutants. Aphidicoline, a DNA polymerase inhibitor, also prevented the appearance of viral DNA in the nucleus (F). The sPLA₂ inhibitors tetracain (TC), and oleyloxyethyl phosphorylcholine (OP), also reduced the number of nuclei containing viral DNA (G).

Figure 8 shows nucleic acid sequences of cloned Vlup regions of porcine parvoviras [SEQ ID NO: 50], B19 human parvovirus [SEQ ID NO: 52], and Galleria mellonella densoviras [SEQ ID NO: 54].

Figure 9 shows amino acid sequences of cloned Vlup regions of porcine parvovirus [SEQ ID NO: 49], B19 human parvovirus [SEQ ID NO: 50], and Galleria mellonella densoviras [SEQ ID NO: 52].

Figure 10 shows the 5234 base pair nucleic acid sequence of Acheta domesticus densoviras [SEQ ID NO: 65]. Figure 11 shows the nucleic acid sequence of the genomic region containing the PLA₂ motif for Acheta domesticus densoviras [SEQ ID NO: 64] and Mythimna loreyi densoviras [SEQ ID NO: 62].

Figure 12 shows the amino acid sequence of the region containing the PLA₂ motif for Acheta domesticus densoviras [SEQ ID NO: 63] andMythimna loreyi densoviras [SEQ ID NO: 61]. The conserved histidine (H) and aspartic acid (D) residues are highlighted.

Figure 13 shows the sequences identified by phage display that bind to viral PLA₂.

DETAILED DESCRIPTION OF THE INVENTION

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. For the purposes of the present invention, the following terms and abbreviations are defined below.

The term "agent" is used herein to denote a chemical compound, a mixture of chemical compounds, a biological macromolecule, or an extract made from biological materials such as bacteria, plants, fungi, or animal (particularly mammalian) cells or tissues.

The term "amino acid motif is used herein to denote a characteristic sequence of amino acids the presence of which can serve to identify members of a family of proteins. It is understood by those of skill in the art that variations of one or two amino acids within a motif does not preclude a protein from belonging to the family defined by the motif.

This invention is based on the unexpected finding that PLA₂ activity is present in the capsids of viruses. This invention is also based on the unexpected determinations that this viral PLA₂ activity is essential for infection and that inhibition of this PLA₂ activity decreases the infectivity and/or replication of the viruses. Characterization of Viral PLA^s

The present invention describes a class of PLA₂s found in viruses. These viral PLA₂s have significant differences from known PLA₂s, including stracture (Figure 4), biological function, and amino acid sequences (Figure 2); thus, these viral PLA₂s form a new group of PLA₂s within the PLA₂ superfamily.

The present invention provides viral PLA₂s containing a unique, characteristic sequence profile. Viral PLA₂s comprise the amino acid motif [WY]-[CIVL]-G-x-[GP]-[GKNS]- x(8,14)-D-x(2)-[AC]-x(2)-H-D-x(2)-Y, where the notation [WY] represents alternative amino acids at this position, x is any amino acid, x(2) represents a stretch of 2 amino acids and x(8,14) represents a stretch of between 8 and 14 amino acids. The above motif is characteristic of viral PLA₂ sequences.

In accordance with one aspect of the present invention, there is provided polynucleotides that encode viral PLA₂s containing the amino acid motif [WY]-[CIVL]-G-x-[GP]-[GKNS]- x(8 , 14)-D-x(2)- [AC] -x(2)-H-D-x(2)- Y. The present invention further relates to polypeptides which contain the viral PLA₂ amino acid motif and exhibit phospholipase A₂ activity.

In one embodiment of the present invention, the polypeptide is a variant of a naturally occurring viral PLA₂ which may have a substitution, deletion, or addition of one or more amino acids. In a related embodiment, there is provided polynucleotides that encode such variants.

The present invention further provides variants of the viral PLA₂s that may be inactive due to the substitution of an active site residue, or that may contain slight variations of the above amino acid motif. Analogous variants exist in the mammalian PLA₂s in the form of pseudogenes. The viral PLA₂s of the present invention may be obtained from any virus presently identified and from viruses yet to be identified or may be prepared by recombinant molecular biology techniques or chemical synthesis. I) PLA₂ activity

The viral PLA₂s of the present invention are ultimately defined by their PLA₂ activity. PLA₂ activity is defined as the ability to hydrolyse the fatty acid from the sn-2 position of phospholipids to yield lysophosphatidyl compounds.

One exemplary assay to determine PLA₂ enzyme activity is described. PLA₂ enzyme activity can be established using the E. coli radioactive assay (Elsbach and Weiss (1991) Methods Enzymol 197:24). E. coli phospholipids are metabolically labeled for 3 hours with [¹⁴C]-oleic acid (95% in sn-2 position). The bacteria are then autoclaved for 15min. to inactivate bacterial phospholipases, and washed. The viral sample (1 -1000 pg) is added to the bacteria (10,000 cpm) in standard buffer (50 mM Tris-HCl, pH 8.0, and 10 mM CaCl₂) and incubated for 30min. at 37°C. The reaction is stopped by the addition of ice-cold 1% BSA. After centrifugation, 50% of the supernatant is counted. PLA₂ activity is identified and/or quantified by the release of radioactive fatty acids from the phospholipids into the supernatant.

A mixed micelles assay can also be used to establish PLA₂ activity. The substrates used are 6 μM of either L-3-phosphatidylcholine,l,2-di[l-¹⁴C]oleoyl (specific activity 110 mCi mmol), L-3 -phosphatidylinositol L- 1 -stearoyl-2- [ 1 -¹⁴C] arachidonyl (specific activity 48 mCi/mmol), or phosphatidylethanolamine L-l-palmitoyl,2-[l-¹ C]arachidonyl (specific activity 54.6 mCi/mmol). The assay is based on that described by Manjunath et al, (1994) Biochem J. 303:121, with the following modifications: 1 mM Triton X-100 is used instead of deoxycholate; the total reaction volume is 50 μl and the reaction is stopped after 10 min by adding 80 μl chloroform/methanol (2:1) and 50 μl of saturated KC1 solution. Separation is on silica gel thin layer chromatography (TLC) plates. Optimal separation of the phosphatidylcholine reaction products is obtained using a solvent solution containing chloroform, methanol and water at a ratio of 65:35:4. For phosphatidyl ethanolamine and phosphatidylinositol, chloroform and methanol (87:13) are used as a solvent. The separated products are quantified using a Molecular Dynamics Phosphorlmager SI after drying. PLA₂ activity is identified and/or quantified by the presence of radioactive fatty acids, released from phospholipids, on each TLC plate. 2) Parvoviral PLA₂s

In one embodiment of the present invention, the viral PLA₂ is a parvoviras PLA₂. In a related embodiment the parvovirus PLA₂ is present in the VP1 -unique part (VPlup) of the largest capsid protein of parvovirases.

In accordance with the present invention, the parvovirus PL A₂s comprise the following, more specific version of the above amino acid motif: Y-x-G-P-G-x(12)-D-x(2)-A-x(2)-H-D-x(2)- Y, where x is any amino acid, x(2) represents a stretch of 2 amino acids, x(12) represents a stretch of 12 amino acids and wherein one of the specified amino acids Y, G, P, G, D, A, D, or Y may be exchanged singly for any other amino acid. This invention includes viral PLA₂s obtained from known parvovirases, including, but not limited to, mammalian parvovirases, such as canine parvoviras (CPV), mink enteritis virus (MEV), porcine parvovirus (PPV), bovine parvovirus (BPV), goose parvovirus (GPV), feline panleukopenia virus (FPLV), the human parvoviruses, such as B 19, V9, and RA- 1 , and insect parvovirases, such as Gm DNV; Ml DNV; Jc DNV; Pi DNV; Ds DNV; Cp DNV; Ce DNV; Ad DNV; DNV; and Bm DNV. The parvoviral PLA₂s of the present invention may also be obtained from parvoviruses yet to be identified. The protein sequences determined for 30 different parvoviral PLA₂s are shown in Figure 2.

Preparation of Viral PLAβ

Unless otherwise specified, the viral PLA₂ proteins, peptides or fragments thereof, of the present invention are prepared in such a manner that their intrinsic enzymatic activity is retained. Further, amino acid residues may be deleted, added or substituted for those that appear in the amino acid sequences of the viruses of interest in instant invention. It should also be appreciated that the present invention contemplates amino acid sequences that are equivalent to, or constitute active fragments of, the amino acid sequences for the viral PLA₂ enzymes of the present invention. The proteins of the present invention can be prepared through the use of recombinant techniques, or from viral extracts. In general, viral PLA₂ proteins according to the instant invention can be produced by transformation (transfection, transduction, or infection) of a host cell with all or part of a viral PLA₂-encoding DNA fragment in a suitable expression vehicle. Suitable expression vehicles include: plasmids, phagemids, viral particles, and phage. The entire expression vehicle, or a part thereof, can be integrated into the host cell genome. In some circumstances, it is desirable to employ an inducible expression vector.

Those skilled in the field of molecular biology will understand that any of a wide variety of expression systems can be used to provide a recombinant viral PLA₂ protein of the instant invention. The precise host cell used is not critical to the instant invention. A viral PLA₂ protein can be produced in a prokaryotic host (e.g., E. coli or B. subtilis) or in a eukaryotic host (e.g., Saccharomyces or Pichia; mammalian cells, e.g., COS, NIH 3T3, CHO, BHK, 293 , or HeLa cells; or insect cells) using techniques that are standard in the field of molecular biology.

The methods of transformation or transfection and the choice of expression vehicle will depend on the host system selected. Transformation and transfection methods are described, e.g., in Ausubel et al. (1994) Current Protocols in Molecular Biology, John Wiley & Sons, New York. Expression vehicles can be chosen from those provided, e.g. in Cloning Vectors: A Laboratory Manual (Pouwels et al, 1985, Supp. 1987).

The host cells harbouring the expression vehicle can be cultured in conventional nutrient media adapted as necessary for activation of a chosen gene, repression of a chosen gene, selection of transformants, or amplification of a chosen gene. One example of an expression system is the mouse 3T3 fibroblast host cell transfected with apMAMneo expression vector (Clontech, Palo Alto, Calif). pMAMneo provides an RSV-LTR enhancer linked to a dexamethasone-inducible MMTV-LTR promotor, together with an SV40 origin of replication, which allows replication in mammalian systems, a selectable neomycin gene, and SV40 splicing andpolyadenylation sites. DNA encoding a viral PLA₂ protein can be inserted into the pMAMneo vector the appropriate orientation to allow expression of the protein. The recombinant viral PLA₂ protein can then be isolated from the expression system using standard techniques. Other host cells that can be used in conjunction with the pMAMneo expression vehicle include COS cells and CHO cells (ATCC Accession Nos. CRL 1650 and CCL 61, respectively).

Alternatively, viral PLA₂ polypeptides can be produced as fusion proteins. For example, the expression vector pUR278 (Rutheretα . (1983)E δOJ 2, 1791) can be used to create lacZ fusion proteins, or the pGΕX vectors can be used to create glutathione S-transferase (GST) fusion proteins. In general, GST-fusion proteins are soluble and can be easily purified from lysed cells by adsorption to glutathione-agarose beads, followed by elution in the presence of free glutathione. The pGΕX vectors are designed to provide tlirombin or factor Xa protease cleavage sites so that the cloned target gene product can be released from the GST moiety.

Specific initiation signals may also be required for efficient translation of inserted nucleic acid sequences. These signals include the ATG initiation codon and adjacent sequences. In cases where an entire native viral PLA₂ gene or cDNA, including its own initiation codon and adjacent sequences, is inserted into the appropriate expression vector, no additional translational control signals may be needed. In other cases, exogenous translational control signals, including, perhaps, the ATG initiation codon, must be provided. Furthermore, the initiation codon must be in phase with the reading frame of the desired coding sequence to ensure translation of the entire insert. These exogenous translational control signals and initiation codons can be of a variety of origins, both natural and synthetic. If necessary, the efficiency of expression can be enhanced by the inclusion of appropriate transcription enhancer elements and / or transcription terminators (Bittner et al. (1987) Methods in Enzymol 153, 516).

Alternatively, a viral PLA₂ protein can be produced from a stably-transfected mammalian cell line. A number of vectors suitable for stable transfection of mammalian cells are available to the public, see, e.g. Pouwels et al. (supra). Methods for constructing such cell lines are also publicly available and known to those skilled in the art, see for example Ausubel et al. (supra). In one example, cDNA encoding the viral PLA₂ protein can be cloned into an expression vector that includes the dihydrofolate reductase (DHFR) gene. Integration of the plasmid and, therefore, the viral PLA₂ protein-encoding gene into the host cell chromosome can then be selected for by including an appropriate amount of methotrexate in the cell culture medium (as described in Ausubel et al, supra). This dominant selection can be accomplished in most cell types.

Fusion proteins can be readily purified by utilising an antibody or other binding partner specific for the fusion protein being expressed. For example, a system described in Janknecht etal. (1981) Proc. Natl Acad. Sci. USA 88:8972, allows for the ready purification of non-denatured fusion proteins expressed in human cell lines. In this system, the gene of interest is subcloned into a vaccinia recombination plasmid such that the open reading frame of the gene is translationally fused to an amino-terminal tag consisting of six histidine residues. Extracts from cells infected with the recombinant vaccinia virus are loaded onto a Ni²⁺ nitriloacetic acid-agarose column, and the histidine-tagged proteins are selectively eluted with imidazole-containing buffer.

Alternatively, a viral PLA₂ or a portion thereof, can be fused to an immunoglobulin Fc domain. Such a fusion protein can be readily purified using a protein A column.

It will be apparent to one skilled in the art that the viral PLA₂ peptides disclosed herein can be created by designing nucleic acid sequences that encode for viral PLA₂, but which differ, by reason of the redundancy of the genetic code, from the sequences disclosed herein. Accordingly, the degeneracy of the genetic code further enables major variations in the nucleotide sequence of a nucleic acid molecule but does not broaden the scope of the present invention since the amino acid sequence of the encoded protein remains unchanged. Based upon the degeneracy of the genetic code, variant nucleic acid sequences may be derived from the nucleic acid sequences disclosed herein. These variant nucleic acid sequences can be produced by modifying or synthesizing nucleic acid sequences. Variant nucleic acid sequences include deletion, addition, substitution, or a combination thereof, of different nucleotides. A viral PLA₂ protein sequence of the present invention can be identified using the criteria set out above and can be chemically synthesized by methods known to those of skill in the art. Such methods include, but are not limited to, exclusive solid phase synthesis, partial solid phase synthesis, fragment condensation or classical solution synthesis (Merrifield (1963) J Am. Chem. Soc. 85:2149; Merrifield (1986) Science 232:341). Further, viral PLA₂ peptides can be isolated and purified by standard purification methods including chromatography (e. g. ion exchange, affinity, and sizing column chromatography or high performance liquid chromatography), centrifugation, differential solubility, or by other standard techniques familiar to a worker skilled in the art. Once synthesized, the authenticity of the resulting peptides can be verified using the assay procedures disclosed herein.

Inactive Viral PLAβ

The present invention also includes viral PLA₂ proteins, peptides or fragments thereof, the sequence of which produces an inactive protein (i.e. one that no longer possesses PLA₂ activity), or has been manipulated to produce an inactive protein.

Once a viral PLA₂ protein sequence has been identified using the criteria set out above, the nucleic acid sequence encoding the protein can be isolated. Standard molecular biology techniques can then be employed to alter the nucleic acid sequence and, thus the protein sequence that it encodes. Methods of altering nucleic acid sequences are well-known in the art, for example, site-directed mutagenesis or PCR-based techniques can be employed.

Naturally inactive mammalian sPLA₂ proteins, generated from pseudogenes, are known. An amino. acid motif: HDXXY (where X = any amino acid) is found in the active site of all known sPLA₂ proteins (Dennis (1994) J Biol. Chem. 269:13057-13060). In the inactive sPLA₂ proteins, the conserved aspartic acid residue (D), subsequent to the conserved histidine residue (H) in the above motif, is frequently mutated to a basic amino acid. The HDXXY motif is also found in viral PLA₂s, therefore, one example of an inactive viral PLA₂ protein would be one in which the aspartic acid residue in this motif is exchanged for a basic amino acid such as, lysine, arginine or histidine..

It is envisioned that such inactive viral PLA₂ proteins or peptides will retain their ability to induce a specific immune response and that they will, therefore, be useful in the development of vaccines.

Uses of Viral PLAβ

The viral PLA₂s of the present invention can be used in manufacturing processes, in the clinical diagnosis and treatment of diseases, and in basic research settings.

1) Research At present, purified bee venom PLA₂ is commonly used in research, for example as a reference standard when determining the PLA₂ enzyme activity contained in various preparations of isolated enzyme. The viral PLA₂ of PPV, however, has a 40 to 100-fold higher specific activity than that of bee venom PLA₂ which makes it better suited to various research applications. In addition, while some parvoviras PLA₂s demonstrate specificity for certain phospholipids (e.g. GmDNV PLA₂ is specific for phosphatidylinositol), others have a broad substrate specificity thus making their use widely applicable.

2) Screening for viral PLA₂ inhibitors

The viral PLA₂s of the present invention are also useful for screening compounds in order to identify agents capable of inhibiting PLA₂ activity. Useful inhibitory agents are identified with a range of assays employing viral PLA₂s or nucleic acids encoding viral PLA₂s. As examples, protein binding assays, nucleic acid binding assays, gel shift assays, cell-based assays, mixed micelle assays, and the like are useful approaches.

In one embodiment of the present invention, there is provided a screening assay in which a candidate inhibitor is added to a solution containing a viral PLA₂ and a labelled phospholipid substrate, and the inhibition of PLA₂ activity is then determined. In order to determine whether the inhibitory activity of the candidate inhibitor compound is specific, PLA₂ activity in the presence of the candidate inhibitor, is compared to the following: a) the PLA₂ activity in the absence of the candidate compound, b) in the presence of a well-characterized PLA₂ inhibitor, c) in the presence of a PL A₂ agonist, d) in the presence of activators and inhibitors of other phospholipase enzymes, for example PLC, and e) in the presence of both the candidate inhibitor agent and the well-characterized PLA₂ inhibitor. PLA₂ activity can be determined according to the methods described herein. Such in vitro screening procedures are especially useful in identifying candidate agents that can be subsequently tested in vivo for their ability to inhibit viral PLA₂s.

In another embodiment of the present invention, the in vitro assay is an automated, cost- effective, high-throughput screening protocol that can be used to survey a large number of test compounds for their ability to inhibit viral PLA₂ activity.

Potential inhibitory compounds are screened from large libraries of synthetic or natural compounds. Numerous means are currently used for random and directed synthesis of saccharide, peptide, and nucleic acid based compounds. Synthetic compound libraries are commercially available from a number of companies including Maybridge Chemical Co.

(Trevillet, Cornwall, UK), Comgenex (Princeton, N.J.), Brandon Associates (Merrimack,

N.H.), and Microsource (New Milford, Conn.). A rare chemical library is available from Aldrich (Milwaukee, Wis.). Combinatorial libraries are also available and can be prepared according to standard procedures. Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant, and animal extracts are available from, e.g., Pan Laboratories

(Bothell, Wash.) or MycoSearch (North Carolina), or are readily producible. Additionally, natural and synthetically produced libraries and compounds are readily modified through conventional chemical, physical, and biochemical means. The agent library can be naive or can be composed of structural analogs of known PLA₂ inhibitors, or can be a combination of both.

3) Gene Therapy Viral phospholipase A₂ has been shown to be critical for the transfer of the viral genome from the late endosome to the nucleus (see Figure 7). Therefore, the present invention provides a method of improving viral-based vectors for gene therapy. An example would be the adeno- associated virus (AAV) vector currently envisioned for gene therapy. Inclusion of the viral PLA₂ gene into known gene therapy vectors can optimize the transfer of the therapeutic gene- carrying genome to the nucleus of target cells. If necessary, the viral PLA₂ gene may be modified for this purpose, for example, by site-directed mutagenesis, for optimal effectiveness and specificity.

4) Viral PLA₂-Binding Peptides The present invention also provides for polypeptides and peptides that bind the viral PLA₂s.

One exemplary method of identifying such peptide is by phage display techniques. Phage display libraries of random short peptides are commercially available, e. g. from New England

Biolabs, Inc., which are utilized through an in vitro selection process known as "panning".

In its simplest form, panning involves incubating the library of phage displayed peptides with a plate, or bead, coated with the target molecule, washing away unbound phage particles and finally eluting the specifically bound phage. For the purposes of the present invention, the target molecule is a viral PLA₂, or fragment thereof.

In one embodiment of the present invention, the target molecule is porcine parvovirus PLA₂ and the peptides displayed by phage display library are 12-mers.

The peptide(s) displayed by the specifically-binding phage are then isolated and sequenced by standard techniques known to those of skill in the art. In some instances the binding strength of the isolated peptide is then tested using standard techniques.

In one embodiment of the present invention, there is provided polynucleotides which encode the viral PLA₂-binding peptide. These polynucleotides can be cloned and fused with a heterologous nucleic acid. In a related embodiment, the resulting fusion gene is used to produce a fusion protein comprising the viral PLA₂-binding peptide and a heterologous protein. As discussed herein, a worker skilled in the art would readily appreciate that the polynucleotides of the present invention can be cloned with the appropriate regulatory sequences for expression.

In one embodiment of the present invention, there is provided a fusion protein comprising a viral PLA₂-binding peptide and thioredoxin. The thioredoxin fusion protein can be used in an ELISA assay for detection of viral PLA₂s. In this assay the viral PLA₂-binding peptide of the fusion protein binds the viral PLA₂ in a sample, and the resulting complexes are detected using anti-thioredoxin antibodies. The antibodies are either labelled directly, or are detected with a.secondary antibody that is labelled, for example with an enzyme in the case of ELISA assays. Such an assay is useful for detecting virus in a sample from a subject suspected of having a viral infection.

Furthermore, peptides that bind to the viral PLA₂s can be directly labelled by techniques known in the art and used as probe molecules. As such, labelled viral PLA₂-binding peptides are deemed to be within the scope of those skilled in the art in view of the teaching provided herein.

Alternatively, peptides can be selected that, by binding to the viral PLA₂ specifically inhibit the activity of the protein. The inhibitory activity of the peptides is determined using assays as known in the art and as described herein. Inhibitory viral PLA₂-binding peptides can be used therapeutically to reduce or eliminate viral infectivity. One embodiment of the present ideation provides pharmaceutical compositions comprising one or more viral PLA₂-binding peptides or polynucleotides encoding such peptides and a pharmaceutically acceptable diluent or excipient.

Anti-viral Agents

The present invention further provides a class of anti- viral agent that selectively inhibits viral

PLA₂ activity. Viral PLA, activity is required in order for the viras to enter a host nucleus; thus, inhibition of viral PLA₂ activity blocks the ability of a viras to infect a host cell. Since the sequence of the viral enzyme is very different from that of the host enzymes, specific inhibitors can be developed that do not impair host PLA₂ enzyme activities. The present invention provides anti-viral PLA₂ agents that inhibit viral PLA₂ activity but that are nontoxic to the host.

1) Selection of Inhibitors (Active Agents)

The anti-viral PLA₂ agents of the present invention are initially selected on the basis of the agent's capacity for inhibiting viral PLA₂ in vitro. Inhibition of PLA₂ activity can be determined using assays that monitor phospholipase activity including the screening assay of the present invention. This initially selected agent is then be administered to a cultured host cell population, which is subsequently exposed to a viras. The capacity of the agent to produce a detectable reduction in the infectivity and / or replication of the virus in the treated cell culture, in comparison to an untreated cell culture, is determined according to standard procedures known in the art, for example by measuring plaque forming units. Agents capable of reducing infectivity and / or replication of the virus are thereby identified as active agents.

In addition to exhibiting anti- viral activity, the anti- viral PLA₂ agents must also be selective. A selective anti-viral PLA₂ agent produces a preferential inhibition of viral PLA₂ as compared to inhibition of mammalian or host PLA₂s. Typically, the anti- viral PLA₂ agent required to produce inhibition of 50% of viral PLA₂ catalytic activity is at least one order of magnitude lower than the concentration required to produce inhibition of 50% of the catalytic activity of phospholipases other than viral PLA₂, including mammalian (host) PLA₂, PLA„ PLC and PLD. Two exemplary methods that can be used to determine the specificity of potential anti-viral PLA₂ agents are described briefly below, although it will be appreciated by those skilled in the art that alternative methods can also be employed.

In one method PLA₂ enzyme activity is measured using a phospholipase assay familiar to one skilled in the art. An example of a suitable phospholipase assay for this procedure is described in detail in Example III. Briefly, isolated virus preparations are homogenized and centrifuged in a manner familiar to one skilled in the art of enzyme purification and supernatants are obtained. PLA₂ activity in the supernatant is then measured in the absence and presence of various potential anti-viral PLA₂ agents. A similar procedure is used to determine the activity of mammalian or host cell PLA₂s, PLAjS, PLCs and PLDs, in the absence and presence of the potential anti- viral agents. The selection of an anti- viral PLA₂ agent is determined by comparing the kinetic parameters of both the viral PLA₂ and the host cell PLA₂. An example of a potentially useful anti-viral PLA₂ agent for clinical trials is one that inhibits viral PLA₂ activity by at least 50 % with an IC₅₀ at least one order of magnitude lower than that which inhibits the host cell PLA₂.

A second method to determine the activity of potential anti-viral PLA₂ agents is by immunofluorescence assay as described in Example VII and shown in Figure 5. A preparation of isolated viras is labelled with a fluorescent marker and then administered to a host cell culture. Entry and infection of the host cells by the viras is monitored by tracking this fluorescent tag over aperiod of time in the presence and absence of a potential anti-viral PLA₂ agent. The ability of the viras to infect the cell, and thus the capacity of an anti-viral PLA₂ agent to inhibit infectivity, is a function of the amount of fluorescence tag that is viewed within the cell. The higher the amount of fluorescence the weaker the inhibitory capacity of the anti- iral PLA₂ agent.

2) Antisense Oligonucleotides

The present invention contemplates antisense oligonucleotides designed to inhibit viral PLA₂ expression, wherein the antisense oligonucleotides can be complementary to all or part of the viral PLA₂ sequences, such as antisense oligonucleotides to the viral PLA₂ gene or mRNA. Such complementary antisense polynucleotides may include nucleotide substitutions, additions, deletions, or transpositions, so long as specific hybridization to the relevant target sequence is retained as a functional property of the polynucleotide. Antisense oligonucleotides include soluble antisense RNA or DNA oligonucleotides which can hybridize specifically to viral PLA₂ mRNA species and prevent transcription of the mRNA species and/or translation of the encoded polypeptide (Ching et al., (l 989) Proc. Natl. Acad. Sci. U.S.A. 86:10006; Broder et al, (1990) Ann. Int. Med. 113:604; Loreau et al, (1990) FEBS Letters 274:53; Holcenberg et al, WO91/11535; U.S. Ser. No. 07/530,165; WO91/09865; WO91/04753; WO90/13641; and EP 386563). In one embodiment of the present invention, the antisense oligonucleotides of the present invention exhibit little, or no, inhibition of expression of non- viral PLA₂s.

Antisense molecules are generally targeted to specific nucleic acids. "Targeting" an antisense compound to a particular nucleic acid, in the context of this invention, is a multistep process. The process usually begins with the identification of a nucleic acid sequence whose function is to be modulated. In the present invention, the target is a nucleic acid molecule encoding the viral PLA₂ gene. The targeting process also includes determination of a site or sites within this gene for the antisense interaction to occur such that the desired effect, e.g., detection or modulation of expression of the protein, will result. Within the context of the present invention, examples of intragenic sites for antisense interaction are the regions encompassing the translation initiation and the termination codon of the open reading frame (ORF) of the gene. The terms "translation initiation codon" or "start codon" can encompass several codon sequences. In the context of the invention, "start codon" and "translation initiation codon" refer to the codon, or codons, that may be present in vivo to initiate translation of an mRNA molecule transcribed from a gene encoding a viral PLA₂ gene, regardless of the sequence(s) of such codons.

The open reading frame (ORF) or "coding region," which is known in the art to refer to the region between the translation initiation codon and the translation termination codon, also can be targeted effectively. Other target regions include the 5' untranslated region (5'UTR) and the 3 ' untranslated region (3 'UTR). The 5'UTR is known in the art to refer to the portion of an mRNA in the 5' direction from the translation initiation codon, and thus includes nucleotides between the 5' cap site and the translation initiation codon of an mRNA, or corresponding nucleotides on the gene. The 3'UTR is known in the art to refer to the portion of an mRNA in the 3' direction from the translation termination codon, and thus includes nucleotides between the translation termination codon and 3' end of an mRNA, or corresponding nucleotides on the gene. The 5' cap of an mRNA comprises an N⁷-mefhylated guanosine residue joined to the 5'-most residue of the mRNA via a 5'-5' triphosphate linkage. The 5' cap region of an mRNA is considered to include the 5' cap stracture itself, as well as the first 50 nucleotides adjacent to the cap. The 5' cap region, therefore, can also be a target region for the antisense molecule.

Antisense oligonucleotides are chosen that are sufficiently complementary to the target, i.e., hybridize sufficiently well and with sufficient specificity, to give the desired effect. In the context of this invention, "hybridization" means hydrogen bonding, which may be Watson- Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding, between complementary nucleoside or nucleotide bases. Adenine and thymine, for example, are complementary nucleobases which pair through the formation of hydrogen bonds. "Complementary, " as used herein, refers to the capacity for precise pairing between two nucleotides: if a nucleotide at a certain position of an oUgonucleotide is capable of hydrogen bonding with a nucleotide at the same position of a DNA or RNA molecule, then the oUgonucleotide and the DNA or RNA are considered to be complementary to each other at that position. The oUgonucleotide and the DNA or RNA are complementary to each other when a sufficient number of corresponding positions in each molecule are occupied by nucleotides that can hydrogen bond with each other such that stable and specific binding occurs between the oUgonucleotide and the DNA or RNA target. It is understood in the art that the sequence of an antisense compound need not be 100% complementary to that of its target nucleic acid to be specifically hybridizable. An antisense compound is specifically hybridizable when binding of the compound to the target DNA or RNA molecule interferes with the normal function of the target DNA or RNA to cause a loss of utility, and when there is a sufficient degree of complementarity to avoid non-specific binding of the antisense compound to non-target sequences under conditions in which specific binding is desired, i.e., under physiological conditions in the case of in vivo assays or therapeutic treatment, and in the case of in vitro assays, under conditions in which the assays are performed.

In the context of this invention, the term "oUgonucleotide" refers to an oligomer or polymer of ribonucleic acid (RNA) or deoxyribonucleic acid (DNA) or mimetics thereof. This term includes oligonucleotides composed of naturally-occurring nucleobases, sugars and covalent internucleoside (backbone) linkages as well as oligonucleotides having non-naturally- occurring portions which function similarly. Such modified or substituted oligonucleotides are often preferred over native forms because of desirable properties such as, for example, enhanced cellular uptake, enhanced affinity for nucleic acid target and increased stability in the presence of nucleases.

Examples of modified or substituted antisense compounds useful in this invention include oligonucleotides containing modified backbones or non-natural internucleoside linkages. As defined in this specification, oligonucleotides having modified backbones include those that retain a phosphorus atom in the backbone and those that do not have a phosphorus atom in the backbone. For the purposes of this specification, and as sometimes referenced in the art, modified oligonucleotides that do not have a phosphorus atom in their internucleoside backbone can also be considered to be oligonucleosides. Other oUgonucleotide mimetics include those in which both the sugar and the internucleoside linkage of the nucleotide units are replaced with novel groups, while the base units are maintained for hybridization with the target nucleic acid. One such oUgonucleotide mimetic that has been shown to have excellent hybridization properties is referred to as a peptide nucleic acid (PNA). In PNA compounds, the sugar-backbone of an oUgonucleotide is replaced with an amide-containing backbone, in particular an aminoethylglycine backbone, see for example, Nielsen et al, Science (1991) 254:1497.

Modified oligonucleotides containing one or more substituted sugar moieties and / or one or more nucleobase substitutions are also comprehended by the present invention. The present invention also includes antisense compounds which are chimeric compounds. "Chimeric" antisense compounds or "chimeras," in the context of this invention, are antisense compounds, particularly oligonucleotides, which contain two or more chemically distinct regions, each made up of at least one monomer unit, i. e. one nucleotide in the case of an oUgonucleotide compound. Chimeric oligonucleotides typically contain at least one region wherein the oUgonucleotide is modified so as to confer upon the oUgonucleotide increased resistance to nuclease degradation, increased cellular uptake, and / or increased binding affinity for the target nucleic acid. The antisense compounds used in accordance with this invention typically comprise from about 8 to about 100 nucleobases. In one embodiment of the present invention, the antisense compounds comprise from about 8 to about 50 nucleobases. In a related embodiment, the antisense compounds comprise from about 8 to about 30 nucleobases. In another related embodiment, the antisense compounds comprise from about 15 to about 30 nucleobases . The antisense compounds can be conveniently and routinely made through the well-known technique of solid phase synthesis. Equipment for such synthesis is sold by several vendors including, for example, Applied Biosystems (Foster City, CA). Other means for such synthesis known in the art can be additionally or alternatively employed. Similar techniques using phosphorothioates and alkylated derivatives have been employed to produce oligonucleotides.

The antisense compounds used in accordance with the present invention can be synthesized in vitro. The present invention also includes genetic vector constructs designed to direct the in vivo synthesis of antisense molecules.

3) Antibodies

Another example of anti- viral PLA₂ agents are antibodies raised against specific epitopes of viral PLA₂. Viral PLA₂s are significantly different from other PLA₂s in both their sequences and structure. Antibodies, therefore, can be raised against specific viral PLA₂ epitopes to produce PLA₂ inhibitors that are specific to viral PLA₂s and that do not interact with, or inhibit the activity of, non-viral PLA₂.

For the production of antibodies, various hosts including goats, rabbits, rats, mice, humans, and others can be immunized by injection with viral PLA₂ protein, or with any fragment or oligopeptide thereof that has immunogenic properties. Depending on the host species, various adjuvants may be used to increase immunological response. Such adjuvants include, but are not limited to, Freund's, mineral gels such as aluminum hydroxide, and surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, Keyhole limpet hemolysin (KLH), and dinitrophenol. Examples of adjuvants used in humans include, BCG (bacilli Calmette-Guerin) and Corynebacterium parvum. The oligopeptides, peptides, or fragments used to induce antibodies to viral PLA₂ can have an amino acid sequence consisting of as little as about 5 amino acids. In one embodiment of the present invention, amino acid sequences of at least about 10 amino acids are used. These oligopeptides, peptides, or fragments can be identical to a portion of the amino acid sequence of the natural protein that contains the entire amino acid sequence of a small, naturally occurring molecule. If required, short stretches of viral PLA₂ amino acids can be fused with those of another protein, such as KLH, and antibodies to the chimeric molecule can be produced.

Monoclonal antibodies to viral PLA₂ can be prepared using techniques that provide for the production of antibody molecules by continuous cell lines in culture. These include, but are not limited to, the hybridoma technique, the human B-cell hybridoma technique, and the EBV-hybridoma technique (See, e.g., Kohler, G. etal. (1975) Nature 256:495-497; Kozbor, D. et al. (1985) J. Immunol. Methods 81:31-42; Cote, R. J. et al. (1983) Proc. Natl. Acad. Sci. 80:2026-2030; and Cole, S. P. et al. (1984) Mol. Cell Biol. 62:109-120.)

For example, the monoclonal antibodies according to the invention can be obtained by immunizing animals, such as mice or rats, with purified viral PLA₂. Spleen cells isolated from the immunized animals are then immortalized using standard techniques. Those isolated immortalized cells whose culture supernatant contains an antibody that causes an inhibition of the activity of viral PLA₂ with an IC₅₀ of less than 100 ng/ml are then selected and cloned using techniques that are familiar and known to one skilled in the art. The monoclonal antibodies produced by these clones are then isolated according to standard protocols.

The immortalization of the spleen cells of the immunized animals can be carried out by fusing these cells with a myeloma cell line, such as P3X63-Ag 8.653 (ATCC CRL 1580) according to the method in (1980) J. oflmm. Meth. 39:285-308. Other methods known to a person skilled in the art can also be used to immortalize spleen cells. In order to detect immortalized cells that produce the desired antibody against the viral PLA₂, a sample of the culture supernatant is tested using an ELISA assay for reactivity with the viral PLA₂. In order to obtain those antibodies that inhibit the enzymatic activity of viral PLA₂, the culture supernatant of clones that produce antibodies that bind to viral PL A₂ is additionally examined for inhibition of PLA₂ activity using an appropriate assay, such as those described herein. Those clones whose culture supernatant shows the desired inhibition of viral PLA₂ activity are expanded and the antibodies produced by these clones are isolated according to known methods.

In addition, techniques developed for the production of "chimeric antibodies," such as the splicing of mouse antibody genes to human antibody genes to obtain a molecule with appropriate antigen specificity and biological activity, can be used (Morrison, S. L. et al. (1984) Proc. Natl Acad. Sci. 81:6851-6855; Neuberger, M. S. et al. (1984) Nature 312:604-608; and Takeda, S. et al. (1985) Nature 314:452-454). Alternatively, techniques described for the production of single chain antibodies can be adapted, using methods known in the art, to produce viral PLA₂-specific single chain antibodies. Antibodies with related specificity, but of distinct idiotypic composition, can be generated by chain shuffling from random combinatorial immunoglobulin libraries (see for example, Burton D.R. (1991) Proc. Natl. Acad. Sci. 88:10134-10137).

Antibodies can also be produced by inducing in vivo production in the lymphocyte population or by screening immunoglobulin libraries or panels of highly specific binding reagents as disclosed in the literature (Orlandi, R. et al. (1989) Proc. Natl. Acad. Sci. 86: 3833-3837; Winter, G. et al. (1991) Nature 349:293-299).

Antibody fragments which contain specific binding sites for viral PLA₂ can also be generated. For example, such fragments include, but are not limited to, F(ab')2 fragments produced by pepsin digestion of the antibody molecule and Fab fragments generated by reducing the disulfide bridges of the F(ab')2 fragments. Alternatively, Fab expression libraries can be constructed to allow rapid and easy identification of monoclonal Fab fragments with the desired specificity (see for example, Huse, W. D. et al. (1989) Science 246:1275-1281).

Various immunoassays can be used for screening to identify antibodies having the desired specificity. Numerous protocols for competitive binding or immunoradiometric assays using either polyclonal or monoclonal antibodies with established specificities are well known in the art. Such immunoassays typically involve the measurement of complex formation between viral PLA₂ and its specific antibody. Examples of such techniques include enzyme-linked immunosorbent assays (ELISAs), radioimmunoassays (RIAs), and fluorescence activated cell sorting (FACS). Alternatively, two-site, monoclonal-based immunoassay utilizing monoclonal antibodies reactive to two non-interfering viral PLA₂ epitopes, or a competitive binding assay can be used (see Maddox, D. E. et al. (1983) J Exp. Med. 158:1211-1216). These and other assays are well known in the art (see for example, Hampton, R. et al. (1990) Serological Methods: A Laboratory Manual, APS Press, St Paul, Minn., Section IV; Coligan, J. E. et al. (1997, and periodic supplements) Current Protocols in Immunology, Greene Pub. Associates and Wiley-Interscience, New York, N.Y.; Maddox, D. E. et al. (1983) J Exp. Med. 158:1211-1216).

Use of Anti-Viral PLA₂ Inhibitors

The identified anti- iral PLA₂ agents of the present invention can be used as commercial reagents as standards in toxicological or pharmaceutical evaluations related to PLA₂s.

The identified anti- viral PLA₂ agents of the present invention can also be used in therapeutic applications in animals and humans. The present invention describes methods of decreasing the infectivity and/or replication of a viras in a subject by administering an anti- viral PLA₂ agent to the subject. In one embodiment, the virus is a parvoviras, including mammalian parvoviruses, such as canine parvovirus (CPV), mink enteritis viras (MEV), porcine parvovirus (PPV), bovine parvovirus (BPV), goose parvovirus (GPV), feline panleukopenia viras (FPLV), and human parvovirases, including adeno-associated viras, B19, V9 (Nguyen etal, (1999)J Clin. Microbiol 37:2483), andRA-1 (Stierle etal, (1987) Ann. Rheum. Dis. 46:219); and insect parvoviruses including densoviruses for the following hosts: Gm Galleria mellonella; Ml Mythimna loreyi; Jc Junonia coenia; Pi Pseudoplusia includens; Ds Diatraea saccharalis; Cp Culexpipiens; Ce Casphalia extranea; Ad Acheta domesticus; PfPeriplanetafuliginos; and Bm Bombyx mori. Anti-viral PLA₂ agents that selectively block viral PLA₂ activity, including chemical compounds, antisense oligonucleotides, and antibodies, can be used in the methods of the present invention.

1) Antisense Oligonucleotides The antisense oligonucleotides of the present invention inhibit production of the viral PLA₂ polypeptides, thus preventing the viras from infecting the host. Compositions containing a therapeutically effective dosage of viral PLA₂ antisense oligonucleotides can be administered for treatment of viral infections.

Viral PLA₂ antisense compounds can be used as research reagents and diagnostics. As an example, antisense oligonucleotides, which are able to inhibit gene expression with exquisite specificity, may be used to elucidate the function of particular genes in the viral cycle. Antisense compounds can also be used to distinguish between functions of various members of viral biological pathways.

The specificity and sensitivity of antisense is also harnessed by those of skill in the art for therapeutic uses. Antisense oligonucleotides have been employed as therapeutic moieties in the treatment of disease states in animals and man. Antisense oligonucleotides have been safely and effectively administered to humans and numerous clinical trials are presently underway. It is thus established that oligonucleotides can be useful therapeutic modalities that can be configured to be useful in treatment regimes for treatment in vitro (i. e. in cell and tissue culture) and in vivo (i.e. in whole animals, especially humans).

The present invention also includes pharmaceutical compositions and formulations which include the antisense compounds of the present invention. In one embodiment of the present invention, there is provided a composition comprising one or more antisense oligonucleotides and a diluent. In an alternative embodiment the composition additionally comprises one or more compounds designed to target the antisense oUgonucleotide to the affected tissue. The pharmaceutical compositions of the present invention may be administered in a number of ways depending upon whether local or systemic treatment is desired and upon the area to be treated. Administration may be topical (including ophthalmic and to mucous membranes including vaginal and rectal delivery); pulmonary, e.g. by inhalation or insufflation of powders or aerosols, including by nebulizer; intratracheal; intranasal; epidermal and transdermal; oral or parenteral. Parenteral administration includes intravenous, intraarterial, subcutaneous, intraperitoneal or intramuscular injection or infusion; or intracranial, e.g., intrathecal or intraventricular, administration.

2) Administration of Purified or Synthetic Anti- Viral PLA₂ Inhibitors and Antibodies The anti- viral PLA₂ agents of the present invention can be delivered alone or in combination, and can be delivered along with a pharmaceutically acceptable vehicle. Ideally, such a vehicle would enhance the stability and/or delivery properties. The invention also provides for pharmaceutical compositions containing the active factor or fragment or derivative thereof, which can be administered using a suitable vehicle such as liposomes, microparticles or microcapsules. In various embodiments of the invention, it may be useful to use such compositions to achieve sustained release of the active component.

When the anti- viral PLA₂ agents of the present invention are employed for the treatment of viral infections in a subject, they can be formulated into oral dosage forms such as tablets, capsules and the like. The compounds can be administered alone or in combination with conventional carriers, such as magnesium carbonate, magnesium stearate, talc, sugar, lactose, pectin, dextrin; starch, gelatin, tragacanth, methylcellulose, sodium carboxymethyl-cellulose, low melting wax, cocoa butter and the like. Diluents, flavoring agents, solubilizers, lubricants, suspending agents, binders, tablet-disintegrating agents and the like can also be employed. The compounds can be encapsulated with or without other carriers. In all cases, the proportion of active ingredients in said compositions both solid and liquid will be at least to impart the desired activity thereto on oral administration. The anti- viral PLA₂ agents can also be injected parenterally, in which case they are used in the form of a sterile solution containing other solutes, for example, enough saline or glucose to make the solution isotonic. For administration by inhalation or insufflation, the anti- viral PLA₂ agents can be formulated into an aqueous or partially aqueous solution, which can then be utilized in the form of an aerosol.

For topical use, the anti- viral PLA₂ agents can be formulated in the form of dusting powders, creams or lotions in pharmaceutically acceptable vehicles, which are applied to the affected portion of the skin.

The dosage requirements of the anti-viral PLA₂ agents will vary with the particular compositions employed, the route of administration, the severity of the symptoms presented and the particular subject being treated. Such dosage requirements can be determined by standard clinical techniques, known to a worker skilled in the art. Treatment can be initiated with small dosages less than the optimum dose of the compound and thereafter increased until the optimum effect under the circumstances is reached. In general, the anti- viral PLA₂ agents of the present invention are most administered at a concentration that will afford effective results without causing any harmful or deleterious side effects. The anti-viral PLA₂ agents can be either administered as a single unit dose, or the dosage can be divided into convenient subunits administered at suitable times throughout the day.

3) Vaccines

Compositions for use as a vaccine against a virus generally comprise an immunizing amount of a viral PLA₂, or an immunogenic fragment thereof, as an antigen in a pharmaceutically acceptable vehicle. The present invention also provides for inactive viral PLA₂ proteins, peptides, or fragments thereof, which retain their ability to induce a specific immune response, for use as vaccines. For example, such vaccines can be prepared as injectables, such as liquid solutions or suspensions. Solid forms for solubilization or resuspension in a liquid before injection also can be prepared, or the preparation can be emulsified. The active or inactive antigenic capsids for vaccination can be mixed with excipients that are pharmaceutically acceptable and compatible with the active capsids. Examples of suitable excipients are water, saline, dextrose, glycerol, ethanol, or the like, and combinations thereof. In addition, if desired, the vaccine can contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents, or adjuvants, such as aluminum hydroxide, which enhance the effectiveness of the vaccine. The vaccines are conventionally administered parenterally by injection, for example, subcutaneously or intramuscularly.

The present invention further provides for inactive recombinant live virus vaccines. Methods of generating recombinant virus particles are well-known in the art. Since viral phospholipase A₂ has been shown to be critical for the transfer of the viral genome from the late endosome to the nucleus, recombinant virus particles containing a genetically engineered PLA₂ gene encoding an inactive PLA₂ protein, will be non-infective. Such recombinant viras particles could, therefore, be combined with a pharmaceutically acceptable vehicle as described above and used to immunize a subj ect against subsequent infection with the active form of the virus.

The vaccines are administered in a manner compatible with the dosage formulation, and in such amount as will be therapeutically effective and immunogenic. The quantity to be administered depends on the subject to be treated, the capacity of the subject's immune system to synthesize antibodies, and the degree of protection desired. Precise amounts of active ingredient required to be administered depend on the judgment of the practitioner and are peculiar to the subject. Suitable regimes for initial administration and booster shots are also variable, but are typified by an initial administration followed in one or two week intervals by a subsequent injection or other administration.

4) Gene Therapy

A viral PLA₂ peptide or biologically active fragments thereof, may also be employed in accordance with the present invention by expression of such proteins in vivo, which is often referred to^" as "gene therapy."

Thus, for example, cells from a patient may be engineered with a polynucleotide (DNA or RNA) encoding a polypeptide ex vivo, with the engineered cells then being provided to a patient to be treated with the polypeptide. Such methods are well-known in the art. For example, cells may be engineered by procedures known in the art by use of a retroviral particle containing RNA encoding viral PLA₂ or a biologically active fragment thereof.

Similarly, cells may be engineered in vivo for expression of a polypeptide in vivo by, for example, procedures known in the art. As known in the art, a producer cell for producing a retroviral particle containing RNA encoding viral PLA₂, or a biologically active fragment thereof, may be administered to a patient for engineering cells in vivo and expression of the polypeptide in vivo. These and other methods for administering viral PLA₂, or a biologically active fragment thereof, by such method should be apparent to those skilled in the art from the teachings of the present invention. For example, the expression vehicle for engineering cells may be other than a retroviras, for example, an adeno viras which may be used to engineer cells in vivo after combination with a suitable delivery vehicle.

Within the expression vehicle, the nucleic acid sequence encoding the polypeptide of the present invention is under the control of one or more suitable promoters. Suitable promoters which can be employed include, but are not limited to, adenoviral promoters, such as the adenoviral major late promoter; or heterologous promoters, such as the cytomegalo virus (CMV) promoter; the respiratory syncytial viras (RS V) promoter; inducible promoters, such as the MMT promoter, the metallothionein promoter; heat shock promoters; the albumin promoter; the ApoAI promoter; human globin promoters; viral thymidine kinase promoters, such as the Herpes Simplex thymidine kinase promoter; retroviral LTRs (including the modified retroviral LTRs hereinabove described);the SV40 promoter; the ²-actin promoter; and human growth hormone promoters. Alternatively, the promoter can be the native PLA₂ gene promoter.

When the expression vehicle is a retroviras, a retroviral plasmid vector is employed to transduce packaging cell lines to form producer cell lines. Examples of packaging cells which may be transfected include, but are not limited to, the PE501, PA317, E-2, E-AM, PA12, T19-14X, VT-19-17-H2, ECRE, ECRIP, GP+E-86, GP+envAml2₅ and DAN cell lines as described in Miller, Human Gene Therapy, Vol. 1, pgs. 5-14 (1990). The vector can transduce the packaging cells through any means known in the art. Such means include, but are not limited to, electroporation, the use of liposomes, and CaPO₄ precipitation. In one alternative, the retroviral plasmid vector can be encapsulated into a liposome, or PTH to a lipid, and then administered to a host.

The transduced producer cell line generates infectious retroviral vector particles that include the nucleic acid sequence(s) encoding the polypeptides. Such retroviral vector particles then can be employed to transduce eukaryotic cells, either in vitro or in vivo. The transduced eukaryotic cells will express the nucleic acid sequence(s) encoding the polypeptide. Eukaryotic cells which can be transduced include, but are not limited to, embryonic stem cells, embryonic carcinoma cells, hematopoietic stem cells, hepatocytes, fibroblasts, myoblasts, keratinocytes, endothelial cells, and bronchial epithelial cells.

The viral vector employed can, in one embodiment, be an adenoviral vector that includes essentially the complete adenoviral genome (Shenk et al, (1984) Curr. Topics Microbiol Immun. 111(3): 1-39). Alternatively, the viral vector can be a modified adenoviral vector in which at least a portion of the adenoviral genome has been deleted. The viruses used in the constraction of viral vectors are generally rendered replication-defective to remove the undesirable effects of viral replication on the target cells.

The DNA sequences encoding the anti-viral PLA₂ agents employed in the present invention can be either cDNA or genomic DNA. DNA encoding either the entire anti- viral PLA₂ agent, or a portion thereof, can be used. Due to the degeneracy of the genetic code, other DNA sequences that encode substantially the same anti- viral PLA₂ agent or a functional equivalent can also be used. Multiple gene copies can also be used.

In order to produce the gene constructs of the invention, recombinant DNA and cloning methods, which are well known to those skilled in the art, can be utilized (see Sambrook et al, Molecular Cloning, A Laboratory Manual, 2d ed. (New York: Cold Spring Harbor Laboratory Press, 1989), including the use of restriction enzymes, site-directed mutagenesis, ligation, homologous recombination, and transfection techniques. It will be appreciated that administration of the viral vectors of the present invention for gene therapy will be by procedures well-established in the pharmaceutical arts, e.g. by direct delivery to the target organ, tissue or site, intranasally, intravenously, intramuscularly, subcutaneously, intradermally and through oral administration, either alone or in combination.

The dosages administered will vary from subject to subject and will be determined by the level of decrease of infectivity and/or replication of the particular virus balanced against any risk or deleterious side effects.

The methods of the present invention will be particularly useful when the subject is infected by a particular virus. As an example, parvovirus B19 infection may lead to fifth disease in normal individuals, transient aplastic crisis in patients with underlying hemolysis, and chronic anemia due to persistent infection in immunocompromised patients. B19 infection in pregnancy can lead to hydrops fetalis and fetal loss and/or congenital infection. B19 has also been associated with inflammation and autoimmune diseases, such as rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), adult-onset Still's disease, and polyarthritis. It is envisioned, therefore, that the anti-viral PLA₂ agents of the present invention can be used to decrease the infectivity and/or replication of B19 in subjects with these disorders.

Improvement over Current Tools

The viral PLA₂s of the present invention are an improvement over PLA₂ standards used currently in manufacturing, clinical, and research settings. At present, bee venom is commonly used as a standard in measuring PLA₂ activity. The viral PLA₂ of PPV, however, has a 41 -fold higher specific activity than bee venom. This high activity makes PPV PLA₂ ideal for use as a PLA₂ standard. The present invention overcomes the shortcomings and drawbacks often associated with treatment of viral infections, through the identification of a novel family of viral PLA₂ genes and nucleic acid sequences, amino acid sequences, clones, vectors, antisense nucleotide sequences, and cell lines. The improvement over current tools lies in the aspects of the present invention that can be used to specifically inhibit the infectivity and / or replication of many pathogenic viruses known to cause disease in humans. The instant application describes for the first time the inhibition of a newly characterized class of phospholipase A₂ enzyme found in many viruses. Inhibition of these viral-specific enzymes is less toxic to the host since host-specific enzymes are not inhibited. This specificity is largely due to the significant differences in the sequences of viral versus non- viral PLA₂s. Thus, the present invention provides a powerful new anti- viral tool, the use of which will not affect host PLA₂s and phospholipase enzymes, but will dramatically decreases the viral PLA₂. These viral specific PLA₂s are critical components for the replication of the virus and may further be required for the pathologies associated with viral infection.

The present invention is described in further detail in the following non-limiting examples. It is to be understood that the examples described below are not meant to limit the scope of the present invention. It is expected that numerous variants will be obvious to the person skilled in the art to which the present invention pertains, without any departure from the spirit of the present invention.

EXAMPLES Materials and Methods

Expression of VPlup fragments: Expressed VPlups were obtained as follows: the pBADTBX vector was constructed from the pB AD/TOPO Thiofusion expression vector (Invitrogen) by inserting a TEV protease site followed by a polylinker (Bglϊi, Xbal) downstream of the thioredoxin gene. The vector contains the sequence for a His-tag downstream of this sequence. VPlup regions from porcine parvovirus [SEQ ID NO: 50], B19 human parvovirus [SEQ ID NO: 52], Galleria mellonella densoviras [SEQ ID NO: 54], Bombyx mori densoviras [SEQ ID NO: 58], Casphalia extranea densoviras [SEQ ID NO: 56] and adeno-associated virus [SEQ ID NO: 60] were cloned into the BgHl-Xbal sites to obtain the respective fusion proteins. These proteins could be purified using the His-tag on a Ni-agarose column. Subsequent cleavage by TEV protease removed the thioredoxin moiety from the fusion protein. PLA₂ activity of the fusion protein, however, was not significantly affected by the presence of thioredoxin. Usual yields were about 1 mg/litre.

Polyclonal Anti- Vlup Antibodies:

Using the above expression vectors containing the Vlup regions, PLA₂ was expressed and purified as described. About 250 mg of the fusion protein was injected into rabbits with adjuvants (subcutaneously and intramuscularly) according to standard protocols. Immunization was repeated after three weeks. The polyclonal antibodies that were generated were isolated by standard techniques.

Assaying PLA₂ Activity:

PLA₂ enzyme activity was established using the E. coli radioactive assay (Elsbach and Weiss (1991) Methods Enzymol 197:24). E. coli phospholipids were metabolically labelled for 3 hours with [¹⁴C]-oleic acid (95% in sn-2 position). The bacteria were then autoclaved for 15min. to inactivate bacterial phospholipases, and washed. The viral sample (1-1000 pg) was added to the bacteria (10,000 cpm) in standard buffer (50 mM Tris-HCl, pH 8.0, and 10 mM CaCl₂) and incubated for 30min. at 37°C. The reaction was stopped by the addition of ice- cold 1% BSA. After centrifugation, 50% of the supernatant was counted.

The mixed micelles assay was also used to establish PLA₂ activity. The substrates (usually from New England Nuclear/DuPont) were 6 μM of either L-3-phosphatidylcholine,l,2-di[l- ¹⁴C]oleoyl (specific activity 110 mCi/mmol), L-3 -phosphatidylinositol L-l-stearoyl-2-[l- ¹⁴C]arachidonyl (specific activity 48 mCi/mmol), or phosphatidylethanolamine L-l- palmitoyl,2-[l-¹⁴C]arachidonyl (specific activity 54.6 mCi/mmol). This assay was based on that described by Manjunath et al, (199 ) Biochem J. 303:121, with the following modifications: 1 mM Triton X-100 was used instead of deoxycholate; the reaction total volume was 50 μl and was stopped after 10 min by adding 80 μl chloroform/methanol (2:1) and 50 μl of saturated KC1 solution. Separation was on silica gel thin layer chromatographic plates. Optimal separation of the phosphatidylcholine reaction products was obtained using a solvent solution containing chloroform, methanol and water (65:35:4). For phosphatidylethanolamine and phosphatidylinositol, chloroform and methanol (87: 13) were used as solvent. The separated products were quantified using a Molecular Dynamics Phosphorlmager SI after drying. Bee venom PLA₂ was purchased from Sigma- Aldrich Co. (Cat. No. P9279). Only the regression lines of activity vs. dilution with a correlation coefficient r^O.98 were used to calculate the amount of protein to hydrolyze 50% of the substrate in the mixed micelles assay. Activity was then expressed as the μmol phospholipid hydrolyzed during the lOmin. assay per ng of enzyme.

Alternatively, the first-order rate constant, k, was calculated from reaction progress curves using the integrated first-order rate equation [P] = S₀(l -e^"1*), where P is product concentration and S is substrate concentration, and in which k =( _cat/_JK_M)_appE₀ where E₀ is the total enzyme concentration. The value o{(k_c K_i^_Αpp represents the catalytic efficiency of the enzyme.

MIMICAssay:

The MIMIC assay is described in Payan et al, (1997) J. Virol Methods 65:299 and Haberhausen et al, (1998) J. Clin. Microbiol 36:628. The primers used for MIMIC PCR were (positions 836-862 inNADL-2) 5'-AGTGGGTATCGCTACTAACCTACACTC [SEQ ID NO : 47] and (positi ons 1 207 - 1 1 8 1 in NADL -2) 5 ' - GATCTGTCATCATCCAGTCTTCTATGC [SEQ ID NO: 48]. The competing MIMIC amplicon contained a deletion from positions 863-903 and was cloned into a pBluescript plasmid.

Immunofluorescence:

For immunofluorescence studies, the 3C9 monoclonal antibody (ATCC CRL-1745) was used as primary antibody (1 hour incubation). Goat anti-mouse IgG, labelled with fluorescein isothiocyanate was obtained from Sanofi Diagnostics Pasteur (Redmond, WA) and used as secondary antibody (incubation 1 hour), following standard techniques. Confocal Immunofluorescence:

The double labelling of internalized PPV with LAMP-2 was performed using a porcine polyclonal anti-PPV antibody and the AC 17 mouse anti-canine LAMP-2 monoclonal antibody (previously reported to recognize mink LAMP-2; Hariri et al, (2000) Mol. Biol. Cell 11 :255). Appropriate controls were performed to ensure that the antibody labelling was specific. In situ hybridization was performed using DIG-conjugated anti-VP 1 up and anti-NS 1 probes, and FITC-conjugated anti-DIG antibody sandwich labelling according to the manufacturer's instructions (Roche Molecular Biochemicals). Nuclear labelling was performed by the addition of 0.02 mg/ml propidium iodide to paraformaldehyde-fixed cells after labelling. Confocal images were acquired using the 63X PlanApochromat objective of a Leica TCS SP confocal microscope equipped with argon and krypton laser sources.

Virus Inactivation: PPV, at a concentration of about 10¹⁰ GE/ml, was treated with β-propiolactone (BPL) at 0.1 % for 8 hours at 18°C. BPL reacts preferentially with purines, with the main targets being N- 1 at adenosine, N-3 at cytidine, and N-7 at guanosine. Depurination may be an important consequence (Brown et al, (1974) J Virol. 14:840; Drinkwater et α/., (1980) Biochemistry 19:5087). These conditions completely inactivated the virus. The viral capsid was not affected and remained fully reactive to the different monoclonal antibodies (obtained from ATCC).

EXAMPLE I: PROTEIN SEQUENCE COMPARISONS

The parvoviras genome is depicted in Figure 1. The right-half of the parvovirus genome contains a large ORF coding for a nested set of capsid proteins (VPs) from alternative in- frame initiation codons so that these VPs differ by their N-terminal extensions. Initiation codons are determined by alternative splicing (eg. , VP 1 and VP2 of porcine parvovirus (PPV) and human parvovirus B19) or a leaky-scanning mechanism (e.g. VP1-4 of Galleria mellonella densoviras, GmDNV). The VPs can be subdivided into 3 domains: common C- terminal domains required for capsid formation (60 copies per capsid); a small hinge-(like) domain; and unique N-terminal extensions. The capsid-forming domain consists of a β-barrel in which β-strands (black boxes: αA and B are helices) are connected by loops (Ll-4). These loops form most of the surface of the capsids, may contain allotropic determinants (ats), and intertwine with neighboring subunits. Only the common domains are required to obtain stable capsids from expression vectors (Hernando et al, (2000) Virology 267:299; Le Gall-Recule etα/. (1996), JGen Virol 77:2159; Miyamuraetα/., (1994) Proc Natl Acad Sci USA 91:8507; Wong etal. , (1994) J Virol 68:4690; Brown et al. , (1994) Virology 198:477; Martinez etal , (1992) Vaccine 10:684). The known 3D-stracture of 4 different parvovirases, solved by X-ray crystallography, is restricted to these domains (Simpson et al, (1998) Structure 6:1355; Agbandje-McKenna et al, (1998) Structure 6:1369; Agbandje et al, (1993) Proteins 16:155; Tsao et al, (1991) Science 251:1456). The hinge-regions for the group of parvoviruses consisting of PPV, Minute Viras of mice (MVM), mink enteritis parvoviras (MEV), canine parvovirus (CPV), and feline panleukemia parvoviras (FPV) contains a flexible polyglycine stretch that may enable the unique N-terminal extensions to be externalized through the 5 -fold channel during maturation or early during infection (Cotmore et al, (1999) Virology 254:169). The N-terminal extensions of B19 VPs, and probably of GmD V, are permanently on the outside of the virion, and their hinge-like regions have a high content of glycine, threonine and serine. About 100 amino acids upstream of the start of VP is a conserved domain (cd). The shaded boxes in Figure 1 represent the domains (Vlups) cloned in expression vectors (VPlup: PPV- VPlup amino acids 2-174 [SEQ ID NO: 50]; B19-VPlup amino acids 2-240 [SEQ ID NO: 52]; and GmDNV-VPlup amino acids 1-378 [SEQ ID NO: 54]).

Various protein sequences of the conserved domain (cd) region of the VPlup region of the largest capsid protein of 34 human, animal, and insect parvovirases were compared by sequence alignment (Figure 2). The following parvovirus VPlups were included in the sequence alignment: Galleria mellonella densoviras (Gm DNV) [SEQ ID NO: 1]; Mythimna loreyi densoviras (Ml DNV) [SEQ ID NO: 2]; Junonia coenia densoviras (Jc DNV) [SEQ ID NO: 3]; Pseudoplusia includens densoviras (Pi DNV)[SEQ ID NO: 4]; Diatraea saccharalis densoviras (Ds DNV) [SEQ ID NO: 5]; Culex pipiens densoviras (Cp DNV)[SEQ ID NO: 6]; Periplanetafuliginos densoviras (P/DNV) [SEQ ID NO: 7]; Acheta domesticus densoviras G4(f DNV)[SEQ ID NO: 8]; Casphalia extranea densoviras (Ce DNV) [SEQ ID NO: 9]; Bombyx mori densoviras (Bm DNV) [SEQ ID NO: 10]; canine parvovirus [SEQ ID NO: 11]; mink enteritis parvovirus [SEQ ID NO: 12]; mouse parvoviras 1 [SEQ ID NO: 13]; feline panleukopenia parvoviras [SEQ ID NO: 14]; Minute Virus of Mice (MVM) [SEQ ID NO: 15]; Kilham rat parvovirus (K.Rat PV) [SEQ ID NO: 16]; porcine parvovirus [SEQ ID NO: 17]; Muscovy duck parvovirus (Mduck PV) [SEQ ID NO: 18]; goose parvoviras [SEQ ID NO: 19]; bovine parvoviras [SEQ ID NO: 20]; simian parvovirus [SEQ ID NO: 21]; chipmunk parvoviras [SEQ ID NO: 22]; Adeno-Associated Viras 2 (AAV2) [SEQ ID NO: 23]; Adeno-Associated Viras 3B (AAV3B) [SEQ ID NO: 24]; Adeno-Associated Viras 4 (AAV4) [SEQ ID NO: 25]; Adeno-Associated Virus 5 (AAV5) [SEQ ID NO: 26]; Adeno-Associated Viras 6 (AAV6) [SEQ ID NO: 27]; LuIII parvoviras (isolated from tissue culture) [SEQ ID NO: 28]; HI parvovirus (isolated from tissue culture) [SEQ ID NO: 29]; human B19 parvoviras [SEQ ID NO: 30].

Representatives of sPLA₂s included in the alignment are: IA - Naja naja snake venom PLA₂ (amino acids 47 - 81 [SEQ ID NO: 31] and amino acids 115 - 126 [SEQ ID NO: 32]); IB - human pancreatic PLA₂ (amino acids 43 - 77 [SEQ ID NO: 33] and amino acids 116 - 127 [SEQ ID NO: 34]); IIA - human synovial fluid PLA₂ (amino acids 40 - 74 [SEQ ID NO: 35] and amino acids 106 - 117 [SEQ ID NO : 36]); IIB - gaboon viper snake venom PLA₂ (amino acids 18 - 52 [SEQ ID NO: 37] and amino acids 81 - 92 [SEQ ID NO: 38]); EC - rat PLA₂ (amino acids 48 - 82 [SEQ ID NO: 39] and amino acids 118 - 129 [SEQ ID NO: 40]); III - Bee venom PLA₂ (amino acids 29 - 69 [SEQ ID NO: 41] and amino acids 87 - 98 [SEQ ID NO: 42]); V - human PLA2 (amino acids 40 - 74 [SEQ ID NO: 43] and amino acids 106 - 117 [SEQ ID NO: 44]); and X - human PLA₂ (amino acids 61 - 95 [SEQ ID NO: 45] and amino acids 128 - 139 [SEQ ID NO: 46]).

This analysis revealed the presence of a short stretch of high similarity, although in some cases the coding sequence was interrupted by an intron. Analysis of the 34 parvoviras sequences in this region also revealed a fully conserved HDXXY motif in all but four of the sequences (Aleutian Disease Viras and those of the Brevidensovirus genus). This HDXXY motif is also present in the catalytic site of secreted PLA₂s (sPLA₂s) (Figure 2).

In the sPLA₂ family (Dennis (1994) J Biol. Chem. 269:13057-13060), the conservedH48 is assisted by D99 to polarize a catalytic water molecule that hydrolyzes the phospholipid ester. The conserved D49 and carbonyl oxygens from G30 and G32 bind a calcium ion involved in the stabilization of the transition state. The conserved calcium-binding loop motif YXGXG is also present in VPlup, although not at the same sequence distance as in sPLA₂ (Fig. 2). Although sPLA₂s contain 5-8 disulfide bridges, which are a basis for their classification, these are absent in the parvoviras VPlup.

The comparison of VPlup sequences with those of sPLA₂ revealed major differences (in addition to disulfide bridges) that suggested vPLA₂ should be considered as a new group of the PLA₂ superfamily. The region connecting the HD helix and the D helix (Dijkstra et al, (19$l)Nature 289:604-606; White etal, (1990) Science 250:1560-1563; Thunnissen etal, (1990) Nature 347:689-691; Scottetal, (1991) Science 254:1007-1010; Scott etal, (1990) Science 250:1563-1566) was found to be minimal in vPLA₂ (Fig.2). Although, the VPlup from the vertebrate B19 parvovirus showed some sequence identity (e.g., NPYTH) to the group IB pancreatic phospholipase, significant homology was also observed (Fig.2) between the vPLA₂ and group III PLA₂ (e.g., bee venom) at the amino-terminal and in the C-terminal helices which form the hydrophobic channel for the fatty acid chain (Scott et al, (1991) Science 254:1007-1010). The observed sequence homology would support the idea that vPLA₂ has a similar fold to the group III PLA₂ domain with connecting loop residues of the group I-II PLA₂ resulting in an overall different 3D structure.

Searches of the databases Prosite PS00118 and PS00119, PRINTS PR00389, Pfam PF00068 and ProDom PD000303 using standard motifs used to recognize PLA₂ failed to identify parvoviral phospholipase A₂ (pvPLA₂). Most probably this failure was due to the fact that these standard motifs rely heavily on the presence of cysteines which are necessary for disulfide bond formation and which are absent in pvPLA₂. _β

Therefore, the conservation of the catalytic site and the Ca²⁺-binding loop was used to develop new motifs that allowed us to detect known and potential PLA₂s. Searches conducted using the pattern [WY]-[CιVL]-G-x-[GP]-[GKNS]-x(8, 14)-D-x(2)-[AC]-x(2)-H-D-x(2)-Y, and allowing for 2 mismatches using, e.g. the PATTLNPROT program (PBIL (http://npsa- pbil.ibcp.fr/)), or Pattern and Profile Searches (ExPASy (http://www.expasy.ch )), identified enzymatically active sPLA₂s from all sPLA₂ groups, albeit with high background. Refined, more specific motifs recognized subsets of sPLA₂ representing specific sPLA₂ groups, with high specificity and sensitivity. In the above motif, the one-letter notation of amino acids is used, bracketed amino acids (eg [WY]) represent alternative amino acids at that position, x represents any amino acid, x(2) represents a stretch of 2 amino acids and x(8, 14) a stretch of between 8 and 14 amino acids.

For pvPLA₂, the specific motif is Y-x-G-P-G-x(12)-D-x(2)-A-x(2)-H-D-x(2)-Y. A search of the TrEMBL protein sequence database conducted using this motif detected all 102 vPLA₂ sequences, of which 5 had 1 mismatch. No additional pvPLA₂s were detected when 2 mismatches were allowed. Only when 3 or more mismatches were allowed were non-specific hits generated.

The general search patterns also permitted the detection of potential sPLA₂ and PLA₂ in other viruses. Virus infections have been reported to modify intracellular second-messenger pathways, lipid metabolism and arachidonic acid metabolism through pathways that normally are associated withPLA₂(Abubakaretα/. (1990) Biochem. Biophys. Res. Commun.166: 953; Shibutani et al, (1997) J. Clin. Invest. 100: 2054) thus indicating the importance of identifying potential PLA₂s in other viruses.

EXAMPLE II: PLA₂ ACTIVITY IN VPlup

The PLA₂ activity in the VPlup region of three divergent parvovirus genera was measured. Human B19, porcine parvoviras (PPV), and the insect parvoviras GwDNV were chosen as typical representatives to demonstrate the presence of viral PLA₂ (vPLA₂) and to identify its role in the viral cycle.

PLA₂ activity of VPlup was established using the E. coli radioactive assay and the mixed micelles assay, as described above, using sPLA₂s from snake venom, bee venom, and pig pancreas as controls. Although the E. coli radioactive assay is more sensitive than the mixed micelles assay, the modified mixed micelles assay was found to be particularly reproducible. PLA₂ activity in purified virus was only detected when high concentrations of PPV virions (>5 μg/ml) were assayed due to the fact that the PLA₂ domain resides predominantly inside the capsid at this stage. Alkali-denaturation and renaturation to disrupt the capsids and expose the VPlup increased the PLA₂ activity 50-100 fold to levels close to that of expressed VPlup. A heat-shock of 2min. at 70°C also released some VPlup as the PLA₂ activity increased about 20-50 times using this method. Polyclonal antibodies raised against PPV VPlup significantly reduced PLA₂ activity of VPlup exposed after dissociation of capsids or by heat shock, but did not affect the activity of bee venom PLA₂.

With the mixed micelles assay, expressed VP 1 up of PPV had a 41 -fold higher PL A₂ specific activity (relative molar amounts of enzyme required to hydrolyze 50% of the substrate) than bee venom sPL A₂ (which had a specific activity 1360 U/mg) . B 19 and GmDNV VP 1 up were 13.5 and 88.0 times less active, respectively, than bee venom PL A₂ with detection thresholds in the low pg to ng range. The activity and pH optimum depended on the substrate. Although PPV and B 19 vPLA₂ had similar activities at their respective pH optima (8.0 and 4.5) in the E. coli assay, B19 vPLA₂ had the same pH optimum as PPV vPLA₂ (pH 8.0) in the mixed micelles assay, but with a 550-fold lower activity.

The optimum Ca²⁺ concentration was similar to the extracellular (and some intracellular compartments) free Ca²⁺ concentration of about 1.2 mM (Evenas et al, (1998) Curr Opin Chem Biol 2:293). A residual activity of 10% at 10 μM Ca²⁺ could be measured. Addition of EDTA or EGTA (chelator of Ca²⁺) reduced activity of PPV and B 19 vPLA₂ to below detectable levels in the mixed micelles assay (Figure 3). No activity was observed at the resting cell cytoplasmic Ca²⁺ concentration of 0.1 μM, although this concentration may increase up to 10 μM in activated cells (Evenas et al, (1998) Curr Opin Chem Biol 2:293) or infected cells (Hallett et al, (1982) Biochem J. 206:671; Hartshorn et al, (1988) J Immunol. 141:1295; Irurzun et al, (1995) J Virol 69:5142).

Reducing agents were not expected to influence the enzyme activity due to the absence of disulfide bridges. In fact, an increase in activity was observed in the presence of 2- mercaptoethanol (see Table 1). Table 1. PLA₂ activity ( % of activities) (parallel E. coli radioactive assay used unless noted otherwise)

B19 PPV pH optimum (E. coli assay) 4.5 8 pH optimum (mixed micelles assay) 6 8

Ca²⁺ l μM 9.6 % 8.6 % l mM 105.9 % 68.0 %

10 mM 100 % 100 %

2-Mercaptoethanol

10 mM 148 % 189 % lOO mM 154 % 161 %

Monoclonal antibody to C-terminus of PPV VPlup 100 % 6.7 %

(at about 0.5 μg/50 μl)¹

Inhibitors²

IC₅₀, manoalide, pH 8.0 3.8 ±0.4μM 2.0 ±0.3μM

IC,_n, oleyloxyethylphosphoryl choline, pH 8.0 >20 μM 11.1 ±0.9 μM determined by use of a mixed micelles assay.

²The IC₅₀ is the inhibitory concentration leading to 50% inhibition.

The values of (k_c K_M)_Αpp for the PLA₂ activity of the expressed Vlups was also determined from the mixed micelle assays and are shown in Table 2.

Table 2. K^^ Values

Phospholipase A₂ Source K_M)_app / M-

Bee venom (1.09±0.14)xl0⁵

Porcine PV Vlup (71.9±9.4)xl0⁵

B19 PV Vlup (2.5±0.2)xl0⁴

GmDNV Vlup (0.4±0.03)xl0⁴

BmDNW Vlup ~0.5xl0⁵

CeDNV Vlup ~0.5xl0⁵

AAV VPlup -0.75x10⁵ Extended regions of PPV VPlup, each containing the conserved pvPLA₂ domain, were also expressed and their PLA₂ activity was determined (Figure 3B). VP 1 up from Ml to S 174 was used throughout this work and was assigned the relative specific activity of 1.0. Sequences within this expressed peptide but outside the PLA₂ motif (dark-grey in the figure) contributed to the activity as shown by the relative specific activities. The activities of these extended Vlup regions indicated that sequences outside the conserved domain had a large impact on the catalytic efficiency of the enzyme.

The PLA₂ activity of untreated virions (0.2 μg), virions after dissociation and after heat shock was also measured, using bee venom (18 ng) as a control. See Figure 3D. Samples in lanes 3, 5 and 7 were treated with anti-VP 1 up antibody (200 ng). This antibody reduced viral, but not bee venom, PLA₂ activity.

EXAMPLE III: SPECIFICITY OF PLA₂ ACTIVITY

The specificity of the vPLA₂ reaction was determined by thin layer chromatography (TLC) after hydrolysis of phosphatidylcholine substrate (PL) into fatty acid (FA) and lysophosphatidylcholine (LPL) by expressed vPLA₂ in the mixed micelles assay. PLA₂ activity was analyzed with a Molecular Dynamics Phosphorlmager (%: fraction hydrolyzed in % = ((FA+LPL)/(FA+PL+LPL))xl 00). PLA₂s used in the assay were: PPV PLA₂(0.6 ng), B19 PLA₂ (250 ng), GmDNV PLA₂ (350 ng) and bee veno PLA₂ (15 ng). Thioredoxin (2,000 ng) was used as a negative control. The results are shown in Figure 3 A. The 3C9 monoclonal antibody, which binds to the C-terminus of PPV- VPlup, reduced PPV PLA₂ activity of VP lup to 18% of the original activity after adding 200 ng of antibody (lane 2), but not that of B19 (lane 4). Adding EGTA to a final concentration of 5 mM abolished PLA₂ activity (lane 5).

Following hydrolysis, TLC showed that the radioactive label, if present only in the sn-2 oleic acid, moved from the phospholipids to the fatty acid (free oleic acid) spots, and, when present at both sn- 1 and sn-2 positions, to both the lysophosphatidyl and fatty acid spots ( Figure 3 A), thus confirming the specificity of the PLA₂ activity.

The impact of Ca²⁺ concentration, pH and different substrates on the activity of pvPLA₂ was also investigated. Since 1 mM Ca²⁺, 50 mM Tris-HCl, pH 8.0, and phosphatidylcholine (PC) were used in standard assays, these were set at 100% relative activity. The results of these assays are shown in Figure 3C. PPV can be seen to efficiently hydrolyse both phosphatidylethanolamine (PE) and phosphatidylinositol (PI), the alternative substrates tested.

EXAMPLE IV: INHIBITING PLA₂ ACTIVITY

PLA₂ inhibitors manoalide (MA) and oleyloxyethylphosphorylcholine (OP) inhibited the VPlup PLA₂ activity in the E. coli assay. The MA concentration leading to 50% inhibition of PLA₂ was 3.8±0.4 for B19 and 2.0±0.3 μM for PPV. Using OP, >20 and 11.1±0.9 μM, respectively, was required to achieve the same degree of inhibition.

EXAMPLE V: 3-D STR UCTURE OF vPLA₂

The 3D-stractures of a large number of group I/II sPLA2 and group III sPLA2 have been solved (e.g., Renetseder et al, (1985) J Biol. Chem. 260:11627; Yuan and Tsai (1999) Biochim. Biophys. Ada. 1441:215; Dijkstra et al, (1981) Nature 289:604); Sekar and Sundaralingam (1999) Acta. Crystallogr. D. Biol. Crystallogr. 55:46; van den Berg et al, (1995) Embo J. 14:4123; Dijkstra et al, (1978) J Mol Biol. 124:53). This permitted the prediction of the B 19/PP V vPL A₂ stracture, which then served to select potentially critical amino acids for site-directed mutagenesis (Figure 4). This sequence and structure alignment indicated that neither the position numbering of group I/II nor that of group III could be maintained and resulted in an adapted PPV numbering for vPLA₂. EXAMPIE VI: SITE-DIRECTED MUTAGENESIS TO CREATE PLA₂ MUTANT PARVOVIRUSES

The 3-D stracture of vPLA₂ was used to identify potentially critical amino acids in this enzyme. From this stracture, residues 21 (proline), 35 (aspartic acid), 41 (histidine), 42 (aspartic acid), 63 (aspartic acid) and 88 (lysine) were selected for site-directed mutagenesis experiments.

Both the PPV-VPlup and the virus itself were submitted to site-directed mutagenesis (Werner et al, (1994) Gene 151:119; Kuipers et α/., (1990) Prot. Eng. 3:599). The mutants of the infectious clone of PPV (NADL-2 strain in pUC19 (Bergeron, et al, (1996) J Virol 70:2508; Maxwell (1993) J Gen. Virol. 74:1175; Brownstein, et al., (1992) J Virol 66:3118; Gardiner and Tattersall (1988) J Virol 62:1713; Corsini, et al, (1996) Adv Virus Res 47:303; Berns (1990) Microbiol Rev 54:316)) were transfected into PT cells and large amounts were produced (despite their often low infectivity). Particular attention was devoted to detect possible revertants by monitoring the infectivity in two successive infection assays. The concentration of full viras particles, expressed as genome equivalents or GE per μl, was determined by a MIMIC assay. The infectivity of the suspensions was measured with the fluorescent-focus assay. The number of fluorescent nuclei in a well was counted using immunofluorescence 20 hours after infection (before fluorescent nuclei from a secondary infection could appear). The relative infectivity of the mutants was determined from the number of full particles, as established by GE in the MIMIC assay, required to obtain one fluorescent focus unit (ffu), after comparing it to the wild-type (wt) viras. Dilutions were used that gave at least 25 ffu (relative error <0.2). For wt PPV, 232 full particles were required per ffu, and this value represents 100%. The value for the relative specific infectivity of each mutant represents the result of five independent assays.

The PLA₂ activity of the wt and mutant VPlups was determined as shown in Table 3. Hydrolysis of the substrate was measured with a Phosphorhnager. Different amounts of expressed pvPLA2 were assayed and the degree of hydrolysis was measured. In this assay, 0.46 ng wt PPV pvPLA₂ fusion protein was required to achieve 50% hydrolysis. The relative amount of each mutant required to obtain the same degree of hydrolysis yielded the relative specific activities. Only the regression lines of activity vs dilution with a correlation coefficient r^O.98 were used to calculate the amount of protein to hydrolyze 50% of the substrate in the mixed micelles assay.

Both the enzyme activity and viral infectivity decreased significantly when amino acids in the catalytic site (H41 and D42) were mutated (Table 3), whereas back-mutations restored the original infectivity/activity (the back-mutations served as a control to confirm the integrity of rest of genome). Among potential aspartic acids corresponding to D99 in sPLA₂, D63 was fully conserved in vPLA2 (Figure 2), and mutation of this residue was found to decrease strongly both infectivity and activity, indicating that the relative position of the predicted α-helices would be correct (Figure 4).

Site-directed mutagenesis of the aspartic acid residue at position 35, one of the very few amino acids that is conserved among all sPLA₂ and vPLA₂ to glutamic acid (D35E) or asparagine (D35N), had an impact that was comparable to the D63E or D63N mutations. This amino acid may be critical for the positioning of the Ca ⁺-loop by 3 hydrogen bonds to this loop.

A surprising observation was the conservation of P21 among the vPLA₂ but not among the sPLA₂. When P21 was mutated to amino acids that occur at that position in sPLA₂ (R, W, L), both the infectivity and the activity were strongly reduced. It is not known whether mutations to P21 in sPLA₂ would increase their activities. This residue has a versatile role (Kuipers et al, (1990) Protein Eng 3:599.) such as involvement in binding and orienting monomeric substrate, binding of the enzyme to micellar substrates and possibly shielding the catalytic site from excess water. P21 may be required in the case of vPLA₂ to compensate for the 3 -amino acid insertion between the Ca²⁺-binding loop and the α-helix containing H41. K88 is conserved among parvoviruses, and in bee venom (K85) where it is involved in receptor binding (Nicolas et al, (1997) J Biol. Chem. 272:7173). The potential role of this residue as a co-receptor in parvovirases could not be established since conservative mutagenesis (K88R) in VPlup already decreased activity strongly (no receptor involved). Nevertheless, the presence of this conserved homologue of the bee venom PLA₂ receptor- binding helix is intriguing.

Table 3. Relative Specific Activity and Infectivity of pv PLA₂ Mutants

EXAMPLE VII: IMMUNOFLUORESCENCE OF INFECTED CELLS The observation that, upon transfection, wild-type and mutant infectious clones are both effective in producing virions indicated that vPLA₂ is required at some stage prior to replication, i.e. during entry. In order to assess the critical step(s), cells were infected with wt or mutant virus (P21L and P21 W) at a concentration of 0.5 μg/ml and tracked by immunofluorescence. Viras was adsorbed to nonconfluent cells for 5 hours at 4°C to ensure synchronized entry when the cold medium was replaced by medium at 37°C (time- point 0 hours).

The following stages could be distinguished for wt by immunofluorescence (Figure 5): 0 hrs: strong, patchy staining on the cellular membrane; 4 hrs: virus entry with cytoplasmic, granular and perinuclear staining; 8 hrs: first nuclear staining; and 12 and 18 hrs: strong nuclear staining. Prior addition of lysosomotropic NH₄C1 reduced infection in a dose- dependent fashion (10, 20, and 50 mM resulted in 20-, 100- and 100,000-fold reduction in infectivity). At 20 mM NH₄C1, the first nuclear staining was delayed to 18 hrs. Although the entry was fast (5-30 min), a decrease in infectivity could be obtained up to about 4 hrs post-infection by adding NH₄C1 or neutralizing antibodies suggesting a long stay in vesicular compartments. These results are consistent with the observation that parvoviras enters the cell via receptor-mediated endocytosis (Parker and Parrish (2000) J Virol 74:1919; Basak and Turner (1992) Virology 186:368; Vihinen-Ranta et al, (1998) J Virol. 72:802). It has been observed (Parker and Parrish (2000) J Virol. 74:1919) that canine parvovirus (CPV) co-localizes with transferrin in perinuclear endosomes, suggesting that parvoviras infection is complex and involves multiple vesicular components. Biotinylation of virus prior to infection and detection by streptavidin- fluorescein, which only detects incoming virus, showed perinuclear staining after 4, 8, 12, 18 hrs but no nuclear staining. In contrast, immunofluorescence showed nuclear staining from about 8 hrs. It also indicated that infectivity, as measured by ffu, was reduced to about 50% by biotinylation. This permuclear accumulation was not due to biotinylation since the same localization was obtained after inactivation of the virus without affecting the capsid (see Methods and Materials), infection, or subsequent immunofluorescence at 4, 8, 12, and 18 hrs. Although this suggests that only the viral genome is delivered from the perinuclear zone, perhaps from vesicles, into the nucleus, it remains possible that a few virus particles entering the nucleus, which are below the detection level, are responsible for the infection.

Several differences were observed between the mutants and the wt viras. In contrast to the wt virus, the mutants showed a rather diffuse staining early during entry. Like the wt, the mutants became perinuclear after 4 hrs and could not be distinguished at this point from wt. Mutants, however, remained perinuclear for long periods (at least 18 hrs) whereas wt virus- infected cells showed a strong nuclear staining starting after 8 hrs. Occasionally, an infected nucleus was obtained after 18 hrs with the mutants. NH₄C1 exacerbated the effect of the mutations. Although the incoming virus still became perinuclear at about 4 hrs post- infection, it remained there without leading to infection. Single-stranded parvoviral genomes entering the nucleus are normally converted into double-stranded DNA by cellular DNA polymerase (P. Tattersall and S.F. Cotmore inP. Tijssen, ed., Handbook of Parvoviruses, vol. 1 (Boca Raton: CRC Press, 1990) 123). This dsDNA would yield productive infections as was shown with mutant or wt infectious clones. Taken together, these results indicate that PLA₂ mutants experience difficulties in transferring their genome from the perinuclear localization into the nucleus.

EXAMPLE VIII. CONFOCAL IMMUNOFLUORESCENCE OF INFECTED CELLS

The binding and entry of ³⁵S -labeled PPV were studied by confocal immunofluorescence using 4xl0⁵ cells and 5xl0⁹ wild type virus particles. Equivalent amounts of mutant viruses were used for the binding studies, and twice as much for the entry studies. Experiments were performed in quadruplicate. The results are shown in Figure 6A (binding) and 6B (entry). Viruses that were either bound to cells or had entered into cells were measured by a liquid scintillation method after performing washing or antibody-stripping procedures, respectively. pvPLA₂ mutations had no effect on virus binding to the cells or entry. Both binding and entry of virus were time- and dose-dependent (not shown). Figure 6C shows the relative radioactivity from experiments in which 10⁶cpm^3SS-labeled PPV particles (wt or mutant) were added to 2xl0⁷ cells in Petri dishes and about 20% was recovered in the cells. After cellular fractionation, the distribution of virus over cytosolic and nuclear fractions was measured by liquid scintillation. Relative radioactivity was calculated as follows: [nuclear fraction-bound activity]/[cellular fraction-bound activity]. Standard errors were calculated from three experiments.

In addition, co-localization of PPV and LAMP-2 was studied by confocal microscopy. Anti- PPV antibodies were labeled with FITC, giving green fluorescence, and anti-LAMP-2 antibodies with Texas Red, giving red fluorescence. Co-localization of PPV and LAMP-2 resulted in yellow staining. The results are shown in Figure 6D. Both wt and mutant viruses, as well as capsids lacking PLA₂ showed extensive co-localization with LAMP-2 in the late endosomes/lysosomes.

Taken together the above results indicate that parvoviruses do not require vPLA₂ activity for binding to the cell surface or initial stages of entry.

EXAMPLE IX. IN SITU HYBRIDIZATION OF INFECTED CELLS

Figure 7 shows the distribution of viral DNA in infected cells. Incoming viral DNA was detected in the cytoplasm by in situ hybridization 4 (A) and 8 (B) hours post-infection (p.i.), whereas replicating DNA in the nucleus was found at 12 hours p.i. (C). At 12 hours p.i., no DNA could be detected in the nucleus in the case of the HD (D) and P21^pvW mutants. Aphidicoline, a DNA polymerase inhibitor used at 2 μg/ml, also prevented the appearance of viral DNA in the nucleus (F). The sPLA₂ inhibitors tetracain (TC, at 100 μM), and oleyloxyethyl phosphorylcholine (OP, at 20 μM), also reduced the number of nuclei containing viral DNA (G).

EXAMPLE X. IDENTIFICATION OF VIRAL VLA₇-BINDING PEPTIDES Four peptides that specifically bind to the PLA₂-containing VPlup of PPV using a phage display library. The Ph.D.™ Phage Display Peptide Library kit was obtained from New England Biolabs and used according to the manufacturer's instructions, with the exception that Linbro/Titertek (Cat. No. 76-231-05) plastic plates were used in the panning procedure to avoid high background.

The sequences peptides displayed on the phage that specifically bound to PLA₂ (p6, p7, p8 and p 10) are shown in Figure 13. The nucleic acid sequences encoding these 12 amino acid peptides were subsequently cloned downstream of thioredoxin in the pBADTBX vector using the BgKl and Xbαl cloning sites, and expressed as fusion proteins.

The fusion proteins were used in ELISA assays to determine their ability to bind the PLA₂- containing VP 1 up of PPV. The titers obtained in ELISA with these fusion proteins and using anti-thioredoxin antibodies as secondary antibody were all, except for p 10, about 3000. P 10 was about 5x weaker.

The invention being thus described, it will be obvious that the same may be varied in many ways. Such variations are not to be regarded as a departure from the spirit and scope of the invention, and all such modifications as would be obvious to one skilled in the art are intended to be included within the scope of the following claims.

Claims

THE EMBODIMENTS OF THE INVENTION IN WHICH AN EXCLUSIVE PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:

1. An isolated viral polypeptide that has phospholipase A activity.

2. The isolated viral polypeptide according to Claim 1 , wherein said polypeptide comprises an amino acid motif:

[WY]-[CrVL]-G-x-[GP]-[GKNS]-x(8,14)-D-x(2)-[AC]-x(2)-H-D-x(2)-Y wherein:

[] indicates the presence of one of the enclosed amino acids at this position; x is any amino acid; x(2) represents a stretch of 2 amino acids; x(8,14) represents a stretch of between 8 and 14 amino acids.

3. The isolated polypeptide according to Claim 2 wherein said amino acid motif is:

Y-x-G-P-G-x(12)-D-x(2)-A-x(2)-H-D-x(2)-Y wherein: x(12) represents a stretch of 12 amino acids; and wherein one of the specified amino acids Y, G, P, G, D, A, D, or Y may be exchanged singly for any other amino acid.

4. The polypeptide according to any one of Claims 1 - 3 wherein said viral polypeptide is a parvoviral polypeptide.

5. The polypeptide according to any one of Claims 1 - 3 wherein said parvoviral polypeptide is derived from Galleria mellonella densoviras, Mythimna loreyi densoviras, Junonia coenia densoviras, Pseudoplusia includens densoviras, Diatraea saccharalis densoviras, Culexpipiens densovirus, Periplaneta fuliginos densoviras, Acheta domesticus densovirus, Casphalia extranea densovirus, Bombyx mori densoviras, canine parvovirus, mink enteritis parvovirus, mouse parvovirus 1, feline panleukopenia parvovirus, Minute Viras of Mice, Kilham rat parvoviras, porcine parvovirus, Muscovy duck parvovirus, goose parvoviras, bovine parvoviras, simian parvoviras, chipmunk parvoviras, Adeno-Associated Virus 2, Adeno-Associated Virus 3B, Adeno- Associated Virus 4, Adeno-Associated Virus 5, Adeno-Associated Virus 6, LuIII parvovirus, HI parvovirus, or human B19 parvovirus.

6. The polypeptide according to Claim 5 wherein the parvoviras is human B19, porcine parvoviras, Galleria mellonella densoviras, Casphalia extranea densovirus, Bombyx mori densovirus or Adeno-Associated Virus.

7. An isolated viral polypeptide comprising an amino acid sequence as set out in SEQ ID NO: 55; SEQ ID NO: 57 or SEQ ID NO: 59.

8. An isolated viral polypeptide that has phospholipase A₂ activity and comprising an amino acid sequence as set out in SEQ ID NO:49; SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:57 or SEQ ID NO:59.

9. The polypeptide according to any one of Claims 1- 8 wherein said polypeptide is chemically synthesized.

10. The polypeptide according to any one of Claims 1-9 wherein said polypeptide fused to a heterologous polypeptide.

11. A polypeptide that is an inactive variant of a viral polypeptides wherein said viral polypeptide comprises an amino acid motif:

[WY]-[CIVL]-G-x-[GP]-[GKNS]-x(8,14)-D-x(2)-[AC]-x(2)-H-D-x(2)-Y wherein:

12. An isolated polynucleotide encoding a viral polypeptide that has phospholipase A₂ activity.

13. The isolated polynucleotide according to Claim 13 , wherein said polypeptide comprises an amino₍ acid motif:

[WY]-[CIVL]-G-x-[GP]-[GKNS]-x(8,14)-D-x(2)-[AC]-x(2)-H-D-x(2)-Y wherein:

14. The isolated polynucleotide according to Claim 14 wherein said amino acid motif is: Y-x-G-P-G-x(12)-D-x(2)-A-x(2)-H-D-x(2)-Y wherein: x(12) represents a stretch of 12 amino acids; and wherein one of the specified amino acids Y, G, P, G, D, A, D, or Y may be exchanged singly for any other amino acid.

15. The isolated polynucleotide according to any one of Claims 12 - 14, wherein said polynucleotide is DNA, cDNA, RNA or genomic DNA.

16. The isolated polynucleotide according to anyone of Claims 12 - 15, wherein said polynucleotide is double stranded or single stranded.

17. The isolated polynucleotide according to any one of Claims 12 - 16, wherein said polynucleotide is fused to a heterologous polynucleotide.

18. The isolated polypeptide according to Claim 17 wherein said heterologous polynucleotide encodes a heterologous polypeptide.

19. An isolated polynucleotide comprising a nucleic acid sequence as set out in SEQ ID NO:56; SEQ ID NO:58 or SEQ ID NO:60.

20. An isolated polynucleotide encoding a viral polypeptide that has phospholipase A₂ activity and comprising a nucleic acid sequence as set out in SEQ ID NO:50; SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58 or SEQ ID NO:60.

21. An antisense oUgonucleotide complementary to the polynucleotide according to any one of Claims 12 - 16, 19 or 20.

22. Use of the antisense oUgonucleotide according to Claim 21 to inhibit viral replication and / or infectivity in a subject.

23. A vector comprising the isolated polynucleotide according to any one of Claims 12 - 20.

24. The vector according to Claim 23 wherein said polynucleotide is operatively linked to one or more regulatory sequences.

25. A method of producing a host cell comprising genetically engineering cells with the vector according to Claim 23 or 24.

26. The host cell produced according to Claim 25.

27. A host cell comprising the vector according to Claim 23 or 24.

28. A host cell comprising the isolated polynucleotide according to any one of Claims 12 - 20.

29. A method of producing a recombinant virus comprising genetically engineering viral particles with the polynucleotide, or fragment thereof, according to Claims 12 - 16, 19 or 20, or with the vector according to Claim 23 or 24.

30. The recombinant virus produced according to Claim 29.

31. An antibody that specifically binds to the polypeptide of any one of Claims 1 - 9.

32. The antibody according to Claim 17 or 18, wherein said antibody is polyclonal.

33. The antibody according to Claim 17 or 18, wherein said antibody is monoclonal.

34. Use of a polypeptide according to any one of Claims 1- 9 as an in vitro standard for measuring phospholipase A₂ activity.

35. A method of screening compounds in order to identify an agent that inhibits viral phospholipase A₂ activity, comprising the steps of: i) incubating a viral phospholipase A₂ and a phospholipid substrate with a candidate agent under conditions whereby, but for the presence of the agent said viral phospholipase would hydrolyse said phospholipid, and ii) measuring the inhibition of viral phospholipase A₂ activity.

36. An anti- viral agent that selectively inhibits the activity of a viral phospholipase isolated according to the method of Claim 35.

37. The anti-viral agent according to Claim 36, wherein said anti-viral agent is capable of producing a detectable decrease in infectivity and / or replication of a viras.

38. The anti- viral agent according to Claim 36 or 37, wherein said anti-viral agent is an antisense oUgonucleotide that binds specifically to mRNA encoding said viral phospholipase.

39. The anti- viral agent according to Claim 38 wherein said antisense oUgonucleotide is about 8 to about 100 nucleobases.

40. The anti- viral agent according to Claim 39 wherein said antisense oUgonucleotide is about 8 to about 50 nucleobases.

41. The anti-viral agent according to Claim 40 wherein said antisense oUgonucleotide is about 15 to about 30 nucleobases.

42. The anti- viral agent according to Claim 36 or 37, wherein said anti-viral agent is an antibody wherein said antibody is monoclonal or polyclonal.

43. A method of identifying an anti-viral agent that selectively inhibits the activity of a viral phospholipase A₂, comprising: i) selecting a candidate molecule on the basis of the capacity of the candidate molecule to inhibit viral phospholipase A₂ in an in vitro assay employing a predetermined amount of a standardized preparation of viral phospholipase A₂; ii) treating a cultured host cell population with the candidate molecule; iii) exposing the host cell population to a viras; iv) determining the reduction in the infectivity and / or replication of the virus; v) determining the growth of the host cell population; and vi) comparing the growth to the growth of an untreated cultured host cell population that has been exposed to a viras; wherein a reduction in the infectivity and / or replication of the virus and increased growth of the host cell population in comparison to the untreated host cell population is indicative of selective inhibition of the viral phospholipase A₂.

44. An anti- viral agent that selectively inhibits the activity of a viral phospholipase isolated according to the method of Claim 43.

45. The anti- viral agent according to Claim 44, wherein said anti-viral agent is capable of producing a detectable decrease in infectivity and / or replication of a virus in vivo.

46. The anti-viral agent according to Claim 44 or 45, wherein said anti-viral agent is an antisense oUgonucleotide.

47. The anti-viral agent according to Claim 44 or 45, wherein said anti-viral agent is a monoclonal antibody.

48. Use of the anti- viral agent according to any one of Claims 36 - 42 or 44 - 47 to decrease the infectivity and / or replication of a viras in a subject.

49. The use according to Claim 48 wherein said virus is a parvovirus.

50. Use of the anti-viral agent according to any one of Claims 36 - 42 or 44 - 47 to inhibit a viral phospholipase A₂ in a subject in need of such therapy.

51. The use according to Claim 50 wherein said subject has a viral-associated disease or disorder.

52. The use according to Claim 51 wherein said viral-associated disease is associated with a parvovirus.

53. The use according to Claim 52 wherein said viral-associated disease is rheumatoid arthritis, systemic erythematosus, adult-onset Still's disease or polyarthritis.

54. Use of the anti- viral agent according to any one of Claims 36 - 42 or 44 - 47 to manufacture a medicament to decrease the infectivity and / or replication of a virus in a subject.

55. Use of the anti- viral agent according to any one of Claims 36 - 42 or 44 - 47 to manufacture a medicament to treat a viral-associated disease.

56. Use of the antisense oUgonucleotide according to any one of Claims 38 - 41 to manufacture a medicament to decrease the infectivity and / or replication of a virus in a subject.

57. Use of the antisense oUgonucleotide according to any one of Claims 38 - 41 to manufacture a medicament to treat a viral-associated disease.

58. Use of the isolated polynucleotide according to any one of Claims 12 - 20 for gene therapy.

59. Use of the isolated polynucleotide according to any one of Claims 12 - 20 to improve a gene therapy vector.

60. The use according to Claim 59 wherein said gene therapy vector is based on an Adeno-Associated viras.

61. A peptide that specifically binds a viral phospholipase A₂.

62. The peptide according to Claim 61 wherein said peptide inhibits the viral phospholipase A₂.