METHODS OF IDENTIFYING VIRAL THERAPEUTICS

Abstract: A method for identifying a substance for affecting a viral infection is described. The method comprises: a) providing a lipid globule targeting sequence, as a first component; b) providing a lipid globule, as a second component; c) contacting the two components with a substance to be tested under conditions that would permit the two components to interact in the absence of the substance; and d) determining whether the substance disrupts the interaction between the first and second components; wherein the targeting sequence comprises a hepatitis C virus (HCV) core protein or a fragment, derivative, variant or homologue thereof.

Description: METHODS OF IDENTIFYING VIRAL THERAPEUTICS Field of the invention This invention relates to substances capable of modulating the interaction between viral proteins capable of binding to intracellular lipid globules, cellular adipocyte-specific differentiation-related protein and intracellular lipid globules. The invention also relates to assays for identifying such substances and the use of these substances in affecting viral infection. Background to the invention Hepatitis C virus (HCN) is a major causative agent of chronic hepatitis and liver disease. It is estimated that, worldwide, approximately 300 million individuals are infected with the virus, 20% of whom are likely to develop mild to severe liver disease or carcinoma. Apart from the risk of succumbing to the long term effects of infection, these individuals also represent a large reservoir of virus for future transmissions. To date, the only widely used therapy for HCV is treatment with interferon. However, sustained response is achieved in only about 20% of cases. Moreover, no vaccine currently exists to protect against infection. Since growth of the virus has not been possible to date in tissue culture systems, very little is known also about the molecular events which occur during viral replication. The core protein of HCV is predicted to constitute the capsid of virus particles. From various studies, expression of this protein results in a range of effects on intracellular processes, including a decrease in transcription of genes from HBN and HIV and alterations to apoptosis. There is also evidence from a study on transgenic mice that liver- specific expression of core may be linked to the development of steatosis (fatty liver), a condition commonly found in HCV-infected individuals which is characterised by the accumulation of fat deposits within hepatocytes. Thus, core protein may also influence lipid metabolism within the liver. Other results from studies on human sera suggest that HCV virus particles are found associated with lipoprotein particles which are produced by the liver. It has also been shown that HCV core protein associates with lipid droplets within cells (Barba et al, 1997; Moradpour et al, 1996). The droplets are storage compartments for both triacylglycerols and cholesterol esters which can be used as substrates for oxidation in mitochondria and for the formation of membranes. In specialised cells, stored cholesterol is used for steroid hormone synthesis. Within the liver, lipid droplets also function as a site for storage of precursors of the lipid which is secreted from this organ in the form of lipoprotein particles. Although lipid droplets were identified several decades ago and they can be readily detected by staining methods, very little is known about the processes of assembly, storage and disassembly within the cell. Summary of the Invention We have surprisingly shown that expression of HCV core protein and its resultant association with intracellular lipid droplets results in the loss of a protein, termed adipocyte-specific differentiation-related protein (ADRP), from the droplets. ADRP has been found to associate with lipid droplets in a range of cell types and in certain organs. It has been proposed that ADRP may be required for maintenance of lipid droplets within cells, however the precise function of the protein has not been identified. Furthermore, progressive increases in core expression result in diminishing amounts of ADRP to undetectable levels. We have also identified regions of the HCV core protein which are required for both lipid globule association and down-regulation of ADRP levels. Without wishing to be bound by theory, we believe that displacement of ADRP by HCV core protein may be a factor involved in HCV infection. Thus, the interaction of HCV core with intracellular lipid globules and associated displacement of ADRP represents a target for therapies designed to prevent or reduce the effects and/or progression of HCV infection. Accordingly the present invention provides a method for identifying a substance for affecting a viral infection, which method comprises: (a) providing a lipid globule targeting sequence, as a first component; (b) providing a lipid globule, as a second component; (c) contacting the two components with a substance to be tested under conditions that would permit the two components to interact in the absence of the substance; and (d) determining whether the substance disrupts the interaction between the first and second components; wherein the targeting sequence comprises a hepatitis C virus (HCV) core protein or a fragment, derivative, variant or homologue thereof. Disruption by the candidate substance of the interaction between the first and second component is typically indicative that the substance is capable of affecting viral infection. In a preferred embodiment, the substance to be tested is administered to a cell, the lipid globule targeting sequence is expressed in said cell and the lipid globule is a natural constituent of said cell. The method of the invention may further comprise: (e) administering a virus to a cell in the absence of a said substance which has been determined to disrupt the interaction between the first and second components; (f) administering the virus to the cell in the presence of the said substance; and (g) determining if the said substance reduces or abolishes the susceptibility of the cell to viral infection or the effects of viral infection. The present invention also provides a method for identifying a substance capable of affecting a viral infection, which method comprises determining whether said substance can modulate, for example, up-regulate expression of ADRP in a cell. The present invention also provides the use of a substance in the manufacture of a medicament for use in affecting a viral infection wherein said substance can upregulate expression of ADRP in a mammalian cell. Preferably, in the methods of the invention, the viral infection is a hepatitis infection or other viral infection of the human or animal liver. More preferably the viral infection is an HCV infection. Where cells are used in the methods of the invention, the cells are preferably mammalian cells, more preferably primate cells, for example human cells. Cells which are targeted by the virus of interest are especially preferred for use in the assay methods of the invention. In particular, liver cells are especially preferred. Substances identified by the methods of the invention may be used in methods of affecting viral infection, such as treating or preventing viral infection. Accordingly the present invention provides a substance identified by the method of any one of the preceding claims. Preferably the substance of the invention has not previously been known to affect viral infection. The present invention also provides a process comprising identifying a substance capable of affecting viral infection by the method of the present invention and then either preparing a pharmaceutical composition comprising the substance together with a pharmaceutically acceptable carrier or diluent or modifying the substance to alter its antiviral properties and then, optionally comprising a suitably modifed substance substance together with a pharmaceutically acceptable carrier or diluent. The present invention also provides a substance capable of disrupting an interaction between (i) a lipid globule targeting sequence and (ii) a lipid globule for use in affecting a viral infection, wherein the targeting sequence comprises a hepatitis C virus (HCV) core protein or a fragment, derivative, variant or homologue thereof. Since we show that HCV core protein and fragments thereof can bind to intracellular lipid globules, and also affect the interaction between ADRP and intracellular lipid globules, a protein comprising HCV core protein or fragments thereof may be used to reduce or prevent the effect of HCV core protein produced during the course of HCV infection. Accordingly, the present invention provides a protein comprising a lipid globule targeting sequence for use in preventing or treating a viral infection wherein the targeting sequence comprises an HCV core protein or a fragment, derivative, variant or homologue thereof. Preferably, as with other references to lipid globule targeting sequences above, the targeting sequence comprises amino acids from 125 to 144 and/or 161 to 166 of the HCV core protein as set out in SEQ ID. Nos. 2 and 3, or the equivalent amino acids in other HCV strains/isolates. More preferably, the lipid globule targeting sequence also comprises a hydrophilic amino acid sequence of at least 8 amino acids The present invention further provides a polynucleotide encoding a protein comprising a lipid globule targeting sequence for use in affecting a viral infection. Thus the present invention relates to substances capable of modulating the interaction of viral proteins, for example HCV core protein, with intracellular lipid globules, in particular where the viral proteins function by reducing the amount of ADRP protein present in a virally infected cell and associated with intracellular lipid globules/vesicles. Detailed Description of the Invention Although in general the techniques mentioned herein are well known in the art, reference may be made in particular to Sambrook et al., Molecular Cloning, A Laboratory Manual (1989) and Ausubel et al, Current Protocols in Molecular Biology (1995), John Wiley & Sons, Inc. A. Proteins/Polypeptides The term "protein" includes single-chain polypeptide molecules as well as multiple- polypeptide complexes where individual consituent polypeptides are linked by covalent or non-covalent means. The term "polypeptide" includes peptides of two or more amino acids in length, typically having more than 5, 10 or 20 amino acids. Lipid globule targeting sequences The term "lipid globule targeting sequence" means an amino acid sequence which is capable of association with a lipid globule, preferably a biologically occurring lipid globule such as an intracellular lipid globule as found in adipocytes or a secreted lipid globule as found in mammalian milk. In addition, the lipid globule targeting sequence is preferably capable of association with a lipid globule when linked to a protein of interest such that the protein of interest is also associated with the lipid globule by virtue of being linked to the targeting sequence. Lipid globule association may take place within a non-cellular and/or extracellular environment, such as in an apparatus - for example a tube or vat. Alternatively, it may take place in a cellular environment where the expressed targeting sequence is directed to intracellular lipid droplets or the membranes of such droplets. The ability of an amino acid sequence to associate with/target lipid globules can be assessed either in vitro or in vivo. For example, a candidate targeting sequence may be added to a dispersion of lipid globules (such as a mixture of phospholipid and triacylglycerol) in an aqueous solvent, the mixture sonicated and the degree of partition between aqueous and lipid phases determined by fractionation. Typically fractionation of the mixture would involve increasing the density of the solution with sorbitol or sodium bromide and ultracentrifuging the solution. The lipid complexes migrate to the top of the centrifuge tube and this upper lipid layer is then examined for candidate targeting sequence. Preferably, a suitable lipid globule targeting sequence should partition at least 50:50 lipid:aqueous phase, more preferably at least 75:25, 80:20 or 90:10. Another suitable test may involve introducing a polynucleotide encoding a candidate sequence, optionally linked to a protein of interest, into a milk-producing cell in culture and determining whether, the targeting sequence/protein of interest has been secreted into the culture medium. The immunocytochemical technique illustrated in the Examples may also be used. Lipid globule targeting sequences used according to the present invention are also preferably capable of displacing ADRP from intracellular lipid globules and/or of reducing the levels of ADRP protein in a cell when expressed in, or administered to, the cell (as illustrated for HCV core protein in the Examples). Suitable techniques for determining whether a candidate sequence has these properties are described below. Suitable lipid globule targeting sequences may be obtained from an HCV core protein. The amino acid sequence of the HCV core protein has been obtained for a large number of different HCV isolates. These sequences are readily available to the skilled person. One such sequence, for HCV strain Glasgow, is set out in SEQ ID No. 1. The means for cloning and identifying new HCV strains, and thus obtaining further core sequences, are described in EP-B-318,216. According to the present invention, it is preferred to use fragments of the HCV core protein which are capable of targeting molecules, to which they are linked, to lipid globules. Amino acid numbering for preferred fragments set out below is with reference to SEQ ID. No. 1. However it will be understood that equivalent fragments of the core protein of other HCV strains/isolates may also be used. An HCV core protein-derivable lipid globule targeting sequence of the invention is preferably a minimal amino acid sequence which can target a molecule, typically a protein, to lipid globules. The minimal sequence will typically comprise a hydrophobic amino acid sequence derived from amino acids 120 to 169 of an HCV core sequence, preferably linked to a hydrophilic amino acid sequence of at least 8, preferably 10, more preferably at least 12 amino acids. It is not necessary for the hydrophilic sequence to be contiguous with the hydrophobic sequence. For example, a protein of interest may be placed between the two sequences such that the hydrophilic sequence is at the N-terminus and the hydrophobic sequence is at the C-terminus. The hydrophobic amino acid sequence typically comprises at least 10, preferably at least 15 or 20 contiguous amino acids and has a hydropathy index of at least +40 kJ/mol (determined, for example, theoretically as described by Engelman et al, 1986. The hydrophilic amino acid sequence typically has a hydropathy plot of less than -20 kJ/mol, preferably less than -40 kJ/mol. Preferred HCV core fragments contain amino acids 161 to 166 (SEQ ID. No. 3). It is also preferred to use fragments of the HCV core protein that contain amino acids 125 to 144 (SEQ ID. No. 2). In a preferred embodiment, HCV core protein fragments of the invention contain both amino acids 125 to 144 and amino acids 161 to 166. In a more general sense, a preferred HCV core fragment comprises substantially all of domain 2 of the HCV core polypeptide (approximately amino acids 123 to 174) although variants which lack residues 155 to 161 and/or are truncated at residue 166, 167, 168 or 169 are also within the scope of the present invention. Equivalent domain 2 regions and variants thereof (for example comprising deletions based on equivalent amino acid numbering) in other flaviviruses or pestiviruses are also within the scope of the invention. In an especially preferred embodiment, the lipid targeting sequence of the invention also comprises a hydrophilic amino acid sequence containing amino acids 1 to 8 of the HCV core sequence. Other preferred fragments contain amino acids 1 to 173 or 1 to 169. Since it has also now been shown that amino acids 9 to 43, 49 to 75, 80 to 118 and 155 to 161 are not required for lipid association, preferred HCV core protein fragments of the invention lack one or more of these sequences. Suitable fragments will be at least about 5, e.g. 10, 12, 15 or 20 amino acids in size and preferably have less than 100, 90, 80, 70, 60 or 50 amino acids. In a preferred aspect, fragments contain an HCV epitope. Lipid globule targeting sequences of the invention, for example HCV core protein sequences and fragments thereof, may, however, be part of a larger polypeptide, for example a fusion protein. In this case, the additional polypeptide sequences are preferably polypeptide sequences with which the lipid globule targeting sequence of the invention is not normally associated. It will be understood that lipid globule targeting sequences of the invention are not limited to sequences obtained from HCV core protein but also include homologous sequences obtained from any source, for example related viral proteins, cellular homologues and synthetic peptides, as well as variants or derivatives thereof. Thus, the present invention covers variants, homologues or derivatives of the targeting sequences of the present invention, as well as variants, homologues or derivatives of the nucleotide sequence coding for the targeting sequences of the present invention. In the context of the present invention, a homologous sequence is taken to include an amino acid sequence which is at least 60, 70, 80 or 90% identical, preferably at least 95 or 98% identical at the amino acid level over at least 5, preferably 8, 10, 15, 20, 30 or 40 amino acids with an HCV core protein lipid targeting sequence, for example as shown in the sequence listing herein. In particular, homology should typically be considered with respect to those regions of the targeting sequence known to be essential for lipid globule association rather than non-essential neighbouring sequences. Although homology can also be considered in terms of similarity (i.e. amino acid residues having similar chemical properties/functions), in the context of the present invention it is preferred to express homology in terms of sequence identity. Homology comparisons can be conducted by eye, or more usually, with the aid of readily available sequence comparison programs. These commercially available computer programs can calculate % homology between two or more sequences. % homology may be calculated over contiguous sequences, i.e. one sequence is aligned with the other sequence and each amino acid in one sequence directly compared with the corresponding amino acid in the other sequence, one residue at a time. This is called an "ungapped" alignment. Typically, such ungapped alignments are performed only over a relatively short number of residues (for example less than 50 contiguous amino acids). Although this is a very simple and consistent method, it fails to take into consideration that, for example, in an otherwise identical pair of sequences, one insertion or deletion will cause the following amino acid residues to be put out of alignment, thus potentially resulting in a large reduction in % homology when a global alignment is performed. Consequently, most sequence comparison methods are designed to produce optimal alignments that take into consideration possible insertions and deletions without penalising unduly the overall homology score. This is achieved by inserting "gaps" in the sequence alignment to try to maximise local homology. However, these more complex methods assign "gap penalties" to each gap that occurs in the alignment so that, for the same number of identical amino acids, a sequence alignment with as few gaps as possible - reflecting higher relatedness between the two compared sequences - will achieve a higher score than one with many gaps. "Affine gap costs" are typically used that charge a relatively high cost for the existence of a gap and a smaller penalty for each subsequent residue in the gap. This is the most commonly used gap scoring system. High gap penalties will of course produce optimised alignments with fewer gaps. Most alignment programs allow the gap penalties to be modified. However, it is preferred to use the default values when using such software for sequence comparisons. For example when using the GCG Wisconsin Bestfit package (see below) the default gap penalty for amino acid sequences is -12 for a gap and -4 for each extension. Calculation of maximum % homology therefore firstly requires the production of an optimal alignment, taking into consideration gap penalties. A suitable computer program for carrying out such an alignment is the GCG Wisconsin Bestfit package (University of Wisconsin, U.S.A.; Devereux et al, 1984, Nucleic Acids Research 12:387). Examples of other software than can perform sequence comparisons include, but are not limited to, the BLAST package (see Ausubel et al, 1999 ibid- Chapter 18), FASTA (Atschul et al, 1990, J. Mol. Biol, 403-410) and the GENEWORKS suite of comparison tools. Both BLAST and FASTA are available for offline and online searching (see Ausubel et al, 1999 ibid, pages 7-58 to 7-60). However it is preferred to use the GCG Bestfit program. Although the final % homology can be measured in terms of identity, the alignment process itself is typically not based on an all-or-nothing pair comparison. Instead, a scaled similarity score matrix is generally used that assigns scores to each pairwise comparison based on chemical similarity or evolutionary distance. An example of such a matrix commonly used is the BLOSUM62 matrix - the default matrix for the BLAST suite of programs. GCG Wisconsin programs generally use either the public default values or a custom symbol comparison table if supplied (see user manual for further details). It is preferred to use the public default values for the GCG package, or in the case of other software, the default matrix, such as BLOSUM62. Once the software has produced an optimal alignment, it is possible to calculate % homology, preferably % sequence identity. The software typically does this as part of the sequence comparison and generates a numerical result. Homologous sequences may be obtained using standard cloning techniques or by database searches, for example using the Blast suite of programs and any one of the SEQ ID. Nos. Listing herein as the query. Lipid globule targeting sequences of the invention, for example HCV core protein sequences, variants, homologues and fragments thereof, may be modified for use in the present invention. Typically, modifications are made that maintain the hydrophobicity/ hydrophilicity of the sequence Amino acid substitutions may be made, for example from 1, 2 or 3 to 10, 20 or 30 substitutions provided that the modified sequence retains the ability to target molecules to lipid globules. Amino acid substitutions may include the use of non- naturally occurring analogues, for example to increase blood plasma half-life of a therapeutically administered polypeptide. Conservative substitutions may be made, for example according to the Table below. Amino acids in the same block in the second column and preferably in the same line in the third column may be substituted for each other: The terms "variant", "homologue" or "derivative" in relation to the targeting sequence of the present invention include any substitution of, variation of, modification of, replacement of, deletion of or addition of one (or more) amino acids from or to the sequence providing the resultant amino acid sequence has a lipid globule targeting activity, preferably having at least the same activity of the targeting sequences presented in the sequence listings. Proteins of the invention comprising lipid globule targeting sequences are typically made by recombinant means, for example as described below. However they may also be made by synthetic means using techniques well known to skilled persons such as solid phase synthesis. Proteins of the invention may also be produced as fusion proteins, for example to aid in extraction and purification. Examples of fusion protein partners include glutathione-S-transferase (GST), 6xHis, GAL4 (DNA binding and/or transcriptional activation domains) and β-galactosidase. It may also be convenient to include a proteolytic cleavage site between the fusion protein partner and the HCV core protein sequence and/or between the HCV core protein sequence and the protein of interest to allow removal of fusion protein sequences. Preferably the fusion protein will not hinder the lipid targeting effect of the lipid globule targeting sequence. The targeting sequence may be linked to either the N-terminus or the C-terminus of the fusion protein partners or proteins of interest Proteins of the invention may be in a substantially isolated form. It will be understood that the protein may be mixed with carriers or diluents which will not interfere with the intended purpose of the protein and still be regarded as substantially isolated. A protein of the invention may also be in a substantially purified form, in which case it will generally comprise the protein in a preparation in which more than 90%, e.g. 95%, 98% or 99% of the protein in the preparation is a protein of the invention. Adipocyte-specific differentiation related protein In another embodiment of the in vitro assay methods of the present invention, ADRP may be added to the mixture to compete with a lipid globule targeting sequence. ADRP protein (also known as adipophilin) can be obtained in a number of ways, for example by purification from mammalian milk or mammalian cell lines (Heid et al, 1998). Alternatively, ADRP can be obtained by recombinant means, as described above for lipid globule targeting sequences. The nucleotide sequence of human ADRP, for example, is given in US Patent No. 5,739,009 and shown in SEQ ID. No. 4. Since ADRP is capable of binding lipid globules, it can also be considered to be a lipid globule targeting sequence within the scope of the invention. Consequently, the disclosure above and below relating to lipid globule targeting sequences also generally applies, where appropriate, to ADRP sequences, for example the use of fragments, homologues, derivatives and variants. In particular, ADRP sequences may be modified for use in the assays of the present invention as described above. B. Polynucleotides and vectors. Polynucleotides of the invention comprise nucleic acid sequences encoding the lipid globule targeting sequences of the invention. It will be understood by a skilled person that numerous different polynucleotides can encode the same polypeptide as a result of the degeneracy of the genetic code. In addition, it is to be understood that skilled persons may, using routine techniques, make nucleotide substitutions that do not affect the polypeptide sequence encoded by the polynucleotides of the invention to reflect the codon usage of any particular host organism in which the polypeptides of the invention are to be expressed. Polynucleotides of the invention may comprise DNA or R A. They may be single- stranded or double-stranded. They may also be polynucleotides which include within them synthetic or modified nucleotides. A number of different types of modification to ohgonucleotides are known in the art. These include methylphosphonate and phosphorothioate backbones, addition of acridine or polylysine chains at the 3' and/or 5' ends of the molecule. For the purposes of the present invention, it is to be understood that the polynucleotides described herein may be modified by any method available in the art. Such modifications may be carried out in order to enhance the in vivo activity or life span of polynucleotides of the invention. The terms "variant", "homologue" or "derivative" in relation to the nucleotide sequence coding for the lipid targeting sequence of the present invention include any substitution of, variation of, modification of, replacement of, deletion of or addition of one (or more) nucleic acid from or to the sequence providing the resultant nucleotide sequence codes for a protein having lipid targeting activity, preferably having at least the same activity of the targeting sequences presented in the sequence listings. As indicated above, with respect to sequence homology, preferably there is at least 75%, more preferably at least 85%, more preferably at least 90% homology to the sequences shown in the sequence listing herein. More preferably there is at least 95%, more preferably at least 98%), homology. Nucleotide homology comparisons may be conducted as described above. The present invention also encompasses nucleotide sequences that are capable of hybridising selectively to the sequences presented herein, or any variant, fragment or derivative thereof, ' or to the complement of any of the above. Nucleotide sequences are preferably at least 15 nucleotides in length, more preferably at least 20, 30, 40 or 50 nucleotides in length. The term "hybridization" as used herein shall include "the process by which a strand of nucleic acid joins with a complementary strand through base pairing" (Coombs J (1994) Dictionary of Biotechnology, Stockton Press, New York NY) as well as the process of amplification as carried out in polymerase chain reaction technologies as described in Dieffenbach CW and GS Dveksler (1995, PCR Primer, a Laboratory Manual, Cold Spring Harbor Press, Plainview NY). Polynucleotides of the invention capable of selectively hybridising to the nucleotide sequences presented herein, or to their complement, will be generally at least 70%, preferably at least 80 or 90% and more preferably at least 95% or 98% homologous to the corresponding nucleotide sequences presented herein over a region of at least 20, preferably at least 25 or 30, for instance at least 40, 60 or 100 or more contiguous nucleotides. Preferred polynucleotides of the invention will comprise regions homologous to nucleotides 715 to 774 and/or nucleotides 826 to 840 of SEQ ID No. 1, preferably at least 80 or 90%) and more preferably at least 95% homologous to nucleotides 715 to 774 and/or nucleotides 826 to 840 of SEQ ID No. 1. The term "selectively hybridizable" means that the polynucleotide used as a probe is used under conditions where a target polynucleotide of the invention is found to hybridize to the probe at a level significantly above background. The background hybridization may occur because of other polynucleotides present, for example, in the cDNA or genomic DNA library being screening. In this event, background implies a level of signal generated by interaction between the probe and a non-specific DNA member of the library which is less than 10 fold, preferably less than 100 fold as intense as the specific interaction observed with the target DNA. The intensity of interaction may be measured, for example, by radiolabelhng the probe, e.g. with P. Hybridization conditions are based on the melting temperature (Tm) of the nucleic acid binding complex, as taught in Berger and Kirnmel (1987, Guide to Molecular Cloning Techniques, Methods in Enzymology, Vol 152, Academic Press, San Diego CA), and confer a defined "stringency" as explained below. Maximum stringency typically occurs at about Tm-5°C (5°C below the Tm of the probe); high stringency at about 5°C to 10°C below Tm; intermediate stringency at about 10°C to 20°C below Tm; and low stringency at about 20°C to 25°C below Tm. As will be understood by those of skill in the art, a maximum stringency hybridization can be used to identify or detect identical polynucleotide sequences while an intermediate (or low) stringency hybridization can be used to identify or detect similar or related polynucleotide sequences. In a preferred aspect, the present invention covers nucleotide sequences that can hybridise to the nucleotide sequence of the present invention under stringent conditions (e.g. 65°C and O.lxSSC { lxSSC = 0.15 M NaCl, 0.015 M Na 3 citrate pH 7.0). Where the polynucleotide of the invention is double-stranded, both strands of the duplex, either individually or in combination, are encompassed by the present invention. Where the polynucleotide is single-stranded, it is to be understood that the complementary sequence of that polynucleotide is also included within the scope of the present invention. Polynucleotides which are not 100%) homologous to the sequences of the present invention but fall within the scope of the invention can be obtained in a number of ways. Other HCV core protein variants of the HCV core protein sequence described herein may be obtained for example by probing DNA libraries made from a range of HCV infected individuals, for example individuals from different populations. In addition, other viral, or cellular homologues particularly cellular homologues found in mammalian cells (e.g. rat, mouse, bovine and primate cells), may be obtained and such homologues and fragments thereof in general will be capable of selectively hybridising to the sequences shown in the sequence listing herein. Such sequences may be obtained by probing cDNA libraries made from or genomic DNA libraries from other animal species, and probing such libraries with probes comprising all or part of SEQ ID. No. 1 under conditions of medium to high stringency. Similar considerations apply to obtaining species homologues and allelic variants of ADRP. Variants and strain/species homologues may also be obtained using degenerate PCR which will use primers designed to target sequences within the variants and homologues encoding conserved amino acid sequences within the lipid globule targeting sequences of the present invention. Conserved sequences can be predicted, for example, by aligning the HCV core protein amino acid sequences from several HCV isolates. Such HCV sequence comparisons are widely available in the art. The primers will contain one or more degenerate positions and will be used at stringency conditions lower than those used for cloning sequences with single sequence primers against known sequences. Alternatively, such polynucleotides may be obtained by site directed mutagenesis of characterised lipid globule targeting sequences, such as SEQ ID. No. 1. This may be useful where for example silent codon changes are required to sequences to optimise codon preferences for a particular host cell in which the polynucleotide sequences are being expressed. Other sequence changes may be desired in order to introduce restriction enzyme recognition sites, or to alter the property or function of the polypeptides encoded by the polynucleotides. Polynucleotides of the invention may be used to produce a primer, e.g. a PCR primer, a primer for an alternative amplification reaction, a probe e.g. labelled with a revealing label by conventional means using radioactive or non-radioactive labels, or the polynucleotides may be cloned into vectors. Such primers, probes and other fragments will be at least 15, preferably at least 20, for example at least 25, 30 or 40 nucleotides in length, and are also encompassed by the term polynucleotides of the invention as used herein. Polynucleotides such as a DNA polynucleotides and probes according to the invention may be produced recombinantly, synthetically, or by any means available to those of skill in the art. They may also be cloned by standard techniques. In general, primers will be produced by synthetic means, involving a step wise manufacture of the desired nucleic acid sequence one nucleotide at a time. Techniques for accomplishing this using automated techniques are readily available in the art. Longer polynucleotides will generally be produced using recombinant means, for example using a PCR (polymerase chain reaction) cloning techniques. This will involve making a pair of primers (e.g. of about 15 to 30 nucleotides) flanking a region of the lipid targeting sequence which it is desired to clone, bringing the primers into contact with mRNA or cDNA obtained from an animal or human cell, performing a polymerase chain reaction under conditions which bring about amplification of the desired region, isolating the amplified fragment (e.g. by purifying the reaction mixture on an agarose gel) and recovering the amplified DNA. The primers may be designed to contain suitable restriction enzyme recognition sites so that the amplified DNA can be cloned into a suitable cloning vector Polynucleotides of the invention can be incorporated into a recombinant replicable vector. The vector may be used to replicate the nucleic acid in a compatible host cell. Thus in a further embodiment, the invention provides a method of making polynucleotides of the invention by introducing a polynucleotide of the invention into a replicable vector, introducing the vector into a compatible host cell, and growing the host cell under conditions which bring about replication of the vector. The vector may be recovered from the host cell. Suitable host cells include bacteria such as E coli, yeast, mammalian cell lines and other eukaryotic cell lines, for example insect Sf9 cells. Preferably, a polynucleotide of the invention in a vector is operably linked to a control sequence that is capable of providing for the expression of the coding sequence by the host cell, i.e. the vector is an expression vector. The term "operably linked" means that the components described are in a relationship permitting them to function in their intended manner. A regulatory sequence "operably linked" to a coding sequence is ligated in such a way that expression of the coding sequence is achieved under condition compatible with the control sequences. A particularly preferred vector for use in the present invention comprises the control sequences naturally associated with ADRP genes, such as the human ADRP gene. Typically, the control sequences are operably linked to a reporter gene. Such a vector may be used in the assays described below for identifying modulators of ADRP expression. The control sequences may be modified, for example by the addition of further transcriptional regulatory elements to make the level of transcription directed by the control sequences more responsive to transcriptional modulators. Vectors of the invention may be transformed or transfected into a suitable host cell as described below to provide for expression of a protein of the invention. This process may comprise culturing a host cell transformed with an expression vector as described above under conditions to provide for expression by the vector of a coding sequence encoding the protein, and optionally recovering the expressed protein. Alternatively, vectors comprising the control sequences naturally associated with ADRP genes operably linked to a reporter gene as a reporter construct may be transformed or transfected into a host cell, for example for use in assays of the invention which measure the effect of candidate substances on transcription from the reporter construct in a cellular environment. The vectors may be for example, plasmid or virus vectors provided with an origin of replication, optionally a promoter for the expression of the said polynucleotide and optionally a regulator of the promoter. The vectors may contain one or more selectable marker genes, for example an ampicillin resistance gene in the case of a bacterial plasmid or a neomycin resistance gene for a mammalian vector. Vectors may be used, for example, to transfect or transform a host cell. Control sequences operably linked to sequences encoding the protein of the invention include promoters/enhancers and other expression regulation signals. These control sequences may be selected to be compatible with the host cell for which the expression vector is designed to be used in. The term promoter is well-known in the art and encompasses nucleic acid regions ranging in size and complexity from minimal promoters to promoters including upstream elements and enhancers. The promoter is typically selected from promoters which are functional in mammalian, cells, although prokaryotic promoters and promoters functional in other eukaryotic cells may be used. The promoter is typically derived from promoter sequences of viral or eukaryotic genes. For example, it may be a promoter derived from the genome of a cell in which expression is to occur. With respect to eukaryotic promoters, they may be promoters that function in a ubiquitous manner (such as promoters of D-actin, D-actin, tubulin) or, alternatively, a tissue-specific manner (such as promoters of the genes for pyruvate kinase). Tissue-specific promoters specific for liver cells are particularly preferred, for example hepatitis B viral promoters, apoliprotein All promoters human serum amyloid P component promoters or human protein C gene promoters. They may also be promoters that respond to specific stimuli, for example promoters that bind steroid hormone receptors. Viral promoters may also be used, for example the Moloney murine leukaemia virus long terminal repeat (MMLV LTR) promoter, the rous sarcoma virus (RSV) LTR promoter or the human cytomegalovirus (CMV) IE promoter. As discussed above ADRP promoters are particularly preferred for use in reporter constructs. It may also be advantageous for the promoters to be inducible so that the levels of expression of the heterologous gene can be regulated during the life-time of the cell. Inducible means that the levels of expression obtained using the promoter can be regulated. In addition, any of these promoters may be modified by the addition of further regulatory sequences, for example enhancer sequences. Chimeric promoters may also be used comprising sequence elements from two or more different promoters described above. Host cells Vectors and polynucleotides of the invention (and reporter gene constructs described below) may be introduced into host cells for the purpose of replicating the vectors/polynucleotides and/or expressing the proteins of the invention encoded by the polynucleotides of the invention. Although the proteins of the invention may be produced using prokaryotic cells as host cells, it is preferred to use eukaryotic cells, for example yeast, insect or mammalian cells, in particular mammalian cells. Particularly preferred cells are those with substantial amounts of intracellular lipid droplets/globules, for example adipocytes. Cells which are targeted by a particular virus which it is desired to treat, for example liver cells, are also preferred. Vectors/polynucleotides of the invention may introduced into suitable host cells using a variety of techniques known in the art, such as transfection, transformation and electroporation. Where vectors/polynucleotides of the invention are to be administered to animals, several techniques are known in the art, for example infection with recombinant viral vectors such as herpes simplex viruses and adeno viruses, direct injection of nucleic acids and biolistic transformation. D. Protein Expression and Purification Host cells comprising polynucleotides of the invention may be used to express proteins of the invention. Host cells may be cultured under suitable conditions which allow expression of the proteins of the invention. Expression of the proteins of the invention may be constitutive such that they are continually produced, or inducible, requiring a stimulus to initiate expression. In the case of inducible expression, protein production can be initiated when required by, for example, addition of an inducer substance to the culture medium, for example dexamethasone or IPTG. Proteins of the invention can be extracted from host cells by a variety of techniques known in the art, including enzymatic, chemical and/or osmotic lysis and physical disruption. Although a large number of different purification protocols may be used, given the ability of the HCV core proteins of the invention to target proteins of interest to lipid globules, a preferred extraction/purification protocol involves centrifuging cell homogenates at high speed (for example 100,000 g for 60 mins at 2 to 4°C) and removing the resulting layer of floating lipids. This will function as a primary purification step. Further purification can then be performed if necessary using, for example, column chromatography such as ion- exchange or affinity chromatography. Cells which secrete lipid globules may also conveniently be used and the lipid globules harvested from the culture supernatant. Proteins associated with the membrane surrounding fat globules can be fractionated into soluble and insoluble fractions by extraction with 1% (w/v) Triton X-100/1.5 M NaCl/ lO mM Tris (pH 7.0), by extraction with 1.5% (w/v) dodecyl β-D maltoside/ 0.75 M aminohexanoic acid/ 10 mM Hepes (pH 7.0) or by sequential extraction with these two detergent-containing solutions (Patton and Huston, 1986). Suspension of the fat globule components in the detergent-containing solution can be achieved by using an all-glass homogenizer, and keeping on ice for 30 to 60 min, after which insoluble and soluble materials can be separated by centrifugation for 60 min at 2°C and 150,000 g. The above conditions can be modified to analyse whether core protein or a fusion protein-containing core as a component is attached to fat globules. Other detergents, both ionic and non-ionic, along with salt solutions at various concentrations could be used to derive the proteinaceous material from fat globules. The incubation times and temperatures may be optimised by empirical means. E. Assays 1. Assays for substances that disrupt the interaction between a lipid globule targeting sequence and a lipid globule i. Candidate substances A substance which disrupts an interaction between the lipid globule targeting sequence and lipid globule may do so in several ways. It may directly disrupt the binding of the lipid globule targeting sequence to the lipid globule by, for example, binding to the lipid globule targeting sequence and masking or altering the site of interaction with the lipid globule. Alternatively, the candidate substance may compete for binding sites on the lipid globule surface and displace the lipid globule targeting sequence. Candidate substances of these types may conveniently be screened by in vitro binding assays as, for example, described below. Candidate substances may also be screened using an "in vivo" whole cell assay as described below. The term "in vivo' is intended to encompass experiments with cells in culture as well as experiments with intact multicellular organisms. A substance which can bind directly to the lipid globule targeting sequence may also inhibit an interaction between the lipid globule targeting sequence and the lipid globule by altering the subcellular localisation of the lipid globule targeting sequence thus preventing the two components from coming into contact within the cell. This can also be tested in vivo using, for example the in vivo assays described below. Alternatively, instead of preventing the association of the components directly, the substance may suppress or enhance the biologically available amount of lipid globule targeting sequence (for example a viral protein component). This may be by inhibiting expression of the viral protein comprising the lipid globule targeting sequence, for example at the level of transcription, transcript stability, translation, post-translational processing or post-translational stability. An example of such a substance would be antisense RNA which suppresses the amount of HCV core protein mRNA translated into protein. Suitable candidate substances include viral peptides, which in some cases may especially be of from about 5 to 20 amino acids in size, based on, for example, lipid globule targeting motifs found within the HCV core protein, or variants of such peptides in which one or more residues have been substituted. Peptide fragments of ADRP may also be used, including modified variants thereof. Peptides from panels of peptides comprising random sequences or sequences which have been varied consistently to provide a maximally diverse panel of peptides may be used. Suitable candidate substances also include antibody products (for example, monoclonal and polyclonal antibodies, single chain antibodies, chimeric antibodies and CDR-grafted antibodies) which are specific for the lipid globule targeting sequence or surface constituents of the lipid globules. Furthermore, combinatorial libraries, peptide and peptide mimetics, defined chemical entities such as organic and inorganic compounds, ohgonucleotides, and natural product libraries may be screened for activity as inhibitors of an interaction between the lipid globule targeting sequence and a lipid globule in assays such as those described below. The candidate substances may be used in an initial screen in batches of, for example 10 substances per reaction, and the substances of those batches which show inhibition tested individually. Candidate substances which show activity in in vitro screens such as those described below can then be tested in in vivo systems, such as mammalian cells which will be exposed to the inhibitor and tested, for example, for susceptibility to viral infection. ii. Assays The assays of the invention may be in vitro assays or in vivo assays, for example using cell lines or an animal model, such as mice or chimpanzees. ln vitro assay systems One type of in vitro assay for identifying substances which disrupt an interaction between the lipid globule targeting sequence and a lipid globule involves measuring in the presence or absence of a candidate substance the association of a targeting sequence with lipid globules formed in vitro. Binding of targeting sequences to lipid globules may be assessed by adding a targeting sequence to a dispersion of lipid globules (such as a mixture of phospholipid and triacylglycerol) in an aqueous solvent, sonicating the mixture and determining by fractionation the degree of partition between aqueous and lipid phases. Typically fractionation of the mixture would involve increasing the density of the solution with sorbitol or sodium bromide and ultracentrifuging the solution. The lipid complexes migrate to the top of the centrifuge tube and this upper lipid layer is then examined for targeting sequence. This is performed in the presence or absence of the candidate substance whose inhibitory activity it is desired to test. Typically, a candidate substance is deemed to inhibit the lipid globule targeting sequence if it causes a reduction of at least 50%, preferably at least 60, 70, 80 or 90%) in the amount of targeting sequence associated with the lipid phase in the absence of the candidate substance. The candidate substance may be pre-incubated with the lipid globule targeting sequence or with a lipid globule or added to the reaction mixture after pre-incubation of the lipid globule targeting sequence with a lipid globule. Another type of in vitro assay for identifying substances which disrupt an interaction between the lipid globule targeting sequence and a lipid globule or a lipophilic surface involves the following: Proteins incorporating the lipid globule targeting sequence, for example a fragment of HCV core protein, and optionally ADRP, may be translated in vitro from RNA transcripts encoding these polypeptides. Reactions would be supplemented with membranes originating from tissue culture cells (e.g. microsomal membranes) or a lipophilic surface which had been artificially created (e.g. a liposome) to which the in vitro translated proteins can bind. Binding may be assessed by purification of the lipid components of reactions (e.g. by centrifugation). Alternatively a chip format (for example BIAcore™ - Biacore AB) wherein the chip has a lipid surface may be used to assess binding. The ability of candidate substances to disrupt association of the targeting sequence and/or ADRP with lipid can be examined by adding to reactions candidate substances during synthesis of the proteins. Thus, the effects of substances on core/lipid globule targeting sequences and ADRP association with lipid can be compared. Preferably, a suitable inhibitor of a lipid globule targeting sequence is capable of inhibiting binding of viral lipid targeting sequences such as HCV core to lipid globules without affecting binding of ADRP. In vivo assay systems In vivo assays for identifying compounds that disrupt an interaction between the lipid globule targeting sequence and a lipid globule typically involve administering a candidate substance to a cell which expresses a lipid globule targeting sequence, for example a fragment of HCV core protein, and determining if the ability of the targeting sequence to associate with intracellular lipid globules has been affected, for example reduced or abolished. Thus the association of the targeting sequence with intracellular lipid globules is determined in the absence of the candidate substance and in the presence of the candidate substance and the results compared. Association of the lipid globule targeting sequence with intracellular globules is typically determined by immunofluorescence microscopy using an antibody which recognises the targeting sequence and an antibody or stain which recognises intracellular lipid globules or a surface component of the globules. If the targeting sequences is able to bind to the lipid globules, then the targeting sequence will substantially co-localise with the lipid globules. A suitable procedure is described in the Examples. In addition, since we show that HCV core protein can displace ADRP from lipid globules, the co-localisation of endogenous ADRP with lipid globules may also be determined in the presence and absence of the candidate substance. A candidate substance is generally considered to be capable of disrupting the interaction between the lipid globule targeting sequence and intracellular lipid globules if, as determined by immunofluorescence microscopy, less than 50%. of the detectable targeting sequence protein co-localises with intracellular lipid globules, preferably less than 60%, more preferably less than 70, 80 or 90%. Preferably, a suitable inhibitor of a lipid globule targeting sequence is capable of disrupting the interaction of viral lipid targeting sequences such as HCN core with intracellular lipid globules/surface components without affecting the interaction of ADRP with intracellular lipid globules/surface components. It will be appreciated by the skilled person that other techniques are available for determining localisation of proteins and lipid within intact cells and that these techniques are also applicable to the assays of the present invention. The candidate substance, i.e. the test compound, may be administered to the cell in one or more ways. For example, it may be added directly to the cell culture medium or injected into the cell. Alternatively, in the case of polypeptide candidate substances, the cell may be transfected with a nucleic acid construct which directs expression of the polypeptide in the cell. Preferably, the expression of the polypeptide is under the control of a regulatable promoter, for example so that expression is induced shortly before it is desired to administer the candidate substance. Another suitable test may involve introducing a polynucleotide encoding a targeting sequence of the invention, optionally linked to a protein of interest, into a milk-producing cell in culture and determining whether, the targeting sequence/protein of interest has been secreted into the culture medium. This would be carried out in the presence or absence of the candidate substance. 2. Assays for substances that are capable of modulating ADRP expression in cells i. Candidate substances Substances that modulate ADRP expression, preferably up-regulate ADRP expression so that the levels of ADRP protein in a cell are increased, may do so by one or more of several mechanisms. For example, a substance may increase levels of transcription from endogenous ADRP genes, stabilise ADRP mRNA levels and/or stabilise ADRP protein. Transcription may be increased from endogenous ADRP genes by, for example, the use of a transcriptional activator that activates transcription from endogenous ADRP genes or a substance that modifies the effect of a transcriptional inhibitor of endogenous ADRP genes. Thus tests for modulation of ADRP expression include determining the effect of a candidate substance on ADRP mRNA levels and/or ADRP protein levels. The term "modulate" in the context of the ADRP expression means a change or alteration in the levels of ADRP mRNA and/or ADRP protein. Suitable candidate substances include substances known to modulate cellular transcription and/or lipid metabolism. Examples of substances which may affect ADRP expression include non-steroidal anti-inflammatory drugs such as ibuprofen and indomethacin, and drugs known to affect lipid metabolism such as fibrates and thiazolidinediones. Furthermore, combinatorial libraries, peptide and peptide mimetics, in particular peptides from panels of peptides comprising random sequences or sequences which have been varied consistently to provide a maximally diverse panel of peptides, defined chemical entities such as organic and inorganic compounds, ohgonucleotides, and natural product libraries may be screened for activity as modulators of ADRP expression in assays such as those described below. The candidate substances may be used in an initial screen in batches of, for example 10 substances per reaction, and the substances of those batches which show inhibition tested individually. In addition, candidate substances which show activity in in vitro screens such as those described below can then be tested in in vivo systems, such as mammalian cells which will be exposed to the inhibitor and tested, for example, for susceptibility to viral infection. ii. Assays The assays of the invention may be in vitro assays or in vivo assays, for example using cell lines or an animal model. In vitro assay systems An in vitro assay system of the invention typically measures the effect on transcription from a polynucleotide construct comprising an ADRP promoter linked to a polynucleotide whose transcribed, and optionally translated, product is capable of detection, for example the naturally occurring ADRP coding sequence or a reporter gene such as luciferase. Techniques for detecting and quantitating transcription products are well known in the art and include, for example hybridisation to labelled probes and direct quantitation of transcribed products by the use of labelled nucleotides which are incorporated during transcription. Translated products may also be detected using well known techniques such as SDS-PAGE and Western blotting, or in the case of biologically active products, suitable assays for detecting said activity (such as CAT assays or chemiluminescence assays). A suitable in vitro assay may for example, be conducted using extracts of mammalian cells, typically supplemented with buffers and nucleotide mixes. Reporter constructs comprising polynucleotides containing ADRP promoter constructs linked to a reporter gene may be added to the mix, or endogenous genomic DNA used. The effect of a candidate substance on ADRP expression may be determined by measuring levels of transcription from the ADRP promoter construct (endogenous or otherwise) in the presence and absence of the candidate substance and comparing the results. Preferably, a control promoter construct should also be tested to ensure that any effect on ADRP expression is specific to the ADRP promoter and is not simply the result of general transcriptional inhibition. A candidate substance is typically considered to modulate ADRP expression if the levels of transcriptional are altered by at least 30%, preferably at least 50, 60, 70, 80 or 90%>. Any affect on the control promoter should preferably be accounted for when calculating changes in ADRP transcription. In vivo assay systems Modulation of ADRP expression can also conveniently be measured in vivo, typically using mammalian cell lines. As with in vitro systems, both reporter constructs and endogenous ADRP genes may be used. In a preferred embodiment, the assay uses mammalian cells stably transfected with a polynucleotide comprising a reporter construct which contains an ADRP promoter operably linked to a reporter gene, for example chloramphenicol transferase (CAT) or luciferase. The cell also preferably comprises a stably transfected control promoter sequence operably linked to a second reporter gene which can be distinguished from the first reporter gene. Typically, levels of transcription from the ADRP construct and the control construct are measured in the absence of a candidate substance and then measured in the presence of a candidate substance. The effect of the candidate substance on transcription from the ADRP reporter construct (or endogenous ADRP gene) can then be determined, taking into account any general effect on transcription as indicated by the result obtained for the control reporter construct. In another embodiment, ADRP expression is measured by determining the amount of ADRP protein in cells before administration of the candidate substance and after administration of the candidate substance. As described above, protein levels are typically measured by analysing cell extracts by SDS-PAGE and detecting the ADRP protein using Western blotting. Alternatively, the cells used in the assay of the invention may comprise a reporter construct containing an ADRP promoter operably linked to a nucleotide sequence encoding a detectable polypeptide product, such as CAT. In a particularly preferred embodiment, the detectable product encodes an enzyme which can cleave a cellular or exogenously added substance causing a detectable change in the absorption spectrum or emission spectrum of the cell or cell medium at a particular wavelength. This will facilitate the large-scale screening of candidate substances in, for example a microtitre plate assay format. Administration of candidate substances to mammalian cell lines and generation of host mammalian cell lines comprising reporter constructs can be carried out as described above. 3. Testing candidate substances for anti-viral activity Candidate substances that are identified by the method of the invention as disrupting an interaction between a lipid globule targeting sequence and a lipid globule, or modulating ADRP expression may be tested for their ability to, for example, reduce susceptibility of cells to viral infection. Such compounds could be used therapeutically to affect viral infection, for example to prevent or treat viral infection. Typically, an assay to determine the effect of a candidate substance identified by a method of the invention on the susceptibility of cells to viral infection comprises: (a) administering a virus, for example HCV, to a cell, in the absence of the candidate substance; (b) administering the virus to the cell in the presence of the candidate substance; and (c) determining if the candidate substance reduces or abolishes the susceptibility of the cell to viral infection. The candidate substance may be administered before, or concomitant with, the virus to establish if infection is prevented. Alternatively, the candidate substance may be administered subsequent to viral infection to establish if viral infection can be treated using the candidate substance. Administration of candidate substances to cells may be performed as described above. The assay is typically carried out using mammalian cell lines, but an animal model could be employed instead, such as chimpanzees in the case of HCV. The virus is contacted with cells, typically cells in culture. The cells may be cells of a mammalian cell line, in particular mammalian cells susceptible to infection by the virus in the absence of the candidate substance, for example in the case of HCV, liver cells. Techniques for assaying infectivity of viruses are well-known in the art. As well as using plaque assays, levels of viral infection can be determined by using recombinant viruses which comprise a reporter gene, for example lacZ. The use of a histochemically detectable reporter gene is especially preferred when experiments are performed with animals. In the case of HCV, plaque assays cannot be used. A suitable method for determining the level of HCV production in an infected animal is to perform RT-PCR analysis of the amount of positive-strand nucleic acid material in cells or serum. In addition, the presence of negative-strand RNA in cells, as determined by RT-PCR, is taken to mean that there is active viral replication. In a preferred embodiment of the above-described assays, lipid globule targeting sequence and derivatives thereof are used in an experimental system to study normal cellular interactions. For example, derivatives of viral proteins such as HCV core protein or derivatives of ADRP, including deletion, insertion and substitution mutants, can be used to disrupt an interaction between ADRP and lipid globules. This can be tested in vitro or in vivo using the assays described above. The interaction between ADRP and lipid globules can also be disrupted in vivo by introducing a lipid globule targeting sequence or derivatives thereof, including deletion, insertion and substitution mutants, into cells in vivo, preferably mammalian cells, more preferably human cells. Lipid globule targeting sequences and their derivatives can be introduced into the cells using techniques described above, for example transfection of nucleic acid constructs encoding lipid globule targeting sequences, or using viral vectors. The effect of this disruption can be determined as described above. Any in vitro data obtained may be used to assist in the rational design of lipid globule targeting sequences for use in the in vivo studies. In addition, the precise regions/amino acid residues of lipid globule targeting sequences which bind to lipid globules can determined by in vitro binding studies using lipid globule targeting sequence derivatives. This will also assist in the rational design of lipid globule targeting sequence derivatives for use in the in vivo studies. Thus viral lipid globule targeting sequences, which are readily distinguished from cellular constituents, may be used as a tool to investigate intracellular lipid metabolism. F. Therapeutic Uses Substances capable of disrupting an interaction between a viral lipid globule targeting sequence and an intracellular globule may be used to affect a viral infection in a human or animal, in particular to treat or prevent a viral infection. Such substances may have been identified by the assay methods of the invention or otherwise. Substances that are capable of modulating ADRP expression may also be used to affect a viral infection in a human or animal, in particular to treat or prevent a viral infection. Such substances may have been identified by the assay methods of the invention or otherwise For example, the non-steroidal anti-inflammatory drugs ibuprofen and indomethacin have been shown to stimulate ADRP expression. Consequently, the present invention provides the use of ibuprofen and/or indomethacin in the manufacture of a medicament for use in affecting a viral infection, preferably an HCV infection. G. Compositions/Administration Proteins of the invention and substances identified or identifiable by the assay methods of the invention may preferably be combined with various components to produce compositions of the invention. Preferably the compositions are combined with a pharmaceutically acceptable carrier or diluent to produce a pharmaceutical composition (which may be for human or animal use). Suitable carriers and diluents include isotonic saline solutions, for example phosphate-buffered saline. The composition of the invention may be administered by direct injection. The composition may be formulated for parenteral, intramuscular, intravenous, subcutaneous, intraocular or transdermal administration or inhalation. Typically, each protein may be administered at a dose of from 0.01 to 30 mg/kg body weight, preferably from 0.1 to 10 mg/kg, more preferably from 0.1 to 1 mg/kg body weight. Polynucleotides/vectors encoding polypeptide components for use in affecting viral infections may be administered directly as a naked nucleic acid construct, preferably further comprising flanking sequences homologous to the host cell genome. When the polynucleotides/vectors are administered as a naked nucleic acid, the amount of nucleic acid administered may typically be in the range of from 1 μg to 10 mg, preferably from 100 μg to 1 mg. Uptake of naked nucleic acid constructs by mammalian cells is enhanced by several known transfection techniques for example those including the use of transfection agents. Example of these agents include cationic agents (for example calcium phosphate and DEAE-dextran) and lipofectants (for example lipofectam™ and transfectam™). Typically, nucleic acid constructs are mixed with the transfection agent to produce a composition. Preferably the polynucleotide or vector of the invention is combined with a pharmaceutically acceptable carrier or diluent to produce a pharmaceutical composition. Suitable carriers and diluents include isotonic saline solutions, for example phosphate- buffered saline. The composition may be formulated for parenteral, intramuscular, intravenous, subcutaneous, intraocular or transdermal administration or inhalation. The routes of administration and dosages described are intended only as a guide since a skilled practitioner will be able to determine readily the optimum route of administration and dosage for any particular patient and condition. The invention will be described with reference to the following Examples which are intended to be illustrative only and not limiting. The Examples refer to the following Figures. Brief Description of the Figures Figure 1 shows (a) the sequence of HCV core protein (amino acids 1 to 195) and (b) a hydropathy plot for the HCV core protein amino acid sequence. Figure 2 shows Western blots probed with antibodies to HCV core protein Figure 3 shows confocal microscopy images of the intracellular localisation of core proteins and lipid droplets. Figure 4 shows confocal microscopy images of cells illustrating the effect of expression of HCV core proteins on the ability to detect ADRP in BHKC13 cells. Figure 5 shows confocal microscopy images of cells illustrating the effect of expression of HCV core protein on the abundance of ADRP. Figure 6 shows Western blots probed with antibodies to HCV core protein and ADRP Figure 7 shows a Western blot probed with antibodies to HCV core protein. Figure 8 shows Western blots probed with antibodies to (a) HCV core protein and (b) E2 protein. Detailed Description of the Figures Figure 1 a. Nucleotide sequence and predicted amino acid sequence for the core coding region of HCV strain Glasgow (supplied by M. McElwee and R. Elliott, personal communication). Numbers at the end of each line give either the nucleotide sequence number or amino acid residue. Basic amino acids are circled. Same as corresponding sequences shown in SEQ ID. No. 1. b. Proposed domain structure and hydrophobicity plot of the predicted core amino acid sequence from HCV strain Glasgow. Figure 2 - Analysis of the core proteins made by the pSFV and pgHCV constructs. A. Western blot analysis of extracts prepared from cells which were harvested 20 hours after electroporation. Aliquots of extracts containing the same number of cell equivalents were analysed with antibody JM122. The samples were from cells electroporated with RNA from the following constructs: lane 1, pSFV.1-195; lane 2, pSFV.1-173; lane 3, pSFV.1-169; lane 4, pSFV.1-153; lane 5, pSFV.Δ155-161; lane 6, pSFV.Δ161-166; lane 7, no RNA. Arrows denote the forms of core which have (labelled C) and have not (labelled UC) been cleaved at the internal processing site. B. In vitro translation of core proteins. Products of reactions were electrophoresed on a 10%) polyacrylamide gel and detected by autoradiography. The samples were from reactions containing the following constructs: lane 1, pgHCV.1-195; lane 2, pgHCV.1-173; lane 3, pgHCV.l-153. Figure 3 - Confocal images of the intracellular localisation of core proteins and lipid droplets. BHK C13 cells were harvested 20 hours after electroporation and fixed with 4% paraformaldehyde, 0.1%> Triton X-100. Indirect immunofluorescence was performed with antibody JM122 and an anti-mouse secondary antibody conjugated with FITC. Lipid droplets were stained with oil red O. Since the epitope recognised by JM122 lies in the region of core removed in pSFV.Δ49-75, anti-core rabbit antisera 308 was used along with an anti-rabbit secondary antibody to analyse cells expressing this variant of core protein. Lipid droplets were stained with oil red O. Panels A, D, F, H, J, L, N, P, R, T, V and X show the distributions of core protein. Panel b shows the location of lipid droplets only. Panels C, E, G, I, K, M, O, Q, S, U, W and Y are merged images of core protein and lipid droplets. Cells were electroporated with RNA from the following constructs: panels A-C, pSFV.1-195; panels D and E, pSFV.1-173; panels F and G, pSFV.1-153; panels H and I, pSFV.1-153; panels J and K, pSFV.Δ2-43; panels L and M; pSFV. Δ9-43; panels N and O; pSFV.Δ49-75; panels P and Q; pSFV.Δ80-l 18; panels R and S, pSFV.Δ125-144; panels T and U, pSFV.Δl 145-154; panels I and W, pSFV. Δ155-161; panels X and Y, pSFV.Δlόl- 166. Figure 4 - Effect of expression of core proteins on the ability to detect ADRP in BHKC13 cells by confocal microscopy. Cells were harvested 20 hours after electroporation and fixed with methanol. ADRP was detected with anti-ADRP antibody and core protein with 308 antisera. Secondary antibodies were an anti-mouse IgG conjugated with FITC (for anti-ADRP) and anti-rabbit IgG conjugated with Cy5 (for 308 antisera). Panels A, D, G, J, M and P are images of ADRP localisation. Panels B, E, H, K, N, and Q are images of core distribution. Panels C, F, I, L, O and R show the merged images of core and ADRP distributions. Cells were electroporated with RNA from the following constructs: panels A, B and C, pSFV.1-195; panels D, E and F, pSFV.1-173; panels G, H and I, pSFV.1-169; panels J, K and L, pSFV.1-153; panels M, N and O, pSFV.Δ155-161; panels P, Q and R, pSFV.Δ161-166. Figure 5 - Effect of expression of core proteins on the ability to detect ADRP in MCA RH7777 cells by confocal microscopy. Cells were examined as described in the legend for Figure 4. Figure 6 - Effect of expression of core protein on the abundance of ADRP. BHK C13 cells were electroporated with RNA from pSFV.1-195 and pSFV.1-153 and extracts were prepared at the times indicated following electroporation. Aliquots of cell extracts were electrophoresed on 10% polyacrylamide gels and then the proteins were transferred to nitrocellulose membrane for Western blot analsysis. The upper panels show membranes probed with JM122 antibody while, in the lower panels, membranes were probed with anti-ADRP antibody. Bands corresponding to core proteins, expressed from pSFV.1-195 and pSFV.1-153, and ADRP are arrowed. Figure 7 - Analysis of the core proteins made by the domain 2 variants. Western blot analysis was performed on extracts prepared from cells which were harvested 20 hours after electroporation. Aliquots of extracts containing the same number of cell equivalents were analysed with antibody JM122. The samples were from cells electroporated with RNA from the following constructs: lane 1, pSFV.1-195; lane 2, pSFV.1-169; lane 3, pSFV.1-153; lane 4, pSFV.Δ125-144; lane 5, pSFV.1-124, 145-169; lane 6, pSFV.Δ125- 134; lane 7, pSFV.Δ135-144; lane 8, pSFV.Δ145-154; lane 9, pSFV.Δ155-161; lane 10, pSFV.Δ161-166; lane 11, pSFV.Δ125-144, Δ161-166; lane 12, pSFV.1-124, 145-160, 167- 169. Arrows denote the forms of core which have (labelled C) and have not (labelled UC) been cleaved at the internal processing site and p21 denotes the mature form of core protein. Numbers below each lane represent the approximate amount of cleaved product generated from each of the core variants Figure 8 - Analysis of the effect of linked El and E2 sequences and the protease inhibitor, MG132, on core protein stability and maturation. Western blot analysis was performed on extracts prepared from electroporated cells which had been treated or not treated with MG132. Aliquots of extracts containing the same number of cell equivalents were analysed with antibody JM122 (in A) and an anti-E2 monoclonal antibody, ALP98 (in B). Samples in even- and odd-numbered lanes had either been treated or not treated respectively with MG132. Samples were from cells electroporated with RNA from the following constructs: lanes 1 and 2, pSFV.Δ125-144; lanes 3 and 4, PSFV.CΔI 25 -I44E1E2; lanes 5 and 6, pSFV.1-124,145-169; lanes 7 and 8, pSFV.CElE2 G!a . The positions of core either cleaved (C) or not cleaved (UC) at the internal processing site are indicated (in A) as is p21 (in A) and glycoprotein E2 (in B). EXAMPLES MATERIALS AND METHODS Cell Lines Baby hamster kidney (BHK) C13 cells were maintained in Glasgow modified Eagle's medium supplemented with 10% newborn calf serum, 100 IU/ml penicillin/streptomycin and 5% tryptose phosphate broth. The rat hepatoma cell line, MCA RH7777, was maintained in minimal essential Eagle's medium supplemented with 20% foetal bovine serum, 100 IU/ml penicillin/streptomycin, 1 x non-essential amino acids and 2 mM L-glutamine. Immunological Reagents Antibody JM122 was a mouse monoclonal antibody raised against a purified fusion protein, expressed in bacteria, which was composed of the N-terminal 118 amino acid residues of core protein encoded by HCV strain Glasgow linked to a histidine tag. Antisera 308 was raised in rabbits against a branched peptide ([A/PJKPQRKTKRNT [I/N]RRPQDVKFPGG) 8 K 7 A. The peptide consists of residues 5-27 of core protein encoded by HCV strain Glasgow (SEQ ID. No. 1, Figure IA). The two degenerate sites at positions 1 and 12 were introduced to obtain antisera which would be reactive against core proteins from other isolates. API 25 (anti-ADRP) was obtained from Cymbus Biotechnology Ltd. ALP98 recognises a linear epitope in HCV glycoprotein E2 and was supplied by Dr. A. Patel. Secondary antibodies were obtained from Sigma with the exception of Cy5 conjugated goat anti-rabbit IgG which was obtained from Amersham. Construction of Core Deletion and Truncation Mutants Plasmids containing the coding region for the core protein of HCV strain Glasgow were obtained by combining fragments from two constructs called core.pTZlδ and 5'-ΔNS2 (provided by M. McElwee and R. Elliott). Core.pTZ18 possesses nucleotide residues 337-915 of the HCV strain Glasgow genome and 5'-ΔNS2 contains residues 1-2895. DNA fragments from these plasmids were combined in a vector called pGEMl. The resultant plasmid contained nucleotide residues 337-2895 of the HCV strain Glasgow genome and therefore encodes the core, El and E2 proteins of this isolate. For further cloning purposes, Bglll sites were introduced at EcoRI and Hindlll sites which lie upstream and downstream from the HCV sequences respectively. The resultant plasmid was called pgHCV.CElE2. Construction of a derivative plasmid, pgHCV.1-195 was achieved by inserting an oligonucleotide (GCTGAGATCTA) that had both a translational stop codon and the sequences for a Bgl II enzyme site between a Fsp I enzyme site at residue 925 in the HCV genome and a Hind III enzyme in the pGEM backbone. Thus, pgHCV.1-195 encodes the N-terminal 195 amino acids of HCV strain Glasgow. The nucleotide and predicted amino acid sequence of this region of HCV strain Glasgow is shown in SEQ ID. No. 1. From pgHCV.1-195, the following series of constructs were made which had various regions of the HCV coding region removed (Table 1). Table 1 Construction of HCV Core Mutants a Numbers in brackets refer to nucleotide positions in Fig. la b These enzyme sites were located in the pGEMl vector 0 This site was incorporated into the oligonucleotide (italicised in Table) used to construct pgHCV/Δ 135-144 Plasmid pSFV.C Δ i 2 s-i 44 ElE2 was made by replacing a BssHII/BstEII fragment in pSFV.CElE2Gi a (Figure. IA, nucleotide residues 141-502) with the corresponding DNA fragment from pgHCV. Δ125-144. The construction of pSFV.CElE2Gia is described in Patel et al. (1999) and expression from this construct produces the three HCV structural proteins (core, El and E2) of strain Glasgow. A similar strategy was employed using pSFV.1-169 to construct truncated variants of core in which internal sequences also were deleted (e.g. pgHCV.1-160, 167-169 lacked residues 161-166 and the coding sequence terminated following codon 169). To create constructs with two internal deletions, the oligonucleotide used to make pgHCV. Δ 125-144 was inserted between the Clal/BstXI sites of plasmids pgHCV.1-160,167-169 and pgHCV.Δl 61-166 (Table 1 and Figure IA). The resultant plasmids were called pgHCV.1-124, 145-160, 167-169 and pgHCV.Δ125-144, Δ161-166. For expression in tissue culture cells, Bgl II DNA fragments carrying the relevant HCV sequences were prepared from the pgHCV plasmid series and inserted into the Bam HI site of a Semliki Forest virus vector, pSFVl. The resultant plasmids were termed the pSFV. series (e.g. pSFV.1-195). In Vitro Translation Proteins were translated in vitro using a coupled transcription/translation kit supplied by Promega. Reactions used 1 μg of DNA as template and were carried out according to manufacturer's instructions. In Vitro Transcription Prior to electroporation, RNA was transcribed in vitro from the appropriate pSFV plasmid which had been linearised at a Spe I enzyme site. Typical reactions were carried out in a volume of 20 μl and contained 40 mM Tris (pH 7.5), 6 mM MgCl 2 , 2mM spermidine, 10 mM NaCl, 1 mM DTT, 1 mM ATP, 1 mM CTP, 1 mM UTP, 0.5 mM GTP, 1 mM m 7 G(5')ppp(5')G cap analogue, 50 units Rnasin, 50 units SP6 RNA polymerase and 2 μg linearised DNA. Reactions were performed at 37°C for 2 hours. Products of the reaction were analysed by agarose gel electrophoresis to examine the quality and quantity of RNA synthesised prior to use in electroporations. Preparation of Cells Competent for Electroporation Cells were washed and treated with trypsin for detachment from tissue culture containers. Detached cells were suspended in 20 ml of growth medium and centrifuged at 100 g for 5 min at room temperature. Cell pellets were suspended in 50 ml of PBSA and centrifuged as previously. Pellets were suspended in PBSA at a final concentration of about 2 x 10 7 cells/ml. Electroporation of Cells, Treatment with MG132 and Preparation of Cell Extracts 0.8 ml of competent cells were mixed with in vitro transcribed RNA in an electroporation cuvette (0.4 cm gap) and pulsed twice at either 1.2 kV, 25 μF (for BHK C13 cells) or 0.36 kV, 960 μF (for MCA RH7777 cells). Between pulses, the cell/RNA suspension was gently mixed. Following electroporation, cells were diluted in growth medium and seeded onto either 35mm tissue culture dishes or coverslips in 24- well tissue culture plates and then incubated at 37°C. To treat cells with MG132, cells were incubated for 6 hours after electroporation at 37°C and the media was replaced with fresh media containing the protease inhibitor at a final concentration of 2.5 μg/ml. Incubation was continued at 37°C for a further 10 hours before the cells were harvested. To prepare extracts, electroporated cells were harvested by removing the growth medium and washing the cell monolayers with PBS. Cells were scraped into PBS and pelleted by centrifugation at 100 g for 5 mins at 4°C. The cell pellet was solubilised in sample buffer consisting of 160 mM Tris (pH 6.7), 2% SDS, 700mM β-mercaptoethanol, 10% glycerol, 0.004% bromophenol blue. Alternatively, sample buffer was added directly to cells which had been washed with PBS. Cells were solubilised at a concentration of approximately 4 x 10 cell equivalents per ml sample buffer. Samples were heated to 100°C for 5 mins to fully denature proteins and nucleic acids. SDS-PAGE and Western Blot Analysis Samples were prepared for electrophoresis and proteins were separated on polyacrylamide gels cross-linked with 2.5%) (wt wt) N,N'-methylene bisacrylamide using standard techniques. Polypeptides were detected either by autoradiography or by staining using Coomassie brilliant blue. For Western blot analysis, proteins were separated on polyacrylamide gels and transferred to nitrocellulose membrane using standard techniques. The nitrocellulose membrane was blocked in 3% gelatin, 20 mM Tris (pH 7.5), 500 mM NaCl for at least 6 hours at 37°C prior to incubation with the primary antibody. Incubations with the primary antibody (diluted to 1/500 for ADRP antibody, 1/1000 for JM122 and 1/2000 for ALP98) were performed in 1% gelatin, 20 mM Tris (pH 7.5), 500 mM NaCl, 0.05% Tween 20 at either room temperature or 37°C for approximately 3-4 hours. Following extensive washing with 20 mM Tris (pH 7.5), 500 mM NaCl, 0.05% Tween 20, the membrane was incubated for 2 hours at room temperature with anti-mouse IgG conjugated with horse radish peroxidase in the same solution as for the primary antibody and at a dilution of 1/1000. Bound antibody was detected by enhanced chemiluminescence. Indirect Immunofluorescence and Staining of Lipids Cells on 13 mm coverslips were fixed in either methanol at -20°C or 4% paraformaldehyde, 0.1% Triton X-100 (prepared in PBS) at 4°C for 30 mins. Following washing with PBS and blocking with PBS/CS (PBS containing 1% newborn calf serum), cells were incubated with primary antibody (diluted in PBS/CS at 1/200 for JM122 antibody, 1/1000 for 308 antisera and 1/100 for ADRP antibody) for 2 hours at room temperature. Cells were washed extensively with PBS/CS and then incubated with conjugated secondary antibody (either anti-mouse or anti-rabbit IgG raised in goat) for 2 hours at room temperature. Cells were washed extensively in solutions of PBS/CS followed by PBS and finally H 2 O before mounting on slides using Citifluor. Samples were analysed using a Leiss LSM confocal microscope. Following incubation with both antibodies and washing, lipid droplets were stained in paraformaldehyde-fixed cells by briefly rinsing coverslips in 60%> propan-2-ol followed by incubation with 0.5ml 60% propan-2-ol containing oil red O for 1.5-2 mins at room temperature. Coverslips were briefly rinsed with 60% propan-2-ol, washed with PBS and H 2 O and mounted as described above. The oil red O staining solution was prepared from a saturated stock of approximately 1% oil red O dissolved in propan-2-ol. Before staining, the stock was diluted with H 2 O and then filtered. RESULTS Example 1 - Predicted Domain Structure of the HCV Core Protein Previous studies have highlighted the overall basic nature of the HCV core protein. Closer examination of the predicted amino acid sequences for strain Glasgow and other HCV strains reveals that the basic residues are clustered at the N-terminal end of the protein (Bukh et al., 1994). These clusters correspond to hydrophilic regions which are found between residues 1-122 as predicted from hydropathicity profiles; the overall basic amino acid content in this region is approximately 24% (Figure IA, B). Beyond residue 122, there are very few basic residues and the hydrophobicity content is generally higher, in particular between amino acids 123 and 145 (Figure IA, B). Another area of yet higher hydrophobicity is found from residue 170 to 190. This latter region corresponds to the signal sequence for El which is removed during processing of core protein to the mature form (see Example 2). Based on this evidence, we propose that core protein can be divided into three domains (Figure IB); the N-terminal region (domain 1 ; residues 1-122), a central domain (domain 2; residues 123-174) and the signal sequence for El (domain 3; residues 175-191). These limits for the domains are somewhat arbitrary, for example the C-terminal end of the central domain would be defined by the cleavage event which gives the mature form of the protein and as yet, the precise cleavage site has not been unambiguously characterised. Therefore, the mature core protein would consist of domains 1 and 2. Example 2 - Expression of HCV core protein and variants in tissue culture cells Presently, there is no system available for propagating HCV in tissue culture cells. Therefore, expression of HCV gene products necessitates the use of heterologous expression systems. For short-term expression in mammalian cells, a variety of viral vectors have been utilised including vaccinia virus, Sendai virus and adenovirus. A further alternative is the Semliki Forest virus (SFV) system in which in vitro transcribed RNA, that encodes the SFV replication proteins as well as a heterologous protein but not the SFV structural proteins, is introduced into tissue culture cells. Introduction of nucleic acid into cells may be achieved by several routes but, in the examples given, the method of choice is electroporation. BHK cells were electroporated with RNA from the series of pSFV constructs. 20 hours following electroporation, cells were harvested and extracts prepared. Samples were electrophoresed on a 10% polyacrylamide gel and, following electrophoresis, the proteins were transferred to nitrocellulose membrane for Western blot analysis. Probing the membrane with the core-specific monoclonal antibody JM122 revealed a major single species in each sample which corresponds to core protein. The apparent molecular weights of the proteins made by pSFV.1-195 and two truncated variants, pSFV.1-173 and pSFV.1-169, are approximately 21 kDa and are almost identical (Figure 2A, lanes 1-3). Cleavage between the core and El coding regions occurs between residues 191 and 192. However, there is additional data which reveals that the core protein is further processed by cleavage around residue 174 (Moradpour et al, 1996) this cleavage site will be referred to as the internal processing site. The precise residue at which this second cleavage event occurs is not known. Hence, in agreement with Figure 2 A, lanes 1-3, it would be predicted that the three constructs named above would generate products of similar molecular weights. Additional evidence for a cleavage event close to residues 169-173 occurring within tissue culture cells is shown in Figure 2B. Here, polypeptides translated in vitro from the pGEMl versions of 3 core variants reveal that the unprocessed species made from pgHCV.1-195 is larger than that from pgHCV.1-173 (compare lanes 1 and 2). As would be predicted from the coding sequences for the third truncated form of core, the major species synthesised from pSFV.1-153 has a lower apparent molecular weight than that from pSFV.1-195 (Figure 2B, compare lanes 1 and 4). The major species made by the internal deletion mutants pSFV.Δ155-161 and pSFV.Δ161-166 are intermediate in size between those produced by pSFV.1-195 and pSFV.1-153 (Figure 2A, lanes 5 and 6). Again, this agrees well with predictions based on the number of amino acids removed in these variants (7 in pSFV.Δ155-161 and 6 in pSFV.Δ161-166). It is also evident that there is a significant amount of material produced by the two internal deletion variants which has a higher molecular weight than the fully processed form of core. This presumably represents reduced cleavage at the internal processing site which may result from the removal of certain residues in these mutants which are necessary for fully efficient processing. To conclude therefore, the core proteins and its variants(data not shown) produced by the SFV constructs can be detected by a core-specific antibody and their apparent molecular weights are in agreement with predictions from the nucleotide sequences and previously published data. The mature core protein will be referred to as p21 and, from Example 1 above, it consists of domains 1 and 2. Example 3 - The Intracellular Distribution of the Native Core Species and its Variants Previous studies have revealed that the HCV core protein can associate with lipid droplets within the cytoplasm of cells (Barba et al, 1997; Moradpour et al, 1996). This conclusion was arrived at by combining the techniques of immune electron microscopy with the ability to stain lipid with osmium tetroxide. However, this method suffers from the disadvantages that it is time-consuming and osmium tetroxide can stain other biological molecules (e.g. proteins) in addition to lipid. Therefore, we developed a method for detecting proteins firstly by indirect immunofluorescence followed by staining of lipid droplets with the oil-soluble colourant oil red O. Combined with the method of confocal microscopy, it is possible to visualise the intracellular localisations of core protein and lipid droplets separately and together. A typical example is shown in Figure 3, panels A-C. Here, BHK C13 cells have been electroporated with pSFV.1-195 RNA and, following incubation at 37°C for 20 hours, the cells have been examined by both indirect immunofluorescence and staining with oil red O. In panel A, the core protein produced by pSFV.1-195 is seen to locate to vesicular structures in the cytoplasm. Panel B reveals the distribution of lipid droplets in the same cell. By merging these data (panel C), it is evident that core protein is sited around the lipid droplets. These data therefore agree with previously published results for constructs expressing the full-length coding region of core. Core protein also associated with lipid droplets in a rat hepatoma cell line called MCA RH7777 (data not shown). Results with the constructs which produce truncated forms of core protein indicated that proteins consisting of 173 and 169 amino acids of the core coding region also locate to droplets (Figure 3, panels D-G). By contrast, expressing only the N-terminal 153 residues results in loss of localisation to droplets and a diffuse cytoplasmic distribution is observed (Figure 3, panels H and I). Thus, residues of core protein between amino acids 154 and 169 are required for localisation to droplets. Further analysis with the variants containing internal deletions showed that lipid droplet association was not prevented by removing various segments spanning residues 2 and 118 from within domain 1 (Figure 3, panels J-Q). The most notable feature from these variants was that there was an increase in the relative sizes of some lipid droplets in a proportion of cells. Such an example is shown for mutant Δ2-43 (panels J and K) and there was apparent fusion of droplets also with mutant Δ9-43 (panels L and M). These phenomena were reproducible over several experiments. Nonetheless, with each of the domain 1 variants, association of core protein at the surface of lipid droplets was not abolished. By contrast, lipid droplet association was not detected for proteins which lacked various regions between residues 125 and 166 within domain 2 (Figure 3, panels R-Y). Indeed, the only sequences dispensable in this portion of core protein lay between amino acids 155 and 161 (panels V, W). From these data we conclude that domain 2 contains critical elements which are necessary for lipid droplet association. By contrast, no specific sequences in domain 1 apparently play an essential part in this localisation of core protein although they may contribute through less specific interactions (e.g. neutralisation of the negative charge on phospholipids at the surface of droplets by basic amino acids in the N-terminal region). Example 4 - Effect of Localisation of Core Protein on the Lipid Droplet Associated Protein ADRP At present, there are few proteins identified in mammalian cells which are known to associate with lipid droplets. One protein which has been recently identified is adipocyte differentiation related protein (ADRP) which is ubiquitously expressed in a number of tissue culture cell lines; ADRP mRNA has also been detected in a range of tissue types in mice. To examine whether the localisation of core to lipid droplets had any affect on ADRP, BHK C13 cells were electroporated with the series of pSFV constructs expressing core protein and its variants. An example of the data is shown in Figure 4. Panels A to C show images of three cells following electroporation with pSFV.1-195, only one of which contains core protein (Panel B). Immunofluorescent results with the ADRP antibody (panel A) reveal that ADRP is located on vesicular structures, consistent with its previously assigned association with lipid droplets. The protein is readily detected in the cells which do not express core protein, however, it is considerably reduced in abundance in the core-expressing cell. Observations from this and a series of other experiments consistently revealed that cells expressing core protein from pSFV.1-195 either lacked or contained barely detectable amounts of ADRP. Nonetheless, some cells in which both core protein and ADRP were present also were found; in general, such cells gave reduced fluorescence for the core protein. Hence, it was concluded that the loss of ADRP was related to the levels of expression of core in individual cells. Results with the variants of core which continue to locate to lipid droplets gave identical data (see panels D-I and M-O). Thus, the majority of cells expressing core protein from constructs pSFV.1-173, pSFV.1-169 and pSFV.Δ 155-161 contained quantities of ADRP which were barely detectable. By contrast, ADRP continued to be readily found in cells producing core proteins from pSFV.1-153 and pSFV.Δ161-166, the variants which do not associate with lipid droplets (see panels J-L and P-R). Thus, association of core protein with lipid droplets correlates with a loss of ability to detect ADRP by immunofluorescence. Any cell type specificity for this affect was tested by performing identical experiments in the rat hepatoma cell line, MCA RH7777. In these cells, core protein and its variants gave identical results for their ability to associate with lipid droplets and this again correlated with the levels of ADRP detected in core-expressing cells (Figure 5). Thus the effect of core protein on ADRP is not cell-type specific. Example 5 - The Ability of Core Protein to Associate with Lipid Droplets Induces a Loss of ADRP The immunofluorescence data revealed that the association of core protein and its variants with lipid droplets led to an inability to detect ADRP. It was possible that this was due to masking of ADRP by core. To examine directly the effect of core protein on the levels of ADRP, Western blot analysis was performed on cell extracts prepared at various times following electroporation with either pSFV.1-195 or pSFV.1-153 RNA. In parallel, immunofluorescence analysis was also performed on these cells and this revealed that expression of the core protein produced by the two RNAs was apparent in greater than 90%) of cells. Analysis with antibody JM122 indicated that core protein could be detected from both constructs at 10 hours following electroporation and peaked by about 20 hours (Figure 6). The abundance of core protein produced by the two constructs was very similar by this time-point. From analysis of these samples with the ADRP-specific antibody, it is apparent that there is no change in the abundance of ADRP following electroporation with the pSFV.1-153 RNA. A third set of cells in this experiment which was electroporated with SFV RNA which expresses the HCV El and E2 proteins also showed no reduction in ADRP levels with time. By contrast, there is a rapid reduction in ADRP levels to barely detectable quantities which mirrors the rise in core protein made from pSFV.1-195. From staining of polyacrylamide gels with Coomassie brilliant blue, there were approximately equivalent amounts of protein in all samples. In addition, probing the membranes with another antibody for an endoplasmic reticulum-specific protein, calnexin, indicated that both pSFV.1-195 and pSFV.1-153 samples had similar quantities of this protein at the various times following electroporation. This affect of core expression on ADRP was consistently found in other experiments. Thus, the association of core protein with lipid droplets directly correlates with a specific reduction in the abundance of this protein in cells. Example 6 - Sequences Required for Lipid Droplet Association also Facilitate the Production of p21 To further investigate the possible functions of residues required for lipid droplet association, we analysed the intracellular levels of p21 made by the core constructs. Removal of segments in domain 1 between residues 9 and 118 had no significant effect on the amount of cleaved product detected (data not shown). With mutants in domain 2, we found that deletion of residues between 125 and 144 dramatically reduced the amount of p21 produced to only about 25% of the abundance of p23 (Figure 7, lane 4). To further define the sequences in this region which affected cleavage, two other constructs were made and these showed that deletion of residues from 125-134 reduced cleavage efficiency to a greater extent than removal of residues from 135-144 (-30% as compared to 50%, Figure 7, lanes 6 and 7). The removal of other sequences between residues 145-166 also gave reduced amounts of p21 but the reductions were less pronounced (Figure 7, lanes 8- 10). Furthermore, the levels of p21 from one mutant which lacked residues 125-144 and 161-166 were extremely low (lane 11), suggesting that more than one element may be important in domain 2 for generating p21. These data were obtained with constructs in which the HCV coding region was truncated at residue 195. Since maturation of core is presumed to occur at the ER, the reduced level of processing to generate p21 could be a consequence of impaired association of ribosomes actively synthesising the core protein with the rough endoplasmic reticulum. This may result from a lack of residues downstream from the core/El cleavage site as well as sequences in domain 2 which also could stabilise interactions at the site where processing occurs. Example 7 - Identification of Sequences which Modulate the Stability of p21 Protein During analysis of the effect of sequences on levels of p21 produced, it was observed that the amount of core protein detected in extracts from cells electroporated with pSFV/1-124, 145-169 RNA was much lower than that made in cells electroporated with pSFN/1-169 RΝA (Figure 7, compare lanes 2 and 5); a similar result was obtained also with pSFV.l- 124, 145-160, 167-169 (Figure 7, lane 12). This could suggest that either translation is inhibited as a result of the removal of these sequences or that the protein produced was unstable and proteolytically digested. To determine whether translation was affected, this mutant was linked to the coding sequences for El and E2, generating plasmid pSFV/ C Δ I25 - I44 E1E2. In this way, any effects on translation could be monitored by examining the abundance of either glycoprotein. Given that removal of residues 125-144 apparently reduced the amount of p21 produced by cleavage of the core precursor protein (Figure 7, compare lanes 1 and 4), this construct also would address whether generation of p21 was affected by the these sequences on linked expression with El and E2. For comparative purposes, levels of HCV structural proteins also were determined from pSFV/CElE2 RΝA which expresses unmodified core, El and E2 of strain Glasgow. Western blot analysis showed that p21 and E2 proteins were readily detected in extracts of pSFV/CElE2-electroporated cells (Figure 8A and B, lane 7). The amount of E2 made by PSFV/CΔI25- 1 44E1E2 was similar, however, substantially less core protein was present (Figure 8A and B, compare lanes 3 and 7). Similar relative patterns of expression for core and envelope glycoproteins were observed visually by indirect immunofluorescence (data not shown). Indeed, in pSFV/C Δ ι 25 -ι 4 ElE2-electroporated cells, HCV glycoproteins were readily identified but there was no fluorescent signal for core protein. This indicates that translation of the structural proteins is not inhibited by deletion of residues 125-144 and that the lack of core protein made by PSFV/C Δ I 25 - I44 E1E2 was a consequence of enhanced degradation. To further examine this possibility, electroporated cells were seeded in duplicate and one culture was treated with the protease inhibitor, MG132. Compared with untreated cells, there was no effect on the amount of E2 detected in MG132-treated samples (Figure 8B, lanes 3, 4, 7 and 8). Similar amounts of p21 were found also in treated and untreated samples from pSFV/CElE2-electroporated cells (Figure 8 A, lanes 7 and 8). However, there was a noticeable rise (approximately 3-5 fold) in the abundance of core protein in pSFV/C Δ i 2 _-i 44 ElE2-electroporated cells treated with MG132 (Figure 8A, lanes 3 and 4). In immunofluorescence studies, it was apparent also that core protein was detectable in pSFV/C A i 2 s-i 44 ElE2-electrporated cells treated with MG132 (data not shown). The size of core protein detected in the pSFV/C Δ i 25 -i44ElE2-electroporated cells corresponded to that of a product which had been cleaved at around residue 172 (Fig. 8α, compare lanes 1 and 2 with 3 and 4). This is in contrast to the significantly reduced amount of cleaved product obtained with construct pSFV/Δ125-144 in which the HCV coding region is terminated at residue 195 (Figure 7, lane 4; Figure 8 A, lanes 1 and 2). Such evidence suggests that the reduced amounts of cleaved product made by this construct are a consequence of both the removal of residues 125-144 and the absence of any substantial El and E2 coding sequences. However, the over-riding conclusion from these experiments is the impact of residues 125 to 144 on the stability of core protein during the normal maturation process. Example 8 - In Vitro Binding Assay to Test Effects of Candidate Substances Synthesis of lipid targeting sequences and ADRP using in vitro transcription/translation Constructs suitable for in vitro synthesis of mRNA are produced by placing nucleotide sequences encoding core protein (e.g. pgHCV.1-195) or any other protein with core lipid globule targeting sequences (e.g. pgHCV. Δ43-119) and ADRP are placed under the control of a suitable bacterial or bacteriophage RNA polymerase promoter e.g. SP6 or T7. RNA transcripts are prepared from each of these constructs using standard methods and the yields of RNA determined. To produce in vitro translated protein, the in vitro synthesised RNA is added to an extract capable of synthesising polypeptides e.g. a reticulocyte lysate prepared from rabbits or an extract from wheat germ; proteins may be radiolabelled by including an isotopically labelled amino acid such as 35 S-methionine. Assessing binding of proteins to lipid Membranous material prepared from animal cells (e.g. microsomal membranes) or synthetically prepared mixtures of triacylglycerol or cholesterol and phospholipid are mixed with the translated proteins and incubated. Lipid fractions are then recovered by centrifugation. The incorporation of proteins into lipid fractions may be determined either directly by SDS-PAGE of radiolabelled proteins, or indirectly by using antibodies which are functional in Western blot and/or ELISA procedures. Combinations of RNA from core protein and ADRP may be mixed in the same reaction in various proportions to test the relative affinity of each protein for lipid. An example of a combination would be 9 parts pgHCV.1-195 RNA to 1 part ADRP RNA. Candidate substances at a range of concentrations, such as from 1 μM to 100 mM, are added to reactions and the effect of these substances on protein binding to lipid analysed using the methods indicated above. A suitable candidate substance is typically a substance that enhances or does not significantly impair lipid association of ADRP but which reduces or abolishes binding of core to lipid. Example 9 - In Vivo Binding Assay to Test Effects of Candidate Substances BHK C13 cells are electroporated with SFV encoding HCV core protein (for example SFV.1-195) as described above. At various time points after electroporation, candidate substances are added to cells at concentrations which are not cytotoxic (as determined with mock electroporated cells, for example). Following incubation at 37°C, cells are harvested and cell extracts prepared. Extracts are analysed by Western blot analysis (antibody JM122) to examine the abundance of core protein and determine the efficiency of cleavage at the internal processing site. The relative abundance of core protein in treated as compared to untreated cells could also be quantitated by ELISA. In addition, cells are fixed and examined by a combination of indirect immunofluorescence (for the core and ADRP proteins) and oil red O staining (for lipid droplets) as described above. This can be used to determine whether the intracellular distribution and abundance of core and ADRP has been altered by the presence of the candidate substances. Western blot analysis may also be used to confirm whether the candidate substance had any affect on ADRP levels. A suitable candidate substance is typically a substance that prevents or reduces core association with droplets, causes reduced levels of core protein and/or impaired cleavage at the internal processing site. By contrast, the candidate substance should preferably not affect the intracellular distribution of ADRP and its abundance is typically either similar to or higher than in cells that do not express the core protein. Example 10 - Assessing anti-viral affects of candidate substances in transgenic animals expressing core or in chimpanzees In transgenic animals expressing liver-specific core protein or animals (chimpanzees) infected with HCV, test substances are added at concentrations which were not toxic to the host. Transgenic animals expressing core protein in a liver-specific manner Transgenic mice which give liver-specific expression of core protein are known in the art. Expression of core protein is associated with the development of steatosis and hepatocellular carcinoma in two lines of such animals (Moriya et al, 1998 and Moriya et al, 1997); both pathologies are associated with HCV infection. Similar transgenic mice may be produced with similar phenotypes and used to examine the effect of substances which prevent core association with lipid droplets on these pathological changes. The effect of candidate substances on core protein localisation and levels may be determined in transgenic animals using cells obtained by liver biopsies and tested using the techniques described in Example 9. Alternatively, or in addition, the effect of a candidate substance of the development of steatosis and hepatocellular carcinoma in these animals may be determined. The efficacy of candidate substances may be measured by a reduction in the pathological changes which occur e.g. reduced hepatosteatosis and significant delays or prevention of the onset of carcinoma. Chimpanzees infected with HCV The effect of a substance on HCV replication in infected chimpanzees is assessed by RT-PCR analysis of sera taken at regular intervals from the animal. Biopsy material from the liver may also be tested for the presence of negative-strand HCV RNA by RT-PCR using standard techniques (see Conry-Cantilena, 1997 for review and references contained therein). Efficacy of the candidate substance is assessed by the reduction in the levels of HCV RNA as measured in either or both assays. A reduction in viral titre by a factor of at least 2 to 3 logs is indicative of an anti-viral effect. All publications mentioned in the above specification are herein incorporated by reference. Various modifications and variations of the described methods and system of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in molecular biology or related fields are intended to be within the scope of the following claims. References Atschul et al, 1990, J Mol Biol: 403-410. Altschul et al, 1994, Nature Genetics 6: 119-129. Barba, G. et al, 1997. Proc. Natl. Acad. Sci. 94: 1200-1205 Brasaemle et al, 1997, J. Lip. Res, 38; 2249-2263 Bukh et al, 1994, Proc Natl Acad Sci USA 91; 8239-8243. Claverie & States (1993) Computers and Chemistry 17:191-201 Conry-Cantilena, 1997, Tibtech 15: 71-76 Devereux et al, 1984, Nucleic Acids Research 12: 387 Engelman et al, 1986, Ann Rev, Biophys. Chem 15: 343 Heid et al, 1998, Cell Tiss Res 294: 309-321 Moradpour, D. et al, 1996, Virology 222; 51-63 Moriya, K. et al, 1998, Nature Medicine 4; 1065-1067 Moriya, K. et al, 1997, J. Gen. Virol. 78; 1527-1531 Patton, S. and Huston, G.E., 1986, Lipids 21; 170-174 Wootton & Federhen, 1993, Computers and Chemistry 17:149-163

Claims: CLAIMS 1. A method for identifying a substance capable of affecting a viral infection, which method comprises: (a) providing a lipid globule targeting sequence, as a first component; (b) providing a lipid globule, as a second component; (c) contacting the two components with a substance to be tested under conditions that would permit the two components to interact in the absence of the substance; and (d) determining whether the substance disrupts the interaction between the first and second components; wherein the targeting sequence comprises a hepatitis C virus (HCV) core protein or a fragment, derivative, variant or homologue thereof. 2. A method according to claim 1 wherein the substance to be tested is administered to a cell, the lipid globule targeting sequence is expressed in said cell and the lipid globule is a natural constituent of said cell. 3. A method according to claim 1 or 2 further comprising: (e) administering a virus to a cell in the absence of a said substance which has been determined to disrupt the interaction between the first and second components; (f) administering the virus to the cell in the presence of the said substance; and (g) determining if the said substance reduces or abolishes the susceptibility of the cell to viral infection or the effects of viral infection. 4. A method according to any one of claims 1 to 3 wherein the lipid globule targeting sequence comprises amino acids from 125 to 144 and/or 161 to 166 of the HCV core protein. 5. A method for identifying a substance for treating or preventing a viral infection, which method comprises determining whether said substance can upregulate expression of adipocyte-specific differentiation related protein (ADRP) in a mammalian cell. 6. A method according to any one of claims 1 to 5 wherein the viral infection is a hepatitis infection or other viral infection of the human or animal liver. 7. A method according to any one of claims 2 to 6 wherein said cell is a liver cell. 8. A substance identified by the method of any one of the preceding claims. 9. A substance according to claim 7 wherein the substance has not previously been known to affect viral infection. 10. A substance capable of disrupting an interaction between (i) a lipid globule targeting sequence and (ii) a lipid globule for use in affecting a viral infection, wherein the targeting sequence comprises a hepatitis C virus (HCV) core protein or a fragment, derivative, variant or homologue thereof. 11. A protein comprising a lipid globule targeting sequence for use in preventing or treating a viral infection wherein the targeting sequence comprises an HCV core protein or a fragment, derivative, variant or homologue thereof 12. A protein according to claim 11 wherein the targeting sequence comprises amino acids from 125 to 144 and/or 161 to 166 of the HCV core protein. 13. A polynucleotide encoding a protein according to claim 11 or 12 sequence for use in affecting a viral infection. 14. Use of a protein according to claim 11 or 12, or a polynucleotide according to claim 13 in the manufacture of a medicament for use in affecting a viral infection 15. Use of a substance in the manufacture of a medicament for use in affecting a viral infection, wherein the substance has been identified by the method of any one of claims 1 to 7. 16. Use of a substance in the manufacture of a medicament for use in affecting a viral infection wherein said substance can upregulate expression of ADRP in a mammalian cell.

Inventors: HOPE GRAHAM, MCLAUCHLAN JOHN, HOPE, GRAHAM, MCLAUCHLAN, JOHN
Applicants: HOPE GRAHAM, MCLAUCHLAN JOHN, MEDICAL RES COUNCIL, HOPE, GRAHAM, MCLAUCHLAN, JOHN, MEDICAL RESEARCH COUNCIL
Application year: 1999
Main classification: G01N33
IPC codes: A61K31, A61K38, A61K45, A61P31, C07K14, G01N33