WO2000014216A1

WO2000014216A1 - Method for selecting peptides inhibiting viral surface protein binding to cell surface receptor

Info

Publication number: WO2000014216A1
Application number: PCT/US1999/019755
Authority: WO
Inventors: Zhen-Yong Keck; Kenneth G. Hadlock
Original assignee: Zk Pharmaceuticals, Inc.
Priority date: 1998-09-04
Filing date: 1999-08-25
Publication date: 2000-03-16
Also published as: AU5589399A; EP1109898A1; CA2341156A1

Abstract

A method is provided for screening a library of tester peptides for those tester peptides which inhibit viral infection of a target cell by a vector capable of infecting the target cell in the absence of the tester peptide through binding of a viral surface protein or viral structural protein expressed by the vector to a receptor protein expressed on a surface of the target cell. According to the method, a library of tester peptides are expressed in the target cells. The target cells are then contacted with the vector and those target cells which are not infected by the vector are selected. The method may include additional rounds of negative selection to establish a resistance of the selected target cells to infection as well as positive rounds of selection.

Description

METHOD FOR SELECTING PEPTIDES INHIBITING VIRAL SURFACE PROTEIN BINDING TO CELL SURFACE RECEPTOR

BACKGROUND OF THE INVENTION

Field of the Invention

The present invention relates to methods and compositions of matter for identifying peptides which inhibit the binding of a viral surface protein to a cell surface receptor.

Description of Related Art

Cells utilize a variety of mechanisms for controlling the entry of compositions into and out of the cell. These compositions range from small molecules, such as sugars, fats, and proteins to viruses and cells. One particular mechanism used by cells to control the entry of compositions into cells involves binding to a cell surface receptor. For example, viruses typically include a viral surface protein (VSP) which the virus uses to bind to a cell via a cell surface receptor. The first step in viral infection is typically the binding of the VSP on the virus to the cell surface receptor. One approach to inhibiting and preventing viral infection involves the use of compounds which inhibit the binding of a VSP on the virus to a cell surface receptor. This may be accomplished by designing compounds which bind to the VSP or to the cell surface receptor. This approach is frequently limited by the fact that most cell surface receptors have not been isolated or characterized.

A need currently exists for compounds which inhibit the infection of cells by viruses. In particular, a need exists for improved methods for identifying compounds which inhibit infection, as well as improved methods for identifying and characterizing receptors involved in viral infections. These and other needs are addressed by the present invention. SUMMARY OF THE INVENTION

The present invention relates to compositions and methods for screening a library of tester peptides for those tester peptides in the library which inhibit binding of a viral surface protein or viral structure protein to a receptor protein.

In one embodiment, the invention relates to the tester peptides used in the methods of the present invention. A tester peptide may include an amino acid sequence having the general formula:

VS- A

where:

VS is a sequence of amino acids which is used to inhibit binding of a viral surface protein to a target cell receptor; and

A is an anchor peptide which anchors the tester peptide to a surface of the target cell. Alternatively, the tester peptide may include an amino acid sequence having the general formula:

SP-VS- A

where:

SP is a sequence of amino acids which encode a signal peptide, also commonly referred to as a secretion signal or leader peptide.

Alternatively, the tester peptide may include an amino acid sequence having the general formulas:

SP-L1-VS-L2-A or

L1-VS-L2-A where

SP, VS and A are the same as are indicated above;

L1 is a linker sequence 3 - 100 amino acids in length, more preferably 5- 35 amino acids in length; and L2 is a linker sequence 3 - 100 amino acids in length, more preferably 5- 35 amino acids in length.

It is noted that the tester peptide will generally be expressed in a cell with a signal peptide (SP). However, the SP may be cleaved from the tester peptide by the cell, hence the different embodiments of the tester peptide.

According to any one of the above embodiments, VS preferably has an amino acid sequence from 1-40 amino acids in length, more preferably 1-30 amino acids in length, and most preferably 1-20 amino acids in length. A may be a naturally occurring protein or a non- naturally occurring protein which has been modified to include a linker sequence of between about 1-20 amino acids in length, preferably 1-10 amino acids in length. The linker serves to link the VS to A.

In another embodiment, the invention relates to a library of tester peptides where the amino acid sequence forming at least one of SP, VS, A, L1 , and L2 is varied within the library. According to this embodiment, one or more of the SP, VS, A, L1 , and L2 may be completely or partially randomized to generate a library of tester peptides which can be screened for those sequences that inhibit infection of a target cell under conditions suitable for vector infection. The sequences may also be partially variable, e.g., part of the VS may remain fixed while another part of the VS is varied.

In another embodiment, the invention relates to a target cell which expresses a tester peptide.

In yet another embodiment, the invention relates to a library of target cells. Each target cell in the library expresses a tester peptide. Cumulatively, the library of target cells express a library of tester peptides where the amino acid sequence forming at least part of the tester peptide is varied within the library.

In yet another embodiment, the invention relates to a method for screening a library of tester peptides for those tester peptides which inhibit viral infection of a target cell by a vector capable of infecting the target cells in the absence of the tester peptide through binding of a viral surface protein or structural protein expressed on the vector to a receptor protein expressed on a surface of the target cell. According to one embodiment of the method, a library of tester peptides are expressed by the target cells. The target cells are then contacted with the vector under conditions suitable for infection. Those target cells which are not infected by the vector are then selected.

According to the above method, the sequence of the receptor protein may either be known or unknown at the time that the method is performed. As described herein in greater detail, a feature of the present invention is the use of the tester peptides identified as inhibiting infection to identify and characterize previously unknown receptors involved in viral infection. The library of tester peptides may have any one of the following general formulas: VS-A, SP-VS-A, SP-L1-VS-L2-A, and SP-L1-VS-L2-A, each of which is described elsewhere in greater detail.

Also according to the above method, the vector may encode a selectable marker which is expressed by the target cell when the target cell is infected. Accordingly, the step in the method of selecting those target cells which are not infected by the vector may include selecting those target cells which do not express a selectable marker encoded by the vector.

Also according to the above method, expression of the tester peptides in the target cells may optionally be under the control of a regulatable promoter (e.g., inducible or repressible promoter). In such instances, a level of expression of the tester peptides may be modified in order to determine whether a resistance of a target cell to infection is dependent on a level of expression of the tester peptide by the target cell.

The above described method may include one or more rounds of selection. For example, the method may further include taking those target cells selected as not being infected by the vector and contacting them with a second vector expressing the viral surface protein or viral structural protein, the vector being capable of infecting the target cell in the absence of the tester peptide. Those target cells which are not infected by the second vector are then selected. The second vector used in the second round of selection may be the same or different than the vector used in the earlier round of selection. Also, the second vector may optionally also encode a second selectable marker which is the same or different than a selectable marker used in the earlier round of selection. In yet another embodiment, the invention relates to a method for screening a library of target cells which appear to be resistant to infection for those cells whose viral infectivity is dependent on tester peptide expression. According to one embodiment of this method, expression of the tester peptides in target cells selected for their resistance to being infected by a vector is reduced or discontinued. The target cells are then contacted with a vector which should be capable of infecting the target cells in the absence of the tester peptide. Those target cells which are infected by the vector are then selected. The level of expression of the tester peptides may be reduced or discontinued by having expression of the tester peptides be under the control of a regulatable promoter (e.g., inducible or repressible promoter). In such instances, a level of expression of the tester peptides may be modified in order to determine whether a resistance of a target cell to infection is dependent on a level of expression of the tester peptide by the target cell. BRIEF DESCRIPTION OF THE FIGURES

Figure 1 illustrates a flow diagram for an embodiment of a method for screening a library of tester peptides for those tester peptides in the library, which inhibit binding of a viral surface protein or a viral structure protein (VSP) to a receptor protein (R).

Figure 2 illustrates several different mechanisms by which the tester peptide can inhibit binding.

Figure 3 illustrates a flow diagram for an embodiment of a method for determining whether a tester peptide expressed by a target cell, which remains uninfected after one or more rounds of selection, is responsible for preventing infection.

Figure 4 illustrates examples of signal peptides that may be used in the present invention.

Figure 5 illustrates examples of anchor peptides A that may be used in the present invention.

Figures 6A-6D illustrates four different embodiments of a vector, which may be used as a construct for transforming a target cell with a sequence encoding a tester peptide.

Figures 7A-7C illustrate different embodiments of a vector encoding a VSP, which may be carried by a pseudoviral particle.

Figure 8 illustrates a graph showing infection rates as a function of tester peptide expression levels.

DETAILED DESCRIPTION

The present invention relates to a method for screening a library of tester peptides for those tester peptides in the library which inhibit binding of a viral surface protein or viral structure protein to a receptor protein. The method utilizes the fact that infection of a target cell expressing a receptor protein is mediated by the binding of a viral surface protein or a viral structure protein (VSP) to the receptor protein. The present invention also relates to the use of the tester peptides identified by the above method to isolate and characterize cell surface receptors.

Figure 1 illustrates a flow diagram of an embodiment of a method according to the present invention. During a first round of selection, most of the target cells express one type of tester peptide. Each tester peptide expressed on the cell surface by the target cells include an amino acid sequence having the general formula:

SP-VS- A

where: SP is a sequence of amino acids, which encode a signal peptide, also commonly referred to as a secretion signal or leader peptide;

A is an anchor peptide that anchors the tester peptide to a surface of the target cell.

In this illustration, VS is shown to vary within the tester peptide.

As illustrated in Figure 1 , the variable sequence (VS) is secreted from the cell while the anchor protein (A) remains attached to the cell surface, thereby anchoring the overall tester peptide to the cell surface. The tester peptide is expressed with a secretion signal or leader peptide (SP) which typically is cleaved prior to secretion of the tester peptide from the cell.

The target cells are contacted with a vector which contains a viral surface protein or viral structure protein capable of mediating infection of the target cells under conditions suitable for the vector to infect the target cells. Once contacted with the vector, those target cells, which are not infected by the vector are separated from those target cells which are infected.

At least some of the target cells which are not infected by the vector are not infected due to the tester peptide inhibiting binding of the viral surface protein or viral structure protein on the retroviral vector to the receptor protein on the target cell. As a result, the concentration of tester peptides which inhibit binding of the viral surface protein to the receptor protein is significantly higher among the tester peptides expressed by target cells which are not infected than among the initial library of different tester peptides.

Viruses and host cell lines

A variety of different types of target (host) cells for different viruses may be used in the present invention. For a particular target virus, any type of host cell line can be used so long as the cell line is susceptible to infection by the target virus. For RSV (Respiratory syncytial virus) preferred host cells include, but not limited to Hep-2, MK, PrCK, PrBEK, MRC-5 Vero, and HeLa (ATCC VR-1302). Hep-2 is a particularly preferred host cell line. For HSV-1 (Herpes simplex type 1), preferred host cell lines include, but not limited to CE (CAM, Y.S.), M (I.C.), Rab (cornea), Rab K TC, Vero, and human fibroblast cells. CE (CAM, Y. S.) and Rab K TC. (ATCC VR-260) are particularly preferred host cell lines. For Coxsackievirus A7, preferred host cell lines include, but not limited to, AGMKK TC, RhMKK TC and WI-38 (ATCC VR-673) cells. This list is not intended to be exhaustive of all the possible target viruses or host cells involved in the selection but, merely, to be exemplary thereof.

Mechanisms of inhibition by a tester peptide

A tester peptide may inhibit binding of the viral surface protein or viral structure protein (VSP) on the retroviral vector to the receptor protein on the target cell by a variety of different mechanisms, all of which are intended to fall within the scope of this invention. For example, the tester peptide can bind to a binding region of either the VSP or the receptor. The tester peptide can also bind to a region of either the viral surface protein / viral structure protein or the receptor which is distinct from the natural binding region of either protein but which nonetheless inhibits the two proteins from binding to each other. A tester peptide need not be attached to the cell surface of the target cells but can be secreted to the culture medium from target cells in the absence of an anchor peptide A.

Figure 2 illustrates several different mechanisms by which the tester peptide can inhibit binding. Other mechanisms for inhibiting binding may also occur.

Selection of target cells expressing tester peptides that inhibit VSP binding to cell surface receptor

As illustrated in Figure 1 , the uninfected target cells isolated during the first round of selection may be subjected to one or more additional rounds of selection (shown in the figure as Selection Round 2). In these additional rounds of selection, uninfected target cells are contacted with a retroviral vector. Those target cells which remain uninfected by the retroviral vector are selected from those target cells which become infected. Since the selection is for the uninfected target cells, these rounds of selection are referred to as a negative screen as opposed to the positive screen described below. By performing these one or more additional rounds of selection, the concentration of target peptides among the uninfected target cells which inhibit binding of the VSP to the receptor protein is continually increased relative to their concentration in the initial library of tester peptides.

The construction of the retroviral vector used in each of the one or more additional rounds of selection may be the same as the construction of the retroviral vector used in other rounds of selection or may have a different construction. For example, it may be desirable to use different selectable markers in different rounds of selection as described below in detail. The target cell could remain uninfected for many reasons other than inhibition by the tester peptide during the negative screen. To eliminate such false positive cells, the present invention also relates to a method for determining whether a tester peptide expressed by a target cell, which remains uninfected after one or more rounds of selection, is related to the cell being resistant to infection. According to the method, the target cells are manipulated so that expression of the tester peptide is halted. This may be achieved by repressing expression of the tester peptide or no longer activating expression of the tester peptide. The target cell is then contacted with a retroviral vector expressing the viral surface protein or viral structure protein under conditions suitable for the retroviral vector to infect the target cell. If a target cell, which is not infected during the first or later rounds of negative selection, is infected when expression of the tester peptide is stopped, such infection suggests that the tester peptide does function to prevent infection by the retroviral vector. Since this round of screen is for the selection of the infected target cells, this round of selection is referred as a positive screen.

Figure 3 illustrates a flow diagram for an embodiment of the method. This round of positive screen is carried out such that the nonpermissive cells that have been selected by the negative screen are rendered susceptible to the retroviral vector expressing the VSP. As illustrated in Figure 3, uninfectable target cells, for example, the products of the method shown in Figure 1 , are grown up under conditions where tester peptides are not expressed. The uninfectable target cells are then contacted with a retroviral vector. Infection of target cells is then detected to determine whether there is a correlation between expression of tester peptide and infectability. Those target cells, which become infected, may be separated from those target cells, which do not become infected.

To further confirm the inhibitory function of the tester peptide during these cycles of screen, the present invention also relates to a method for determining whether a tester peptide expressed by a target cell is responsible for the reverse of viral susceptibility. The levels of expression of the tester peptide is controlled in a dose-dependent manner in order to determine whether there is a correlation between the level of expression of the tester peptide and the frequency of infection of the target cell. In another embodiment of the method, a competition assay is performed using purified VSP to compete with the binding of the VSP carried by the retroviral vector to the tester peptide. These assays is intended to ensure that the tester peptides selected from these rounds of negative and positive screens are responsible for preventing the target cell from retroviral infection.

Characterization of the tester peptides and cloning of novel receptors

Once a cell is found to be resistant to infection, the tester peptide expressed by the cell may be characterized. For example, the sequence encoding the tester peptide may be amplified by PCR amplification and then sequenced.

The tester peptides identified by the method of the present invention can be used to identify and isolate novel cell surface receptors. For example, the tester peptide may inhibit infection by binding to the VSP and thus may have a degree of sequence homology to the receptor. Using the nucleic acid sequence encoding the tester peptide, it is possible to screen a cDNA library of the target cells for a sequence encoding a receptor which has homology to the tester peptide. DNA sequences isolated from the cDNA library may then be used as a probe for hybridization and/or a primer for PCR amplification to clone the novel receptor(s).

The tester peptides

In addition to the above methods, the present invention also relates to the tester peptides used in the above methods. A tester peptide expressed on the surface of the target cells generally has the formula SP-VS-A prior to being secreted from the cell and VS-A after being secreted from the cell where SP is a sequence of amino acids that encode a signal peptide, also commonly referred to as a secretion signal or leader peptide;

A is a peptide that can be used to anchor the tester peptide to a cell surface of the target cell.

According to the present invention, SP may be any amino acid sequence that encodes a signal peptide (secretion signal or leader sequence) capable of causing the tester peptide to be secreted from the cell to the cell surface or to the culture medium. Examples of signal peptides that may be used in the present invention include, but are not limited to those amino acid sequences shown in Figure 4.

According to the present invention, VS may be an amino acid sequence from 1-40 amino acids in length, more preferably 1-30 amino acids in length, and most preferably 1-20 amino acids in length. The sequence of VS may be completely or partially randomized to generate a library which can be screened for those sequences that inhibit infection of a target cell under conditions suitable for vector infection. The sequence of VS may also be partially variable, i.e., part of the VS may remain fixed while the other part being variable. VS may be assayed on the cell surface of the target cell as general formula SP-VS-A described; VS may also be assayed in culture medium in the absence of A. Alternatively, the sequence of VS may be held constant when it is desired to screen for anchor sequences (A) which in combination with the VS sequence inhibit infection of a target cell under conditions suitable for vector infection. In these instances, VS is preferably a sequence that is known to inhibit infection. Once a suitable anchor peptide is selected, the anchor peptide may then be used in a method to screen for variable sequences which inhibit infection.

According to the present invention, A is a peptide that can be used to anchor the tester peptide to a cell surface of the target cell. The anchor peptide may be a naturally occurring protein or a non-naturally occurring protein which has been modified to include a linker sequence of between about 1-20 amino acids in length, preferably 1-10 amino acids in length. The linker serves to link the VS to a peptide which functions as an anchor peptide. A may be fixed within a library in order to screen for those VS which inhibit infection of a target cell under conditions suitable for retroviral vector infection. Alternatively, VS may be held constant while the sequence of A is varied partially or completely when it is desired to screen for anchor sequences which in combination with the VS sequence inhibit infection of a target cell under conditions suitable for retroviral vector infection. Examples of A that may be used in the present invention include, but are not limited to those amino acid sequences shown in Figure 5.

In another embodiment, the tester peptide has the general formula SP-L1 -VS-L2-A prior to being secreted from the cell and L1-VS-L2-A after being secreted from the cell where

SP, VS and A are the same as are indicated above; L1 is a linker sequence 3 - 100 amino acids in length, more preferably 5- 35 amino acids in length; and L2 is a linker sequence 3 - 100 amino acids in length, more preferably 5- 35 amino acids in length.

L1 is a sequence that links the SP to the VS. Examples of L1 that may be used include, but are not limited to, amino acids known to form an alpha helical structure; amino acids known to form a beta sheet structure; amino acids that are highly disordered in their secondary structure (random coil peptides); and amino acids which form a signature peptide structure or domain that is found repeatedly in nature including: helix-turn-helix protein domain; extracellular domain of immunoglobulin superfamily. L2 is a sequence used to link the VS to the A. L2 may be used for distancing the VS from the cell surface. L2 is preferably between about 3 and 100 amino acids in length. L2 may have a variable, partially variable, or fixed sequence.

Constructs encoding a tester peptide

The present invention also relates to a construct encoding a tester peptide, which is capable of infecting a target cell. The construct may be a retroviral vector, plasmid, or other viral vector. Examples of commercially available vectors include, but not limited to the retroviral vector pLNCX (Clonetech, K1060-C, GenBank accession number M28247), the plasmid vectors pcDNA3.1(V790-20), pRC/CMV (V750- 20) or pRC/RSV (V780-20) from Invitrogen, and other viral vectors such as adenoviral vector systems (Quantum Biotechnologys, Inc; Berkner, K.L., Biotechniques 6: 616-629, 1988). Preferably, a retroviral vector is used in this invention.

In preferred embodiments the nucleic acid encoding the tester peptide is under transcriptional control of a promoter. The particular promoter that is employed to control the expression of the tester peptide is not believed to be important, so long as it is capable of expressing the nucleic acid in the target cell. In various embodiments, the human cytomegalovirus (CMV) immediate early gene promoter, the SV 40 early promoter and the Rous Sarcoma Virus long terminal repeat (RSV) can be used to obtain high-level expression of the tester peptide. The CMV promoter is preferred in this invention. The vector may also contain an Escherichia coli origin of replication and E. coli antibiotic resistance genes for propagation and antibiotic selection in bacteria. Many E. coli origins are known, including colE1 , pMB1 and pBR322, The pBR322 origin of replication is preferably used in this invention. Many E. coli drug resistance genes are known, including the tetracycline resistance gene, chloramphenoicol resistance gene and the ampicillin resistance gene. In one particular embodiment, the ampicillin resistance gene is used in the vector. The vector may also further comprise an IRES (internal ribosome entry site) sequence between two coding regions to allow translation of two open reading frames from one transcript. The vector may further comprise a multiple cloning site (MCS) to facilitate both cloning and PCR amplification. Many kinds of MCS combinations are known, including the MCS derived from pBluescripts, pGEM, MCS pCDNA3, and pUC19. In one particular embodiment, the MCS derived from pGEM (Promega) is used. The vector further comprises a retroviral packaging signal ( ψ ) in order to package the viral particles.

Since the variable sequence portion of the tester peptide needs to be secreted from the cell, the construct preferably also encodes a signal peptide positioned on the construct for causing secretion of the variable sequence from the cell. The construct also preferably includes a selectable marker, which may be used to confirm whether a target cell has been transfected by a construct encoding a tester peptide as well as to confirm the proper expression of the tester peptides. Many such selectable marker genes are known, including but are not limited to, drug resistance genes such as neomycin resistance gene; cell surface markers such as ICAM, Lyt2 and CD40L gene; and fluorescence genes such as EGFP, EYFP, and EBFP. In one particular embodiment, the selectable marker is green fluorescence protein (EGFP). The vector preferred also includes a bacterial dual promoter

(activator / repressor expression system), which regulates expression of the tester sequence in mammalian cells under the control of tetracyclines (Gossenm. and Bujard, H. 1992, Proc.Natl. Acad. Sci. USA, 89, 5547-5551 ; Clonetech RevTet-off system, K1626-1). Any other kind of inducible mammalian gene expression systems may also be used. For examples, factors such as heat shocks, steroid hormones, heavy metals, phorbol ester, the adenovirus E1A element, interferon, or serum, can be used to induce the expression of mammalian genes. This enables a level of expression of the tester peptide to be altered during the method illustrated in Figure 3. As discussed in the above method, altering a level of expression of the tester peptide allows one to perform a positive screen to determine whether a tester peptide expressed by a target cell which remains uninfected after one or more rounds of selection is related to preventing infection. In one particular embodiment, the promoter is a tetracycline (Tc)-inducible promoter. The present invention also relates to a library of constructs capable of infecting a target cell encoding different tester peptides. In the library, the tester peptide is varied. It is preferred that the tester peptide be varied in the library with regard to the variable sequence (VS). Alternatively, the tester peptide can be varied in regard to the anchor peptide (A).

The viral particle

The present invention also relates to an infectious agent (native particle, pseudoparticle, viral particle, or virion, non-replicating viral particle) that contains a viral surface protein or viral structure protein on its surface that mediates infection of a target cell. The vector can be the retroviral vector pLNCX (Clonetech, K1060-C, GenBank accession number M28247), the plasmid vectors pcDNA3.1(V790-20), pRC/CMV (V750-20) or pRC/RSV (V780-20) from Invitrogen, and other viral vectors such as adenoviral vector systems (Quantum Biotechnologys, Inc; Berkner, K.L., Biotechniques 6: 616-629, 1988). Preferably, a retroviral vector is used in this invention. The infectious agent can bind to the receptor on the target cell and enter into the cell expressing a tester peptide on its surface, depending on the viral susceptibility of the target cell.

In preferred embodiments, the nucleic acid encoding the VSP is under transcriptional control of a promoter. The particular promoter that is employed to control expression of the VSP is not believed to be important, so long as it is capable of expressing the nucleic acid in the target cell. In various embodiments, the human cytomegalovirus (CMV) immediate early gene promoter, the SV 40 early promoter and the Rous Sarcoma Virus long terminal repeat (RSV) can be used to obtain high- level expression of the tester peptide. The CMV promoter is most preferred in this invention. The vector may also contain an Escherichia coli origin of replication and an E. coli drug resistance genes for propagation and antibiotic selection in bacteria. Many of E. coli origins are known, including colE1 , pMB1 and pBR322, The pBR322 origin of replication is preferably used in this invention. Many suitable E. coli antibiotic resistance genes are known including, for example, the tetracycline resistance gene, chloramphenoicol resistance gene and the ampicillin resistance gene. In one particular embodiment, the ampicillin resistance gene is used in the vector. The vector may also further comprise an IRES (internal ribosome entry site) sequence between two coding regions to allow translation of two open reading frames from one transcript. The vector may further comprise a multiple cloning site (MCS) to facilitate both cloning and PCR amplification. Many kinds of MCS combinations are known including, for example, the MCS derived from pBluescripts, pGEM, MCS pCDNA3, and pUC19. In one preferred embodiment, the MCS derived from pGEM (Promega) is used. The vector further comprises a retroviral packaging signal ( ψ ) in order to package the viral particles.

The vector also comprises a selectable marker, which enables target cells infected by the virion to be separated from uninfected target cells. Many suitable selectable markers are known including, for example, the neomycin resistance gene, cell surface markers ICAM, Lyt2 and CD40L gene, fluorescence gene such as EGFP, EYFP and EBFP, secreted alkaline phosphatase (SEAP), and any kind of gene encoding a protein that signals apoptosis and causes programmed cell death, such as the CAR1 gene (Cell 1996, 87, 845-855). In one particular embodiment, the selectable markers are yellow fluorescence protein (EYFP; Clonetech 6068-1), CAR1 gene, and a SEAP (Clonetech 6052-1) on three different vectors. The target cells

The present invention also relates to target cells comprising a sequence encoding the tester peptide as well as target cells, which express the tester peptide on its surface. The present invention also relates to a library of target cells which encode and / or express different tester peptides. The different tester peptides are introduced using a library of constructs as described above. In the library, it is preferred that most target cells express only one type of tester peptide. This can be accomplished, for example, by adjusting M.O.I, (multiplicity of infection) ratios of the different recombinant viruses such that only one copy of the peptide expression cassette is incorporated into each cell.

Kits for Selecting Tester Peptides

The present invention also relates to various kits for performing the methods of the present invention. In one embodiment, the kit comprises a library of constructs encoding different tester peptides and a particle expressing a viral surface protein or viral structure protein. In one embodiment, the kit comprises a library of target cells encoding and / or expressing different tester peptides and particles expressing a VSP.

EXAMPLES

1. SYNTHESIS OF LIBRARY OF CONSTRUCTS ENCODING TESTER PEPTIDES

Expression of a library of tester peptides in target cells may be achieved by transfecting or infecting target cells with constructs or retroviruses encoding a combinatorial library of tester peptides using known methods such as the one described in the manufacture manual from Clontech (K1060-C). The library of constructs may be formed by first synthesizing all of the combinations of oligonucleotide sequences that may be used in the tester peptides. For example, the potential combinations for a peptide 18 amino acids in length is about 18²⁰ , where 20 different amino acids are possible. Oligonucleotide synthesis is commercially available from BRL, Keystone, etc. To control the orientation of the oligonucleotide in the vector during cloning, the combinations of oligonucleotide sequences are synthesized with a different restriction enzyme recognition site on each end, for example, EcoRI at 5' end and BamHI at 3' end. The oligonucleotides may be synthesized in complementary pairs and anneal into double stranded fragments prior to ligation into the vector using known methods. Molecular Cloning, A Laboratory Manual, Second Edition, J. Sambrook, et al., Cold Spring Harbor Laboratory Press (New York, 1989) chapters 1 1.5 and 1.53.

The following describes how to prepare a series of retroviral constructs where the variable sequence VS is varied within the library. According to this example, a series of specific vectors are constructed based on a modified retroviral vector (Clonetech pLNCV vector, K1060-C) that contains 5' and 3' LTRs, retroviral packaging signal (ψ), CMV promoter; MCS (multicloning site); tTA regulatory element, TRE inducible promoter; EGFP, and Neo^r selection markers, and pBR322 origin of replication and E coli Amp^r gene for propagation and antibiotic selection in bacteria. Tetracycline-inducible tTA regulatory element is a fusion of TetR and the VP16 activation domain. tTA binds the TRE and activates transcription in the absence of Tc or a Tc derivative doxycycline (Dox). As will be discussed herein, the tetracycline-inducible tTA regulatory element is used as a positive peptide screen where altering the level of expression of tester peptide is used to evaluate the effect the tester peptide has on preventing infection.

Double stranded oligonucleotides (as described above) are ligated into the following specific vectors to construct plasmid libraries using a known method as described in chapter 1.53 of Molecular Cloning, A Laboratory Manual, Second Edition, J. Sambrook, et al., Cold Spring Harbor Laboratory Press (New York, 1989), chapters 11.5 and 1.53.

Oligonucleotide Library 1

As illustrated in Figure 6A, a library of different oligonucleotides may be ligated inframe downstream of a signal peptide (SP) on the vector that will direct the expressed peptides to be secreted out of the cells into the media.

Oligonucleotide Library 2

As illustrated in Figure 6B, a library of different oligonucleotides may be ligated inframe upstream of a transmembrane domain and in frame downstream of a signal peptide on the vector. The expressed peptides will be secreted and attached onto the cell surface.

Oligonucleotide Library 3

As illustrated in Figure 6C, a library of different oligonucleotides may be ligated inframe downstream of signal peptide and a specific gene, which encode a specific secondary structure protein, such as a hairpin or V-loop structure. The expressed peptides can be secreted into the culture media as a part of the carrier protein with a certain secondary structure.

Oligonucleotide Library 4

As illustrated in Figure 6D, a library of different oligonucleotides may be ligated inframe either downstream or upstream of a specific gene which encodes a specific secondary structure protein, such as a hairpin or V-loop structure and immediate inframe of transmembrane domain. The expressed peptides will be secreted onto the cell surface. For propagation, the above described constructs may be transformed into E. coli competent cells by using methods described in the manufacture manual from BRL (18263-012) or Promega (L2011 ). The plasmid DNA is prepared from at least 1 million transformed cells (Amp") for each library using known methods. For example, large scale preparation of plasmid DNA can be performed by using commercially available columns, such as a plasmid Maxiprep Kit (12162) from Qiagen or Wizard™ Maxipreps DNA Purification System (A7270) from Promega. To package replication-incompetent retroviral particles containing genes encoding the tester peptide library, the plasmid constructs described above are transfected into retrovirus packaging cells (e.g. RetroPack PT67 cell line, Clontech K1060-X) and propagated. The packaged viruses are then titered. These steps may be performed using a method described in the manufacture manual from Clontech (K1060-C).

2. TRANSFECTED TARGET CELLS EXPRESSING TESTER PEPTIDES

A target cell which expresses a tester peptide may be formed by transfecting a target cell with a construct as described in Example 1. It is important to obtain a population of the target cells wherein every single target cell contains at least one copy of the construct and the combinatorial peptides are properly expressed inside of the target cells. Therefore, the construct preferably contains a selectable marker that enables one to separate those target cells that are transfected with the construct from those target cells that were not transfected with the construct. Since the oligonucleotide sequence encoding the tester peptide is cloned upstream of the selection marker in the constructs utilizing an IRES, the selection marker can also serve as an indicator of the proper expression of the tester peptide inside of the target cells. One selectable marker which may be used is green fluorescence protein (EGFP) which may be used to sort cells via fluorescence- activated cell sorting (FACS), based on the specific fluorescence of EGFP, i.e. those cells transformed with the construct should express the green fluorescence protein and therefore be selected based on their fluorescence. Other selectable markers may also be used and are intended to fall within the scope of the present invention. A library of target cells which express different tester peptides may be formed by transfecting a target cell line with a library of constructs as described in Example 1. By controlling the ratio of constructs to cells, it is possible to have most of target cells transfected by a single construct and thus express a single type of tester peptide.

3. RETROVIRAL PARTICLE EXPRESSING VIRAL SURFACE PROTEIN OR VIRAL STRUCTURE PROTEIN TOGETHER WITH SELECTION MARKERS

A retroviral particle expressing a VSP used in the method of the present invention may either be a pseudoparticle, a complete replication competent particle or nonreplicating virus. An advantage for using pseudoparticle is the increased infectivity. An advantage for using a nonreplicating virus is that only one copy of the viral particle of interest is present in the infected the cells through the entire selection process, since it is incapable of making additional virus in the target cell. The retroviral particle preferably includes a sequence encoding a selectable marker, which is expressed in an infected cell. The selectable marker may be any marker that enables an infected cell to be separated from uninfected cells. For example, the selectable marker may be a color marker, such as green or yellow fluorescence protein, which would enable uninfected cells to be separated from infected cells by cell sorting methods such as FACS. Since EGFP is preferably used for the selection of the target cells transfected with constructs encoding the tester peptides, as described in section 2, a yellow fluorescence protein (EYFP) is preferably used for this round of selection here. The selectable marker could also be a gene that encodes secreted alkaline phosphatase (SEAP). The advantage of a SEAP marker is that the secreted SEAP enzyme is assayed directly from the culture medium without dependence on cell lysates, which is well suited for the dose dependent expression assays. Additional examples of suitable selectable markers include antibiotic resistance genes, such as the Neo^r gene.

Figures 7A-7C illustrate three different embodiments of a vector, which may function as a pseudo retroviral particle encoding a VSP. The series of specific vectors are constructed based on a basic retroviral vector (pLNCX, Clonetech) which contains 5' and 3' LTRs, CMV promoter, MCS, Neo^r selection markers, and pBR322 origin of replication and E. coli Amp^r gene for propagation and antibiotic selection in bacteria. The VSP gene is ligated into the following specific vectors with either restriction enzyme cloning sites or the blunt ends to construct viral pseudoparticle. The cloning may be performed using known methods such as those described in Molecular Cloning, A Laboratory Manual, Second Edition, J. Sambrook, et al., Cold Spring Harbor Laboratory Press (New York, 1989), chapter 1.53. Construct 1

As illustrated in Figure 7A, the VSP gene is ligated into a retroviral vector containing EYFP gene as a selection marker to monitor the viruses binding and the entry to the target cell.

Construct 2

As illustrated in Figure 7B, the VSP gene is ligated into a retroviral vector containing CAR1 gene as a selection marker to initiate apoptosis/programmed cell death.

Construct 3 As illustrated in Figure 7C, the VSP gene is ligated into a retroviral vector containing secreted alkaline phosphatases (SEAP). The secreted SEAP enzyme can be assayed directly in the culture medium and is suitable for dose-dependent transcriptional regulation studies.

4. METHOD FOR SCREENING A LIBRARY OF TESTER

PEPTIDES FOR THOSE TESTER PEPTIDES IN THE LIBRARY WHICH INHIBIT BINDING OF A VSP TO A RECEPTOR PROTEIN.

The following example describes a preferred embodiment of a method for screening a library of tester peptides. Cell culture technique employed below may be performed using known protocols as described in Volume 1 of Cells, A laboratory Manual, D.L., Spector., et al., Cold Spring Harbor Laboratory Press (New York, 1998).

Step 1. Pre-selection of target cells transfected with constructs encoding the tester peptides [EGFP (+)]

In this round of selection, a viral susceptible cell line is infected with retroviral particles containing a degenerate oligonucleotide library encoding the tester peptides using a known method, e.g., the manufacture manual from Clontech (K1060-C). This viral construct will have the ability to regulate the expression of the peptide with tetracycline. The degenerate oligonucleotide library may vary within the library with regard to the sequences encoding the anchor peptide A, the variable sequence VS, linker L1 , and/or linker L2. The quantity of retroviral particles used relative to the cells is preferably such that the cells are infected with a single retroviral particle. Those cells which are infected may be selected with FACS based on expression of the green fluorescence protein [e.g., EGFP (+)]. This pre-selection by FACS is important for eliminating false positives during the cycles of screen, especially the first two rounds of negative screens where uninfected cells are selected.

Step 2. Infection of the target cells [EGFP (+)] with pseudoviral particles containing VSP Tissue culture plates or flasks are seeded with the FACS preselected EGFP (+) cells from step 1 encoding the degenerate library of tester peptides. On day 3, duplicate plates are made and two days later the plates are infected with pseudoviral particles containing the VSP and a yellow fluorescence protein (EYFP) selection marker. In parallel, target cells are mock infected with media containing an infection agent as the control. The target cells [EGFP (+) from step 1] would remain permissive to the pseudoviral particles containing the VSP and EYFP unless the tester peptide blocks the interaction between the receptor on the target cells and the VSP. The selection pressure corresponding to EYFP selection marker is set for selecting those target cells that have become nonpermissive to the pseudoviral particles containing the VSP and EYFP. Step 3. Selection of nonpermissive target cells [EGFP (+) / EYFP (-)] — negative selection round 1

On day 7, the plates are trypsinized and the cells from step 2 are selected with FACS for those cells which express green fluorescence protein and which do not express yellow fluorescence protein [EGFP (+) / EYFP (-)]. Cells which express both green and yellow fluorescence protein [EYFP (+) / EGFP (+)] are discarded. These are the cells that remain susceptible to the infection of pseudoviral particles without the inhibition by the tester peptides. Cells that express green fluorescence protein and do not express yellow fluorescence protein [EGFP (+) / EYFP (-)] correspond to cells that were infected in step 1 but were not infected in step 2. These target cells may have become nonpermissive to the pseudoviral particles due to the inhibitory effect of the tester peptide that blocks the interaction between the receptor on the target cells and the VSP. [EGFP (+) / EYFP (-)] cells are selected and will be further tested to confirm that expression of the tester peptide prevents infection of the cells.

Step 4. Selection of target cells [EGFP (+) / EYFP (-) / vital]— negative selection round 2 Tissue culture plates or flasks are seeded with the EGFP (+) /

EYFP (-) cells from step 3. Two days later, duplicate plates are made and the plates are infected with pseudoviral particles containing the VSP and the CAR1 gene encoding a protein that signal apoptosis and causes programmed cell death. In parallel, target cells are mock infected with media containing an infection agent as control. The target cells [EGFP (+) / EYFP (-) from step 3] would remain permissive to the pseudoviral particles containing the VSP and CAR1 gene as selection marker unless the tester peptide blocks the interaction between the receptor on the target cells and the viral surface protein. Selection pressure corresponding to cell death / vital marker of CAR1 is set for selecting those target cells [EGFP (+) / EYFP (-)] that continue remain nonpermissive to pseudoviral particles containing the VSP and CAR1 genes. By using different selection markers in the two rounds of negative selection, the population of the false positive cells is minimized. Two days later, the plates are trypsinized and the live cells are cultured in fresh medium. After two passages the vital cells are selected, and they correspond to cells that were not infected with pseudoviral particles containing the VSP and CAR1 genes. These cells [EGFP (+) / EYFP (-) / vital] are further tested to confirm that expression of the tester peptide prevents infection of the cells.

Step 5. Selection of permissive target cells with repressed expression of the target peptide [EGFP (+) / EYFP (-) / vital / SEAP (+)] — positive screen 1

Tissue culture plates or flasks are seeded with the vital cells [EGFP (+) / EYFP (-) /vital] from step 4. Two days later, duplicate plates are made and the plates are infected with pseudoviral particles containing the VSP and the SEAP gene as a selection marker in the presence of Tc or Dox. In parallel, target cells are mock infected with media containing an infection agent as control. As indicated in the construct design (Fig. 6), tetracycline-inducible tTA regulatory element is a fusion of TetR and the VP16 activation domain, and tTA binds to the TRE and activates transcription in the absence of Tc or Dox. For the first two rounds of negative selection (steps 2-4), degenerated oligonucleotides are transcribed and the tester peptides are expressed by target cells in the absence of Tc or Dox. Adding Tc or Dox to the culture in this step is intended to repress the expression of the inhibitory tester peptide, thus rendering the nonpermissive cells susceptible to the viral infection. In this positive screen, infected cells that are susceptible to the pseudoviral particles containing the VSP and SEAP genes in the presence of Tc or Doc are selected. Therefore, a selection pressure corresponding to SEAP levels is set for selecting those target cells

[EGFP (+) / EYFP (-) / vital] that have regained their susceptibility to the pseudoviral particles under conditions that expression of tester peptide is repressed. The secreted SEAP may be assayed according to a protocol described in Clontech manufacture manual (6052-1). The cells that show SEAP activity are selected, and they correspond to cells that were infected with pseudoviral particles containing VSP and the SEAP gene. These cells [EGFP (+) / EYFP (-) / vital / SEAP (+)] will be further tested to confirm that expression of the tester peptide prevents infection of the cells.

Step 6. Selection of target cells with Tc/Dox-dose-dependent expression of SEAP — positive selection 2

This method is designed for further confirmation analysis of those cells selected after two rounds of negative screen and one round of positive screen. These cells should be [EGFP (+) / EYFP (-) /vital / SEAP (+)] after these screens. As indicated in the construct design (Figs. 6A-6D) where the transcription of the tester peptide is regulated in a Tc or Dox dose-dependent manner, i.e., the level of tester peptide expression can be controlled by the concentration of Tc or Dox in the culture medium. Based on this feature, it would be a good indication that expression of the tester peptide prevents infection of the cells when infectivity or susceptibility of the target cells is Tc or Dox dose- dependent.

Tissue culture plates or flasks are seeded with the live cells [EGFP (+) / EYFP (-) / vital / SEAP (+)] into multiple plates and Tc or Dox at different concentrations is added into the culture medium to regulate the level of inhibitory peptide transcribed and expressed. The cells are then infected with pseudoviral particles containing VSP and SEAP in the presence of Tc or Dox. In parallel, target cells are mock infected with media containing an infection agent as a control.

Two days later, the cells culture containing different concentration of Tc or Dox are collected and SEAP is assayed as described in Clontech manufacture manual, 6052-1. The cells that show SEAP activity in a dose-dependent manner are selected. These cells correspond to cells that were infected with pseudoviral particles containing VSP and SEAP and the level of infectivity correlates with the degree of expression of the inhibitory peptide. The secreted SEAP enzyme can be assayed directly from the culture medium without dependence on cell lysates, which is suitable for dose-dependent assays. As illustrated in Figure 10, if expression of the tester peptide affects the infectibility of the cell, one should see an increase in infection rates as less tester peptide is expressed.

Step 7. The competition assay — positive selection 3

The cells selected from step 6 could be further tested with a competition assay to confirm that they express the inhibitory peptides that block the protein-protein interaction between the VSP and the target cell receptor. If the infection of the pseudoviral particles containing the VSP is directly mediated by the target cell receptor, excess of the free VSP should block the entry of pseudoviral particles into the target cell.

In this round of selection, tissue culture plates or flasks are seeded with the cells [EGFP (+) / EYFP (-) /vital / SEAP (+)-dose dependent] from step 6. Two days later, the cells are split into multiple plates and the purified VSP at different concentrations is added into the culture medium to compete with the availability of the cell receptor to the pseudoviral particles containing the same VSP. The cells are then infected with pseudoviral particles containing the VSP and SEAP in the presence of Tc or Dox. In parallel, target cells are mock infected with media containing an infection agent as control.

To express and purify the VSP, the gene encoding the VSP may be inserted into a suitable expression vector and the VSP protein expressed in vitro can be purified by using commercially available expression systems, for example, pGEM plasmid (P2991) from Promega or BAC-to-BAC™ Baculovirus Expression system (10359-016) from Invitrogen. The cell culture containing different concentration of purified VSP is collected and secreted alkaline phosphatase (SEAP) is assayed as described above to monitor the degree of inhibition of pseudoviral particles infection. The cells that show SEAP activity in competitor- dose-dependent manner are selected. These cells correspond to cells that were infected with pseudoviral particles containing the VSP and SEAP and the level of infectivity correlates with the concentration of the competitor.

Finally, the cells expressing SEAP marker in Tc/Dox and /or competitor dose-dependent manner are selected and the cell cultures are grown up for sequence identification of the inhibitory peptide(s). In the above described method, steps 2-4 correspond to two negative screen cycles. In these negative screen cycles, degenerated tester peptides are expressed that are either secreted into the culture medium or attached to the cell membrane. Some of these peptides will recognize and bind onto the cell surface receptors or the VSP and block the interactions between the cell receptor and the VSP. The negative selection is that the cells that remain susceptible to the target viruses (without indication of inhibitory peptide interaction) are discarded. The cells that have become nonpermissive to the viruses (an indication that the loss of the susceptibility to the viruses might be due to the inhibitory effect of the target peptide) are selected.

Steps 5 corresponds to a positive screen where transcription of the tester peptides is turned off. The nonpermissive cells that have been selected by the previous two negative round screen should at this round reverse back to their susceptibility to the target viruses. The positive selection is that the cells that remain nonpermissive to the viruses (not responding to the repression of transcription of the tester peptide) are discarded. The cells that have reversed back susceptibility to the viral particles (an indication that the loss of the susceptibility to the viruses might be due to the inhibitory effect of the tester peptide) are selected. Steps 6-7 correspond to two kinds of confirmation analysis that the cells selected from both the negative and positive screens are tested for Tc or Dox dose-dependence and viral surface protein competition. The assays are designed to confirm that the selected tester peptide indeed blocks the protein-protein interaction between the virus and the target cell receptor. Any other type of confirmation assays could be employed as well, e.g., deletion or mutation of the degenerated oligonucleotides or competition assays using tester peptide as competitors.

5. METHOD FOR DETERMINING SEQUENCE OF TESTER PEPTIDE IDENTIFIED IN METHOD OF SECTION 4 AND SUBSEQUENT CLONING OF THE VIRAL RECEPTOR

Once target cells have been selected which are resistant to infection when a tester peptide is expressed and are susceptible to infection when expression of the tester peptide is repressed. The sequence of the tester peptide of interest can be determined as follows. DNAs from selected cells are extracted using known methods described in Chapter 14.5 of Molecular Cloning: A Laboratory Manual. Second Edition, J. Sambrrok, et al., Cold Spring Harbor Laboratory Press (New York, 1989). To amplify the variable sequences present in the DNA samples, PCR may be performed with the primers that are complementary to the flanking region of the variable sequence, for example the known multiple cloning sites MCS, by using known methods described in Chapter 14.5 of Molecular Cloning: A Laboratory Manual. Second Edition, J. Sambrrok, et al., Cold Spring Harbor Laboratory Press (New York, 1989). The PCR products will be subcloned to a bacterial vector and individual clones will be selected to separate the mixture of the oligonucleotides and sequenced. Subcloning may be performed using methods described in Chapter 1.53 of Molecular Cloning: A Laboratory Manual. Second Edition, J. Sambrrok, et al., Cold Spring Harbor Laboratory Press (New York, 1989). DNA sequencing may be performed on automated DNA sequencing machine. Sequencing reaction may be performed by using a PRISM™ Ready Reaction Dye Deoxy™ Terminator cycle sequencing kit from Perkin Elmer.

The amplified variable sequences may then be expressed as a peptide which is then labeled with biotin by using commercially available reagents. This biotin-labeled peptide is used as an affinity label that binds to the receptor(s) expressed by a cDNA library. A cDNA library may be expressed by using any kind of known methods, for example, SuperscriptTM choice system for cDNA synthesis (18090-019) from BRL or Riboclone cDNA synthesis systems: AMVRT & M-MLV(H-) (C1001) from Promega.

Alternatively, the deduced peptides can also be labeled with radio isotopes by using commercially available kits, e.g., BRL Radioactive Labelling (18428-011), to probe a Western blot of the whole cell membrane proteins as described in chapter 18.60 of Molecular Cloning: A Laboratory Manual. The distinct band(s) on the nitrocellulose paper is cut off and the bound protein can either be used to generate antibodies (as described in chapter 18.3 and 18.11 of Molecular Cloning: A Laboratory Manual) or to be sequenced (as described above). The antibody may be used to screen the expression library (as described in Chapter 12.16 of Molecular Cloning: A Laboratory Manual) and the deduced nucleotides are used to screen cDNA library as described in commercially available manufacture manual from SuperscriptTM choice system for cDNA synthesis (18090-019) from BRL or Riboclone cDNA synthesis systems: AMVRT & M-MLV(H-)(C1001) from Promega.

The foregoing description of a preferred embodiment of the invention has been presented for purposes of illustration and description. It is not intended to be exhaustive or to limit the invention to the precise forms disclosed. Obviously, many modifications and variations will be apparent to practitioners skilled in this art. It is intended that the scope of the invention be defined by the following claims and their equivalents.

Claims

What is claimed is:

1. A method for screening a library of tester peptides expressed on surfaces of target cells for those tester peptides which inhibit viral infection of a target cell by a vector capable of infecting the target cell in the absence of the tester peptide through binding of a viral surface protein or viral structural protein expressed by the vector to a receptor protein expressed on a surface of the target cell, the method comprising: expressing the library of tester peptides on the surface of the target cells; contacting the target cells with the vector; and selecting those target cells which are not infected by the vector.

2. A method according to claim 1 wherein the tester peptide includes an amino acid sequence having the general formula:

VS- A

where: VS is a sequence of amino acids which is used to inhibit binding of a viral surface protein to a target cell receptor; and A is an anchor peptide that anchors the tester peptide to a surface of the target cell.

3. A method according to claim 2 wherein at least a portion of the amino acid sequence forming at least one of VS and A varies within the library.

4. A method according to claim 2 wherein at least a portion of the amino acid sequence forming VS varies within the library.

5. A method according to claim 2 wherein at least a portion of the amino acid sequence forming A varies within the library.

6. The method according to claim 2 wherein VS has an amino acid sequence between about 1-40 amino acids in length.

7. The method according to claim 2 wherein VS has an amino acid sequence between about 1-30 amino acids in length.

8. The method according to claim 2 wherein VS has an amino acid sequence between about 1-20 amino acids in length.

9. A method according to claim 1 wherein the tester peptide includes an amino acid sequence having the general formula:

SP-VS- A

where: VS is a sequence of amino acids which is used to inhibit binding of a viral surface protein to a target cell receptor; A is an anchor peptide that anchors the tester peptide to a surface of the target cell; and SP is a sequence of amino acids which encodes a signal peptide.

10. A method according to claim 1 wherein the tester peptide includes an amino acid sequence having the general formula:

L1-VS-L2-A

where: VS is a sequence of amino acids which is used to inhibit binding of a viral surface protein to a target cell receptor; A is an anchor peptide that anchors the tester peptide to a surface of the target cell; L1 is a linker sequence 3 - 100 amino acids in length; and L2 is a linker sequence 3 - 100 amino acids in length.

11. A method according to claim 1 wherein the tester peptide includes an amino acid sequence having the general formula:

SP-L1-VS-L2-A

where: VS is a sequence of amino acids which is used to inhibit binding of a viral surface protein to a target cell receptor; A is an anchor peptide that anchors the tester peptide to a surface of the target cell; L1 is a linker sequence 3 - 100 amino acids in length; L2 is a linker sequence 3 - 100 amino acids in length; and SP is a sequence of amino acids which encode a signal peptide.

12. The method according to claim 1 wherein the sequence of the receptor protein to which the viral surface protein or viral structural protein expressed by the vector binds is not known at the time the method is performed.

13. The method according to claim 1 wherein the vector encodes a selectable marker which is expressed by the target cell when the target cell is infected, the step of selecting those target cells which are not infected by the vector including the step of selecting those target cells which do not express the selectable marker encoded by the vector.

14. The method according to claim 1 wherein expression of the tester peptides by the target cells is under control of a regulatable promoter.

15. The method according to claim 14 wherein the regulatable promoter is an inducible promoter, the method further including the step of inducing expression of the tester peptide.

16. The method according to claim 1 wherein the regulatable promoter is a repressible promoter, the method further including the step of not repressing expression of the tester peptide.

17. A method for screening a library of tester peptides on surfaces of target cells for those tester peptides which inhibit viral infection of a target cell by vectors capable of infecting the target cell in the absence of the tester peptide through binding of a viral surface protein or viral structural protein expressed by the vectors to a receptor protein expressed on a surface of the target cell, the method comprising: expressing the library of tester peptides on the surfaces of the target cells; contacting the target cells with a first vector capable of infecting the target cells in the absence of the tester peptide; selecting those target cells which are not infected by the vector; taking the selected target cells and contacting them with a second vector capable of infecting the target cell in the absence of the tester peptide; and selecting those target cells which are not infected by the second vector.

18. The method according to claim 17 wherein the first vector encodes a first selectable marker which is expressed by the target cell when the target cell is infected, the step of selecting those target cells which are not infected by the first vector including the step of selecting those target cells which do not express the first selectable marker; and the second vector encodes a second selectable marker which is expressed by the target cell when the target cell is infected, the step of selecting those target cells which are not infected by the second vector including the step of selecting those target cells which do not express the second selectable marker.

19. A method for screening a library of tester peptides on the surfaces of target cells for those tester peptides which inhibit viral infection of a target cell by vectors capable of infecting the target cell in the absence of the tester peptide through binding of a viral surface protein or viral structural protein expressed by the vectors to a receptor protein expressed on a surface of the target cell, the method comprising: expressing the library of tester peptides on the surfaces of the target cells; contacting the target cells with a first vector capable of infecting the target cells in the absence of the tester peptide; selecting those target cells which are not infected by the vector; reducing a level of expression of the tester peptides in the selected target cells; contacting the selected target cells with a second vector capable of infecting the target cell in the absence of the tester peptide; and selecting those target cells which are infected by the second vector.

20. The method according to claim 19 wherein the step of reducing a level of expression includes discontinuing expression of the tester peptides.