WO2007062063A2

WO2007062063A2 - Recombinant viral compositions and uses thereof

Info

Publication number: WO2007062063A2
Application number: PCT/US2006/045104
Authority: WO
Inventors: Sean Whelan; David K. Cureton
Original assignee: President And Fellows Of Harvard College
Priority date: 2005-11-23
Filing date: 2006-11-22
Publication date: 2007-05-31
Also published as: WO2007062063A3

Abstract

The invention is directed to a modified vesicular stomatitis virus (VSV). the VSV is characterized by a fusion protein comprising a reporter molecule fused to a viral protein. The invention further includes genetically engineered or DNA constructs encoding such a fusion protein, either alone or in combination with one or more VSV genes and genes that encode proteins involved in attachment of VSV or another virus, bacteria or protein to a host cell, internalization of VSV or another virus into a host cell or release of VSV or another virus into the cytoplasm of a host cell. The VSVs of the invention can be used in methods for rapidly detecting viral attachment and entry and can be used, for example, in methods of rapidly identifying new antiviral therapeutics and monitoring the presence of antibodies.

Description

RECOMBINANT VIRAL COMPOSITIONS AND USES THEREOF FIELD OF THE INVENTION

The invention relates to novel viral compositions and their use. The invention provides modified vesicular stomatitis virus (VSV) compositions which are useful in methods for rapidly detecting viral attachment and entry as well as in methods of rapidly identifying new antiviral therapeutics, and diagnostic assays that measure antibody levels against a range of viruses.

GOVERNMENT SUPPORT

The invention was supported, in whole, or in part, by NIH grant number ROl AI059371. The Government has certain rights in the invention.

BACKGROUND OF THE INVENTION

The development of inhibitors directed against specific viral targets has become a focus of antiviral research. However, the expedited identification of such inhibitors relies on the use of efficient, high-throughput drug-screening assays. Therefore, there is a continual need to develop more rapid and efficient drug-screening assays, particularly in the high hazard antiviral field. In addition, there is significant need for easy diagnostic assays that determine whether individuals have been exposed to, or possess neutralizing antibodies to a variety of agents.

SUMMARY OF THE INVENTION

The invention is directed to a modified vesicular stomatitis virus (VSV). The VSV is characterized by a fusion protein comprising a reporter molecule fused to a viral protein. The VSV preferably comprises a nucleic acid molecule that encodes the fusion protein. The VSVs of the invention can be used in methods for rapidly detecting viral attachment and entry and can be used, for example, in methods of rapidly identifying new antiviral therapeutics. Where the virus further comprises a ligand (such as a viral, bacterial or non-viral cellular attachment protein, mammalian receptor ligands, antibodies, or antigen binding fragments thereof), the method can be used to screen modulators (e.g., antagonists and agonists) of cellular receptors. The invention further includes genetically engineered or DNA constructs encoding such a fusion protein, either alone or in combination with one or more VSV genes and genes that encode proteins involved in attachment of VSV or another virus, bacteria or protein to a host cell, internalization of VSV or another virus into a host cell or release of VSV or another virus into the cytoplasm of a host cell. Further, the invention includes plasmids and other vectors comprising such constructs, host cells transformed with the vectors, cells infected by the VSV, cells which are capable of producing the VSV of the invention or any such construct, plasmid or other vector of the invention and cells or cell lines capable of producing the VSV of the invention or any such construct, plasmid or other vector of the invention. In one aspect, the invention provides a recombinant DNA construct encoding a fusion protein, wherein the fusion protein comprises a reporter molecule fused to a VSV polypeptide selected from the group consisting of: nucleocapsid protein (N), the phosphoprotein (P), the matrix protein (M), the glycoprotein (G) and the KNA-dependent RNA polymerase (L), or fragment thereof. In one embodiment, the recombinant DNA construct encodes a reporter molecule that is an enzyme which can be detected by contacting the reporter molecule with colorimetric or fluorigenic substrates. In one embodiment, reporter enzyme is selected from the group that consisting of: oxidases, luciferases, peptidases, glycosidases, peroxidases, and phosphatases. In one embodiment, the reporter molecule is Renilla luciferase. In one embodiment, the DNA construct encodes a reporter molecule which is a fluorescent protein. In one embodiment, the fluorescent reporter is Green Fluorescent Protein (GFP) or Reef Coral Fluorescent Protein (RFCP).

In one aspect, the invention provides a nucleic acid vector comprising a DNA construct of the invention.

In one aspect, the invention provides a modified VSV comprising a nucleic acid vector comprising a DNA construct of the invention. In one embodiment, the virus contains a construct that comprises a nucleic acid vector comprising a DNA construct of the invention which further comprises a nucleic acid that encodes at least one viral cell attachment protein. In one embodiment, the at least one viral cell attachment protein is a VSV cell attachment protein. In one embodiment, the at least one cell attachment protein is a viral cell attachment protein derived from a virus that is not VSV. In one embodiment, the at least one viral attachment protein is derived from a virus of a genus selected from the group consisting of: Retroviridae, Togaviridae, Flaviridae, Coronoviridae, Rhabdoviradae, Filoviridae, Paramyxoviridae, Orthomyxoviridae, Bunyaviridae, Arena viridae, Herpesviridae, and Poxviridae. In one embodiment, the at lease one viral attachment protein is derived from a negative- strand RNA virus. In one embodiment, the at least one viral attachment protein is derived from a virus of a genus selected from the group that consists of: Respirovirus, Morbillivirus, Rubulavirus, Henipavirus, Avuvlavirus, Pneumovirus, Metapneumovirus, Lyssavirus, Ephemerovirus, Marburgvirus, Ebolavirus, Bronavirus, Influenzavirus A, Influenzavirus B, Influenzavirus C, Orthobunyavirus, Hantavirus, Nairovirus, Phlebovirus, and Arenavirus. In one embodiment, the virus of the invention the virus contains a construct that comprises a nucleic acid vector comprising a DNA construct of the invention which further comprises a nucleic acid that encodes at least one non-viral protein. In one embodiment, the at least one non-viral protein is a mammalian ligand to a cellular receptor or a bacterial attachment protein. In one embodiment, the virus of the invention contains a fusion protein which is a functional peptide fused to a viral protein. In one embodiment, the functional peptide is a fluorescent lanthanide binding peptide. In one embodiment, the virus contains a fusion protein comprising a selectable marker fused to the viral protein. In one embodiment, the selectable marker is neomycin.

In one aspect, the invention provides a host cell transformed with a vector of the invention.

In one aspect, the invention provides a cell comprising a virus of the invention or one or more DNA constructs that encode the viral proteins of a virus of the invention.

The invention is further directed to high-throughput methods for screening compounds or other agents, or improved derivatives thereof, for antiviral properties using recombinant VSV of the invention and/or the cell lines stably replicating the VSV constructs, plasmids or other vectors of the invention. That is, in one aspect, the invention provides methods of screening/identfying modulators of viral attachment and/or entry. In one embodiment, the invention provides a method of screening for modulators of cell attachment, comprising the steps of: a) providing at least one control cell sample; b) providing at least one test cell sample; c) contacting at least one test cell sample of step (b), with at least one candidate agent; d) infecting at least one control cell sample of step (a) and the at least one test cell sample of step (c) with a virus of the invention; and e) assaying the cell samples of step (d) to detect the activity of the reporter molecule, wherein a reduction of the level of reporter molecule activity in the at least one test cell sample contacted with the at least one candidate agent relative to the level of reporter molecule activity observed in the at least one control cell sample not contacted with the at least one candidate agent is indicative of antagonist activity of cell attachment by the candidate agent and wherein an increase in the level of reporter molecule activity in the at least one cell sample contacted with the at least one candidate agent relative to the level of reporter molecule activity observed in the at least one control cell not contacted with the at least one candidate agent is indicative of agonist activity of cell attachment. In one embodiment, the invention provides a method of screening for inhibitors of viral infectivity, comprising the steps of: a) providing at least one control cell sample; b) providing at least one test cell sample; c) contacting the at least one test cell sample of step (b), with the at least one candidate inhibitor of viral infectivity; d) infecting the at least one control cell sample of step (a) and the at least one test cell sample of step (c) with a virus of the invention; and e) assaying the cell samples of step (d) to detect the activity of the reporter molecule, wherein a reduction in the level of reporter molecule activity in the at least one test cell sample contacted with the at least one candidate inhibitor relative to the level of reporter molecule activity observed in the at least one control cell sample not contacted with at least one candidate inhibitor is indicative of inhibition activity of the candidate inhibitor. The invention also provides viral inhibitors or other therapeutics identified by the screening methods of the present invention. The inhibitors can be provided as pharmaceutical compositions. The invention also provides a means of measuring the presence of anti-virus antibodies in samples. Accordingly, in one aspect, the invention provides methods of determining the presence or amount of a VSV immunoreactive polypeptide in a sample. In one embodiment, the invention provides a method for determining the presence or amount of a VSV immunoreactive polypeptide in a biological sample, comprising the steps of: a) providing at least one control cell sample; b) providing at least one test cell sample; c) contacting the at least one test cell sample of step (b), with the biological sample; d) infecting the at least one control cell sample of step (a) and the at least one test cell sample of step (c) with a virus of the invention; and e) assaying the a cell samples of step (d) to detect the activity of the reporter molecule, wherein a reduction of the level of reporter molecule activity in the at least one test cell sample contacted with the biological sample relative to the level of reporter molecule activity observed in the at least one control cell sample not contacted with the biological sample is indicative of the presence of the vesicular stomatitis virus immunoreactive polypeptide in the biological sample. In one embodiment, the invention provides a method for determining the presence or amount of a VSV immunoreactive polypeptide in a sample, the method comprising: a) providing the sample; b) contacting the sample with a virus of the invention; and c) determining the presence or amount of the virus bound to a VSV immunoreactive polypeptide, thereby determining the presence or amount of the vesicular stomatitis virus immunoreactive polypeptide in the sample.

In one aspect, the invention provides a kit comprising in one or more containers, a virus of the invention and instructions for using the contents therein.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is a schematic diagram of the replication cycle of vesicular stomatitis virus.

Figure 2 shows one embodiment of an assay of the invention useful to measure viral entry and attachment in cells using luciferase as an enzymatic reporter. Panel A shows a schematic diagram of a VSV construct encoding a Renilla luciferase-phosphoprotein fusion polypeptide (REN-P) of the invention and a scheme for a Renilla luciferase assay. Panel B shows a graph of relative light units (RLU) as a function of time (minutes) observed in a screening assay using a virus comprising a REN- P construct of the structure illustrated in panel A. Panel C shows a graph of RLU as a function of time (minutes) observed in a screening assay using a virus comprising a REN-P construct of the structure illustrated in panel A. Bafilomycin-A (BAF); cyclohexamide (CHX).

Figure 3 shows other embodiments of an assay of the invention useful to measure viral entry and attachment using a fluorescent reporter or an enzymatic reporter. Panel A is a schematic illustration of select embodiments of the genomes of LUC and eGFP reporter viruses of the invention. Panel B is an epifluorescence micrograph of HeIa cells infected with VSV-eGFP, 7 hours prior to imaging. Panel C is a bar graph of the percent cells GFP+ as a function of the MOI (pfu) where cells were infected at an MOI varying from 0.01 to 60 and the percent of eGFP expressing cells (GFP+) was determined by flow cytometry. Panel D is a graph of the RLU (1000's) as a function of MOI (pfu) where luciferase activity was quantified following lysis of cells and measuring relative luminescence.

Figure 4 shows a schematic diagram of an embodiment of an assay of the invention useful to study the entry of enveloped viruses. Reporter virus lacking the G gene but expressing luciferase (REN-P) is useful to infect BHK cells expressing any desired envelope protein from an expression plasmid (pEnv). Most envelopes are efficiently incorporated into the surface of budding virus particles which can be used to infect fresh cells employing the entry mechanism conveyed by the foreign envelope protein. Lymphocytic choriomemingitis (LCMV); Nipah (NiV); respiratory syncytial (RS); Ebola (EBO); Marburg (MBG); West Nile (WNV).

Figure 5 shows a schematic diagram of an embodiment of an assay of the invention useful to study the entry of enveloped viruses. Reporter virus lacking the G gene but expressing eGFP is useful to infect BHK cells expressing any desired envelope protein from an expression plasmid (pEnv). Most envelopes are efficiently incorporated into the surface of budding virus particles which can be used to infect fresh cells employing the entry mechanism conveyed by the foreign envelope protein. Lymphocytic choriomemingitis (LCMV); Nipah (NiV); respiratory syncytial (RS); Ebola (EBO); Marburg (MBG); West Nile (WNV).

Figure 6 shows one embodiment of a viral attachment and internalization assay. Panel A shows an image of an SDS-PAGE gel illustrating virus bound to CHO cells in a viral attachment assay. The viral proteins are indicated at the left of the gel, alongside the marker lane which represents input virus (I). Phosphoimage analysis shows that approximately 4% of the input inoculum binds cells. Panel B shows an image of an SDS-PAGE gel illustrating the assay of viral internalization in CHO cells. The cells were treated with pro K to remove unbound virus. To measure internalization, cells were warmed to 37°C prior to pro K treatment. Proteinase K= pro K

Figure 7 shows an enzymatic assay for entry fusion and uncoating of virus. Panel A shows a schematic diagram of VSV-REN-P genome, depicting the location of the REN-P fusion. Panel B shows SDS-PAGE gel analysis of purified VSV (rVSV) and VSV-REN-P. Panel C is a graph of RLU as a function of viral titer showing purified VSV-REN-P contains a functionally active renilla luciferase. Different amounts of virus were treated with dual glow luciferase reagent (Promega) and RLU measured using a luminometer. Panel D shows a graph of RLU as a function of time in minutes. CHO-cells were infected with VSV-REN-P at an MOI of 25 in the presence of puromycin (+PUR) or ammonium chloride (+NH₄Cl) as indicated, and renilla levels monitored at 15-30 min intervals by conversion of Enduren. Panel E shows a graph of RLU as a function of MOI (pfu) demonstrating that Renilla activity depends on input inoculum. MOI was varied from 0-50 and RLU measured at 300 min pi. RLU = relative luminescence. PFU = plaque forming units.

Figure 8 shows a graph of RLU as a function of MOI (pfu) prepared with data obtained using an enzymatic assay to study viral attachment. VSV-REN-P was bound to cells on ice for 2h, unbound virus was removed and cell associated RLU determined following lysis of the cell associated virus.

DETAILED DESCRIPTION OF THE INVENTION

The invention relates to novel viral compositions and their use. The invention provides modified vesicular stomatitis virus (VSV) compositions which are useful in methods for rapidly detecting viral attachment and entry as well as in methods of rapidly identifying new antiviral therapeutics. The patent and scientific literature referred to herein establishes the knowledge that is available to those with skill in the art. All United States patents and published or unpublished United States patent applications cited herein are incorporated by reference. All published foreign patents and patent applications cited herein are hereby incorporated by reference. All other published references, documents, manuscripts and scientific literature cited herein are hereby incorporated by reference.

In one aspect, the invention provides a modified VSV that comprises a reporter molecule. In one embodiment, the modified VSV comprises a reporter molecule fused to a viral protein, or fragment thereof. In one embodiment, the size of a viral protein fragment that is fused to a reporter molecule of the invention is at least about 1% of the length of the mature wild-type viral polypeptide sequence. In one embodiment, the size of a viral protein fragment that is fused to a reporter molecule of the invention is at least about 10% of the length of the mature wild-type viral polypeptide sequence. In one embodiment, the size of a viral protein fragment that is fused to a reporter molecule of the invention is at least about 25% of the length of the mature wild-type viral polypeptide sequence. In one embodiment, the size of a viral protein fragment that is fused to a reporter molecule of the invention is at least about 50% of the length of the mature wild-type viral polypeptide sequence. In one embodiment, the size of a viral protein fragment that is fused to a reporter molecule of the invention is at least about 75% of the length of the mature wild-type viral polypeptide sequence. In one embodiment, the size of a viral protein fragment that is fused to a reporter molecule of the invention is at least about 90% of the length of the mature wild-type viral polypeptide sequence. In one embodiment, the viral protein may also be a derivative or homologue of wild-type viral protein. That is, the fusion protein may contain a derivative or homologue of wild-type viral, protein comprises at least one substitution, deletion or addition of amino acid(s) relative to the wild- type viral protein. The VSV can be used to monitor attachment of such a virus to a cell, and/or entry (internalization arid fusion) of such a virus into such a cell and/or uncoating of such a virus and/or replication of such a virus. The modified VSV can be produced, for example, from recombinant DNA constructs engineered to encode a reporter molecule fused to a viral protein, or fragment thereof. The constructs are then incorporated into suitable cloning plasmids or vectors that are transfected into host cells and virus recovered. Suitable vectors are known in the art and commercially available. The host cells express or otherwise produce the recombinant VSV particles that can be purified by a variety of methods known in the art.

Therefore, in accordance with the present invention there may be employed conventional molecular biology, microbiology, and recombinant DNA techniques within the skill of the art. Such techniques are explained fully in the literature. See, e.g., Maniatis et al, "Molecular Cloning: A Laboratory Manual," (1989); Ausubel, Ed., "Current Protocols in Molecular Biology," Volumes I-III (1994); Celis, Ed., "Cell Biology: A Laboratory Handbook," Volumes I-III (1994); Coligan, Ed., "Current Protocols in Immunology," Volumes I-III (1994); Gait, Ed., "Oligonucleotide Synthesis" (1984); Hames et al, Eds., "Nucleic Acid Hybridization" (1985); Hames et al, "Transcription and Translation" (1984); Freshney, Ed., "Animal Cell Culture" (1986); IRL Press, "Immobilized Cells and Enzymes" (1986); and Perbal, "A Practical Guide To Molecular Cloning" (1984). The invention provides that the nucleic acid encoding the reporter molecule may be positioned at any location within the viral genome which is advantageous for reporter molecule expression.

Li one embodiment, the virus is infectious and includes the structural proteins necessary for attachment, entry, uncoating and, optionally, replication and/or viral packaging. However, it may be desirable to modify (or attenuate) the virus, or its genome, to limit its ability to replicate and/or package viral particles after the initial viral entry. For example, this could be achieved by deleting genes that are essential for replication such as the viral polymerase gene (L), and/or the nucleocapsid protein (N) or by deleting the attachment protein gene (G). .

VSV has a non-segmented negative-stranded RNA genome of 11,161 nucleotides that encode five viral proteins: The nucleocapsid protein (N), the phosphoprotein (P), the matrix protein (M), the glycoprotein (G) and the RNA-dependent RNA polymerase (L). The replication cycle (Figure 1) should be considered a continuum of events, however, it can be divided into three stages which include, attachment, entry and uncoating; gene expression; and assembly and budding.

The invention provides a recombinant DNA construct encoding a fusion protein, wherein said fusion protein comprises a reporter molecule fused to a VSV polypeptide selected from the group consisting of: phosphoprotein, nucleocapsid protein, matrix protein, glycoprotein, and large polymerase subunit. In one embodiment, a DNA construct of the invention comprises a gene encoding a reporter molecule fused to the gene encoding the VSV phosphoprotein (P). One of ordinary skill in the art will understand that the term "reporter molecule" refers to a type of protein the presence or amount of which can be assayed or detected, or one which has an activity that can be measured and/or detected. The term "activity" as it is used in the instant invention refers to a function of a protein or enzyme that can be assayed (e.g., catalytic activity, binding to a second protein, ligand or other target, selection marker etc.). One of skill in the art will appreciate that a single protein or enzyme can perform one or a plurality of activities and, thus, is not necessarily limited to a single activity. Further, the activity of a protein may be linked to one or more specific physical regions, domains and/or amino acid sequences on the protein, or a combination of one or more thereof. Also, by joining proteins together, either naturally as with multi-subunit protein complexes or pairs or other multiples of binding partners (e.g., as with transcription factors, receptor-ligand pairs, etc.), or artificially, as with recombinant "fusion proteins" or other covalently linked proteins (i.e., by chemical crosslinking agents, peptide linkers, etc.), different activities can be combined.

Some reporter molecules are enzymes, which can be detected by introducing colorimetric or fluorigenic substrates, i.e., substrates that produce a detectable color or emit a fluorescence signal when acted upon by the reporter (e.g., firefly luciferase, a reporter, catalyzes the cleavage of a fluorescence-emitting substrate such as luciferin). Enzymes that can be used as reporter genes can include oxidases, luciferases, peroxidases (such as horseradish peroxidase) - peptidases (such as caspase-3), glycosidases (such as beta-galactosidase) and phosphatases (such as alkaline phosphatase). Reporters can also include fluorescent proteins, such as green fluorescent protein (GFP), the fluorescent signal of which can be detected directly following illumination with ultraviolet light. Red fluorescent protein or Reef Coral protein can also be used. It will be appreciated by one of ordinary skill in the art that novel, naturally-occurring fluorescent proteins can be isolated from naturally-occurring fluorescence organisms, such as many species of ocean coral and other deep-sea life. It also will be appreciated that novel fluorescent proteins can be engineered (e.g., based on the properties of known fluorescent proteins, for example to fluoresce at a different frequency) by methods known in the art. It will also be appreciated that fluorescent and other reporters useful in the invention, and, in the case of reporter proteins, their corresponding amino acid sequences, the nucleotide sequences that encode them and cloning tools, such as, reporter-encoding vectors, can be obtained from public, non-commercial or commercial sources. Fluorescence can also be provided by fusing the viral protein to peptides, such as those that bind to lanthanide.

Preferred reporter molecules of the invention include, but are not limited to, e.g., Renilla luciferase or eGFP. Other reporter molecules include selectable markers such that cells in which the viral payload is delivered to can be selected using chemicals. For example one could select for neomycin resistance. This would allow the screening of libraries for permissiveness factors for viral attachment, entry, and fusion. In general, the modified virus contains at least one cell (or cellular) attachment protein and, preferably, further comprises a nucleic acid molecule that encodes the cell attachment protein. In a preferred embodiment, the cell attachment protein is a viral glycoprotein, such as the VSV glycoprotein.

In one embodiment, the genome of the virus contains deletions of one or more genes encoding structural proteins of VSV. For example, the gene encoding the VSV glycoprotein (G), the attachment protein of VSV, is deleted or replaced with a heterologous attachment protein. Pseudotyping of the VSV by inserting into the genome or by complementing in producer cells in trans with other, non-VSV viral glycoproteins is, thus, possible. Pseudotyping of VSV with other viral glycoproteins is particularly useful in the investigation of viral attachment and of viral entry pathways and particularly the investigation of inhibitors of viral attachment and/or entry in a high throughput screening format of the source virus without incurring the risks or hazards of the source virus. As used herein "pseudotyping" means that the attachment protein G of VSV is not present in the particle and is instead replaced by one or more foreign envelope proteins. These foreign envelope proteins can be derived from other enveloped viruses or from bacteria, cells, parasites, or other proteins that mediate binding to eukaryotic membranes including through the recognition of specific cellular receptors or markers. Such replacement may be effected by methods known in the art, including, without limitation, homologous recombination between viral genomes (e.g., if two or more different viruses are co-cultured in a cell or cells), recombinant DNA techniques, in vitro long DNA synthesis (i.e., chemical or enzymatic synthesis), and the like.

Examples of viruses useful in pseudotyping include but are not limited to: Retro viridae (e.g. human immunodeficiency viruses, such as HIV-I (also referred to as HTLV-III, LAV or HTLV- πi/LAV, or HTV-III; and other isolates, such as HIV-LP); Togaviridae (e.g., equine encephalitis viruses, rubella viruses); Flaviridae (e.g., dengue viruses, encephalitis viruses, yellow fever viruses); Coronoviridae (e.g., coronaviruses); Rhabdoviradae (e.g., vesicular stomatitis viruses, rabies viruses); Filoviridae (e.g., ebola viruses); Paramyxoviridae (e.g., parainfluenza viruses, mumps virus, measles virus, respiratory syncytial virus); Orthomyxoviridae (e.g., influenza viruses); Bunyaviridae (e.g., Hantaan viruses, bunya viruses, phleboviruses and Nairo viruses); Arena viridae (hemorrhagic fever viruses); Herpesviridae (herpes simplex virus (HSV) 1 and 2, varicella zoster vims, cytomegalovirus (CMV), herpes virus; and Poxviridae (variola viruses, vaccinia viruses, pox viruses). Other viruses suitable for pseudotyping VSV include, but are not limited to, negative-strand viruses such as Respirovirus, Morbillivirus, Rubulavirus, Henipavirus Avuvlavirus, Pneumovirus, Metapneumovirus, Lyssavirus, Ephemerovirus, Marburgviras, Ebolavirus, Bornavirus, Influenzavirus A, Influenzavirus B, Influenzavirus C. Other viruses include but are not limited to, Orthobunyavirus, Hantavirus, Nairovirus, Phlebovirus, or Arenavirus, flaviviruses (Hepatitis C, West Nile, Dengue), Coronaviruses (SARS), Togaviruses (EEE, Rubella), Retroviruses: (HTV), DNA viruses: (Herpes simplex, EBV).

In another aspect of the instant invention, high-throughput methods for screening compounds which can modulate the ability of the modified virus to attach and enter a cell. Where the modified virus contains a viral attachment protein, the invention includes methods for screening antiviral therapies using the modified VSV of the present invention. In one embodiment, the high-throughput methods are cell-based assays, which measure the reporter gene activity, such as GFP or Renilla luciferase, in the presence of a potential antiviral therapeutic, such as a viral inhibitor. Microscopic detection of cells in cell-based assays can be used employing known scanning and data analysis instrumentation, which allows localized detection of a signal, such as to the outside of cells, the cytoplasm, etc., or may detect cell-to-cell spreading of signal indicative of viral release and reinfection. Such cell-based approaches for screening therapeutics, such as the methods provided by the instant invention, screen for potential therapeutics against various activities required during the normal viral infection cycle in a simultaneous manner.

For example, employing a modified VSV which contains a flavivirus cellular attachment protein can be used to screen agents or candidates for antiflaviviral therapy, e.g., an inhibitor of viral attachment, entry, fusion and uncoating.

Thus, the invention includes a method of screening for modulators of virus infection, comprising the steps of: a) contacting at least one cell sample, with at least one candidate agent; b) exposing said cell sample(s) to the virus of the invention; and c) assaying said cell samples to detect the activity of the reporter molecule; wherein the absence of reporter molecule activity in said cell is indicative of antagonist activity of cell attachment and/or entry and/or fusion and/or uncoating by the candidate agent and/or the presence of reporter molecule activity in said cell is indicative of agonist activity of cell attachment. In a preferred embodiment, the method further comprises a control cell sample. Alternatively, the reduction of reporter molecule activity in said cell contacted with at least one candidate agent relative to the level of reporter molecule activity observed in a control cell not contacted with at least one candidate agent is indicative of antagonist activity of cell attachment and/or entry and/or fusion and/or uncoating by the candidate agent. Further, the increase of reporter molecule activity in said cell contacted with at least one candidate agent relative to the level of reporter molecule activity observed in a control cell not contacted with at least one candidate agent is indicative of agonist activity of cell attachment and/or entry and/or fusion and/or uncoating by the candidate agent.

The invention also provides a method of screening for modulators of cell attachment, comprising the steps of: a) providing at least one control cell sample; b) providing at least one test cell sample; c) contacting the at least one test cell sample of step (b), with at least one candidate agent; d) infecting at least one control cell sample of step (a) and at least one the test cell sample of step (c) with a virus of the invention; and e) assaying said cell samples to detect the activity of the reporter molecule, wherein a reduction of the level of reporter molecule activity in the test cell sample contacted with at least one candidate agent relative to the level of reporter molecule activity observed in a control cell sample not contacted with at least one candidate agent is indicative of antagonist activity of cell attachment by the candidate agent and wherein an increase in the level of reporter molecule activity in said cell sample contacted with at least one candidate agent relative to the level of reporter molecule activity observed in the control cell not contacted with at least one candidate agent is indicative of agonist activity of cell attachment.

As used herein, the term "modulator" includes inhibitors and activators, e.g., agonists. Inhibitors, e.g., antagonists, are agents that, e.g., bind to, partially or totally block, decrease, prevent, or delay viral attachment and/or entry and/or fusion and/or uncoating of the virus to, or within, a target, e.g., a cell. Activators, e.g., agonists, are agents that, e.g., bind to, stimulate, increase, open, activate, facilitate, or enhance viral attachment and/or entry and/or fusion and/or uncoating of the virus to, or within, a target, e.g., a cell. Modulators include compounds (agents) such as proteins, e.g., antibodies, peptides, lipids, carbohydrates, polysaccharides, nucleic acids (e.g., RNA or DNA) or combinations of the above, e.g., lipoproteins, glycoproteins, and the like. Modulators also include genetically modified versions of a naturally-occurring polypeptide or nucleic acids, e.g., with altered activity, as well as naturally-occurring and synthetic ligands, antagonists, agonists, small chemical molecules and the like. i

The invention also provides a method of screening for inhibitors of viral infectivity, comprising the steps of: a) providing at least one control cell sample; b) providing at least one test cell sample; c) contacting the at least one test cell sample of step (b), with at least one candidate inhibitor of viral infectivity; d) infecting the at least one control cell sample of step (a) and the at least one the test cell sample of step (c) with a virus of the invention; and e) assaying the cell samples to detect the activity of the reporter molecule, wherein a reduction in the level of reporter molecule activity in the the at least one test cell sample contacted with at least one candidate inhibitor relative to the level of reporter molecule activity observed in the at least one control cell sample not contacted with at least one candidate inhibitor is indicative of inhibition activity of the candidate inhibitor.

Similarly, a virus which substitutes a heterologous cellular attachment protein in place of (or in addition to) the VSV G protein can be used to screen for modulators of the interaction of the heterologous protein with the cellular membrane to which it attaches. In this embodiment, products that can antagonize or agonize the attachment and/or entry and/or fusion and/or uncoating of the virus to the cell can be screened.

The embodiments of the instant invention that comprise detection of fluorescence signals, such as from GFP or luciferase, can be practiced using live cells or fixed cells. The choice of fixed or live cell screens depends on the specific cell-based assay required. Methods for high throughput screening of live or fixed cells by fluorescence-based technologies are generally known in the art.

In one aspect, the invention provides a radiometric assay virus binding assay which is useful to measure viral attachment and internalization. The radioisotopes useful in radiolabeling the virus, e.g., VSV, include, but are not limited to, iodine, tritium, sulfur, phoshorus and technetium. In one embodiment a radiolabeled virus is prepared by incubating infected cells with ³⁵S-methionine. Radiolabeled virus may be purified by standard methods known in the art.

The invention provides a method of screening for modulators of virus infection, comprising the steps of: a) contacting at least one cell sample, with at least one candidate agent; b) exposing said cell sample(s) to the radiolabeled virus of the invention; and c) assaying said cell samples to detect the binding or internalization of the radiolabeled virus to said cell; wherein the reduction of the level of radioactivity in said cell relative to the level of radioactivity observed in a cell in the absence of the at least one candidate agent is indicative of antagonist activity of cell attachment and/or entry and/or fusion and/or uncoating by the candidate agent and/or an increase in the level of radioactivity in said cell relative to the level of radioactivity observed in a cell in the absence of the at least one candidate agent is indicative of agonist activity of cell attachment. In a preferred embodiment, the method further comprises a control cell sample.

The assay methods of the present invention may be applied to test one sample alone or more than one sample individually or in parallel. The assay methods of the present invention are useful in standard and high throughput screening application. The assay methods of the invention may be adapted to multiple formats which include, but are not limited to, 24, 96, and 384 multiwell format.

DIAGNOSTIC APPLICATIONS

In another aspect, the invention provides methods useful for the detection of agents present in a biological sample derived from a subject that bind a viral composition of the invention. Such methods are useful to identify a subject that has been contacted with a virus. A "subject," as used herein, is preferably a mammal, such as a human, but can also be an animal, e.g., domestic animals (e.g., dogs, cats and the like), farm animals (e.g., cows, sheep, pigs, horses and the like) and laboratory animals (e.g., rats, mice, guinea pigs and the like). In one embodiment, the agent is a VSV immunoreactive polypeptide, or fragment thereof such as an antibody, or fragment thereof, that binds one or more components of a viral composition of the invention. As used herein, the term "antibody" means a polypeptide comprising a framework region from an immunoglobulin gene or fragments thereof that specifically binds and recognizes an antigen, e.g., a viral polypeptide. Use of the term antibody is meant to include whole antibodies, including single-chain whole antibodies, and antigen- binding fragments thereof. As used herein, the term "biological sample" means sample material derived from or contacted by living cells. The term "biological sample" is intended to include tissues, cells and biological fluids isolated from a subject, as well as tissues, cells and fluids present within a subject. Biological samples of the invention include, e.g., but are not limited to, whole blood, plasma, semen, saliva, tears, urine, fecal material, sweat, buccal, skin, cerebrospinal fluid, and hair. Biological samples can also be obtained from biopsies of internal organs or from cancers. Biological samples can be obtained from subjects for diagnosis or research or can be obtained from undiseased individuals, as controls or for basic research.

The invention also provides a means of screening samples, e.g., biological sample, for antibodies, e.g., VSV immunoreactive polypeptides, against proteins that are incorporated onto the surface of a viral composition of the invention. Samples can be screened for the presence of antibodies that will modulate the attachment and/or internalization of virus into cells by incubation of virus with the sample prior to exposure to cells and monitoring reporter activity.

In one embodiment, the invention provides a method for determining the presence or amount of a VSV immunoreactive polypeptide in a biological sample, comprising the steps of: a) providing at least one control cell sample; b) providing at least one test cell sample; c) contacting at least one test cell sample of step (b), with a biological sample; d) infecting at least one control cell sample of step (a) and at least one test cell sample of step (c) with a virus of the invention; and e) assaying the cell samples of step (d) to detect the activity of the reporter molecule, wherein a reduction of the level of reporter molecule activity in the at least one test cell sample contacted with the biological sample relative to the level of reporter molecule activity observed in the at least one control cell sample not contacted with the biological sample is indicative of the presence of VSV immunoreactive polypeptide.

In one embodiment, the invention provides a method for determining the presence or amount of a VSV immunoreactive polypeptide in a sample, the method comprising: a) providing the sample; b) contacting the sample with a VSV of the invention; and c) determining the presence or amount of VSV of the invention bound to a VSV immunoreactive polypeptide, thereby determining the presence or amount of the VSV immunoreactive polypeptide in the sample.

The methods of the invention are conducted under conditions suitable for binding a VSV immunoreactive polypeptide and a VSV of the invention. The amount of binding of a VSV of the invention observed in a sample is compared with a suitable control, which can be the amount of binding in the absence of the sample, or the amount of the binding in the presence of non-specific immunoglobulin composition, or both. A 1% increase in the level of binding of a VSV of the invention relative to the level of VSV binding in the control indicates the presence of VSV immunoreactive polypeptide in the sample. The amount of binding can be assessed by any suitable method. Binding assay methods include, e.g., ELISA, enzyme assay, fluorescence assay, cell based assay, and the like. In one embodiment, the presence of VSV immunoreactive polypeptide in a sample indicates that a subject has been contacted with a virus. The subject may have been contacted with a VSV or another virus which bears a component, e.g., coat protein, bound by the immunoreactive polypeptide.

KITS

The methods described herein can be performed, e.g. , by utilizing pre-packaged kits comprising at least one modified VSV composition described herein, which can be conveniently used, e.g., in clinical settings to diagnose subjects exhibiting symptoms or family history of a disease or illness involving a viral infection, or for screening of modulators of viral attachment and/or, entry and/or, fusion and/or, uncoating. In once embodiment, the kit further comprises instructions for use of the at least one modified VSV composition described herein for diagnosis or screening of modulators of viral attachment and/or, entry and/or, fusion and/or, uncoating. In one embodiment, the kit further comprises living or fixed cells.

A better understanding of the present invention and of its many advantages will be had from the following examples, which further describe the present invention and given by way of illustration. The examples that follow are not to be construed as limiting the scope of the invention in any manner, hi light of the present disclosure, numerous embodiments within the scope of the claims will be apparent to those of ordinary skill in the art.

EXAMPLES

EXAMPLE 1. STUDIES USING AN ASSAY OF THE INVENTION USEFUL TO MEASURE VIRAL ENTRY AND ATTACHMENT IN CELLS USING LUCIFERASE AS AN ENZYMATIC REPORTER

Recombinant virus in which the reporter gene Renilla luciferase (REN) was fused to the VSV phosphoprotein gene (P) was prepared such that a fusion protein REN-P was expressed. The virus VSV::ren-P was viable and replicated to high titer. The modified virus can be used to generate viral pseudotypes. Recombinant VSV constructed with the reporter molecule fusion protein can monitor infection of live cells in real time (Figure 2, panel B, VSV). VSV attachment and entry has been successfully monitored in Chinese hamster ovary cells, Baby Hamster Kidney cells, Vero cells, HeIa cells and 293 cells. New viral protein synthesis was inhibited in these cells, and just the input virus particles were detected (Figure 2, panel B, VSV + CHX). Bafilomycin-A (BAF), a chemical that inhibits endosome acidification, prevented VSV fusion (Figure 2, panel B, BAF). Indeed, no luciferase activity was observed above background level in cells treated with BAF (Figure 2, panel B, BAF). Thus, the signal monitored in this assay reflected viral entry, fusion and disassembly.

The quantitative nature of the viral assay system was demonstrated in Figure 2 (panel C), where the number of INPUT infectious particles per cell was varied from 1- 50 in the presence of cyclohexamide. As shown in Figure 2, panel C, the level of luciferase expression in treated cells was positively correlated with the number of input infectious particles. It is noteworthy that the VSV attachment glycoprotein G from this virus was deleted to generate a recombinant that was readily pseudotyped by foreign membrane proteins. The assay of the invention can be performed rapidly. Note that in the graphs in Figure 2, panel B and panel C, the X axis is expressed in minutes. Thus, an assay of the invention is a robust, quantitative and sensitive high throughput method useful to expressed screen for inhibitors of viral attachment, entry, fusion and uncoating.

EXAMPLE 2: STUDIES USING ASSAYS OF THE INVENTION USEFUL TO MEASURE VIRAL ENTRY AND ATTACHMENT IN CELLS USING A FLUORESCENT REPORTER OR ENZYMATIC REPORTER

VSV replication was monitored in HeIa cells by measuring the expression of a viral reporter gene encoding either a fluorescent reporter polypeptide (e.g., eGFP) or an enzymatic reporter (e.g., firefly luciferase). Specifically, recombinant VSV was engineered to express either fluorescent reporter genes such as GFP and RFP or enzymatic reporters such as Renilla and Firefly luciferase. Each reporter gene was under the control of VSV specific transcriptional regulation signals, thus directly linking reporter gene expression to viral gene expression. For maximum expression, the reporter gene was inserted between the leader and N genes of an infectious cDNA clone of VSV (Figure 3, panel A). Epifluorescence of HeIa cells infected with VSV-eGFP, 7 hours prior to imaging is shown in the micrograph in Figure 3, panel B.

To demonstrate that levels of reporter gene expression correlate with dose of infectious virus, the amount of input virus was varied from an MOI of 0.01 to 60. The % of eGFP positive cells was measured by FACS in cells infected with the VSV modified to express eGFP (Figure 3, panel C). The level of luciferase expression in cell infected with VSV modified to express luciferase was measured using a luminometer (Figure 3, panel D). The data presented in both Figure 3, panel C and Figure 3, panel D show that the level of reporter gene expression correlates with virus titer. These assays readily lend themselves to use in HTS assays allowing assessment of virus replication by microcopy using an autoscope, or by luminescence using a luminometer. EXAMPLE 3: MEASUREMENT OF VIRAL INFECTION OF CAENORHABDITIS ELEGANS CELLS USING AN ASSAY OF THE INVENTION

A reporter virus in which eGFP was fused to the amino terminus of the P protein was prepared and studied. This virus was used to show that VSV can infect Caenorhabditis elegans as detailed by Schott et ah, An antiviral role for the RNA interference machinery in Caenorhabditis elegans. Proc. Natl. Acad Sci. USA, 102: 18420-4 (2005).

Materials and Methods:

Recombinant VSV encoding enhanced GFP-P fusion (rVSV::eGFP-P).

A plasmid encoding the rVSV::eGFP-P genome was constructed in several steps:

A. DNA fragments corresponding to the open reading of eGFP and nucleotides 121-1395 and 1399-3841 of the VSV genome were amplified by polymerase chain reaction (PCR) from peGFP-Nl (Clontech technologies, Palo Alto, CA) and the full-length cDNA clone of VSV (pVSVl(+))(Whelan, S. P., Ball, L. A., Barr, J. N., & Wertz, G. T. (1995) Proc. Natl. Acad. Sci. USA 92, 8388-8392), respectively. The three fragments were fused together by PCR and the product ligated into pGEM-T using the pGEM-T Easy Vector System (Promega Corporation, Madison, WI).

eGFP primers:

5'-gaaaaaaactaacagatatcatggtgagcaagggcg-3' (SEQ ID NO.:1), 5'-cttttgtgagattatccttgtacagctcgtccatg-3' (SEQ ID N0.:2) VSV 121-1395 primers: 5'-gcaaatgaggatccagtgg-3' (SEQ ID NO.:3) 5'-cgcccttgctcaccatgatatctgttagtttttttc-3' (SEQ ID NO.:4) VSV 1399-3841 primers:

5'-catggacgagctgtacaaggataatctcacaaaag-3' (SEQ ID NO.: 5), 5'-atctcgaaccagacacctg-3' (SEQ ID N0.:6)

B. The resulting plasmid and pSWINT2 (a plasmid encoding nts 1-3866 of the full-length VSV genome) were digested with BstZ17I and Xbal, followed by ligation of the fragment containing the eGFP-P gene into pSWINT2 (pSWINT2-eGFP).

C. pVSVl(+) was digested with Avrll, SpM, and BgII, and the 7702 bp fragment (corresponding to bps 3717-11418 of pVSVl(+)) was ligated into the Avrll and Sphl sites of pSWINT2-eGFP-P. D. The rVSV::eGFP-P was recovered from plasmid DNA and working stocks were prepared essentially as described in Whelan, S. P., Ball, L. A., Barr, J. N., & Wertz, G. T. (1995) Proc. Natl. Acad. ScL USA 92, 8388-8392. Since the ability of virus stocks to infect C. elegans cells was observed to deteriorate much faster than their ability to infect mammalian cells at 4° C, fresh virus stocks were divided into small aliquots and stored at -70° C.

Plaque Assays. Confluent Vero African green monkey kidney cells in 3 cm wells were exposed to dilutions of virus in 200 μl medium for 1 hour at 37° C with repeated shaking. The cells were then overlaid with 3 ml medium containing 0.25% low gelling temperature agarose. After 30-40 hours incubation at 34° C, the cells were fixed in 10% formaldehyde for 1 hour, the block of medium was removed, and the cells were stained with 0.05% crystal violet in 10% ethanol. Medium from uninfected worm cells produced no plaques. Note that multiplicities of infection based on numbers of green fluorescent foci and spots in both wild-type and rde-l(ne219) worm cell cultures infected with dilute virus were approximately 1/50* of those measured by plaque assay of mammalian cells.

C. elegans Cell Culture. C. elegans embryonic cell isolation and cell culture were performed much as described in Christensen, M., Estevez, A., Yin, X., Fox, R., Morrison, R., McDonnell, M., Gleason, C, Miller, D. M. 3rd, & Strange, K. (2002) Neuron 33, 503-514, with some modifications. In order to prevent bacterial contamination of C. elegans cells, the synchronized worms were cultured in synthetic minimal (S) medium containing E. coli food. The Gravid worms were purified by flotation on ice-cold 30% sucrose to remove fecal matter and debris before bleaching. Halfway through the bleaching, the eggs were transferred to fresh sterile centrifuge tubes in a laminar flow hood and where all subsequent manipulations were performed.

The cells were seeded at 3 x 10⁶ cells/cm² (as determined by counting of Hoechst 33342- stained nuclei on a hemacytometer) in 8-well chambered coverglasses coated with peanut lectin. The cells were incubated at 15° C for storage (up to 20 hours) and 27° C for experiments in a sealed humid chamber. We infected C. elegans cells by replacing the culture medium in each 0.64 cm² well with 200 μl culture medium containing an appropriate dilution of virus, incubating at 27° C for one hour, followed by washing the well one time with fresh medium, then adding 200 μl fresh medium.

GFP Measurement. Unless otherwise stated, cells were incubated at 27° C for 7 days postinfection. Following incubation, the medium was aspirated from the cells and the cells were fixed in 2% formaldehyde in phosphate-buffered saline (PBS) for ten minutes at room temperature. The cells were then rinsed once in PBS and stained for DNA 10 minutes in PBS containing Hoechst 33342 dye. The cells were then rinsed 2 more times in PBS. The cells were imaged in a widefϊeld epifluorescence microscope mounted with a CCD camera. To measure relative amounts of GFP and DNA, linear transect of image pairs of 0.3 x 0.2 mm fields across each well were collected. For each field, we focused on the nuclei in the Hoechst 33342 fluorescence channel, collected an image, and then collected an image blind from the GFP fluorescence channel. Exposure times were set to avoid saturation of pixels by the brightest cells, such that the sum of the recorded image was linearly related to the amount of light reaching the camera. To determine background signal for the Hoechst 33342 and GFP channels, data for unstained and uninfected cells, respectively, were also collected. The filter set used allowed little or no detectable bleedthrough between fluorescence channels. No green fluorescence was visible by eye in uninfected cells, but background signal in the GFP channel due to stray light and fluorescence in the microscope optics (estimated by imaging an empty well), and background signal due to cellular autofluorescence (estimated by imaging uninfected cells; by eye the autofluorescence is distinct from GFP due to the difference in color), were 11% and 8% respectively, of the total captured light after a typical 7-day infection of wild type cells at MOI = 3. Uninfected controls were used in each experiment to correct GFP values for this background signal.

VSV (-) Strand Quantitation. The relative concentrations of VSV (-) strand RNA in C. elegans cell cultures were measured by quantitative PCR of cDNA. Invitrogen ThermoScript™ reverse transcriptase kit was used to make cDNA from RNA extracted from infected C. elegans cells. The Qiagen Quantitect™ SYBR Green PCR kit was used for cDNA amplification. To minimize spurious detection of (-) strand copies of viral transcripts produced by C. elegans RNA-directed RNA polymerase activity, the reverse transcriptase primer (primer VSV P/M forward) were designed such that the cDNA would need to span both the P-M and M-G intergenic regions in order to produce a PCR product with the PCR primers used (primers VSV M/G forward and VSV G/M reverse). Serial dilutions of RNA extracted from infected rde-l(219) cells were compared to RNA extracted from uninfected cells.

VSV P/M forward primer: 5'-tcctgctcggcctgagatac-3' (SEQ K) NO.:7) VSV M/G forward primer: 5'-tcctggattctatcagccactt-3' (SEQ K) NO.: 8) VS V G/M reverse primer; 5'-ccagtttcctttttggttgtgt-3' (SEQ K) NO.:9) Results and Discussion

VSV Infects C. elegans Cells. The VSV genome consists of a (-) sense single-stranded RNA molecule. VSV were engineered to express a fusion of enhanced GFP with the viral phosphoprotein (P). In C. elegans cell culture, the first brightly green fluorescent cells appeared 20 to 24 hours after exposure to virus at a multiplicity of infection (MOI) of 3 (as measured by plaque assay on mammalian cells, see note in Materials and Methods), and fluorescence continued to increase until a peak at roughly seven days post-infection. The VSV P protein is a cofactor for the viral polymerase and a structural component of the virion. To confirm that the fluorescence observed was due to viral gene expression, cells were exposed to the protein synthesis inhibitor cycloheximide at 24 hours postinfection. Such treatment inhibited the further accumulation of green fluorescence, indicating that the viral GFP fusion protein gene was being transcribed and translated in the worm cells (data not shown). To test whether virus was capable of spreading among cells in the culture, infected cells were treated with a neutralizing antibody targeted to the viral attachment glycoprotein (G). This restricted spread of the infection. Spread of virus among cells in the culture is apparent on infection of cells at an input multiplicity of 0.003.

EXAMPLE 4: PSEUDOTYPING ASSAYS OF THE INVENTION USEFUL TO SCREEN FOR INHIBITORS OF THE ATTACHMENT AND ENTRY PATHWAYS OF ENVELOPED VIRUSES.

The present invention provides modified VSV which are pseudotypes of other viruses useful to assay the infectivity thereof. As detailed in Figure 4 and Figure 5, such viral pseudotypes bear the attachment properties dictated by the foreign protein and can be used to perform rapid, high throughput screening for inhibitors of viral attachment and/or, entry and/or, fusion and/or, uncoating. In one embodiment, an important application of this virus is in the study of high-hazard viruses, which can be assayed safely through the use of pseudotypes of the invention.

Such a VSV-based pseudotyping assay system has been successfully exploited. Specifically, the VSV genome was modified by deletion of the G gene, and reporter genes inserted (see generally, Figure 4 and Figure 5). For example, virus bearing the construct shown in Figure 5 was amplified in cells expressing VSV G from transfected plasmids. Recombinant VSV bearing such constructs are useful to express the attachment proteins of other enveloped viruses by expressing them in trans from plasmid DNA. Such recombinant VSV can be successfully employed as pseudotypes for viral attachment. Indeed, such an assay system has been successfully used in a collaborative venture to evaluate the role of endosomal proteolysis in the entry pathway of Ebola virus (Chandran et ah, Endosomal proteolysis of the Ebola virus glycoprotein is necessary for infection. Science, 308: 1643- 5 (2005)). While other systems are known, (Takada et ah, A system for functional analysis of Ebola virus glycoprotein. Proc. Natl. Acad. Sci. USA, 94:14764-9 (1997)), at least one advantage of the assay system of the present invention is the use of P-fusion reporter viruses. These constructs permits analysis of entry in the absence of gene expression as the input particles themselves are fluorescent or contain functional reporter enzymes.

EXAMPLE 5: STUDIES USING A RADIOMETRIC ASSAY OF THE INVENTION

The invention provides a radiolabeled virus binding assay which is useful to measure viral attachment and internalization. Radioactive virus was generated in BHK cells and purified. Briefly, 10 x 10 cm plates of confluent BHK cells were infected with VSV at an MOI of 3, exposed to 5 ml of methionine (met) free media supplemented with [³⁵S]-met from 4 hours post-infection (h.p.i.)- At 12 h.p.i, cell culture media was collected, centrifuged at 10,000xg to pellet cell debris, filtered through a 0.45 μm filter and virus recovered by centrifugation (Ty 50 rotor at 21,000 rpm for 90 min at 4°C). The viral pellet was resuspended overnight on ice (500 μl NTE buffer, 100 mM NaCl, 10 mM Tris HCl pH 8.0, 1 mM EDTA) and layered onto a linear 15-45% sucrose gradient. Virus was separated by centrifugation, visualized using a hand held lamp, collected by side puncture and recovered following centrifugation through a 10% sucrose NTE cushion. This pellet was resuspended overnight in 400 μl of NTE buffer and virus titer determined. The radiolabeled virus binding assay is useful to identify small molecules that target the attachment step of viral infection.

An example of a virus attachment and internalization assay performed in CHO cells is shown in Figure 6. Cells were grown to an approximate density of 1 x 10⁶ cells well^"1 and shifted to 4°C to inhibit endocytosis. Virus was added at an MOI of 100 and allowed to bind on ice for 2h. The cells were then washed three times with ice cold PBS to remove unbound particles, and the quantity of cell associated virus determined by scintillation counting, or following SDS-PAGE and phosphoimage analysis (Figure 6, panel A). To measure internalization of virus, bound virus was treated with proteinase K (ProK) to liberate the cell-associated virus, or warmed rapidly to 37°C to permit internalization for 2 h, prior to ProK treatment. Internalized virus becomes resistant to Pro K treatment (Figure 6, panel B). Quantitative analysis of the relative radiolabeled virus in the presence and absence of ProK measures the efficiency of internalization, and demonstrated that > 90% of the attached virus was internalized, and that > 70% of the bound virus was removed by Pro K treatment (Figure 6, panel B).

EXAMPLE 6: STUDIES USING AN ASSAY OF THE INVENTION USEFUL TO MEASURE VIRAL ENTRY AND ATTACHMENT IN CELLS USING LUCIFERASE AS AN ENZYMATIC REPORTER

To monitor viral entry and uncoating a novel recombinant VSV was provided wherein the reporter enzyme renilla luciferase (REN) was fused to the viral P protein a structural component of the viral core, to generate VSV-REN-P (Figure 7, panel A). This virus replicates to titers similar to wild-type virus in cell culture, and packages the REN-P fusion protein into viral particles (Figure 7, panel B). Purified viral particles contain a functional renilla, and the activity of the enzyme is viral dose dependent (Figure 7, panel C). Importantly, these studies used Enduren ® (Promega), a modified substrate for REN that contains a blocking group that is only removed in the host cell cytoplasm. Using Enduren ® (Promega) it was demonstrated that a modified VSV of the invention can monitor infection of live cells in real time (Figure 7, panel D). Specifically, in a 96 well plate format, VSV-REN-P was used to infect Chinese hamster ovary (CHO) cells that were loaded with Enduren and treated with the protein synthesis inhibitor puromycin. Under these circumstances renilla activity reflects access of uncoated input particles and thus reports directly on fusion and uncoating. Consistent with this, inhibitors of endosome acidification, such as bafilomycin-Al (BAF- Al) or ammonium chloride (NH₄Cl), prevent access of the viral packaged enzyme to its substrate (Figure 7, panel D). The multiplicity of infection (MOI) was varied from 1-50 in the presence of cycloheximide (Figure 7, panel E), to demonstrate that the assay is dose dependent. To confirm that the REN activity seen in cells was due to enzyme carried into the cell by the input virus, and not through a low level of gene expression in the presence of puromycin, VSV REN-P was treated with a low dose ultra-violet (u.v.) radiation to inhibit transcription of the viral genome (Ball, L. A. and White, C.N., Order of transcription of genes of vesicular stomatitis virus. Proc. Natl. Acad. Sci. USA, 73:442-6 (1976); Whelan , S.P. and Wertz, G.W., Transcription and replication initiate at separate sites on the vesicular stomatitis virus genome. Proc. Natl. Acad. Sci. USA, 99:9178-83 (2002)). A dose of 1230 ergs mm^"2 of u.v. light inhibited transcription, and reduced luminescence to levels observed in the presence of puromycin (data not shown). These data show that modified VSV of the invention is useful in methods of the invention which provide an enzyme based reporter assay for viral entry and uncoating in cells. Moreover, these studies demonstrate that the methods and viral compositions of the present invention are useful to monitor viral entry and uncoating in the absence of new gene expression by treating cells with protein synthesis inhibitors such as puromycin, or by u.v. irradiation of input virus. Further, the compositions and methods of the invention are useful to screen for modulators, e.g., small molecule, inhibitors of these early steps of viral replication.

EXAMPLE 7: STUDIES USING AN ENZYME BASED ASSAY OF THE INVENTION USEFUL TO MONITOR VIRAL ATTACHMENT TO CELLS.

Enzyme based screening assays of the invention are sensitive and readily applied in high throughput screening applications. Studies were performed using VSV-REN-P to monitor virus binding to cells at 4⁰C. Briefly, CHO cells in 96 well plates were chilled on ice to inhibit endocytic transport, and exposed to purified VSV-REN-P at an MOI of 25-500. At 2 h.p.i., the input inoculum was removed and cells were washed twice with ice cold PBS to remove unbound virus. The amount of bound virus was determined following lysis of the cell associated virus and exposure of the renilla enzyme to substrate by treatment of the cells with dual glow luciferase reagent (Promega). Luminescence values were measured using a luminometer and the results shown in Figure 8. These data show a linear, dose-dependent increase in renilla activity with input MOI.

In one embodiment, the invention provides a method to measure viral attachment wherein RLU is dependent upon VSV G. To that end, the effects of pretreatment of VSV with a neutralizing antibody to G, and separately pretreatment of virus with ProK on viral attachment can be assessed. The number of infectious virus particles that bind to the cells are determined by comparing luminescence values to those simultaneously obtained by generating a standard curve from a dilution series of infectious virus. An assay method of the invention for viral attachment is a useful screening method to determine whether molecules target viral attachment.

Test agents that directly inhibit luciferase can be identified by measuring the effect of the test agent on renilla activity by lysis of the modified virus of the invention in the presence of the test agent. If the test agent inhibits the renilla luciferase activity released from the modified virus of the invention directly, then the test agent may have an effect on reporter molecule activity.

EQUIVALENTS

While this invention has been particularly shown and described with references to preferred embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention encompassed by the appended claims.

Claims

CLAIMSWhat is claimed is:

1. A recombinant DNA construct encoding a fusion protein, wherein the fusion protein comprises a reporter molecule fused to a vesicular stomatitis virus polypeptide selected from the group consisting of: nucleocapsid protein (N), the phosphoprotein (P), the matrix protein (M), the glycoprotein (G) and the RNA-dependent RNA polymerase (L), or fragment thereof.

2. The recombinant DNA construct according to claim 1 , wherein the reporter molecule is an enzyme which can be detected by contacting the reporter molecule with colorimetric or fluorigenic substrates.

3. The recombinant DNA construct according to claim 2, wherein the enzyme is selected from the group that consisting of: oxidases, luciferases, peptidases, glycosidases, peroxidases, and phosphatases.

4. The recombinant DNA construct according to claim 3, wherein the reporter molecule is Renilla luciferase.

5. The recombinant DNA construct according to claim 1 , wherein the reporter molecule is a fluorescent protein.

6. The recombinant DNA construct according to claim 5, wherein the fluorescent protein is Green Fluorescent Protein (GFP) or Reef Coral Fluorescent Protein (RFCP).

7. A nucleic acid vector comprising the DNA construct of claim 1.

8. A modified vesicular stomatitis virus comprising the nucleic acid vector of claim 7.

9. The virus according to claim 8, wherein the construct further comprises a nucleic acid that encodes at least one viral cell attachment protein.

10. The virus according to claim 9 wherein, the at least one viral cell attachment protein is a vesicular stomatitis virus cell attachment protein.

11. The virus according to claim 9, wherein the at least one cell attachment protein is a viral cell attachment protein derived from a virus that is not vesicular stomatitis virus.

12. The virus according to claim 11, wherein the at least one viral attachment protein is derived from a virus of a genus selected from the group consisting of: Retroviridae, Togaviridae, Flaviridae, Coronoviridae, Rhabdoviradae, Filoviridae, Paramyxoviridae, Orthomyxoviridae, Bunyaviridae, Arena viridae, Herpesviridae, and Poxviridae,.

13. The virus according to claim 9, wherein the at least one viral attachment protein is derived from a negative-strand RNA virus.

14. The virus according to claim 13, wherein the at least one viral attachment protein is derived from a virus of a genus selected from the group that consists of: Respirovirus, Morbillivirus, Rubulavirus, Henipavirus, Avuvlavirus, Pneumovirus, Metapneumovirus, Lyssavirus, Ephemerovirus, Marburgvirus, Ebolavirus, Bronavirus, Influenzavirus A, Influenzavirus B, Influenzavirus C, Orthobunyavirus, Hantavirus, Nairovirus, Phlebovirus, and Arenavirus.

15. The virus according to claim 8, wherein the virus further comprises a nucleic acid that encodes at least one non-viral protein.

16. The virus according to claim 15, wherein the at least one non-viral protein is a mammalian ligand to a cellular receptor or a bacterial attachment protein.

17. The virus according to claim 8, wherein the fusion protein comprises a functional peptide fused to a viral protein.

18. The virus according to claim 17, wherein the functional peptide is a fluorescent lanthanide binding peptide.

19. The virus according to claim 8, wherein the fusion protein comprises a selectable marker fused to the viral protein.

20. The virus according to claim 19, wherein the selectable marker is neomycin.

21. A host cell transformed with the nucleic acid vector of claim 7.

22. A cell comprising the virus of claim 8 or one or more DNA constructs that encode the viral proteins of the virus of claim 8.

23. A method of screening for modulators of cell attachment, comprising the steps of: a) providing at least one control cell sample; b) providing at least one test cell sample; c) contacting the at least one test cell sample of step (b), with the at least one candidate agent; d) infecting the at least one cpntrol cell sample of step (a) and the at least one test cell sample of step (c) with the virus of claim 8; and e) assaying the cell samples of step (d) to detect the activity of the reporter molecule, wherein a reduction of the level of reporter molecule activity in the test cell sample contacted with the at least one candidate agent relative to the level of reporter molecule activity observed in a control cell sample not contacted with the at least one candidate agent is indicative of antagonist activity of cell attachment by the candidate agent and wherein an increase in the level of reporter molecule activity in the cell sample contacted with the at least one candidate agent relative to the level of reporter molecule activity observed in the control cell sample not contacted with the at least one candidate agent is indicative of agonist activity of cell attachment.

24. A method of screening for inhibitors of viral infectivity, comprising the steps of: a) providing at least one control cell sample; b) providing at least one test cell sample; c) contacting the at least one test cell sample of step (b), with at least one candidate inhibitor of viral infectivity; d) infecting the at least one control cell sample of step (a) and the at least one test cell sample of step (c) with the virus of claim 8; and e) assaying said cell samples of step (d) to detect the activity of the reporter molecule, wherein a reduction in the level of reporter molecule activity in the test cell sample contacted with the at least one candidate inhibitor relative to the level of reporter molecule activity observed in the control cell sample not contacted with the at least one candidate inhibitor is indicative of inhibition activity of the candidate inhibitor.

25. A method for determining the presence or amount of a vesicular stomatitis virus immunoreactive polypeptide in a biological sample, comprising the steps of: a) providing at least one control cell sample; b) providing at least one test cell sample; c) contacting at least one test cell sample of step (b), with the biological sample; d) infecting at least one control cell sample of step (a) and the at least one test cell sample of step (c) with the virus of claim 8; and e) assaying the cell samples of step (d) to detect the activity of the reporter molecule, wherein a reduction of the level of reporter molecule activity in the at least one test cell sample contacted with the biological sample relative to the level of reporter molecule activity observed in the at least one control cell sample not contacted with the biological sample is indicative of the presence of the vesicular stomatitis virus immunoreactive polypeptide in the biological sample.

26. A method for determining the presence or amount of a vesicular stomatitis virus immunoreactive polypeptide in a sample, the method comprising: a) providing the sample; b) contacting the sample with the virus of claim 8; and c) determining the presence or amount of the virus bound to the vesicular stomatitis virus immunoreactive polypeptide, thereby determining the presence or amount of the vesicular stomatitis virus immunoreactive polypeptide in the sample.

27. A kit comprising one or more containers, the virus of claim 8 and instructions for using the contents therein.