Methods of determining susceptibility of cells to reovirus infection and methods for treating cellular proliferative disorders with

[01] This application claims the benefit of the U.S. Provisional application No. 60/844,988, filed on September 15, 2006, which is incorporated herein by reference in its entirety.

FIELD OF THE INVENTION

[02] The present invention relates to methods of identifying the susceptibility of cells to reovirus. The invention also relates to methods of treating cellular proliferative disorders.

BACKGROUND

[03] Viruses capable of selectively replicating in and destroying cancer cells have become the subject of intense interest in the last decade. To effectively use and develop viruses for cancer therapy, the molecular basis for preferential tumor oncolysis by these therapeutic viruses must be understood. At the same time, oncolytic viruses are providing a powerful tool for deciphering unique and critical differences between normal and cancer cells. Oncolytic viruses have shown dependency for growth on the deregulation of important cellular processes, including the interferon-mediated antiviral response, p53 -mediated apoptosis, pRB-mediated cell growth arrest, and mRNA export (Balachandran and Barber, 2000; Balachandran and Barber, 2004; Bischoff et al., 1996; Johnson et al., 2002; O'Shea et al., 2004; Stojdl et al., 2003).

[04] Mammalian reovirus is an RNA virus with inherent preference for replication in transformed cells (Hashiro et al., 1977). Efficient replication and spread of reovirus was shown to require the activation of the Ras signaling pathway(s) (Strong et al., 1998). The Ras oncogene is constitutively activated in 30% of human cancers, while mutated upstream activators and downstream effectors are prevalent in over 80% of human tumors (Bos, 1989). Activated Ras signaling is one of five essential changes necessary for transformation of human cells in culture (Hahn et al., 1999). The Ras GTP-binding proteins function as molecular switches at the hub of over 18 downstream pathways, which together impact critical cellular processes including apoptosis, cell cycle transitions, protein translation, cytoskeletal rearrangement, and intracellular vesicle transport (Malumbres and Pellicer, 1998). To determine which of these important cellular processes impacts the replication of reovirus, which steps of virus replication require changes associated with Ras transformation must first be understood.

[05] Reovirus entry into host cells is mediated through binding to sialic acid and a secondary junction-associated membrane protein (Flint et al., 2004). Following cellular clathrin-dependent endocytosis, reovirus particles are partially uncoated by lysosomal proteases and then traverse the endo/lysosomal membrane. Although reovirus encodes its own RNA-dependent RNA polymerase to transcribe RNA and produce dsRNA genomes, it must use cellular nucleotide pools for biosynthesis. Further, like all viruses, the host translation machinery mediates translation of reovirus proteins. Reovirus uses the host cytoskeleton to establish localized domains of replication. Assembly of reovirus progeny requires encapsidation of ten distinct RNA segments that is still poorly understood. Finally, reovirus depends on apoptosis late during infection for efficient release. There are, therefore, many opportunities for molecular changes associated with cancer to favorably impact the replication of viruses. Indeed, downstream effectors of Ras that cause pleio tropic effects on a cell could in theory enhance each of the above-listed events.

[06] Some viruses have been engineered to be selectively oncolytic by making them dependent on cellular processes known to be deregulated in cancer, such as p53 function, cell cycle control, and the interferon-mediated antiviral response. Since reovirus has inherent preference for replicating in Ras-transformed cells, it offers an excellent tool to discover cellular processes associated with Ras transformation that play unforeseen but critical roles in virus replication and oncolysis.

SUMMARY

[07] The present invention provides methods to determine whether cells are susceptible to reovirus infection. The invention also provides methods for diagnosing proliferative disorders which can be treated with reovirus.

[08] In one aspect the invention provides a method of determining susceptibility of a cell to reovirus infection by measuring uncoating of reoviral coat proteins during entry of reovirus into the cell, comparing uncoating with a cell that does not have an activated Ras pathway (a control cell), and determining whether efficient uncoating of reoviral coat proteins occurred in the cell. Efficient uncoating occurred if uncoating in the cell is greater than uncoating in the cell that does not have an activated Ras pathway. If efficient uncoating has occurred, it indicates that the cell is susceptible to reovirus infection. In one embodiment, measuring uncoating comprises measuring cleavage of the μlc protein to δ. Cleavage of the μlc protein to δ can be measured by western blot analysis. In another embodiment, efficient uncoating of

reoviral coat proteins occurred in the cell if at least two times more μlc protein is cleaved to δ in the cell as compared to the cell that does not have an activated Ras pathway. [09] In yet another embodiment, determining the amounts of (+) and (-) reovirus transcripts can be used as an indirect method to measure uncoating. The amounts of the transcripts can be determined by quantitative RT-PCR or northern blot analyses. In this embodiment efficient uncoating of reoviral coat proteins occurred if the amounts of (+) and (-) reovirus transcripts are two times more in the cell than the amounts the cell that does not have an activated Ras pathway (control cell).

[10] In a second aspect, the invention provides a method of determining susceptibility of a cell to reovirus infection by comparing the infectivity of progeny virus from a cell to infectivity of progeny virus from a cell that does not have an activated pathway (a control cell). The cell is susceptible if the infectivity of progeny virus from the cell is greater than the infectivity of progeny virus from the control cell. In one embodiment, the infectivity of progeny reovirus is determined by the ratio of plaque forming units of the progeny reovirus to the number of particles of progeny reovirus. The number of particles of progeny reovirus can be determined by transmission electron microscopy or by .electrophoresis of the viral proteins and subsequent staining and quantifying viral proteins. In another embodiment, the cell is susceptible if the infectivity of progeny virus from the cell is at least two times greater than the infectivity of progeny virus from the control cell. In yet another embodiment, the progeny virus are purified from the cells at 18 hours post infection. [11] In a third aspect, the invention provides a method of determining susceptibility of a cell to reovirus infection by measuring apoptotic dependent release of progeny reovirus from the cell. This can be done by first quantifying the number of plaque forming units of reovirus obtained from a cell and from a cell that does not have an activated Ras pathway (a control cell), second, measuring apoptosis in the cell infected with reovirus and in a reovirus infected control cell in the presence and absence of an apoptosis inhibitor, and third, comparing the number of plaque forming units of reovirus from the cell and the amount of apoptosis from the reovirus infected cell to the number of plaque forming units from the control cell and the amount of apoptosis in the reovirus infected control cell. The cell is susceptible if the number of plaque forming units correlates with the amount of apoptosis in the reovirus infected cell and in the control cell, and if the number of plaque forming units and amount of apoptosis is higher in the cell than in the cell that does not have an activated Ras pathway. In one embodiment, the amount of apoptosis is quantified by FACS analysis. In another embodiment, the amount of apoptosis is at least 2 or 5 times higher in the cell than in the cell

that does not have an activated Ras pathway. In yet another embodiment, the apoptosis inhibitor is zVAD-fmk.

[12] In embodiments of the above aspects, the cell is a cancer cell, or the cell is obtained from a tumor in an animal.

[13] In a fourth aspect, the invention provides a method of treating a proliferative disorder in an animal comprising the steps of (1) providing a biological sample from the animal, wherein the sample comprises cells, (2) determining susceptibility of cells in the sample to reovirus infection by measuring uncoating of reoviral coat proteins during entry of reovirus into the cells; measuring the infectivity of progeny reovirus obtained from the cells and/or measuring apoptosis dependent release of progeny virus from the cells, and comparing uncoating, infectivity of progeny reovirus and/or apoptosis dependent release of progeny reovirus to that from cells that do not have an activated Ras pathway (control cells). If the cells are susceptible, the animal is administered an effective amount of one or more reoviruses under conditions that result in substantial lysis of cells of the proliferative disorder. In one embodiment, the biological sample is obtained from a tumor.

BRIEF DESCRIPTION OF THE DRAWINGS

[14] Figure 1 shows the effects of Ras transformation on infection of cells by reovirus, the production of infectious progeny and virus spread.

[15] Figure 2 shows the effects of Ras transformation on reovirus uncoating. [16] Figure 3 shows the effects of Ras transformation on viral transcription. [17] Figure 4 shows the effects of Ras transformation on viral translation. [18] Figure 5 shows the effect of Ras transformation on the rate of viral assembly. [19] Figure 6 shows the infectivity of reovirus progeny from non- and Ras-transformed cells.

[20] Figure 7 shows release of infectious reovirus progeny and apoptosis in Ras- transformed cells. [21] Figure 8 shows steps of reovirus infection in non-and Ras-transformed cells.

DETAILED DESCRIPTION

[22] The present invention provides methods of determining the susceptibility of a cell to a reovirus, and methods of treating proliferative disorders in animals. [23] The methods of determining susceptibility of a cell to a reovirus comprise the following steps: (1) providing a cell or cells; (2) infecting the cells with reovirus; (3)

measuring a characteristic of the reovirus or the cells; and (4) comparing the characteristic of the cells or reovirus with a control cell, a control susceptible cell, or control reovirus. The methods of treating proliferative disorders in animals comprise the following steps: (1) providing a sample from an animal; (2) determining susceptibility of cells in the sample to a reovirus and (3) if the cells are susceptible, administering one or more reoviruses to the animal.

I. CELLS

[24] The cells can be any mammalian cells, including cells that can be cultured and cells from a biological sample. Cultured cells can be established cancer cell lines, or cells obtained from a biological sample, including a biopsy, or progeny of the cells. Cultured cells include, without limitation, those from the NCI-60 collection (Shoemaker, et al., 1988). Other cultured cells include cell lines, such as L929 cells, NIH-3T3 cells, HEK cells, HBL- 100 cells (US Patent No. 7,052,832), cancer cell lines PC-3 and BxPC-3 (Tan, et al. 1986), MDA-MB-231, MDA-MB-468, MDA-MB-435 (Calleau, et al. 1978), MCF-7 (Soule, et al. 1973), T47D (Keydar, et al, 1979), SK-BR3 (US Patent No. 7,052,832), MCFlOCAl (Santner et al. 2001), NMuMG (Owens, 1974), BT474 (Lasfargues et al. 1978), and WSUFSCCL cells (Mohammad, et al., 1993). Cells obtained from biological samples can include, without limitation, cells in blood and other body fluids and cells obtained from tissue samples and biopsies.

[25] Efficient replication and spread of reovirus can require the activation of the Ras signaling pathway(s). Accordingly, cells can also be transformed with Ras. For example, NIH3T3 cells can be transformed with Ras to obtain NIH3T3 cells with an activated Ras pathway.

[26] As used herein, "control cells" are cells that do not have an activated Ras pathway and are not susceptible to reovirus. A cell is not susceptible to reovirus if it does not efficiently produce infectious reovirus progeny, as defined in the Examples. A "control susceptible cell" is a cell that is susceptible to reovirus infection. A cell is susceptible to reovirus infection if it efficiently produces infectious reovirus progeny as defined in the Examples. An example of a control susceptible cell is one with an activated Ras pathway. [27] Genetic alteration of the proto-oncogene Ras is believed to contribute to approximately 30% of all human tumors (Wiessmuller and Wittinghofer, 1994; Barbacid, 1987). The role that Ras plays in the pathogenesis of human tumors is specific to the type of tumor. Activating mutations in Ras itself are found in most types of human malignancies,

and are highly represented in pancreatic cancer (80%), sporadic colorectal carcinomas (40 50%), human lung adenocarcinomas (15 24%), thyroid tumors (50%) and myeloid leukemia (30%) (Millis, et al., 1995; Chaubert, et al., 1994; Bos, 1989). Ras activation is also demonstrated by upstream mitogenic signaling elements, notably by tyrosine receptor kinases (RTKs). These upstream elements, if amplified or overexpressed, ultimately result in elevated Ras activity by the signal transduction activity of Ras. Examples of this include overexpression of PDGFR in certain forms of glioblastomas, as well as in c-erbB-2/neu in breast cancer (Levitzki, 1994; Janes, et al. 1994; Bos, J., 1989).

II. REOVIRUSES

[28] An "oncolytic virus" is a virus that selectively kills neoplastic cells. Killing of neoplastic cells can be detected by any method established in the art, such as determining viable cell count, cytopathic effect, apoptosis of the neoplastic cells, synthesis of viral proteins in the neoplastic cells (e.g., by metabolic labeling, western analysis of viral proteins, or reverse transcription polymerase chain reaction of viral genes necessary for replication), or reduction in size of a tumor.

[29] As used herein, "reovirus" refers to any virus classified in the reovirus genus. The name reovirus (Respiratory and enteric orphan virus) is a descriptive acronym suggesting that these viruses, although not associated with any known disease state in humans, can be isolated from both the respiratory and enteric tracts.

[30] Human reoviruses include three serotypes: type 1 (strain Lang or TlL), type 2 (strain Jones, T2J) and type 3 (strain Dearing or strain Abney, T3D). The three serotypes are easily identifiable on the basis of neutralization and hemagglutinin-inhibition assays. (See, for example, Nibert et al., 1996).

[31] The reovirus may be naturally occurring or modified. The reovirus is "naturally- occurring" when it can be isolated from a source in nature and has not been intentionally modified by humans in the laboratory. For example, the reovirus can be from a "field source", that is, from a human who has been infected with the reovirus. [32] The reovirus may be modified but still capable of lytically infecting a mammalian cell having an active ras pathway. The reovirus may be chemically or biochemically pretreated (e.g., by treatment with a protease, such as chymo trypsin or trypsin) prior to administration to the proliferating cells. Pretreatment with a protease removes the outer coat or capsid of the virus and may increase the infectivity of the virus. The reovirus may be encapsidated in a liposome or micelle (Chandran and Nibert, 1998) to reduce or prevent an immune response

from a mammal which has developed immunity to the reovirus. For example, the virion may be treated with chymotrypsin in the presence of micelle forming concentrations of alkyl sulfate detergents to generate a new infectious subvirion particle.

[33] The reovirus may be a recombinant reovirus resulting from the recombination and/or reassortment of genomic segments from two or more genetically distinct reoviruses. The recombinant reovirus may be from two or more types of reoviruses with differing pathogenic phenotypes such that it contains different antigenic determinants, thereby reducing or preventing an immune response by a mammal previously exposed to a reovirus subtype. Recombinant reoviruses may also exhibit different biological activities (e.g., replication activities in neoplastic cells and biodistribution) compared to the original reoviruses. Recombination and/or reassortment of reovirus genomic segments may occur in nature following infection of a host organism with at least two genetically distinct reoviruses. Recombinant virions can also be generated in cell culture, for example, by co-infection of permissive host cells with genetically distinct reoviruses (Nibert et al. 1996). [34] Accordingly, reoviruses include recombinant reoviruses resulting from reassortment of genome segments from two or more genetically distinct reoviruses, including, without limitation, human reovirus, such as type 1 (e.g., strain Lang), type 2 (e.g., strain Jones), and type 3 (e.g., strain Dealing or strain Abney), non-human mammalian reoviruses, and also including avian reovirus. Reoviruses also include recombinant reoviruses resulting from reassortment of genome segments from two or more genetically distinct reoviruses wherein at least one parental virus is genetically engineered, comprises one or more chemically synthesized genomic segment, has been treated with chemical or physical mutagens, or is itself the result of a recombination event. In addition, reoviruses include recombinant reovirus that has undergone recombination in the presence of chemical mutagens, including but not limited to dimethyl sulfate and ethidium bromide, or physical mutagens, including but not limited to ultraviolet light and other forms of radiation.

[35] Recombinant reoviruses that comprise deletions or duplications in one or more genome segments, that comprise additional genetic information as a result of recombination with a host cell genome, or that comprise synthetic genes are also included. [36] The reovirus may be modified by incorporation of mutated coat proteins, such as for example σl, into the virion outer capsid. The proteins may be mutated by replacement, insertion or deletion. Replacement includes the insertion of different amino acids in place of the native amino acids. Insertions include the insertion of additional amino acid residues into the protein at one or more locations. Deletions include deletions of one or more amino acid

residues in the protein. Such mutations may be generated by methods known in the art. For example, oligonucleotide site directed mutagenesis of the gene encoding for one of the coat proteins could result in the generation of the desired mutant coat protein. Expression of the mutated protein in reovirus infected mammalian cells in vitro such as COS 1 cells will result in the incorporation of the mutated protein into the reovirus virion particle (Turner and Duncan, 1992; Duncan et al., 1991; Mah et al., 1990). Mutant reoviruses can also be naturally occurring and can for example be isolated from persistently infected cells (Kim et al., 2007). In addition, mutant reovirus can be engineered using reverse genetics (Kobayashi et al., 2007).

[37] The reovirus can be a reovirus modified to reduce or eliminate an immune reaction to the reovirus. Such modified reoviruses are termed "immunoprotected reoviruses". Such modifications could include packaging of the reovirus in a liposome, a micelle or other vehicle to mask the reovirus from the mammal's immune system. Alternatively, the outer capsid of the reovirus virion particle may be removed because the proteins present in the outer capsid are the major determinant of the host humoral and cellular responses. [38] In addition to reoviruses, other oncolytic viruses can be used to practice the present invention in the same manner as reoviruses. Oncolytic viruses that do not inhibit PKR function can be used. These viruses can be naturally existing, like the reovirus, or they can be modified or mutated such that a viral factor that inhibits PKR is not functional. [39] A few such oncolytic viruses are discussed below, and a person of ordinary skill in the art can practice the present invention using additional oncolytic viruses according to the disclosure herein and knowledge available in the art. The oncolytic virus may be a member in the family of myoviridae, siphoviridae, podoviridae, teciviridae, corticoviridae, plasmaviridae, lipothrixviridae, fuselloviridae, poxyiridae, iridoviridae, phycodnaviridae, baculoviridae, herpesviridae, adenoviridae, papovaviridae, polydnaviridae, inoviridae, microviridae, geminiviridae, circoviridae, parvoviridae, hepadnaviridae, retroviridae, cyctoviridae, reoviridae, bimaviridae, paramyxoviridae, rhabdoviridae, fϊloviridae, orthomyxoviridae, bunyaviridae, arenaviridae, leviviridae, picomaviridae, sequiviridae, comoviridae, potyviridae, caliciviridae, astroviridae, nodaviridae, tetraviridae, tombusviridae, coronaviridae, glaviviridae, togaviridae, or barnaviridae. As with reovirus, immunoprotected or reassortant viruses of other oncolytic viruses are also encompassed in the present invention. Furthermore, a combination of at least two oncolytic viruses, including reovirus, can also be employed to practice the present invention.

III. CHARACTERISTICS OF REOVIRUSES OR INFECTED CELLS

A. Uncoating

[40] Reovirus entry into host cells is mediated through binding to sialic acid and a secondary junction-associated membrane protein (Flint et al., 2004). Following cellular clathrin-dependent endocytosis, reovirus particles are partially uncoated by lysosomal proteases and then traverse the endo/lysosomal membrane.

[41] Uncoating of reovirus can be measured in a number of ways. One method is to measure the cleavage of coat protein μlc to δ. This can be measured by western blot analysis using anti-reo virus antibodies. Cleavage could also be measured using antibodies specific for μlc and δ. The specific antibodies could also be used in an ELISA to measure amounts of μlc and δ.

[42] Uncoating of reovirus can also be measured by quantifying reovirus RNA. Enhanced efficiency of reovirus uncoating would produce more transcriptionally active virus cores and consequently lead to increased levels of reovirus transcripts. (+) and (-) sense RNA syntheses can be assessed using sense-specific primers for reverse transcription. Primers for different transcripts of reovirus (e.g., S4, S2, Ml, M2 and Ll) can be used in quantitative RT- PCR to determine levels of the transcripts. Transcript levels can also be quantified using northern blot analyses.

[43] "Efficient particle uncoating" as used herein, is removal of reovirus coat proteins during viral entry to a cell that is greater than uncoating observed in non-transformed cells.

B. Infectivitv of Reovirus Progeny

[44] The infectivity of reovirus progeny from infected cells can also be quantified. As used herein, "Progeny virus" means virus produced by cells infected by reovirus. The number of plaque forming units (pfu) and the number of reovirus particles produced by a set number of reovirus infected cells can be quantified. The number of pfu can be quantified using a standard plaque assay. The number of reovirus particles can be quantified by a number of methods, including, without limitation, transmission electron microscopy, immunoblots using reovirus antibodies or an antibody specific for a coat protein, ELISA using reovirus antibodies or an antibody specific for a coat protein and SDS-PAGE followed by protein staining, such as a silver stain.

[45] Once the pfus and number of particles are determined, the pfu/particle ratio can be determined as a measure of infectivity of the progeny reovirus.

C. Apoptosis and Release of Reovirus Progeny

[46] Apoptosis of reovirus infected cells can be correlated to release of reovirus progeny to determine the susceptibility of cells to reovirus infection. Apoptosis can be measured in the presence and absence of an apoptosis inhibitor. As used herein, an "apoptosis inhibitor" is a substance (e.g., a molecule, biomolecule, small organic compound) that inhibits one or more processes in an apoptosis pathway, so that apoptosis of a cell or cells is reduced or eliminated. Apoptosis can be quantified in a number of ways, including, without limitation, treating cells with propidium iodide and annexin-V FITC, with staining quantified by FACS analysis. Other methods include, without limitation, a TUNEL assay and DNA fragmentation. Using these assays, the number or proportion of cells undergoing apoptosis can be determined. A correlation between apoptosis of a cell or cells and release of reovirus progeny from cells exists if more reovirus progeny are released from apoptotic cells than from cells that are not apoptotic.

[47] The amount of progeny reovirus released can be quantified by determining the amount of viral particles and pfus released into the medium. To determine if enhanced release of reovirus is associated with apoptosis, apoptosis can be blocked with an apoptosis inhibitor, such as zVAD-fmk (carbobenzoxy-valyl-alanyl-aspartyl flurormethyl ketone). Release of progeny reovirus from untreated cells and cells treated with zVAD-fmk can be compared.

D. Comparisons to Determine Susceptibility of Cells to Reovirus

[48] Comparison of uncoating, infectivity of progeny reovirus and/or apoptotic release of progeny reovirus from cells being tested for susceptibility to reovirus to uncoating, infectivity of progeny virus and/or apoptotic release of progeny reovirus from cells that are not susceptible, such as NIH3T3 cells and to cells that are susceptible, such as Ras transformed NIH3T3 cells can determine whether the cells are susceptible to reovirus.

IV. BIOLOGICAL SAMPLES

[49] A "biological sample" is a sample collected from a biological subject, such as an animal.

[50] As used herein, the term "subject" refers to any animal (e.g., a mammal), including, but not limited to, humans, non-human primates, rodents, and the like, which is to be the recipient of a particular treatment. Typically, the terms "subject" and "patient" are used interchangeably herein in reference to a human subject.

[51] As used herein, the term "animal" refers to all animals including, without limitation, primates, including humans, other vertebrates such as rodents, non-human primates, ovines,

bo vines, ruminants, lagomorphs, porcines, caprines, equines, canines, felines, aves, etc. and non-vertebrate animals such as insects and nematodes.

[52] A biological sample can be taken from a subject or animal, and includes, without limitation, body fluids, including blood, urine, saliva, etc., tissue, including biopsy tissue, tissue or organs taken from a live animal, and post mortem tissue or organs. The biological sample can be a biopsy taken from a mammal suspected of having a proliferative disorder. [53] A "mammal suspected of having a proliferative disorder" means that the mammal may have a proliferative disorder or tumor or has been diagnosed with a proliferative disorder or tumor or has been previously diagnosed with a proliferative disorder or tumor, the tumor or substantially all of the tumor has been surgically removed and the mammal is suspected of harboring some residual tumor cells.

[54] A "proliferative disorder" is any cellular disorder in which the cells proliferate more rapidly than normal tissue growth. Thus a "proliferating cell" is a cell that is proliferating more rapidly than normal cells. The proliferative disorder, includes but is not limited to neoplasms. A neoplasm is an abnormal tissue growth, generally forming a distinct mass that grows by cellular proliferation more rapidly than normal tissue growth. Neoplasms show partial or total lack of structural organization and functional coordination with normal tissue. These can be broadly classified into three major types. Malignant neoplasms arising from epithelial structures are called carcinomas, malignant neoplasms that originate from connective tissues such as muscle, cartilage, fat or bone are called sarcomas and malignant tumors affecting hematopoietic structures (structures pertaining to the formation of blood cells) including components of the immune system, are called leukemias and lymphomas. A tumor is the neoplastic growth of the disease cancer. As used herein, a "neoplasm", also referred to as a "tumor", is intended to encompass hematopoietic neoplasms as well as solid neoplasms. The proliferative disorder can be selected from the group consisting of lung cancer, prostate cancer, colorectal cancer, thyroid cancer, renal cancer, adrenal cancer, liver cancer, pancreatic cancer, breast cancer and central and peripheral nervous system cancer. Other proliferative disorders include, but are not limited to neurofibromatosis.

V. SUSCEPTIBILITY OF CELLS IN THE BIOLOGICAL SAMPLE TO REOVIRUS

[55] Susceptibility of cells to reovirus is determined as described above and in the examples. Measuring uncoating of reovirus, infectivity of progeny reovirus and/or apoptotic release of reovirus from infected cells and comparison to the characteristics of reovirus from control cells determines susceptibility of the cells.

[56] "Phenotyping" a tumor means classifying a tumor according to its phenotype. For example, tumor phenotypes include ras pathway activation, interferon-resistance, p53- defϊciency and Rb-defϊciency. Phenotypes also include the efficiency of uncoating a reovirus and the ratio of infectious virus particles produced. The phenotypes are not mutually exclusive, namely, a tumor may be phenotyped into more than one class. [57] This invention has been distinctly connected to a Ras-activated pathway. However, many cancers (up to 80%) that be successfully treated by reovirus may not have activated Ras specifically, but other downstream or upstream effectors in the Ras pathway may be mutated and therefore will make the cancer cells susceptible to reovirus infection. In general it has been proposed that up to 80% of cancers could be treated by reovirus. For example a tumor may not have activated Ras, but may have some other mutated downstream Ras-effector that results in efficient reovirus uncoating and therefore reovirus infection. Accordingly, cells without activated Ras may still be susceptible to reovirus. The methods of this invention can identify such cells.

VI. FORMULATION AND ADMINISTRATION

[58] If cells from a biological sample from an animal are found to be susceptible to reovirus infection, reovirus can be administered to the animal. The reovirus can be formulated and then administered.

A. Formulation

[59] This invention includes pharmaceutical compositions which contain, as the active ingredient, one or more of the reoviruses associated with pharmaceutically acceptable carriers or excipients. In making the compositions, the reovirus can be mixed with an excipient, diluted by an excipient or enclosed within such a carrier which can be in the form of a capsule, sachet, paper or other container. When the pharmaceutically acceptable excipient serves as a diluent, it can be a solid, semi-solid, or liquid material, which acts as a vehicle, carrier or medium for the active ingredient. Thus, the compositions can be in the form of tablets, pills, powders, lozenges, sachets, cachets, elixirs, suspensions, emulsions, solutions, syrups, aerosols (as a solid or in a liquid medium), ointments containing, for example, up to 10% by weight of the active compound, soft and hard gelatin capsules, suppositories, sterile injectable solutions, and sterile packaged powders.

[60] Some examples of suitable excipients include lactose, dextrose, sucrose, sorbitol, mannitol, starches, gum acacia, calcium phosphate, alginates, tragacanth, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, sterile water, syrup, and

methyl cellulose. The formulations can additionally include lubricating agents such as talc, magnesium stearate, and mineral oil; wetting agents; emulsifying and suspending agents; preserving agents such as methyl- and propylhydroxy-benzoates; sweetening agents; and flavoring agents. The compositions of the invention can be formulated so as to provide quick, sustained or delayed release of the active ingredient after administration to the patient by employing procedures known in the art.

[61] For preparing solid compositions such as tablets, the reovirus is mixed with a pharmaceutical excipient to form a solid preformulation composition containing a homogeneous mixture of a compound of the present invention. When referring to these preformulation compositions as homogeneous, it is meant that the active ingredient is dispersed evenly throughout the composition so that the composition may be readily subdivided into equally effective unit dosage forms such as tablets, pills and capsules. [62] The tablets or pills may be coated or otherwise compounded to provide a dosage form affording the advantage of prolonged action. For example, the tablet or pill can comprise an inner dosage and an outer dosage component, the latter being in the form of an envelope over the former. The two components can be separated by an enteric layer which serves to resist disintegration in the stomach and permit the inner component to pass intact into the duodenum or to be delayed in release. A variety of materials can be used for such enteric layers or coatings, such materials including a number of polymeric acids and mixtures of polymeric acids with such materials as shellac, cetyl alcohol, and cellulose acetate. [63] The liquid forms in which the compositions may be incorporated for administration orally or by injection include aqueous solutions, suitably flavored syrups, aqueous or oil suspensions, and flavored emulsions with edible oils such as corn oil, cottonseed oil, sesame oil, coconut oil, or peanut oil, as well as elixirs and similar pharmaceutical vehicles. [64] Compositions for inhalation or insufflation include solutions and suspensions in pharmaceutically acceptable, aqueous or organic solvents, or mixtures thereof, and powders. The liquid or solid compositions may contain suitable pharmaceutically acceptable excipients as described herein. The compositions can be administered by the oral or nasal respiratory route for local or systemic effect. Compositions in preferably pharmaceutically acceptable solvents may be nebulized by use of inert gases. Nebulized solutions may be inhaled directly from the nebulizing device or the nebulizing device may be attached to a face mask tent, or intermittent positive pressure breathing machine. Solution, suspension, or powder compositions may be administered orally or nasally, from devices which deliver the formulation in an appropriate manner.

[65] Another formulation utilized in the methods of the present invention employs transdermal delivery devices ("patches"). Such transdermal patches may be used to provide continuous or discontinuous infusion of the reovirus of the present invention in controlled amounts. The construction and use of transdermal patches for the delivery of pharmaceutical agents is well known in the art. See, for example, U.S. Pat. No. 5,023,252, herein incorporated by reference. Such patches may be constructed for continuous, pulsatile, or on demand delivery of pharmaceutical agents.

[66] Other suitable formulations for use in the present invention can be found in Remington's Pharmaceutical Sciences, the disclosure of which is incorporated herein by reference.

[67] The reovirus or the pharmaceutical composition comprising the reovirus may be packaged into convenient kits providing the necessary materials packaged into suitable containers. It is contemplated the kits may also include chemotherapeutic agent.

B. Administration

[68] Administration to an animal can occur via a number of routes and methods. [69] "Administration to a proliferating cell or neoplasm" indicates that the reovirus is administered in a manner so that it contacts the proliferating cells or cells of the neoplasm (also referred to herein as "neoplastic cells"). The route by which the reovirus is administered, as well as the formulation, carrier or vehicle, will depend on the location as well as the type of the neoplasm. A wide variety of administration routes can be employed. For example, for a solid neoplasm that is accessible, the reovirus can be administered by injection directly to the neoplasm. For a hematopoietic neoplasm, for example, the reovirus can be administered intravenously or intravascularly. For neoplasms that are not easily accessible within the body, such as metastases or brain tumors, the reovirus is administered in a manner such that it can be transported systemically through the body of the mammal and thereby reach the neoplasm (e.g., intrathecally, intravenously or intramuscularly). Alternatively, the reovirus can be administered directly to a single solid neoplasm, where it then is carried systemically through the body to metastases. The reovirus can also be administered subcutaneously, parentarelly, intraoccularly, intraperitoneally, topically (e.g., for melanoma), orally (e.g., for oral or esophageal neoplasm), rectally (e.g., for colorectal neoplasm), vaginally (e.g., for cervical or vaginal neoplasm), nasally or by inhalation spray (e.g., for lung neoplasm).

[70] Reovirus can be administered systemically to mammals that are immune compromised or which have not developed immunity to the reovirus epitopes. In such cases,

reovirus administered systemically, i.e. by intraveneous injection, will contact the proliferating cells resulting in lysis of the cells. Where the mammals to be treated have higher titers of anti-reovirus antibodies, more reovirus must be administered to be effective. [71] Immunocompetent mammals previously exposed to a reovirus subtype may have developed humoral and/or cellular immunity to that reovirus subtype. Nevertheless, it has been found that direct injection of the reovirus into a solid tumor in immunocompetent mammals will result in the lysis of the neoplastic cells. On the other hand, when the reovirus is administered systemically to immunocompetent mammals, the mammals may produce an immune response to the reovirus. Such an immune response may be avoided if the reovirus is of a subtype to which the mammal has not developed immunity, or the reovirus has been modified as previously described herein such that it is immunoprotected, for example, by protease digestion of the outer capsid or packaging in a micelle.

[72] Alternatively, it is contemplated that the immunocompetency of the mammal against the reovirus may be suppressed either by the prior or co-administration of pharmaceuticals known in the art to suppress the immune system in general (Cuff et al., "Enteric reovirus infection as a probe to study immunotoxicity of the gastrointestinal tract" Toxicological Sciences 42(2):99 108 (1998)) or alternatively the administration of such immunoinhibitors as anti-antireo virus antibodies. The humoral immunity of the mammal against reovirus may also be temporarily reduced or suppressed by plasmapheresis of the mammal's blood to remove the anti-reovirus antibodies. The humoral immunity of the mammal against reovirus may additionally be temporarily reduced or suppressed by the intraveinous administration of non-specific immunoglobulin to the mammal.

[73] It is contemplated that the reovirus may be administered to immunocompetent mammals immunized against the reovirus in conjunction with the administration of immunosuppressants and/or immunoinhibitors. Such immunosuppressants and immunoinhibitors are known to those of skill in the art and include such agents as cyclosporin, rapamycin, tacrolimus, mycophenolic acid, azathioprine and their analogs, and the like. Other agents are known to have immunosuppressant properties as well (see, e.g., Goodman and Gilman, 7 ^th Edition, page 1242, the disclosure of which is incorporated herein by reference). Such immunoinhibitors also include "anti-antireo virus antibodies", which are antibodies directed against anti-reovirus antibodies. Such antibodies can be made by methods known in the art. See for example "Antibodies: A laboratory manual" E. Harlow and D. Lane, Cold Spring Harbor Laboratory (1988). Such anti-antireovirus antibodies may be administered prior to, at the same time or shortly after the administration of the reovirus.

A2007/001656

An effective amount of the anti-antireovirus antibodies can be administered in sufficient time to reduce or eliminate an immune response by the mammal to the administered reo virus. The terms "immunosuppressant" or "immune suppressive agent" include conventional immunosuppressants, immunoinhibitors, antibodies and conditions such as radiation therapy or HIV infection which result in compromise of the immune system.

[74] The term "substantial lysis" means at least 10% of the proliferating cells are lysed. At least 50% or at least 75% of the cells can also be lysed. The percentage of lysis can be determined for tumor cells by measuring the reduction in the size of the tumor in the mammal or the lysis of the tumor cells in vitro.

[75] An "effective amount" is an amount which is sufficient to achieve the intended purposes. For example, an effective amount of reovirus for the purpose of treating or ameliorating a disease or medical condition is an amount sufficient to result in a reduction or complete removal of the symptoms of a disease or medical condition. The effective amount of a given therapeutic agent will vary with factors such as the nature of the agent, the route of administration, the size and species of the animal to receive the therapeutic agent, and the purpose of the administration. The effective amount in each individual case may be determined empirically by a skilled artisan according to established methods in the art. [76] "Treating or ameliorating" a disease or medical condition means the reduction in the symptoms of a disease or complete removal of the symptoms of a disease or medical condition.

[77] A therapeutic agent is "selective" for a particular disease or medical condition if the agent is more effective for the disease or medical condition than for other diseases or medical conditions. Similarly, a therapeutic agent is selective for a particular group of neoplastic cells if the agent kills the particular group of neoplastic cells with higher efficiency than other neoplastic cells.

[78] The immunosuppressant or immunoinhibitor is administered in an appropriate amount and using an appropriate schedule of administration sufficient to result in immunosuppression or immunoinhibition of the mammal's immune system. Such amounts and schedules are well known to those of skill in the art.

[79] The reovirus is administered in an amount that is sufficient to treat the proliferative disorder (e.g., an "effective amount"). A proliferative disorder is "treated" when administration of reovirus to the proliferating cells effects lysis of the proliferating cells. This may result in a reduction in the rate of growth of the neoplasm, a reduction in size of the neoplasm, or in a complete elimination of the neoplasm. The reduction in size of the

neoplasm, or elimination of the neoplasm, is generally caused by lysis of neoplastic cells

("oncolysis") by the reovirus. The effective amount can be that amount able to inhibit tumor cell growth. The effective amount can be from about 1.0 pfu/kg body weight to about 10 pfu/kg body weight or from about 10 ² pfu/kg body weight to about 10 ¹³ pfu/kg body weight.

For example, for treatment of a human, approximately 10 to 10 plaque forming units of reovirus can be used, depending on the type, size and number of tumors present. The effective amount will be determined on an individual basis and may be based, at least in part, on consideration of the type of reovirus; the chosen route of administration; the individual's size, age, gender; the severity of the patient's symptoms; the size and other characteristics of the neoplasm; and the like. The course of therapy may last from several days to several months or until diminution of the disease is achieved.

[80] The reovirus can be administered in a single dose, or multiple doses (i.e., more than one dose). The multiple doses can be administered concurrently, or consecutively (e.g., over a period of days or weeks). The reovirus can also be administered to more than one neoplasm in the same individual.

[81] The compositions can be formulated in a unit dosage form, each dosage containing an appropriate amount of immunosuppressant or immunoinhibitor and from about 10 pfus to about 10 ¹³ pfus of the reovirus. The term "unit dosage forms" refers to physically discrete units suitable as unitary dosages for human subjects and other mammals, each unit containing a predetermined quantity of reovirus calculated to produce the desired therapeutic effect, in association with a suitable pharmaceutical excipient.

[82] It has been found that the reovirus is effective for the treatment of solid neoplasms in immunocompetent mammals. Administration of unmodified reovirus directly to the neoplasm results in oncolysis of the neoplastic cells and reduction in the size of the tumor in immunocompetent animals. When animals are rendered immunosuppressed or immunodefϊcient in some way, systemic administration of reovirus will be more effective in producing oncolysis.

[83] The reovirus may be administered in conjunction with surgery or removal of the neoplasm. Thus, the reovirus may be administered at or near the site of a solid neoplasm during or following surgical removal of the neoplasm.

[84] The reovirus may be administered in conjunction with or in addition to radiation therapy which renders the mammal immunosuppressed.

[85] The reovirus of the present invention may be administered in conjunction with or in addition to known anticancer compounds or chemotherapeutic agents. Chemotherapeutic

agents are compounds which may inhibit the growth of tumors. Such agents, include, but are not limited to, 5-fluorouracil, mitomycin C, methotrexate, hydroxyurea, cyclophosphamide, dacarbazine, mitoxantrone, anthracyclins (Epirubicin and Doxurubicin), antibodies to receptors, such as herceptin, etopside, pregnasome, platinum compounds such as carboplatin and cisplatin, taxanes such as taxol and taxotere, hormone therapies such as tamoxifen and anti-estrogens, interferons, aromatase inhibitors, progestational agents and LHRH analogs. [86] The reovirus can be used to reduce the growth of tumors that are metastatic. In an embodiment of the invention, a method is provided for reducing the growth of metastatic tumors in a mammal comprising administering an effective amount of a reovirus to an animal.

VII KITS

[87] The materials for use in the method of this invention are ideally suited for the preparation of a kit. Such a kit may comprise a carrier means being compartmentalized to receive one or more container means such as vials, tubes, and the like, each of the container means comprising one of the separate elements to be used in the method. For example, one of the container means may comprise a sample containing reovirus. A second container may comprise ras-transformed cells. A third container may comprise non-transformed cells. The constituents may be present in liquid or lyophilized form, as desired. A kit can also comprise other container means comprising reagents and compounds for use with the methods.

EXAMPLES

[88] The examples, experiments and results described herein are offered to illustrate this invention and are not to be construed in any way as limiting the scope of the present invention.

EXAMPLE 1 Materials and Methods

A. Cell Lines Virus, Molecular Constructs and Reagents

[89] NIH 3T3 cells (American Type Culture Collection, ATCC) were cultivated in Dulbecco's Modified Eagle's Medium (DMEM),10% newborn calf serum (NCS) and antibiotic antimycotic cocktail (AA, Invitrogen). L-929 (ATCC) cells were cultivated as adherent monolayers or in suspension in Minimal Essential Medium (MEM) or Joklik's Modified Eagle's Medium (Sigma-Aldrich), respectively, supplemented with 5% fetal bovine serum (FBS, Invitrogen) and AA. The 293T packaging cell line generously provided by Dr.

Y. Ikeda (Mayo Clinic College of Medicine, Rochester, MN) was cultivated in DMEM, 10% FBS, AA.

[90] Retroviral vectors pBabepuro and activated Ras (RasV12) in pBabepuro were gifts from Dr. C. Der (University of North Carolina, Chapel Hill, NC). Retrovirus was generated in 293T cells following standard procedures. NIH 3T3 cells were infected with retrovirus and 8 μg/mL polybrene. At 48 hpi (hours post infection), cells were selected with 2 μg/ml puromycin (Sigma Aldrich). Pools of selected, puromycin-resistant cells were used for 8 passages. Elevated Ras levels in NIH 3T3 cells expressing RasV12 was confirmed with a Ras activation assay kit according to the manufacturer's instructions (Upstate Biotechnology, Lake Placid, NY).

[91] Reovirus (Serotype 3 Dealing) was propagated in L-929 suspension culture, purified through a CsCl gradient following previously published procedures (Smith et al., 1969).

B. Reovirus Infection and Titers

[92] Ras- or non-transformed cells, seeded on 2% gelatin-coated plates (Sigma-Aldrich) at a density of 5 x 10 ⁴ cells per cm ² , were infected with 2000 reovirus particles/cell for 1 hr at 37° C in DMEM. [Reovirus binds L929 cells 100 times better than NIH 3T3 (unpublished results), and consequently the multiplicity of infection (MOI) determined by plaque titration on L929 does not correspond to the same MOI on NIH 3T3 cells. Therefore, although 2000 reovirus particles/cell corresponds to an MOI of 10 based on L929 titers, only 10-30% of NIH 3T3 cells become infected.] Virus was removed and cells incubated in 0.2mL/cm DMEM, 10% NCS. At indicated hpi, 1/10 volume of media was collected and centrifuged at 12,000 x g for 5 min. One-tenth volume of 1OX RIPA [10% Igepal, 5% sodium deoxycholate, 10 x protease inhibitor cocktail (PIC)] was then added to the wells and total reovirus (cell-associated and released virus) was collected. Media and lysates were flash frozen and stored at -80° C. Reovirus titers were measured on L929 cells by standard plaque assay, or subjected to western blot analysis as described below.

C. Immunofluorescence Microscopy and Fluorescence-Associated Cell Sorting [93] For immunofluorescence, cytospins of trypsin-harvested cells (1 x 10 ⁵ cells/slide) were fixed with methanol for 5 min, incubated with blocking buffer (BB, 5% BSA, 0.1% Triton X-100 in phosphate buffered solution, PBS) and probed with anti-μNS polyclonal rabbit antibody (1/500) and goat anti-rabbit IgG conjugated to Cy2 (1/200, Invitrogen). Washed slides were mounted in 90% glycerol, 100 mM Tris-HCl pH 8, 2.5% w/v DABCO (Sigma Aldrich), 1 μg/mL bisbenzimide H 33342 trihydrochloride (Sigma Aldrich). All

photographs were captured on a Zeiss λxiovert 200 inverted microscope with an λxioCam HRc digital camera under identical capture parameters.

[94] For FACS analysis, harvested cells fixed in 4% paraformaldehyde were blocked and permeabilized in 5% BSA, 0.1% Triton X-100 in PBS, and probed with anti-reovirus polyclonal rabbit antibody (1/10 000 dilution) and goat anti-rabbit IgG conjugated with Cy2 (1/200). Washed cells were quantified with a FACScan flow cytometer (Becton Dickinson) and analyzed using WinDMI Version 2.8 (Scripps Research Institute).

D. Detection of Reovirus Binding. Endocvtosis and Uncoating During Entry

[95] For binding experiments, cells were pre-cooled for 30 min. on ice. Reovirus was added for 1 hr at 4° C or 37° C as above. After 3 PBS washes, cells were collected immediately or incubated at 37° C in DMEM/ 10% NCS or DMEM with 10 μg/mL chymotrypsin (CHT, Sigma-Aldrich) to permit internalization. 20 μg/ml cyclohexaminde (Sigma-Aldrich) was added to prevent de-novo synthesis for samples infected at 37° C. At respective collection time points, cells were scraped in DMEM/ 10% FBS, washed 3 times with 1 mL PBS and lysed with RIPA [50 mM TRIS-HCl (pH 7.5), 150 mM NaCl, 1% IGEPAL, 0.5% sodium deoxycholate, PIC]. Nuclei were removed by centrifugation, and viral proteins visualized by western blot analysis with polyclonal anti-reovirus antibodies (1 :3000) and horse radish peroxidase (HRP)-conjugated goat anti-rabbit antibodies (1 :20000). [96] To measure resistance to cell-surface biotinylation as a measure of endocytosis, reovirus was bound to cells at 4° C as described above. Cells were washed 3 times with PBS and incubated with EZ-Link ^® Sulfo-NHS-SS-Biotin (Pierce) at 1 mg/ml in PBS (pH 8.0) for 30 min at 4° C. Alternatively, cells were incubated at 37° C for 20, 40 or 60 min prior to biotinylation. Reactions were terminated with 50 mM TRIS and cells collected as described above. For immunoprecipitation of biotinylated cell surface proteins, 150 μg of cell lysate was incubated with 25 μL of strep tavidin-conjugated magnetic beads (Dynal, Norway) at 4° C for 1 hr followed by 3 washes with RIPA. Immunoprecipitated biotinylated reovirus proteins were detected by western blotting.

E. Quantitative RT PCR and Northern Analysis

[97] RNA extracted with TRIzol (Invitrogen) was reverse transcribed into cDNA using the Superscript III reverse transcriptase kit (Invitrogen) as per manufacturer's instructions [0.7 μg of total RNA per 20 μL reaction, 0.1 μM gene-specific primers (Table 1) and 2.5 μM oligo(dT) ₂ o]. Brilliant SYBR Green quantitative PCR master mix (Stratagene) was used as per manufacturer's instructions with 6.25 μM of forward and reverse primers. Forty amplification cycles of 95° C, 59° C and 72° C for 30 seconds were completed using the

Mx3000P real-time PCR instrument (Stratagene). Standard curves were generated as shown in Figure 3E.

Table 1 Primers

[98] Reovirus S4 and M2 mRNA were detected by northern blot using the NorthernMax system (Ambion). 0.25 μg total RNA in glyoxal loading dye (Ambion), heated to 50 ⁰ C (denatures mRNA but not dsRNA) or 65° C (denatures mRNA and dsRNA) for 30 min or 5 min, respectively, was separated on a 1% agarose/1 x denaturing gel (Ambion). 18S ribosomal RNA bands were detected with ethidium bromide. RNA was transferred to positively charged nylon membranes (Roche Diagnostics, Germany) and probed with digoxigenin-labeled RNA probes complementary to S4 and M2 (generated with the DIG RNA labeling Kit (Roche) according to manufacturer's instructions with pCDNA3.1 (Invitrogen) containing the S4 or M2 segments). Probed mRNAs were detected with the DIG block and washing buffer kit (Roche) using the anti-digoxigenin-alkaline phosphatase, Fab fragment (Roche). Substrate DDAO phosphate (Molecular Probes) was added (30 min) and signal visualized with the Typhoon 9400. Band intensity was determined using the ImageQuant TL software (Amersham) in all applications. F. Metabolic Radiolabeling and Immunoprecipitation

[99] Cells were metabolically labeled with 100 μCi/mL EasyTag [ ³⁵ S] methionine (Perkin Elmer) in methionine-free DMEM (Invitrogen) for 10, 30 or 60 min and lysed with RIPA. Alternatively, following pulse labeling, monolayers were washed twice and chased for various time intervals in DMEM containing 10% NCS prior to lysis. To isolate cytoskeletal inclusion bodies, cells were incubated with 10 mM Pipes (pH 6.8), 3 mM MgCl ₂ , 100 mM KCl, 30 mM sucrose, 1% triton X-100, 1 mM PMSF, and PIC on ice for 5 min and soluble proteins collected. The remaining cytoskeleton and associated structures were solubilized with RIP A/0.1% SDS.

[100] Metabolically labeled cell lysates were incubated for 1 h at RT with protein A sepharose (Amersham) bound to anti-reovirus rabbit polyclonal antibody or anti-reovirus σl C terminal rabbit polyclonal antibody. Antibody-bound complexes were washed twice with

RIPA. Immunoprecipitated and cell lysate samples were separated by 10% SDS-PAGE and detected by autoradiography on the Typhoon 9400 imager.

G. Western Blotting Analysis

[101] Infected cells were harvested, washed once with PBS and lysed in RIPA. Protein concentrations of cell lysates were determined using a Coomassie protein assay reagent (Pierce). Lysates (15 μg) were separated by 10% SDS-PAGE, electrophoretically transferred to PolyScreen™ transfer polyvinylidene fluoride membranes (Perkin Elmer). The membranes were probed with anti-reo virus polyclonal rabbit antibody (1/10 000 or 1/100 000 dilution), or anti-actin (1/20 000 dilution, Sigma Aldrich), followed by HRP-conjugated goat anti-rabbit IgG (1/10 000, Jackson Laboratories). The immuno-reactive protein bands were detected using enhanced chemiluminescence (Amersham) and visualized with a Typhoon 9400 imager.

H. Analysis of Virus Isolated From Ras-Transformed and Non-Transformed NIH 3T3

Cells

[102] At 18 hpi, 1.0 x 10 ⁷ cells were harvested, washed once with PBS and lysed in 10 mL of RIPA. Cellular and nuclear debris was removed by centrifugation. Reovirus was pelleted through a 30% sucrose cushion for 1 h at 100,000 x g, resuspended in PBS, and used for plaque titration. Diluted purified reovirus was separated by 10% SDS-PAGE electrophoresis and viral proteins detected by silver staining and quantified using the typhoon 9400 imager.

I. Electron Microscopy

[103] At 24 hpi, cells were harvested, pelleted, and fixed for 2 hr at RT in 3% glutaraldehyde, 0.25 M sucrose, 50 niM sodium cacodylate buffer, then post-fixed in 2% OsO ₄ in the same buffer, and incubated overnight in 0.1% aqueous uranyl acetate. Cells were embedded in TAAB resin and dehydrated in ethanol. For negative staining, purified virus preparations were applied to carbon-coated formvar films; contrast was provided using 0.1% uranyl acetate. Micrographs were taken with a JEOL JEM- 1230 transmission electron microscope.

J. Apoptosis Quantification

[104] At 24 hpi, 1.0 x 10 ⁶ cells were cultivated in the presence or absence of 50 μM Z-VaI- Ala-DL-Asp(OMe)-fluoromethylketone (ZVAD-fmk, Calbiochem), harvested and washed once with PBS. Apoptotic cells were labeled with annexin V-FITC and propidium iodide as described in the manufacturer's protocol (BD Pharmingen). Cell death was quantified by flow cytometry using a FACScan flow cytometer and analyzed using WinDMI Version 2.8.

EXAMPLE 2 Effect of Ras Transformation on Reovirus Cell Infection. Infectious Pro ^g en ^y and Virus Spread

[105] It has been shown that constitutive activation of Ras or activated upstream and downstream Ras signaling elements results in enhanced permissiveness of NIH 3T3 cells to reovirus infection (Norman et al., 2004; Strong et al., 1998). Reovirus protein synthesis was significantly higher in Ras-transformed cells at 48 hours post-infection (hpi), and this was presumed to account for the enhanced susceptibility seen with Ras-transformed cells (Strong et al., 1998).

[106] In this example, the progression of reovirus infection in NIH 3T3 cells freshly transformed with activated Ras was followed using immunofluorescence (IF) microscopy and flow cytometry (FACS). At various times post infection (pi), non- and Ras-transformed cells were visualized by IF or FACS. Results are shown in Figure 1. Total cells were detected by nuclear DAPI staining and reovirus protein-expressing cells fluorescent green (Fig. IA). The percent of cells positive for reovirus (as determined by increased fluorescence) is depicted in each panel of Fig. IB. Total (cell -associated and released) infectious virus particles made in non- (•) and Ras-transformed (o) cells were quantified by plaque titration assay at various times post infection. Each data point represents the average of three to six separate determinations ± standard deviation (SD) (Fig. 1C). Plaque titration of reovirus on Ras- and non-transformed cells was carried out as follows: Three days after infection, plaques of reovirus-infected cells were detected by immunohistochemical staining with reovirus-specific serum (Fig. ID). Numbers indicate quantities of productively infected cells within each plaque. Plaque size reflects the efficiency of replication and spread to neighboring cells. Figure IE shows photomicrographs of non- and Ras-transformed cells infected with reovirus. From these results the following observations were made. [107] First, it was consistently noted that within the first 18-24 hpi, there were approximately three times more productively infected Ras-transformed cells than non- transformed cells, despite exposure of equal number of cells to equal amounts of reovirus particles per cell (Figures IA and B). Surprisingly, both infected Ras and non- transformed cells stained at the same relative level of fluorescence intensity, indicating similar levels of reovirus protein expression at the per infected cell level. This suggests that an early event prior to viral transcription and translation is likely facilitated in Ras-transformed cells. [108] Second, while the percentage of fluorescent non-transformed NIH 3T3 cells remained relatively unchanged after 18-24 hpi, there was a drastic increase in the percentage of

fluorescent Ras-transformed cells after this time (Figures IA and IB). The rapid spread of reovirus to neighboring cells after 24 hpi in Ras-transformed cells represented a second round of infection in these cells that was scarcely detectable in non-transformed cells, and established the length of a single round of infection in NIH 3T3 cells to be 18-24 hpi. In congruence with this notion, standard virus plaque titration of reovirus showed very large plaques on Ras-transformed cell monolayers after 3 days of infection but only small plaques or singly infected cells on non-transformed cell monolayers (Fig. ID). By light microscopy, non-transformed cells appeared completely healthy at 48 hpi while the monolayer of Ras transformed cells was largely ablated (supplemental Fig. IE). The minimal spread of infection in non-transformed cell populations may have several explanations, such as an inability on the part of non-transformed cells to generate infectious progeny virus particles, inefficiency of virus release from infected cells, or interferon-mediated resistance of non- transformed cells to infection by progeny reovirus.

[109] Third, infectious reovirus titers were found to be 13-fold higher in Ras-transformed cells than in non-transformed cells by 18 hpi (Figure 1C). Taking into consideration that up to 3 times more Ras-transformed cells were productively infected in the first 18 h (Figure IA and B), activated Ras signaling further increased the yield of infectious progeny virus per infected cell by 4-5 fold. This led to the possibility that virus assembly may be more efficient in Ras-transformed cells or that virion assembly proceeds normally but the virions produced in Ras-transformed cells are somehow more infectious, or both. The difference in infectious reovirus titers between Ras-transformed and non-transformed cells was further amplified over subsequent rounds of infection (Figure 1C, 48 hpi).

[110] Because of the above observations, an in-depth analysis of each step of the reovirus life cycle was undertaken, with the objective of revealing the precise stages where Ras activation plays an important role.

EXAMPLE 3 Effects of Ras Transformation on Reovirus Uncoating [111] When first exposed to the same reovirus dose per cell, 2-3 times fewer non- transformed cells become productively infected than Ras-transformed cells (Figs. IA and B). To determine whether reovirus binds to non- and Ras-transformed cells at different levels, reovirus was allowed to bind to cells at 4° C for 1 hr, and the predominant reovirus protein, μlc, was detected with anti-reovirus antibody by western blot (Fig. 2A). The Reo lane is the purified reovirus marker. The histogram depicts densitometry quantification of μlc relative

to β-actin (0.99 +/- 0.08 SD, n = 4) in non- and Ras-transformed cells (1.01 +/- 0.14 SD, n=4). These results show that reovirus binds to non- and Ras-transformed cells at comparable levels. Consequently, the lower infection levels of non-transformed cells cannot be attributed to differences in virus attachment, since reovirus bound equally well to both non- and Ras-transformed cells (Figure 2A).

[112] To determine if differential rates of endocytosis were responsible for increased infection observed in Ras-transformed cells, the amount of reovirus that was internalized and had therefore become resistant to the membrane impermeable biotinylation reagent, sulfo- NHS-SS-biotin was measured. Virus was allowed to bind to cells at 4° C for 1 hr, the cells were next incubated at 37° C to allow for virus internalization. At various times thereafter, non-endocytosed virus that remained on the cell surface was detected by cell surface biotinylation followed by immunoprecipitation of biotinylated proteins. The results are shown in Fig. 2B. The top panel depicts a western blot of immunoprecipated reovirus detected with anti-reo virus antibody and shows reovirus proteins μlc and 8; relative band intensities (μlc/β-actin) quantified by densitometry are shown below. The results show nearly complete reovirus internalization within 1 hour and similar internalization rates for non- and Ras-transformed cells over sequential 20-minute intervals. [113] To establish a productive infection, internalized reovirions must undergo sequential proteolysis by lysosomal proteases to produce partially uncoated, transcriptionally active cores (Borsa et al., 1981). The degradation of outer coat protein σ3 and cleavage of coat protein μlc to δ are hallmarks of reovirus uncoating and are thought to occur simultaneously as the virus traverses the lysosomal membrane (Nibert et al., 2005). The inability of reovirus to infect several restrictive cell lines has been associated with inefficient uncoating (Golden et al., 2002). Other laboratories have also shown an increase in lysosomal protease activity in conjunction with activated Ras signaling cascades (Collette et al., 2004). To test whether reovirus uncoating is more efficient in Ras-transformed cells than in non-transformed cells, virus was allowed to bind to cells at 4° C for 1 hr, the cells were next incubated at 37° C to allow for virus internalization. Virus uncoating was monitored by following the cleavage of μlc to δ over time by western blot, quantified by densitometry and recorded as % cleavage. The results show that reovirus uncoating is indeed more efficient in Ras-transformed cells (Fig. 2C). The efficiency and rate of cleavage of μlc to δ was notably enhanced by constitutive Ras signaling. Reovirus prebound to cells for 1 hour at 4° C and then internalized at 37 ⁰ C for various lengths of time underwent negligible cleavage in non-transformed cells

even 4 h post binding, while 2/3 of reovirus μlc was cleaved to δ in Ras-transformed cells by this time. This finding therefore reveals a novel means by which Ras signaling impinges on a relative early step in the viral infection process, which is in turn accountable for the increased number of infected cells.

[114] Figure 2D shows the results of an experiment in which cells bound with reovirus were treated with chymo trypsin, and then incubated at 37° C for 4 hr.

EXAMPLE 4 Effect of Ras on Levels of Reovirus RNA and Transcripts [115] Enhanced efficiency of reovirus uncoating would produce more transcriptionally active virus cores and so should lead to increased levels of reovirus transcripts in Ras- transformed cells. Because previous quantification of reovirus transcription using reverse transcriptase PCR failed to show significant differences associated with Ras transformation (Strong et al., 1998), transcript levels were assessed using highly quantitative PCR (QPCR) methods.

[116] Both (+) and (-) sense RNA synthesis were first assessed using sense-specific primers for reverse transcription (Table 1). Positive-sense (+) RNA levels include both mRNA and dsRNA, while negative-sense (-) RNA, as part of the double-stranded genome, is synthesized only within newly assembled virus cores. The reovirus S4 and /or M2 RNAs were arbitrarily chosen for the analysis. The relative levels of reovirus (+) or (-) S4 RNA in infected non- (•) and Ras-transformed (o) cells were detected using QPCR following reverse transcription (Fig. 3A). The histogram represents the mean fold enhancement of reovirus S4 (+) RNAs in Ras-transformed cells over non-transformed cells at 4, 6 and 10 hpi, from four separate experiments. Note that non- and Ras-transformed cells bound with reovirus and then treated with chymotrypsin (CHT) (which equalizes the virus uncoating step in both cell types as shown in Figure 2D) had equal amounts of S4 (+) RNA at 6 hpi. At the initial time point examined (2 hpi), (+) and (-) S4 RNA levels were equivalent (Figure 3A), indicating that the inoculum applied to the two cell populations were equivalent, and that de novo virus-specific RNA synthesis was as yet undetectable. At subsequent time points (4-10 hpi), levels of both (+) and (-) RNA species were elevated significantly in Ras-transformed cells, as compared to those in non-transformed cells (Fig. 3A). An average 4-fold increase in the level of (+) and (-) RNA and was detected by QPCR in Ras-transformed cells from 4 to 10 hpi (Figure 3A). This correlates well with the approximately three times more productively infected Ras- transformed than non-transformed cells during this period (Figures IA and IB). Importantly,

the rates of S4 (+) RNA and (-) RNA synthesis were similar in both cell types after 4 hpi, again suggesting that Ras signaling is not involved in viral transcription or replication. Therefore, the higher level of viral RNA present in Ras-transformed cells is due largely to the more efficient virus uncoating in these cells.

[117] When the medium of infected cells was supplemented with chymotrypsin, which overcomes the uncoating requirement for intracellular proteases (Golden et al., 2002), complete cleavage of μlc to δ was found in both non-transformed and Ras-transformed cells (Figure 2D), and equal amounts of reovirus transcripts were detected at 6 hpi in non- and Ras-transformed cells infected in the presence of chymotrypsin (Fig. 3 A, right bar in bar graph). Ras signaling therefore augments the efficiency of reovirus uncoating, and the subsequent transcriptional activation of input reovirions, with the end result being approximately three times more Ras-transformed cells become productively infected. [118] Northern blotting for S4 and M2 reovirus mRNAs (which encode σ3 and μl, respectively) at 10-24 hpi confirmed the QPCR results. (-) sense probes were used. [Heating RNA samples at 50° C denatures mRNA but not dsRNA, whereas heating to 65° C denatures both mRNA and dsRNA.] Reovirus mRNA fold increases in Ras-transformed over non- transformed cells are indicated and are calculated from serial dilutions of samples (shown in Fig. 3C) when the signal is saturated. Reovirus S4 (-) sense RNA (from dsRNA) synthesized at various times post-infection was detected by heating RNA samples to 65° C and probing with a (+) sense probe using northern blot analysis (Fig. 3D). 18S ribosomal RNA was used throughout as loading control. The Reo lane is dsRNA isolated from purified reovirus and denatured by heating to 65° C. These results consistently demonstrate 2-4 fold more viral mRNA in Ras- than non-transformed cells (Figs. 3B and 3C). Similar differences were found for S2, Ml and Ll transcripts (Fig. 3F). By 48 hpi, reovirus-specific mRNA synthesis (M2 and S4) was increased, on average, 10-fold in Ras-transformed cells (Fig. 3C). The second round of infection (24-48 hpi) that occurs in Ras, but not in non-transformed cells (Figure IA and B), likely accounts for the observed significant amplification of reovirus RNA in Ras- transformed cells.

EXAMPLE 5 Effect of Ras Transformation on Translation Efficiency [119] The rate of reovirus protein translation was measured in Ras and non-transformed NIH 3T3 cells. Cells were metabolically labeled with [ ³⁵ S] methionine for 1 hr then subjected to immunoprecipitation with reovirus-specific antibodies. Figure 4A shows the

results of SDS-PAGE analysis of cell lysates prepared from infected non- or Ras-transformed cells pulse-labeled with [ ³⁵ S]-methionine for 1 h at various times pi. The table below fig. 4A shows relative intensities (Ras/non) of individual protein bands and the average fold increase of overall reovirus protein synthesis in Ras-transformed cells relative to that in non- transformed cells. Figure 4B shows western blot analysis of reovirus proteins in non- and Ras-transformed cells at various times pi. Blots were probed with anti-reovirus antibody (top) or anti-β-actin antibody as loading control (bottom). Figure 4C shows a representative histogram showing approximately three-fold enhancement of both reovirus S4 mRNA and its translation product protein σ3 in Ras-transformed cells relative to those in non-transformed cells at 12, 18, and 24 hpi.

[120] It was consistently found that protein translation was enhanced an average 3-fold in Ras-transformed cells in the first 18 hours of the virus replication cycle (Fig. 4A). In addition, virus protein accumulation detected by western blotting confirmed that in the first 24 hpi, there was no more than a 2-3 fold increase in reovirus proteins within Ras- transformed cells (Fig. 4B). When the 3-4 fold differences in mRNA levels between Ras- transformed and non-transformed cells are taken into consideration, however, there was no enhancement of reovirus protein translation in Ras-transformed cells (Fig. 4C). At 48 hpi, protein synthesis was increased nearly 7-fold in Ras-transformed cells, again reflecting the increase in transcript levels (Figs. 4A and 3B) as the result of second round infection that occurs in these cells (Figs. IA and B). These experiments suggest that reovirus protein synthesis does not play any apparent role in Ras-mediated reovirus permissiveness.

EXAMPLE 6 Effect of Ras Transformation on Virion Assembly Rate [121] The 3-fold differences in reovirus protein levels did not account for the 13-fold increase in total infectious virus production by 18 hpi (Figure 1C). Accordingly, steps of reovirus replication downstream of protein translation were looked at in search of additional effects of Ras signaling that might account for the large enhancement in reovirus titer. Ras- induced cytoskeletal changes could, for example, affect the recruitment of newly synthesized viral proteins to cytoskeleton-associated inclusion bodies (viral 'factories'), the sites of reovirus assembly (Malumbres and Pellicer, 1998; Becker et al., 2001). [122] Virus assembly was assessed by monitoring the shift of newly synthesized soluble (S) viral proteins to insoluble (I) "viral factories". Infected cells were metabolically labeled with [ ³⁵ S]-methionine at 18 hpi for 30 min (pulse) and then chased for up to 6 h. Triton X-soluble (S) and insoluble (I) reovirus proteins were analysed by immunoprecipitation with anti-

reovirus antibody (Fig. 5A). Proteolytic cleavage of reovirus coat protein μl to μlc in non- and Ras-transformed cells was examined. Reovirus proteins were immunoprecipitated with anti-reo virus antibody from cells that were metabolically labeled at 18 hpi for 10 min (pulse) and then chased for up to 8 h (Fig. 5B). Next, the progression of reovirus particle assembly in non- and Ras-transformed cells was studied. At 18 hpi, cells were metabolically labeled for 1 h (pulse) and then chased for up to 4 h (top) and virions immunoprecipitated with anti- σl antibody (bottom) (Fig. 5C). Densitometric analysis of relative protein band intensities and the average fold increase of reovirus-specific protein in Ras-transformed cells are summarized in the table below fig. 5C. Transmission electron microscopy (TEM) was used to detect viral factories and particles in sections of non- and Ras-transformed cell 24 hpi (Fig. 5D).

[123] The results demonstrate that the movement of reovirus proteins into triton X-100 insoluble inclusion bodies was similar in both non- and Ras-transformed cells, as shown by pulse-chase experiments at 18 hpi (Fig. 5A). Electron microscopy of cell sections of reovirus infected non- and Ras-transformed cells at 24 hpi confirmed that viral factories had formed in both cell types with no obvious difference in the morphology of the assembled virions (Fig. 5D).

[124] An important maturation step in the assembly of reovirus is the autocatalytic cleavage of the 76 kD μl outer capsid protein to yield the 72 kD μlc cleavage product (Nibert et al., 2005). Pulse-chase experiments at 18 hpi followed by immunoprecipitation with reovirus- specific antibodies showed that non-transformed and Ras-transformed cells had similar rates of μl to μlc cleavage (Fig. 5B); again suggesting that virus assembly is similar in these two cell types.

[125] One of the final steps in reovirus assembly involves the addition of the receptor binding protein σl to the 12 turrets of the virus particle (Lee et al., 1981). Virion assembly can therefore be assessed by the ability of anti-σl antibodies to pull down virions. To this end, lysates prepared from cells metabolically labeled at 18 hpi for 1 h and then chased for up to 4 h were immunoprecipitated with anti-σl antibody and analysed by SDS-PAGE (Fig. 5C). A 2-to 4-fold difference in the relative amount of assembled reovirus particles was observed in Ras-transformed compared to non-transformed cells, but this difference can, as discussed previously, be readily accounted for by the increased infection rate seen in transformed cells, and attributed here to enhanced uncoating, and therefore resulting increased reovirus RNA and proteins. In addition, assembled virus particles where clearly visible in both cell types by

electron microscopy (Fig. 5D). These experiments suggest that Ras signaling cascades do not impact the efficiency of reovirus assembly, leaving as yet unexplained the 13 -fold difference in titers observed at 18 hpi.

EXAMPLE 7 Infectivitv of Progeny Reovirus from Ras Transformed Cells [126] An apparent lack of congruence between virus production (2-4 fold increase; Fig. 5) and titer (13-fold increase; Fig. 1C) during the first round of infection in Ras-transformed cells could be explained if a greater proportion of assembled virus in these cells was infectious. Even in the highly susceptible L929 mouse fibroblast cells used to propagate reovirus for decades, only 10 ^"2 to 10 ^"3 virus particles assembled are capable of establishing a productive infection (plaque forming unit, pfu). Explanations as to why most assembled reovirus particles fail to establish infection remain as yet unsatisfactory. [127] To address the possibility that virus particles assembled in cells with activated Ras are more infectious, virus was purified from infected cells at 18 hpi (end of first round infection but prior to major virus release) by high-speed centrifugation and determined the effect of activated Ras signaling on the pfu-to-particle ratio. Figure 6A shows negative staining TEM of virus preparations purified from identical number of non- and Ras-transformed cells harvested at 18 hpi. Figure 6B shows silver staining of reovirus capsid proteins of virus preparations. Two-fold serially diluted highly purified reovirus (lanes 1-5) was used as a standard to determine the relative protein concentration of two-fold serially diluted virus preparations from non- (lanes 6-10) or Ras-transformed cells (lanes 11-15). A table showing the infectivity of each virus preparation (pfu/ml) compared to number of virus particles/mL, calculated pfu:particle ratio, and fold more infectious virus particles made in Ras-transformed cells is Figure 6C.

[128] Electron microscopy showed an estimated 2-to 4-fold more particles from Ras- transformed cells than from an equivalent number of non-transformed cells with few (3-4%) empty capsids present in either preparation (Fig. 6A). The total amount of reovirus particles in each preparation was further assessed by measuring the abundance of structural proteins following SDS-PAGE and silver staining (Fig. 6B). Proteins from virions purified from Ras- transformed and non-transformed cells were quantitatively compared to CsCl gradient- purified reovirus with known particle concentration. This approach showed that there were 3.7 times more virus particles made in Ras-transformed cells (Fig. 6C). [129] Virus infectivity was determined by standard virus plaque titration, which revealed that the virus preparation from Ras-transformed cells (4.9 xlO ¹⁰ pfu/mL) was approximately

15 times more infectious than the preparation from non-transformed cells (3.3 xl0 ⁹ pfu/mL, Fig. 6C). The virus particles made in Ras-transformed cells are therefore 4.1 times more active than virus particles assembled in non-transformed cells (Fig. 6C). On average, reovirus progeny made in Ras-transformed cells were 4.14 +/- 0.50 SD (n = 5) fold more infectious than progeny reovirus assembled in non-transformed cells. It was determined that 1/582 and 1/143 virus particles were infectious when assembled in non-transformed cells and Ras-transformed cells, respectively. This is the first time that the Ras signaling pathway(s) has been implicated in the production of virus particles having enhanced infectivity.

EXAMPLE 8 Apoptosis and Release of Infectious Reovirus Progeny from Ras-Transformed Cells

[13Oj Taken together, enhanced reovirus entry and activity of reovirus particles in Ras- transformed cells produce the 13-fold higher reovirus titers during the first round of infection (at 18 hpi, Fig. 1C), and partially account for the amplification of reovirus in subsequent rounds of infection.

[131] This was further examined by determining infectious particles released into the medium and apoptosis. Infectious virus particles released into the medium of non- (•) and Ras-transformed (o) cells were quantified by plaque titration assay at various times pi. Data points represent the average (n = 3-6) ± SD (Fig. 7A). Figure 7B shows virus detected by western blot with anti-reovirus antibody in total (cells and medium, top) and released (medium only, bottom) lysates of non- and Ras-transformed cells at various times pi. Amounts of non- and Ras-transformed cells positive for cell death by propidium iodide (PI, y axis) and annexin-V FITC (AV, x axis) staining were quantified by FACS analysis (Fig. 7B). Cells were mock infected or infected with reovirus for 24 h, in the presence of absence of apoptosis inhibitor zVAD-fmk. The percent of cells localized in each quadrant is indicated (lower left, - PI, - AV; lower right, - PI, + AV; upper left, + PI, - AV; upper right, + PI, + AV). Figure 7D shows a western blot of virus released in medium or of virus in total lysates of non- and Ras-transformed cells at 24 hpi, in the presence or absence of zVAD-fmk. Figure 7E shows FACS analysis of infected non- and Ras-transformed cells at 0, 20 and 48 hpi, in the presence or absence of zVAD-fmk. The percent of cells positive for reovirus (as determined by increased fluorescence) is depicted in each panel. [132] In addition to enhanced reovirus entry and activity of reovirus particles in Ras- transformed cells, however, a large difference in the extent of reovirus released from Ras- transformed and non-transformed cells was found. Titers of extracellular (released) reovirus

from Ras-transformed cells were 200 times higher than those from non-transformed cells at 24 hpi (Fig. 7A). At this time point, a second round infection for the Ras-transformed cells was already well underway, resulting in a 23-fold increase in total (cell associated and extracellular) infectious virus production (Fig. 1C). The efficiency of release is therefore nine times higher in association with Ras activation. Western blot analysis comparing the total virus with released virus produced by non-transformed and Ras transformed cells correlated with the plaque titration data, and confirmed that significantly more virus was released from Ras-transformed cells (Fig. 7B).

[133] Enhanced release of reovirus was associated with an increase in apoptosis of infected Ras-transformed cells. Cell death was quantified by FACS analysis of propidium iodide and annexin V-FITC stained cells. At 24 hpi, significantly more Ras-transformed cells underwent apoptosis (Fig. 7C). When background cell death of mock-infected cells was subtracted, 33.8% and 3.1% death of Ras- and non-transformed cells, respectively was found. Furthermore, the addition of pan-caspase apoptosis inhibitor zVAD-fmk effectively blocked the majority of cell death associated with reovirus infection, and release of virus (Figs. 7C and 7D). Inhibition of apoptosis with zVAD-fmk also significantly reduced the second round infection in Ras-transformed cells at 48 hpi (Fig. 7E). Together, these experiments suggest that enhanced release of reovirus through apoptosis in Ras transformed cells plays an important role in augmenting the spread of reovirus to neighboring cells. [134] Finally, while sufficient titers of reovirus were present in the medium of Ras- transformed cells to initiate a second round of infection by 18 hpi (i.e. titers were a little higher than I X lO ⁵ pfu/ml used initially to infect 30% of cells in Figure 1), reovirus released by non-transformed cells was insufficient for initiation of a second round of infection until approximately 36 hpi (Fig. 7A). Even though the interferon-mediated antiviral response may still play an additional role in preventing re-infection of non-transformed cells after 36 hpi, the effects of enhanced efficiency of uncoating, virus activity, and release can account for a vast increase in the spread of reovirus within Ras-transformed cells.

[135] EXAMPLE 9 Activated Ras Signaling Affects Steps in the Reovirus Life Cycle [136] Figure 8 shows the life cycle of reovirus in non-and Ras-transformed cells. Activated Ras signaling enhances three separate steps in the reovirus life cycle: virus uncoating, infectivity of progeny virions, and virion release. Reovirus binding and internalization are comparable for both non- and Ras-transformed cells, however, virus uncoating is 3 times more efficient in Ras-transformed cells. This results in 3-fold enhancement in virus

transcription, and corresponding enhancements in translation of viral proteins and production of progeny virus in Ras-transformed cells (step A). Viruses produced in Ras-transformed cells are 4-fold more infectious (pfu/particle ratio) than those produced in non-transformed cells (step B). Apoptosis is enhanced in reovirus-infected Ras-transformed cells and results in a 9-fold increase in release of progeny virus. The overall enhancement of approximately 100-fold (3 x 4 x 9) in production of infectious virions and release of progeny virus results in enhanced spread of virus infection in Ras-transformed cells. In contrast, the lack of efficient production and release of infectious progeny virus in non-transformed cells limits the spread of infection in these cells.

[137] Our comparative analysis of reovirus replication from entry to exit in non- versus Ras-transformed cells further defines the nature of Ras-dependent permissiveness. The above examples show that Ras-mediated reovirus oncolysis involves enhancement of three distinct steps in the virus replication cycle: (1) virus uncoating, (2) production of infectious progeny virions, and (3) apoptosis-mediated release of progeny virions (Fig. 8). Because reovirus replication in isogenic cell lines differing only in the presence or absence of an activating dominant mutation in Ras were compared, and because freshly transformed cells were used to exclude possible effects of additional accumulating mutations, it is definite that the differences observed can be attributed directly to Ras signaling. [138] For the first time, the present invention connects Ras activation with enhanced uncoating of reovirus during entry. Reovirus naturally infects humans through the enteric route, where proteolysis of the outer capsid is mediated by extracellular serine proteases (Wolf et al., 1981). In the absence of extracellular proteases, low pH-dependent cysteine proteases such as cathepsin L, cathepsin B and cathepsin S are involved in reovirus disassembly following endocytosis (Ebert et al., 2002). In the context of cancer therapy, where reovirus is administered intravenously or intratumorally, the intracellular and extracellular proteases may play a critical role.

[139] That viral oncolysis might depend in part on the efficiency of virus uncoating has several implications for cancer therapy. Upregulation of lysosomal cathepsins in response to Ras activation has been demonstrated in several cultured cell lines and is often observed in human tumor cells (Chambers et al., 1992; Denhardt et al., 1987). Efficient uncoating of reovirus may, therefore, play a significant role in establishing preferential replication and spread in vivo. Lysosomal protease secretion associated with tumor invasion and metastasis may, in fact, create an extremely favorable condition for rcovirus entry that mimics the natural enteric route of infection (Nomura and Katunuma, 2005). Possibly, selective

oncolysis by reo virus in vivo may ultimately be enhanced by protease-directed cancer treatments. In addition, it should be noted that many viruses (e.g., influenza virus, parvovirus, and adenovirus) require exposure to endocytic proteases for productive infection of cells (Basak and Turner, 1992; Greber et al., 1993; Stegmann et al., 1990). Accordingly, viruses other than reoviruses could be selected or engineered that are absolutely dependent on the precise cocktail of proteases found in cancer cells.

[140] The above examples show for the first time that Ras signaling is required for production of virus progeny that are highly infectious. Virus assembly is a complex process involving a series of orchestrated events; not surprisingly, a majority of progeny viruses are typically not infectious. This is particularly true in the case of RNA viruses having segmented genomes, including the reoviruses (Flint et al., 2004). The infectious activity of reovirus might be correlated with differences in structural components, such as proteins or RNA. The predominant reovirus structural proteins synthesized in Ras-transformed and non- transformed cells were compared by gel electrophoresis and western blotting, but were not appreciably different. For example, cleavage of the μl outer capsid protein was equally efficient in Ras-transformed and non-transformed cells. In addition, the relative amount of the σl receptor binding protein was comparable in both cell types. The virus-associated NTPase, μ2, was also present in similar amounts (data not shown). About the only reovirus structural protein whose level not measured was the RNA-dependent RNA polymerase (λ) due to its low abundance (12 copies per virion), co-migration with other structural proteins, and the absence of a specific antiserum.

[141] Differences may exist in the genomes of reovirus particles packaged in non- versus Ras-transformed cells. The mechanism(s) by which reovirus ensures that a full complement of its genome is incorporated into newly synthesized particles is unknown, and may be affected by Ras signaling.

[142] The final step in the reovirus life cycle is the release of progeny virions. The present invention shows for the first time that virus release is significantly enhanced, mediated through apoptosis and is required for efficient spread of infection in Ras-transformed cells. Cells transformed by constitutive Ras activation are more sensitive to reovirus-mediated apoptosis (Smakman et al., 2005; Strong et al., 1998). Reovirus induces apoptosis via activation of cellular stress kinase, c-Jun N-terminal kinase (JNK), and nuclear transcription factor NF-κB (Clarke et al., 2004; Connolly et al., 2000). Because Ras transformation also results in JNK and NF-κB activation (Pruitt et al., 2002; Finco et al., 1997; Malumbres and Pellicer, 1998), it is conceivable that activation of these kinases is somehow amplified by

reo virus infection, resulting in enhanced apoptosis-mediated release of progeny virus in Ras- transformed cells.

[143] Previous reports have demonstrated that the activation status of double-stranded RNA-activated protein kinase (PKR) plays an important role in determining the permissiveness of cells to reovirus replication (Strong et al., 1998). Since PKR is most notably responsible for regulating translation, it was presumed that the relationship between PKR activation and reovirus replication reflected the effects of PKR on reovirus translation. While a link between PKR inactivation and Ras activation likely exists (Mundschau and Faller, 1994), in view of this invention, however, PKR may impact reovirus replication through as yet uncharacterized mechanism(s). Ras-dependent inactivation of PKR may be important in regulating apoptosis via the NFKB pathway (Zamanian-Daryoush et al., 2000), promoting virus spread and hence, increased viral protein levels. Further study of reo virus- induced apoptosis could provide new avenues for determining the relationships between Ras signaling pathways, PKR activation, the fate of NFKB, and apoptosis. [144] Understanding the signaling components associated with reovirus-mediated apoptosis may reveal new insights into the regulation of cell death in transformed cells, and may provide novel targets for oncolytic therapy. Furthermore, as apoptosis is a common mechanism by which many non-enveloped viruses are released from cells (Flint et al., 2004), the study or Ras-dependant reovirus replication may provide a platform for discovery of additional selectively oncolytic viruses.

[145] Our in vitro model system demonstrates the potential of mammalian reovirus as an oncolytic agent. In non-transformed cells, inefficient uncoating, the production of inactive progeny virions, and inefficient release results in negligible spread and killing by reovirus. In Ras-transformed cells, on the other hand, reovirus efficiently establishes a productive infection, and produces active progeny viruses that are readily released. After just three replication cycles (72 hours), when only minimal spread of reovirus has occurred in non- transformed cells, most Ras-transformed cells have already been infected (Fig. 1). Consequently, three independent Ras-mediated mechanisms account for the potency and selectivity of reovirus in killing tumor cells: enhanced efficiency of virus uncoating, production of a larger proportion of infectious virus progeny and increased release. The enhanced efficiency of these three steps in reovirus replication produces a substantial amplification (about 100-fold) of reovirus production and virus spread in Ras-transformed cells, while non-transformed cells remain relatively unaffected.

[146] Human reovirus is a promising candidate for oncolytic viral therapy and is currently being tested in phase II clinical trials.

EXAMPLE 10 Susceptibility of Cells from a Tissue Sample to Reovirus Oncolysis

[147] A lump is found in a 65 year old woman when she has her regular mammogram. A sample is collected from the lump during biopsy and appears to be a malignant tumor. To determine if the tumor contains cells susceptible to reovirus, the sample is placed in cell culture and incubated with reovirus.

[148] The biopsy sample is minced in DMEM, incubated with reovirus. Uncoating of the reovirus is measured by measuring either degradation of the outer coat protein σ3 or by cleavage of coat protein μlc to δ. The infectiousness of the progeny virus produced from cells in the biopsy sample is determined by measuring the infectivity of the viral particles and comparing to the number of viral particles produced. Release of virus during apoptosis is measured by comparing virus released in the presence and absence of an apoptosis inhibitor.

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OTHER EMBODIMENTS

[206] It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages and modifications are within the scope of the following claims.

[207] All references cited herein are incorporated herein in their entirety.