Methods for the production of HCV, assaying HCV entry, and screening drugs and cellular receptors for HCV

A method for identifying a drug that inhibits HCV propagation comprising:

• (a) (i) infecting a cell with an HCV particle or (ii) transfecting a cell with one or more nucleic acids comprising an HCV nucleic acid, wherein the one or more nucleic acids encode a virus;
• (b) contacting the cell with a drug in a cell culture system;
• (c) incubating the cell culture system at a temperature from about 20° C. to about 34° C.;
• and
• (d) assaying the cell culture supernatant for the presence of infectious HCV particles, wherein the absence or reduction of infectious HCV particles compared to a supernatant from an uninfected or non-transfected cell culture, identifies the drug as a drug that inhibits HCV propagation.

A method for identifying a mutation that modulates HCV infection of cells comprising:

• (a) (i) infecting a cell with an HCV particle comprising a genome having at least one mutation or (ii) transfecting a cell with one or more nucleic acids comprising an HCV genome having at least one mutation;
• (b) incubating the cell in a cell culture system at a temperature from about 20° C. to about 34° C.;
• and
• (c) assaying the cell culture supernatant for the presence of infectious HCV particles, wherein the absence or reduction of infectious HCV particles compared to a supernatant from a cell culture infected or transfected with an unmutated HCV genome indicates that the mutation modulates HCV infection of cells.

A method for detecting HCV E1 and E2 mediated cell fusion comprising:

• (a) co-culturing in a cell culture system an effector cell with a target cell, wherein the effector cell comprises an HCV E1 protein and an HCV E2 protein that are displayed on the effector cell surface;
• (b) incubating the cell culture system at a temperature from about 20° C. to about 34° C.;
• and
• (c) assaying the co-culture for syncytium formation, thereby detecting HCV E1 and E2 mediated cell fusion.

A method for detecting HCV E1 and E2 mediated cell fusion comprising:

• (a) co-culturing in a cell culture system an effector cell with a target cell, wherein the effector cell comprises an HCV E1 protein and an HCV E2 protein that are displayed on the effector cell surface, wherein the E1 protein comprises an E1 ectodomain and an E1 transmembrane domain, and wherein the E2 protein comprises an E2 ectodomain and an E2 transmembrane domain;
• (b) transiently incubating the cell culture system at a pH from about 5.0 to about 5.4;
• and
• (c) assaying the co-culture for syncytium formation, thereby detecting HCV E1 and E2 mediated cell fusion.

A method for detecting HCV E1 and E2 mediated cell fusion comprising:

• (a) co-culturing in a cell culture system an effector cell with a target cell, wherein the effector cell comprises:
• (i) a trigger that activates a reporter,
• (ii) an HCV E1 protein displayed on the effector cell surface, and
• (iii) an HCV E2 protein displayed on the effector cell surface,
• and wherein the target cell comprises the reporter, wherein the E1 protein comprises an E1 ectodomain and an E1 transmembrane domain, and wherein the E2 protein comprises an E2 ectodomain and an E2 transmembrane domain;
• (b) transiently incubating the cell culture system at a pH from about 5.0 to about 5.4;
• and
• (c) assaying the co-culture for reporter activity, wherein detection of reporter activity indicates HCV E1 and E2 mediated cell fusion.

A method for identifying a polypeptide involved in HCV E1 and E2 mediated cell fusion comprising:

• (a) co-culturing in a cell culture system an effector cell with a target cell, wherein the effector cell comprises an HCV E1 protein and an HCV E2 protein that are displayed on the effector cell surface, and wherein the target cell comprises:
• (i) a polypeptide encoded by a library vector that is displayed on the target cell surface,
• (ii) a CD81 protein displayed on the target cell surface, and
• (b) incubating the cell culture system at a temperature from about 20° C. to about 34° C.;
• and
• (c) detecting increased syncytium formation as compared to a control co-culture wherein the target cell does not comprise the library vector, wherein detection of increased syncytium formation indicates that the polypeptide is involved in HCV E1 and E2 mediated cell fusion.

A method for identifying a polypeptide involved in HCV E1 and E2 mediated cell fusion comprising:

• (a) co-culturing in a cell culture system an effector cell with a target cell,
• wherein the effector cell comprises:
• (i) a trigger that activates a reporter,
• (ii) an HCV E1 protein displayed on the effector cell surface, and
• (iii) an HCV E2 protein displayed on the effector cell surface;
• and wherein the target cell comprises:
• (i) the reporter,
• (ii) a polypeptide encoded by a library vector that is displayed on the target cell surface,
• (iii) a CD81 protein displayed on the target cell surface,
• (b) incubating the cell culture system at a temperature from about 20° C. to about 34° C.;
• and
• (c) assaying the co-culture for increased reporter activity as compared to a control co-culture wherein the target cell does not comprise the library vector, wherein detection of increased reporter activity indicates that the polypeptide is involved in HCV E1 and E2 mediated cell fusion.

A method for identifying a mutation that inhibits HCV E1 and E2 mediated cell fusion comprising:

• (a) co-culturing in a cell culture system an effector cell with a target cell capable of HCV E1 and E2 mediated cell fusion, wherein the effector cell comprises:
• (i) a trigger that activates a reporter,
• (ii) an HCV E1 protein displayed on the effector cell surface, and
• (iii) an HCV E2 protein displayed on the effector cell surface, wherein at least the E1 protein or the E2 protein contains a mutation, and wherein the target cell comprises a the reporter;
• (b) incubating the cell culture system at a temperature from about 20° C. to about 34° C.;
• and
• (c) assaying the co-culture for decreased reporter activity as compared to a control co-culture wherein E1 and E2 do not contain the mutation, wherein detection of decreased reporter activity indicates that the mutation inhibits HCV E1 and E2 mediated cell fusion.

A method for identifying a molecule that inhibits HCV E1 and E2 mediated cell fusion comprising:

• (a) co-culturing in a cell culture system an effector cell with a target cell capable of HCV E1 and E2 mediated cell fusion, wherein the effector cell comprises:
• (i) a trigger that activates a reporter,
• (ii) an HCV E1 protein displayed on the effector cell surface, and
• (iii) an HCV E2 protein displayed on the effector cell surface, and wherein the E1 protein comprises an E1 ectodomain and an E1 transmembrane domain, and wherein the E2 protein comprises an E2 ectodomain and an E2 transmembrane domain;
• (b) adding a molecule to the cell culture system;
• (c) transiently incubating the cell culture system at a pH from about 5.0 to about 5.4;
• and
• (d) assaying the co-culture for decreased syncytia formation as compared to a control co-culture that is not exposed to the molecule, wherein detection of decreased syncytia formation indicates that the molecule inhibits HCV E1 and E2 mediated cell fusion.

A method for identifying a molecule that inhibits HCV E1 and E2 mediated cell fusion comprising:

• (a) co-culturing in a cell culture system an effector cell with a target cell capable of HCV E1 and E2 mediated cell fusion, wherein the effector cell comprises:
• (i) a trigger that activates a reporter,
• (ii) an HCV E1 protein displayed on the effector cell surface, and
• (iii) an HCV E2 protein displayed on the effector cell surface, wherein the E1 protein comprises an E1 ectodomain and an E1 transmembrane domain, wherein the E2 protein comprises an E2 ectodomain and an E2 transmembrane domain, and wherein the target cell comprises a repressor of the reporter;
• (b) adding a molecule to the cell culture system;
• (c) transiently incubating the cell culture system at a pH from about 5.0 to about 5.4;
• and
• (d) assaying the co-culture for decreased reporter activity as compared to a control co-culture that is not exposed to the molecule, wherein detection of decreased reporter activity indicates that the drug inhibits HCV E1 and E2 mediated cell fusion.