Replication competent hepatitus C virus and methods of use
AssigneeBoard of Regents, The University of Texas System
InventorsStanley M. Lemon, MinKyung Yi
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Abstract
The present invention provides a replication competent hepatitis C virus that includes a heterologous polynucleotide. The invention also includes methods for modifying a hepatitis C virus polynucleotide, selecting a replication competent hepatitis C virus polynucleotide, detecting a replication competent hepatitis C virus polynucleotide, and identifying a compound that inhibits replication of a hepatitis C virus polynucleotide.
Claims45
A method for identifying a compound that inhibits replication of an HCV RNA, the method comprising:
- contacting a cell comprising a replication competent HCV RNA with a compound, the replication competent HCV RNA comprising a heterologous polynucleotide comprising a first coding sequence encoding a transactivator;
- incubating the cell under conditions wherein the replication competent HCV RNA replicates in the absence of the compound;
- and
- detecting the replication competent HCV RNA, wherein a decrease the replication competent HCV RNA in the cell contacted with the compound compared to the replication competent HCV RNA in a cell not contacted with the compound indicates the compound inhibits replication of the replication competent HCV RNA.
A method for selecting a replication competent HCV RNA, the method comprising:
- incubating a vertebrate cell in the presence of a selecting agent,
- wherein:
- the cell comprises an HCV RNA comprising a first coding sequence encoding a hepatitis C virus polyprotein, and a heterologous polynucleotide;
- the heterologous polynucleotide comprises a second coding sequence encoding a selectable marker that confers resistance to the selecting agent;
- and
- the selecting agent inhibits replication of a cell that does not express the selectable marker;
- and
- detecting a cell that replicates in the presence of the selecting agent, wherein the presence of such a cell indicates the HCV RNA is replication competent.
A method for detecting a replication competent HCV RNA, the method comprising:
- incubating a vertebrate cell comprising an HCV RNA, wherein:
- the HCV RNA comprises a first coding sequence encoding a hepatitis C virus polyprotein, and a heterologous polynucleotide comprising a second coding sequence encoding a transactivator;
- the cell comprises a transactivated coding region and an operator sequence operably linked to the transactivated coding region;
- and
- the transactivated coding region encodes a detectable marker, wherein the transactivator alters transcription of the transactivated coding region;
- and
- detecting the detectable marker, wherein the presence of the detectable marker indicates the cell comprises a replication competent HCV RNA.
A method for detecting a replication competent HCV RNA, the method comprising:
- incubating a vertebrate cell comprising an HCV RNA, wherein:
- the HCV RNA comprises a first coding sequence encoding a subgenomic hepatitis C virus polyprotein, and a heterologous polynucleotide comprising a second coding sequence encoding a transactivator;
- the cell comprises a transactivated coding region and an operator sequence operably linked to the transactivated coding region;
- and
- the transactivated coding region encodes a detectable marker, wherein the transactivator alters transcription of the transactivated coding region;
- and
- detecting the detectable marker, wherein the presence of the detectable marker indicates the cell comprises a replication competent HCV RNA.
A replication competent HCV polynucleotide comprising:
- a first coding sequence encoding a subgenomic hepatitis C virus polyprotein;
- and
- a heterologous polynucleotide comprising a second coding sequence encoding a transactivator, wherein the heterologous polynucleotide is located 5′ of the first coding sequence.
A replication competent HCV polynucleotide comprising a first coding sequence encoding a hepatitis C virus polyprotein, and a heterologous polynucleotide.
A kit comprising:
- a replication competent HCV polynucleotide comprising a heterologous polynucleotide comprising a first coding sequence encoding a transactivator;
- and
- a vertebrate cell comprising a polynucleotide comprising a transactivated coding sequence encoding a detectable marker and an operator sequence operably linked to the transactivated coding sequence, wherein the transactivator interacts with the operator sequence and alters expression of the transactivated coding sequence.
Dependencies are inferred from the claim text.