Degron fusion constructs and methods for controlling protein production

Abstract

Engineered fusion proteins comprising a self-excising degron for controlling protein production are disclosed. In particular, the inventors have constructed fusion proteins comprising a degron connected to a protein of interest through a cleavable linker comprising a hepatitis C virus (HCV) protease site. The degron can be removed from the protein of interest by a cis-encoded HCV protease such that the protein of interest can be produced with minimal structural modification. Clinically available HCV protease inhibitors can be used to block protease cleavage such that the degron is retained after inhibitor addition on subsequently synthesized protein copies. The degron when attached causes rapid degradation of the linked protein. Such fusions of a degron to a protein of interest will be especially useful when control over protein production with minimal structural modification is desired.

1 . 70 . (canceled) 
     
     
         71 . A conditionally replicating viral vector comprising a modified genome of a virus such that production of a polypeptide required for efficient replication of the virus is controllable, wherein the viral vector comprises a nucleic acid encoding a fusion protein comprising: i) the polypeptide required for efficient replication of the virus; ii) a degron, wherein the degron is operably linked to the polypeptide required for efficient replication of the virus when the fusion protein is in an uncleaved state, such that the degron promotes degradation of the polypeptide in a cell; iii) a protease, wherein the protease can be inhibited by contacting said fusion protein with a protease inhibitor; and iv) a cleavable linker that is located between the polypeptide required for efficient replication of the virus and the degron, wherein the cleavable linker comprises a cleavage site recognized by the protease, wherein cleavage of the cleavable linker by the protease releases the polypeptide required for efficient replication of the virus from the fusion protein, such that when the fusion protein is in a cleaved state, the degron no longer controls degradation of the polypeptide required for efficient replication of the virus. 
     
     
         72 . The conditionally replicating viral vector of  claim 71 , wherein the virus is an RNA virus. 
     
     
         73 . The conditionally replicating viral vector of  claim 72 , wherein the virus is a measles virus. 
     
     
         74 . The conditionally replicating viral vector of  claim 73 , wherein the polypeptide required for efficient replication of the virus is a measles virus phosphoprotein. 
     
     
         75 . The conditionally replicating viral vector of  claim 74 , wherein the viral vector comprises a nucleotide sequence having at least 80% identity to the nucleotide sequence of SEQ ID NO:9. 
     
     
         76 . The conditionally replicating viral vector of  claim 71 , wherein the degron comprises an amino acid sequence having at least 80% identity to an amino acid sequence of SEQ ID NO:1. 
     
     
         77 . The conditionally replicating viral vector of  claim 76 , wherein the fusion protein comprises a hepatitis C virus (HCV) nonstructural protein 3 (NS3) protease. 
     
     
         78 . The conditionally replicating viral vector of  claim 71 , wherein the vector is a plasmid. 
     
     
         79 . The conditionally replicating viral vector of  claim 71 , further comprising a multiple cloning site. 
     
     
         80 . The conditionally replicating viral vector of  claim 71 , further comprising a polynucleotide encoding a tag, a detectable label, a targeting sequence, a linker, or any combination thereof. 
     
     
         81 . The conditionally replicating viral vector of  claim 80 , wherein the detectable label is a fluorescent protein or a bioluminescent protein. 
     
     
         82 . The conditionally replicating viral vector of  claim 71 , further comprising an exogenous nucleic acid. 
     
     
         83 . A recombinant virion comprising the conditionally replicating viral vector of  claim 71 . 
     
     
         84 . A method of controlling production of a virus, the method comprising:
 culturing a host cell comprising the conditionally replicating viral vector of  claim 71  under conditions suitable for producing the virus; and   contacting the host cell with a protease inhibitor, such that the polypeptide required for efficient replication of the virus is degraded when production of the virus is no longer desired.   
     
     
         85 . The method of  claim 84 , further comprising removing the protease inhibitor from the host cell, when resuming production of the virus is desired. 
     
     
         86 . The method of  claim 84 , further comprising, prior to the culturing, introducing the conditionally replicating viral vector into the host cell. 
     
     
         87 . The method of  claim 84 , wherein the viral vector comprises a nucleotide sequence having at least 80% identity to the nucleotide sequence of SEQ ID NO:9, wherein production of the virus can be inhibited with a protease inhibitor. 
     
     
         88 . The method of  claim 84 , wherein the degron comprises an amino acid sequence having at least 80% identity to an amino acid sequence of SEQ ID NO:1. 
     
     
         89 . The method of  claim 88 , wherein the fusion protein comprises a hepatitis C virus (HCV) nonstructural protein 3 (NS3) protease. 
     
     
         90 . The method of  claim 84 , wherein the protease inhibitor is selected from the group consisting of simeprevir, danoprevir, asunaprevir, ciluprevir, boceprevir, sovaprevir, paritaprevir and telaprevir.