Compositions and method for identifying enzyme and transport protein inhibitors

Abstract

The invention is directed to compositions to screen for small molecule drugs that inhibit proteases, such as viral proteases, e.g., HIV proteases; and methods for making and using these compositions. The invention provides compositions and methods for identifying compositions, e.g., drug molecules, that can inhibit proteases, e.g., HIV proteases. In alternative embodiments, the invention provides cell-based assays to screen for compositions, e.g., small molecules or drugs, that inhibit or modify the activity of enzymes such as calcium-dependent protein convertases involved in HIV envelop protein processing, including cleavage of the HIV gp160 envelope precursor, resulting in gp120 and gp41 envelope products.

What is claimed is: 
     
         1 . A cell-based method for monitoring the activity of a protease, a viral protease, or an HIV-1 protease (PR), comprising:
 (1) (a) providing a nucleic acid encoding a scaffold protein operatively linked to a transcriptional regulatory unit, wherein the scaffold protein comprises:
 (i) an amino acid motif or subsequence susceptible to cleavage by the protease, viral protease or HIV-1 protease (PR), under physiologic (cell culture) conditions; 
 (ii) a transmembrane domain; 
 (iii) a signal sequence or any amino acid motif that places the scaffold protein on the extracellular surface of the cell; and 
 (iv) a detectable moiety, 
 wherein the amino acid motif or subsequence susceptible to cleavage by the protease, viral protease or HIV-1 protease (PR) is positioned within the scaffold protein such that when the detectable moiety is cleaved away from (off from) the scaffold protein by the protease, viral protease or HIV-1 protease (PR) the remaining subsequence of scaffold protein on the extracellular surface of the cell lacks the detectable moiety; 
   (b) providing a nucleic acid encoding the protease, viral protease or HIV-1 protease (PR) operatively linked to a transcriptional regulatory unit, or a cell that expresses a heterologous or endogenous protease, viral protease or HIV-1 protease (PR);   (c) inserting (transfecting) the nucleic acid of (a) and (b) into the cell if the cell does not already express a heterologous or endogenous protease, viral protease or HIV-1 protease (PR);   (d) co-expressing the nucleic acid of (a) and (b) in the cell, or expressing the nucleic acid of (a) in the cell if the cell already expresses a heterologous or endogenous protease, viral protease or HIV-1 protease (PR); and   (e) determining whether the scaffold protein comprising the detectable moiety is expressed on the extracellular surface of the cell,   wherein an intact scaffold protein comprising the detectable moiety is expressed on the extracellular surface of the cell when the protease, viral protease or HIV-1 protease (PR) is not enzymatically active, and an intact scaffold protein is not or is substantially less expressed on the extracellular surface of the cell when the protease, viral protease or HIV-1 protease (PR) is enzymatically active (the detectable moiety is cleaved off by the protease, viral protease or HIV-1 protease (PR));   (2) the method of (1), wherein the scaffold protein further comprises an endoplasmic reticulum (ER) retention motif or a KDEL (SEQ ID NO:1) motif,   wherein the ER retention motif or KDEL (SEQ ID NO:1) motif is positioned in the scaffold protein such that when PR is active the scaffold will be separated into two pieces, leaving the ER retention motif-comprising or KDEL (SEQ ID NO:1) motif-comprising portion of the polypeptide in the ER and freeing the detectable moiety-comprising portion to the cell's extracellular membrane, and if PR is blocked or inactive, the entire scaffold polypeptide will be retained in the ER, and as a consequence will not be detected on the cell's extracellular surface; or   (3) the method of (1), wherein the scaffold protein further comprises a p2/p7 recognition site imbedded in the cytoplasmic loop of the scaffold.   
     
     
         2 . The method of  claim 1 , further comprising screening for an inhibitor of a protease, a viral protease or an HIV-1 protease (PR) by:
 (a) providing a compound to be screened as an inhibitor of a protease, viral protease or HIV-1 protease (PR), or providing a nucleic acid to be screened as encoding an inhibitor of a protease, viral protease or HIV-1 protease (PR);   (b) contacting a plurality of the cells with the compound or nucleic acid of (a) either before, during and/or after the co-expressing the nucleic acid of claim  1 (a) and claim  1 (b) in the cell; and   (c) determining whether the scaffold protein comprising the detectable moiety is expressed on the extracellular surface of the cell,   wherein an intact scaffold protein comprising the detectable moiety is expressed on the extracellular surface of the cell when the protease, viral protease or HIV-1 protease (PR) is inhibited by: the compound, a composition encoded by the nucleic acid, or a compound present in the cell only because the nucleic acid was expressed, and an intact scaffold protein is not or is substantially less expressed on the extracellular surface of the cell when the protease, viral protease or HIV-1 protease (PR) is enzymatically active (the detectable moiety is cleaved off by the protease, viral protease or HIV-1 protease (PR)) and the enzymatic activity of the protease, viral protease or HIV-1 protease (PR) is not significantly inhibited by: the compound, a composition encoded by the nucleic acid, or a compound present in the cell only because the nucleic acid was expressed.   
     
     
         3 . The method of  claim 1  or  claim 2  further comprising running a negative control comprising dividing the plurality of the cells co-expressing the nucleic acid of (a) and (b) in the cell and not adding the compound to be screened as an inhibitor to one of the divided cell samples. 
     
     
         4 . The method of  claim 1  or  claim 2  further comprising running a positive control comprising dividing the plurality of the cells co-expressing the nucleic acid of (a) and (b) in the cell and adding a known inhibitor of the protease, viral protease or HIV-1 protease (PR) to one of the divided cell samples. 
     
     
         5 . The method of  claim 1 , wherein the amino acid motif or subsequence susceptible to cleavage by the HIV-1 protease (PR) under physiologic (cell culture) conditions comprises SEQ ID NO:3 or SEQ ID NO:4. 
     
     
         6 . The method of  claim 1 , wherein the HIV-1 protease (PR) comprises SEQ ID NO:5 or SEQ ID NO:6. 
     
     
         7 . The method of  claim 1 , wherein the transcriptional regulatory unit comprises a promoter, an inducible promoter or a constitutive promoter. 
     
     
         8 . The method of  claim 1 , wherein the cell is a mammalian cell, a monkey cell or a human cell. 
     
     
         9 . The method of  claim 1 , wherein the scaffold proteins comprise all or part of a mouse Lyt2 or a human CD8 polypeptide. 
     
     
         10 . The method of  claim 1 , wherein the detectable moiety comprises an epitope for an antibody, or a FLAG tag. 
     
     
         11 . The method of  claim 1 , wherein the detectable moiety is detected or measured on the extracellular surface of the cell by a high throughput screen, a flow cytometry or microscope visualization. 
     
     
         12 . The method of  claim 1 , wherein the compound to be screened as an inhibitor of the protease, viral protease or HIV-1 protease (PR) comprises a small molecule, a nucleic acid, a polypeptide or peptide, a peptidomimetic, a polysaccharide or a lipid. 
     
     
         13 . The method of  claim 1 , wherein the compound to be screened as an inhibitor of the protease, viral protease or HIV-1 protease (PR) is a member of a library of compounds to be screened, or a member of a random peptide library or a chemical compound library. 
     
     
         14 . A cell-based method for monitoring the activity of a protease comprising:
 (1) (a) providing a nucleic acid encoding a scaffold protein operatively linked to a transcriptional regulatory unit, wherein the scaffold protein comprises:
 (i) an amino acid motif or subsequence susceptible to cleavage by the protease under physiologic (cell culture) conditions; 
 (ii) a transmembrane domain; 
 (iii) a signal sequence or any amino acid motif that places the scaffold protein on the extracellular surface of the cell; and 
 (iv) a detectable moiety, 
 wherein the amino acid motif or subsequence susceptible to cleavage by the protease is positioned within the scaffold protein such that when the detectable moiety is cleavage away from (off from) the scaffold protein by the protease the remaining subsequence of scaffold protein on the extracellular surface of the cell lacks the detectable moiety; 
   (b) providing a nucleic acid encoding the protease operatively linked to a transcriptional regulatory unit, or a cell that expresses a heterologous or endogenous protease;   (c) inserting (transfecting) the nucleic acid of (a) and (b) into the cell if the cell does not already express a heterologous or endogenous protease;   (d) co-expressing the nucleic acid of (a) and (b) in the cell, or expressing the nucleic acid of (a) in the cell if the cell already expresses a heterologous or endogenous protease; and   (e) determining whether the scaffold protein comprising the detectable moiety is expressed on the extracellular surface of the cell,   wherein an intact scaffold protein comprising the detectable moiety is expressed on the extracellular surface of the cell when the protease is not enzymatically active, and an intact scaffold protein is not or is substantially less expressed on the extracellular surface of the cell when the protease is enzymatically active (the detectable moiety is cleaved off by the protease); or   (2) the method of (1), wherein the scaffold protein further comprises an endoplasmic reticulum (ER) retention motif or a KDEL (SEQ ID NO:1) motif,   wherein the ER retention motif or KDEL (SEQ ID NO:1) motif is positioned in the scaffold protein such that when PR is active the scaffold will be separated into two pieces, leaving the ER retention motif-comprising or KDEL (SEQ ID NO:1) motif-comprising portion of the polypeptide in the ER and freeing the detectable moiety-comprising portion to the cell's extracellular membrane, and if PR is blocked or inactive, the entire scaffold polypeptide will be retained in the ER, and as a consequence will not be detected on the cell's extracellular surface.   
     
     
         15 . The method of  claim 14 , further comprising screening for an inhibitor of a protease by:
 (a) providing a compound to be screened as an inhibitor of a protease, or providing a nucleic acid to be screened as encoding an inhibitor of a protease;   (b) contacting a plurality of the cells with the compound or nucleic acid of claim  14 (a) either before, during and/or after the co-expressing the nucleic acid of claim  14 (a) and claim  14 (b) in the cell; and   (c) determining whether the scaffold protein comprising the detectable moiety is expressed on the extracellular surface of the cell,   wherein an intact scaffold protein comprising the detectable moiety is expressed on the extracellular surface of the cell when the protease is inhibited by: the compound, a composition encoded by the nucleic acid, or a compound present in the cell only because the nucleic acid was expressed, and an intact scaffold protein is not or is substantially less expressed on the extracellular surface of the cell when the protease is enzymatically active (the detectable moiety is cleaved off by the protease) and the enzymatic activity of the protease is not significantly inhibited by: the compound, a composition encoded by the nucleic acid, or a compound present in the cell only because the nucleic acid was expressed.   
     
     
         16 . The method of  claim 14  or  claim 15  further comprising running a negative control comprising dividing the plurality of the cells co-expressing the nucleic acid of (a) and (b) in the cell and not adding the compound to be screened as an inhibitor to one of the divided cell samples. 
     
     
         17 . The method of  claim 14  or  claim 15  further comprising running a positive control comprising dividing the plurality of the cells co-expressing the nucleic acid of (a) and (b) in the cell and adding a known inhibitor of the protease to one of the divided cell samples. 
     
     
         18 . The method of  claim 14 , wherein the transcriptional regulatory unit comprises a promoter. 
     
     
         19 . The method of  claim 18 , wherein the transcriptional regulatory unit comprises an inducible promoter. 
     
     
         20 . The method of  claim 18 , wherein the transcriptional regulatory unit comprises a constitutive promoter. 
     
     
         21 . The method of  claim 14 , wherein the cell is a mammalian cell, a monkey cell or a human cell. 
     
     
         22 . The method of  claim 14 , wherein the scaffold proteins comprise all or part of a mouse Lyt2 or a human CD8 polypeptide. 
     
     
         23 . The method of  claim 14 , wherein the detectable moiety comprises an epitope for an antibody, or a FLAG tag. 
     
     
         24 . The method of  claim 14 , wherein the detectable moiety is detected or measured on the extracellular surface of the cell by a high throughput screen, a flow cytometry or microscope visualization. 
     
     
         25 . The method of  claim 14 , wherein the compound to be screened as an inhibitor of protease comprises a small molecule, a nucleic acid, a polypeptide or peptide, a peptidomimetic, a polysaccharide or a lipid. 
     
     
         26 . The method of  claim 14 , wherein the compound to be screened as an inhibitor of protease is a member of a library of compounds to be screened, or a member of a random peptide library or a chemical compound library. 
     
     
         27 . The method of  claim 14 , wherein the protease is an HIV-1 protease (PR). 
     
     
         28 . The method of  claim 14 , wherein the protease is a viral, a microbial or a mammalian protease. 
     
     
         29 . A cell-based method for monitoring the activity of a cell's ER and/or trans-Golgi network comprising:
 (1) (a) providing a nucleic acid encoding a scaffold protein operatively linked to a transcriptional regulatory unit, wherein the scaffold protein comprises:
 (i) a transmembrane domain; 
 (ii) a signal sequence or any amino acid motif that places the scaffold protein on the extracellular surface of the cell; and 
 (iii) a detectable moiety; 
   (b) inserting (transfecting) the scaffold protein-encoding nucleic acid of (a) into the cell;   (d) expressing the nucleic acid of (a); and   (e) determining whether the scaffold protein comprising the detectable moiety is expressed on the extracellular surface of the cell,   wherein the scaffold protein is expressed on the extracellular surface of the cell when the activity of the cell's ER and trans-Golgi network is functioning; or   (2) the method of (1), wherein the scaffold protein further comprises an endoplasmic reticulum (ER) retention motif or a KDEL (SEQ ID NO:1) motif,   wherein the ER retention motif or KDEL (SEQ ID NO:1) motif is positioned in the scaffold protein such that when PR is active the scaffold will be separated into two pieces, leaving the ER retention motif-comprising or KDEL (SEQ ID NO:1) motif-comprising portion of the polypeptide in the ER and freeing the detectable moiety-comprising portion to the cell's extracellular membrane, and if PR is blocked or inactive, the entire scaffold polypeptide will be retained in the ER, and as a consequence will not be detected on the cell's extracellular surface.   
     
     
         30 . The method of  claim 14 , further comprising screening for an inhibitor of the cell's ER and trans-Golgi network by:
 (a) providing a compound or nucleic acid to be screened as an inhibitor of the cell's ER and trans-Golgi network;   (b) contacting a plurality of the cells with the compound or nucleic acid of (a) either before, during and/or after the co-expressing the nucleic acid of claim  29 (a) in the cell; and   (c) determining whether the scaffold protein comprising the detectable moiety is expressed on the extracellular surface of the cell,   wherein an intact scaffold protein comprising the detectable moiety is expressed (or is substantially expressed) on the extracellular surface of the cell when the cell's ER and trans-Golgi network is not inhibited.   
     
     
         31 . The method of  claim 29  further comprising running a negative control comprising dividing the plurality of the cells co-expressing the nucleic acid of claim  29 (a) in the cell and not adding the compound to be screened as an inhibitor to one of the divided cell samples. 
     
     
         32 . The method of  claim 29  further comprising running a positive control comprising dividing the plurality of the cells co-expressing the nucleic acid of claim  29 (a) in the cell and adding a known inhibitor of the cell's ER and/or trans-Golgi network to one of the divided cell samples. 
     
     
         33 . The method of  claim 29 , wherein the transcriptional regulatory unit comprises a promoter, an inducible promoter or a constitutive promoter. 
     
     
         34 . The method of  claim 29 , wherein the cell is a mammalian cell, a monkey cell or a human cell. 
     
     
         35 . The method of  claim 29 , wherein the scaffold proteins comprise all or part of a mouse Lyt2 or a human CD8 polypeptide. 
     
     
         36 . The method of  claim 29 , wherein the detectable moiety comprises an epitope for an antibody, or a FLAG tag. 
     
     
         37 . The method of  claim 29 , wherein the detectable moiety is detected or measured on the extracellular surface of the cell by a high throughput screen, a flow cytometry or microscope visualization. 
     
     
         38 . The method of  claim 29 , wherein the compound to be screened as an inhibitor of protease comprises a small molecule, a nucleic acid, a polypeptide or peptide, a peptidomimetic, a polysaccharide or a lipid. 
     
     
         39 . The method of  claim 29 , wherein the compound to be screened as an inhibitor of protease is a member of a library of compounds to be screened, or a member of a random peptide library or a chemical compound library. 
     
     
         40 . The method of  claim 29 , wherein the protease is an HIV-1 protease (PR). 
     
     
         41 . The method of  claim 29 , wherein the protease is a viral, a microbial or a mammalian protease. 
     
     
         42 . An isolated, recombinant or synthetic nucleic acid encoding a scaffold protein operatively linked to a transcriptional regulatory unit, wherein the scaffold protein comprises:
 (a) (i) an amino acid motif or subsequence susceptible to cleavage by a protease under physiologic (cell culture) conditions;   (ii) a transmembrane domain;   (iii) a signal sequence or any amino acid motif that places the scaffold protein on the extracellular surface of the cell; and   (iv) a detectable moiety; or   (b) the nucleic acid of (a), wherein the scaffold protein further comprises an endoplasmic reticulum (ER) retention motif or a KDEL (SEQ ID NO:1) motif,   wherein the ER retention motif or KDEL (SEQ ID NO:1) motif is positioned in the scaffold protein such that when PR is active the scaffold will be separated into two pieces, leaving the ER retention motif-comprising or KDEL (SEQ ID NO:1) motif-comprising portion of the polypeptide in the ER and freeing the detectable moiety-comprising portion to the cell's extracellular membrane, and if PR is blocked or inactive, the entire scaffold polypeptide will be retained in the ER, and as a consequence will not be detected on the cell's extracellular surface.   
     
     
         43 . The isolated, recombinant or synthetic nucleic acid of  claim 42 , wherein the protease is an HIV-1 protease (PR). 
     
     
         44 . The isolated, recombinant or synthetic nucleic acid of  claim 42 , wherein the protease is a viral, a microbial or a mammalian protease. 
     
     
         45 . The isolated, recombinant or synthetic nucleic acid of  claim 42 , wherein the scaffold protein comprise all or part of a mouse Lyt2 or a human CD8 polypeptide. 
     
     
         46 . The isolated, recombinant or synthetic nucleic acid of  claim 42 , wherein the detectable moiety comprises an epitope for an antibody, or a FLAG tag. 
     
     
         47 . A vector, expression cassette, cosmid or plasmid comprising the isolated, recombinant or synthetic nucleic acid of any of  claims 42  to  46 . 
     
     
         48 . An isolated, recombinant or synthetic polypeptide encoded by the nucleic acid of  claim 42 . 
     
     
         49 . A cell comprising the isolated, recombinant or synthetic nucleic acid of  claim 42 , the vector, expression cassette, cosmid or plasmid of  claim 47 , or isolated, recombinant or synthetic polypeptide of  claim 48 . 
     
     
         50 . A chimeric polypeptide comprising:
 (1) (i) an amino acid motif or subsequence susceptible to cleavage by the HIV-1 protease (PR) under physiologic (cell culture) conditions;   (ii) a transmembrane domain;   (iii) a signal sequence or any amino acid motif that places the scaffold protein on the extracellular surface of the cell; and   (iv) a detectable moiety,   wherein the amino acid motif or subsequence susceptible to cleavage by the HIV-1 protease (PR) is positioned within the scaffold protein such that when the detectable moiety is cleavage away from (off from) the scaffold protein by the HIV-1 protease (PR) the remaining subsequence of scaffold protein on the extracellular surface of the cell lacks the detectable moiety;   (2) the chimeric polypeptide of (1), wherein the scaffold protein further comprises an endoplasmic reticulum (ER) retention motif or a KDEL (SEQ ID NO:1) motif,   wherein the ER retention motif or KDEL (SEQ ID NO:1) motif is positioned in the scaffold protein such that when PR is active the scaffold will be separated into two pieces, leaving the ER retention motif-comprising or KDEL (SEQ ID NO:1) motif-comprising portion of the polypeptide in the ER and freeing the detectable moiety-comprising portion to the cell's extracellular membrane, and if PR is blocked or inactive, the entire scaffold polypeptide will be retained in the ER, and as a consequence will not be detected on the cell's extracellular surface; or   (3) the chimeric polypeptide of (1), wherein the scaffold protein further comprises a p2/p7 recognition site imbedded in the cytoplasmic loop of the scaffold,   where optionally the p2/p7 recognition sequence comprises or consists of ATIMMQRGN (SEQ ID NO:2), or optionally an exemplary amino-acid sequence of p2/p7 comprises AEAMSQVTNS/ATIMMQRGN/FRNQRKIVKCFNCGKEGHTARNCRAPRKKGCWK CGKEGHQMKDCTERQAN ATIMMQRGN (SEQ ID NO:3).   
     
     
         51 . A chimeric polypeptide comprising:
 (1) (i) an amino acid motif or subsequence susceptible to cleavage by the protease under physiologic (cell culture) conditions;   (ii) a transmembrane domain;   (iii) a signal sequence or any amino acid motif that places the scaffold protein on the extracellular surface of the cell; and   (iv) a detectable moiety,   wherein the amino acid motif or subsequence susceptible to cleavage by the protease is positioned within the scaffold protein such that when the detectable moiety is cleavage away from (off from) the scaffold protein by the protease the remaining subsequence of scaffold protein on the extracellular surface of the cell lacks the detectable moiety; or   (2) the chimeric polypeptide of (1), wherein the scaffold protein further comprises an endoplasmic reticulum (ER) retention motif or a KDEL (SEQ ID NO:1) motif,   wherein the ER retention motif or KDEL (SEQ ID NO:1) motif is positioned in the scaffold protein such that when PR is active the scaffold will be separated into two pieces, leaving the ER retention motif-comprising or KDEL (SEQ ID NO:1) motif-comprising portion of the polypeptide in the ER and freeing the detectable moiety-comprising portion to the cell's extracellular membrane, and if PR is blocked or inactive, the entire scaffold polypeptide will be retained in the ER, and as a consequence will not be detected on the cell's extracellular surface.   
     
     
         52 . A cell-based method for monitoring the activity of any enzyme, e.g., protease or HIV-1 protease (PR), or NS2/NS3 or NS3/NS4A protease of HCV, comprising:
 (1) (a) providing a nucleic acid encoding a scaffold protein operatively linked to a transcriptional regulatory unit, wherein the scaffold protein comprises:
 (i) an amino acid motif or subsequence susceptible to cleavage by the enzyme, e.g., protease or HIV-1 protease (PR), or NS2/NS3 or NS3/NS4A protease of HCV, under physiologic (cell culture) conditions; 
 (ii) a transmembrane domain; 
 (iii) a signal sequence or any amino acid motif that places the scaffold protein on the extracellular surface of the cell; and 
 (iv) a detectable moiety (e.g., a Green Fluorescent Protein (GFP) or a luciferase, or any compound that can be directly or indirectly detected), 
 wherein the amino acid motif or subsequence susceptible to cleavage by the enzyme, e.g., protease or HIV-1 protease (PR), or NS2/NS3 or NS3/NS4A protease of HCV, is positioned within the scaffold protein such that when the detectable moiety is cleaved away from (off from) the scaffold protein by the enzyme, e.g., protease or HIV-1 protease (PR), or NS2/NS3 or NS3/NS4A protease of HCV, the remaining subsequence of scaffold protein on the extracellular surface of the cell lacks the detectable moiety; 
   (b) providing a nucleic acid encoding the enzyme, e.g., protease or HIV-1 protease (PR), or NS2/NS3 or NS3/NS4A protease of HCV, operatively linked to a transcriptional regulatory unit, or a cell that expresses a heterologous or endogenous enzyme, e.g., protease or HIV-1 protease (PR), or NS2/NS3 or NS3/NS4A protease of HCV;   (c) inserting (transfecting) the nucleic acid of (a) and (b) into the cell if the cell does not already express a heterologous or endogenous enzyme, e.g., protease or HIV-1 protease (PR), or NS2/NS3 or NS3/NS4A protease of HCV;   (d) co-expressing the nucleic acid of (a) and (b) in the cell, or expressing the nucleic acid of (a) in the cell if the cell already expresses a heterologous or endogenous enzyme, e.g., protease or HIV-1 protease (PR), or NS2/NS3 or NS3/NS4A protease of HCV; and   (e) determining whether the scaffold protein comprising the detectable moiety is expressed on the extracellular surface of the cell (e.g., by flow cytometry or any high-throughput assay),   wherein an intact scaffold protein comprising the detectable moiety is expressed on the extracellular surface of the cell when the enzyme, e.g., protease or HIV-1 protease (PR), or NS2/NS3 or NS3/NS4A protease of HCV, is not enzymatically active, and an intact scaffold protein is not or is substantially less expressed on the extracellular surface of the cell when the enzyme, e.g., protease or HIV-1 protease (PR), or NS2/NS3 or NS3/NS4A protease of HCV, is enzymatically active (the detectable moiety is cleaved off by the enzyme, e.g., protease or HIV-1 protease (PR), or NS2/NS3 or NS3/NS4A protease of HCV);   (2) the method of (1), wherein the scaffold protein further comprises an endoplasmic reticulum (ER) retention motif or a KDEL motif,   wherein the ER retention motif or KDEL motif is positioned in the scaffold protein such that when the enzyme, e.g., protease or HIV-1 protease (PR), or NS2/NS3 or NS3/NS4A protease of HCV, is active the scaffold will be separated into two pieces, leaving the ER retention motif-comprising or KDEL motif-comprising portion of the polypeptide in the ER and freeing the detectable moiety-comprising portion to the cell's extracellular membrane, and if the enzyme, e.g., protease or HIV-1 protease (PR), or NS2/NS3 or NS3/NS4A protease of HCV, is blocked or inactive, the entire scaffold polypeptide will be retained in the ER, and as a consequence will not be detected on the cell's extracellular surface; or   (3) the method of (1), wherein the scaffold protein further comprises a p2/p7 recognition site imbedded in the cytoplasmic loop of the scaffold.   
     
     
         53 . The method of  claim 52 , further comprising screening for an inhibitor of an enzyme, e.g., protease or HIV-1 protease (PR), or NS2/NS3 or NS3/NS4A protease of HCV, by:
 (a) providing a compound to be screened as an inhibitor of an enzyme, e.g., protease or HIV-1 protease (PR), or NS2/NS3 or NS3/NS4A protease of HCV, or providing a nucleic acid to be screened as encoding an inhibitor of an enzyme, e.g., protease or HIV-1 protease (PR), or NS2/NS3 or NS3/NS4A protease of HCV;   (b) contacting a plurality of the cells with the compound or nucleic acid of (a) either before, during and/or after the co-expressing the nucleic acid of claim  1 (a) and claim  1 (b) in the cell; and   (c) determining whether the scaffold protein comprising the detectable moiety is expressed on the extracellular surface of the cell,   wherein an intact scaffold protein comprising the detectable moiety is expressed on the extracellular surface of the cell when the enzyme, e.g., protease or HIV-1 protease (PR), or NS2/NS3 or NS3/NS4A protease of HCV, is inhibited by: the compound, a composition encoded by the nucleic acid, or a compound present in the cell only because the nucleic acid was expressed, and an intact scaffold protein is not or is substantially less expressed on the extracellular surface of the cell when the enzyme, e.g., protease or HIV-1 protease (PR), or NS2/NS3 or NS3/NS4A protease of HCV, is enzymatically active (the detectable moiety is cleaved off by the enzyme, e.g., protease or HIV-1 protease (PR), or NS2/NS3 or NS3/NS4A protease of HCV) and the enzymatic activity of the enzyme, e.g., protease or HIV-1 protease (PR), or NS2/NS3 or NS3/NS4A protease of HCV, is not significantly inhibited by: the compound, a composition encoded by the nucleic acid, or a compound present in the cell only because the nucleic acid was expressed.   
     
     
         54 . The method of  claim 52  or  claim 53 , further comprising:
 (a) running a negative control comprising dividing the plurality of the cells co-expressing the nucleic acid of (a) and (b) in the cell and not adding the compound to be screened as an inhibitor to one of the divided cell samples; or 
 (b) further comprising running a positive control comprising dividing the plurality of the cells co-expressing the nucleic acid of (a) and (b) in the cell and adding a known inhibitor of the enzyme, e.g., protease or HIV-1 protease (PR), or NS2/NS3 or NS3/NS4A protease of HCV, to one of the divided cell samples. 
 
     
     
         55 . The method of  claim 52 , wherein:
 (a) the amino acid motif or subsequence susceptible to cleavage by the enzyme, e.g., protease or HIV-1 protease (PR), or NS2/NS3 or NS3/NS4A protease of HCV, under physiologic (cell culture) conditions comprises SEQ ID NO:2 or SEQ ID NO:3;   (b) the enzyme, e.g., protease or HIV-1 protease (PR), or NS2/NS3 or NS3/NS4A protease of HCV, comprises SEQ ID NO:4 or SEQ ID NO:5;   (c) the transcriptional regulatory unit comprises a promoter, an inducible promoter or a constitutive promoter;   (d) the cell is a mammalian cell, a monkey cell or a human cell;   (e) the scaffold proteins comprise all or part of a mouse Lyt2 or a human CD8 polypeptide;   (f) the detectable moiety comprises an epitope for an antibody, or a FLAG tag;   (g) the detectable moiety is detected or measured on the extracellular surface of the cell by a high throughput screen, a flow cytometry or microscope visualization;   (h) the compound to be screened as an inhibitor of the enzyme, e.g., protease or HIV-1 protease (PR), or NS2/NS3 or NS3/NS4A protease of HCV, comprises a small molecule, a nucleic acid, a polypeptide or peptide, a peptidomimetic, a polysaccharide or a lipid; or
 (i) the compound to be screened as an inhibitor of the enzyme, e.g., protease or HIV-1 protease (PR), or NS2/NS3 or NS3/NS4A protease of HCV, is a member of a library of compounds to be screened, or a member of a random peptide library or a chemical compound library. 
   
     
     
         56 . A cell-based method for monitoring the activity of an enzyme, e.g., a protease or HIV-1 protease (PR), or NS2/NS3 or NS3/NS4A protease of HCV, comprising:
 (1) (a) providing a nucleic acid encoding a scaffold protein operatively linked to a transcriptional regulatory unit, wherein the scaffold protein comprises:
 (i) an amino acid motif or subsequence susceptible to cleavage by the enzyme, e.g., protease or HIV-1 protease (PR), or NS2/NS3 or NS3/NS4A protease of HCV, under physiologic (cell culture) conditions; 
 (ii) a transmembrane domain; 
 (iii) a signal sequence or any amino acid motif that places the scaffold protein on the extracellular surface of the cell; and 
 (iv) a detectable moiety (e.g., a Green Fluorescent Protein (GFP) or a luciferase, or any compound that can be directly or indirectly detected), 
 wherein the amino acid motif or subsequence susceptible to cleavage by the enzyme, e.g., protease or HIV-1 protease (PR), or NS2/NS3 or NS3/NS4A protease of HCV, is positioned within the scaffold protein such that when the detectable moiety is cleaved away from (off from) the scaffold protein by the enzyme, e.g., protease or HIV-1 protease (PR), or NS2/NS3 or NS3/NS4A protease of HCV, the remaining subsequence of scaffold protein on the extracellular surface of the cell lacks the detectable moiety; 
   (b) providing a nucleic acid encoding the enzyme, e.g., protease or HIV-1 protease (PR), or NS2/NS3 or NS3/NS4A protease of HCV, operatively linked to a transcriptional regulatory unit, or a cell that expresses a heterologous or endogenous protease;   (c) inserting (transfecting) the nucleic acid of (a) and (b) into the cell if the cell does not already express a heterologous or endogenous protease;   (d) co-expressing the nucleic acid of (a) and (b) in the cell, or expressing the nucleic acid of (a) in the cell if the cell already expresses a heterologous or endogenous enzyme, e.g., protease or HIV-1 protease (PR), or NS2/NS3 or NS3/NS4A protease of HCV; and   (e) determining whether the scaffold protein comprising the detectable moiety is expressed on the extracellular surface of the cell (e.g., by flow cytometry or any high-throughput assay),   wherein an intact scaffold protein comprising the detectable moiety is expressed on the extracellular surface of the cell when the enzyme, e.g., protease or HIV-1 protease (PR), or NS2/NS3 or NS3/NS4A protease of HCV, is not enzymatically active, and an intact scaffold protein is not or is substantially less expressed on the extracellular surface of the cell when the enzyme, e.g., protease or HIV-1 protease (PR), or NS2/NS3 or NS3/NS4A protease of HCV, is enzymatically active (the detectable moiety is cleaved off by the protease); or   (2) the method of (1), wherein the scaffold protein further comprises an endoplasmic reticulum (ER) retention motif or a KDEL motif,   wherein the ER retention motif or KDEL motif is positioned in the scaffold protein such that when the enzyme, e.g., protease or HIV-1 protease (PR), or NS2/NS3 or NS3/NS4A protease of HCV, is active the scaffold will be separated into two pieces, leaving the ER retention motif-comprising or KDEL motif-comprising portion of the polypeptide in the ER and freeing the detectable moiety-comprising portion to the cell's extracellular membrane, and if the enzyme, e.g., protease or HIV-1 protease (PR), or NS2/NS3 or NS3/NS4A protease of HCV, is blocked or inactive, the entire scaffold polypeptide will be retained in the ER, and as a consequence will not be detected on the cell's extracellular surface.   
     
     
         57 . The method of  claim 56 , further comprising screening for an inhibitor of a protease by:
 (a) providing a compound to be screened as an inhibitor of the enzyme, e.g., protease or HIV-1 protease (PR), or NS2/NS3 or NS3/NS4A protease of HCV, or providing a nucleic acid to be screened as encoding an inhibitor of the enzyme, e.g., protease or HIV-1 protease (PR), or NS2/NS3 or NS3/NS4A protease of HCV;   (b) contacting a plurality of the cells with the compound or nucleic acid of claim  14 (a) either before, during and/or after the co-expressing the nucleic acid of claim  14 (a) and claim  14 (b) in the cell; and   (c) determining whether the scaffold protein comprising the detectable moiety is expressed on the extracellular surface of the cell,   wherein an intact scaffold protein comprising the detectable moiety is expressed on the extracellular surface of the cell when the enzyme, e.g., protease or HIV-1 protease (PR), or NS2/NS3 or NS3/NS4A protease of HCV, is inhibited by: the compound, a composition encoded by the nucleic acid, or a compound present in the cell only because the nucleic acid was expressed, and an intact scaffold protein is not or is substantially less expressed on the extracellular surface of the cell when the enzyme, e.g., protease or HIV-1 protease (PR), or NS2/NS3 or NS3/NS4A protease of HCV, is enzymatically active (the detectable moiety is cleaved off by the protease) and the enzymatic activity of the enzyme, e.g., protease or HIV-1 protease (PR), or NS2/NS3 or NS3/NS4A protease of HCV, is not significantly inhibited by: the compound, a composition encoded by the nucleic acid, or a compound present in the cell only because the nucleic acid was expressed.   
     
     
         58 . The method of  claim 56  or  claim 57 , further comprising:
 (a) running a negative control comprising dividing the plurality of the cells co-expressing the nucleic acid of (a) and (b) in the cell and not adding the compound to be screened as an inhibitor to one of the divided cell samples; or 
 (b) running a positive control comprising dividing the plurality of the cells co-expressing the nucleic acid of (a) and (b) in the cell and adding a known inhibitor of the enzyme, e.g., protease or HIV-1 protease (PR), or NS2/NS3 or NS3/NS4A protease of HCV, to one of the divided cell samples. 
 
     
     
         59 . The method of  claim 56 , wherein
 (a) the transcriptional regulatory unit comprises a promoter, or the transcriptional regulatory unit comprises an inducible promoter, or the transcriptional regulatory unit comprises a constitutive promoter;   (b) the cell is a mammalian cell, a monkey cell or a human cell; or a lymphocyte, or a T cell, or a CD4- or CD8-expressing cell;   (c) the scaffold proteins comprise all or part of a mouse Lyt2 or a human CD8 polypeptide;   (d) the detectable moiety comprises an epitope for an antibody, or a FLAG tag;   (e) the detectable moiety is detected or measured on the extracellular surface of the cell by a high throughput screen, a flow cytometry or microscope visualization;   (f) the compound to be screened as an inhibitor of protease comprises a small molecule, a nucleic acid, a polypeptide or peptide, a peptidomimetic, a polysaccharide or a lipid;   (g) the compound to be screened as an inhibitor of protease is a member of a library of compounds to be screened, or a member of a random peptide library or a chemical compound library; or   (h) the protease is an HIV-1 protease (PR), or a NS2/NS3 or a NS3/NS4A protease of HCV, or the protease is a viral, a microbial or a mammalian protease.   
     
     
         60 . A cell-based method for monitoring the activity of a cell's ER and/or trans-Golgi network comprising:
 (1) (a) providing a nucleic acid encoding a scaffold protein operatively linked to a transcriptional regulatory unit, wherein the scaffold protein comprises:
 (i) a transmembrane domain; 
 (ii) a signal sequence or any amino acid motif that places the scaffold protein on the extracellular surface of the cell; and 
 (iii) a detectable moiety (e.g., a Green Fluorescent Protein (GFP) or a luciferase, or any compound that can be directly or indirectly detected); 
   (b) inserting (transfecting) the scaffold protein-encoding nucleic acid of (a) into the cell;   (d) expressing the nucleic acid of (a); and   (e) determining whether the scaffold protein comprising the detectable moiety is expressed on the extracellular surface of the cell (e.g., by flow cytometry or any high-throughput assay),   wherein the scaffold protein is expressed on the extracellular surface of the cell when the activity of the cell's ER and trans-Golgi network is functioning; or   (2) the method of (1), wherein the scaffold protein further comprises an endoplasmic reticulum (ER) retention motif or a KDEL motif,   wherein in alternative embodiments the ER retention motif or KDEL motif is positioned in the scaffold protein such that when the enzyme is active the scaffold will be separated into two pieces, leaving the ER retention motif-comprising or KDEL motif-comprising portion of the polypeptide in the ER and freeing the detectable moiety-comprising portion to the cell's extracellular membrane, and if the enzyme is blocked or inactive, the entire scaffold polypeptide will be retained in the ER, and as a consequence will not be detected on the cell's extracellular surface.   
     
     
         61 . The method of  claim 60 , further comprising screening for an inhibitor of the cell's ER and trans-Golgi network by:
 (a) providing a compound or nucleic acid to be screened as an inhibitor of the cell's ER and trans-Golgi network;   (b) contacting a plurality of the cells with the compound or nucleic acid of (a) either before, during and/or after the co-expressing the nucleic acid of claim  29 (a) in the cell; and   (c) determining whether the scaffold protein comprising the detectable moiety is expressed on the extracellular surface of the cell,   wherein an intact scaffold protein comprising the detectable moiety is expressed (or is substantially expressed) on the extracellular surface of the cell when the cell's ER and trans-Golgi network is not inhibited.   
     
     
         62 . The method of  claim 60 , further comprising:
 (a) running a negative control comprising dividing the plurality of the cells co-expressing the nucleic acid of claim  29 (a) in the cell and not adding the compound to be screened as an inhibitor to one of the divided cell samples; or   (b) running a positive control comprising dividing the plurality of the cells co-expressing the nucleic acid of claim  29 (a) in the cell and adding a known inhibitor of the cell's ER and/or trans-Golgi network to one of the divided cell samples.   
     
     
         63 . The method of  claim 60 , wherein
 (a) the transcriptional regulatory unit comprises a promoter, an inducible promoter or a constitutive promoter;   (b) the cell is a mammalian cell, a monkey cell or a human cell; or a lymphocyte, or a T cell, or a CD4- or CD8-expressing cell;   (c) the scaffold proteins comprise all or part of a mouse Lyt2 or a human CD8 polypeptide;   (d) the detectable moiety comprises an epitope for an antibody, or a FLAG tag;   (e) the detectable moiety is detected or measured on the extracellular surface of the cell by a high throughput screen, a flow cytometry or microscope visualization;   (f) the compound to be screened as an inhibitor of protease comprises a small molecule, a nucleic acid, a polypeptide or peptide, a peptidomimetic, a polysaccharide or a lipid;   (g) the compound to be screened as an inhibitor of protease is a member of a library of compounds to be screened, or a member of a random peptide library or a chemical compound library; or   (h) the protease is an HIV-1 protease (PR) or a NS2/NS3 or a NS3/NS4A protease of HCV, or the protease is a viral, a microbial or a mammalian protease.   
     
     
         64 . An isolated, recombinant or synthetic nucleic acid encoding a scaffold protein operatively linked to a transcriptional regulatory unit, wherein the scaffold protein comprises:
 (a) (i) an amino acid motif or subsequence susceptible to cleavage by an enzyme, e.g., the protease, e.g., HIV protease, e.g., HIV-1 protease (PR), or an NS2/NS3 or NS3/NS4A HCV protease, under physiologic (cell culture) conditions;
 (ii) a transmembrane domain; 
 (iii) a signal sequence or any amino acid motif that places the scaffold protein on the extracellular surface of the cell; and 
 (iv) a detectable moiety (e.g., a Green Fluorescent Protein (GFP) or a luciferase, or any compound that can be directly or indirectly detected); or 
   (b) the nucleic acid of (a), wherein the scaffold protein further comprises an endoplasmic reticulum (ER) retention motif or a KDEL motif,   wherein in alternative embodiments the ER retention motif or KDEL motif is positioned in the scaffold protein such that when the enzyme, e.g., the protease, e.g., HIV protease, e.g., HIV-1 protease (PR), or an NS2/NS3 or NS3/NS4A HCV protease, is active the scaffold will be separated into two pieces, leaving the ER retention motif-comprising or KDEL motif-comprising portion of the polypeptide in the ER and freeing the detectable moiety-comprising portion to the cell's extracellular membrane, and if the enzyme, e.g., the protease, e.g., HIV protease, e.g., HIV-1 protease (PR), or an NS2/NS3 or NS3/NS4A HCV protease, is blocked or inactive, the entire scaffold polypeptide will be retained in the ER, and as a consequence will not be detected on the cell's extracellular surface.   
     
     
         65 . The isolated, recombinant or synthetic nucleic acid of  claim 64 , wherein
 (a) the protease is an HIV-1 protease (PR), or an NS2/NS3 or NS3/NS4A protease of HCV, or the protease is a viral, a microbial or a mammalian protease;   (b) the scaffold protein comprise all or part of a mouse Lyt2 or a human CD8 polypeptide; or   (c) the detectable moiety comprises an epitope for an antibody, or a FLAG tag.   
     
     
         66 . A vector, expression cassette, cosmid or plasmid comprising the isolated, recombinant or synthetic nucleic acid of  claim 64 . 
     
     
         67 . An isolated, recombinant or synthetic polypeptide encoded by the nucleic acid of  claim 64 . 
     
     
         68 . A cell comprising the isolated, recombinant or synthetic nucleic acid of  claim 64 , the vector, expression cassette, cosmid or plasmid of  claim 66 , or isolated, recombinant or synthetic polypeptide of  claim 67 . 
     
     
         69 . A chimeric polypeptide comprising:
 (1) (i) an amino acid motif or subsequence susceptible to cleavage by an enzyme, e.g., the protease, e.g., HIV protease, e.g., HIV-1 protease (PR), or an NS2/NS3 or NS3/NS4A HCV protease, under physiologic (cell culture) conditions;   (ii) a transmembrane domain;   (iii) a signal sequence or any amino acid motif that places the scaffold protein on the extracellular surface of the cell; and   (iv) a detectable moiety (e.g., a Green Fluorescent Protein (GFP) or a luciferase, or any compound that can be directly or indirectly detected),   wherein the amino acid motif or subsequence susceptible to cleavage by the enzyme, e.g., the protease, e.g., HIV protease, e.g., HIV-1 protease (PR), or an NS2/NS3 or NS3/NS4A HCV protease, is positioned within the scaffold protein such that when the detectable moiety is cleaved away from (off from) the scaffold protein by the enzyme, e.g., the protease, e.g., HIV protease, e.g., HIV-1 protease (PR), or an NS2/NS3 or NS3/NS4A HCV protease, the remaining subsequence of scaffold protein on the extracellular surface of the cell lacks the detectable moiety;   (2) the chimeric polypeptide of (1), wherein the scaffold protein further comprises an endoplasmic reticulum (ER) retention motif or a KDEL motif,   wherein in alternative embodiments the ER retention motif or KDEL motif is positioned in the scaffold protein such that when the enzyme, e.g., the protease, e.g., HIV protease, e.g., HIV-1 protease (PR), or an NS2/NS3 or NS3/NS4A HCV protease, is active the scaffold will be separated into two pieces, leaving the ER retention motif-comprising or KDEL motif-comprising portion of the polypeptide in the ER and freeing the detectable moiety-comprising portion to the cell's extracellular membrane, and if the enzyme, e.g., the protease, e.g., HIV protease, e.g., HIV-1 protease (PR), or an NS2/NS3 or NS3/NS4A HCV protease, is blocked or inactive, the entire scaffold polypeptide will be retained in the ER, and as a consequence will not be detected on the cell's extracellular surface; or   (3) the chimeric polypeptide of (1), wherein the scaffold protein further comprises a p2/p7 recognition site imbedded in the cytoplasmic loop of the scaffold,   where optionally the p2/p7 recognition sequence comprises or consists of ATIMMQRGN (SEQ ID NO:5), or optionally an exemplary amino-acid sequence of p2/p7 comprises AEAMSQVTNS/ATIMMQRGN/FRNQRKIVKCFNCGKEGHTARNCRAPRKKGCWK CGKEGHQMKDCTERQAN ATIMMQRGN (SEQ ID NO:6).   
     
     
         70 . A chimeric polypeptide comprising:
 (1) (i) an amino acid motif or subsequence susceptible to cleavage by an enzyme, e.g., the protease, e.g., HIV protease, e.g., HIV-1 protease (PR), or an NS2/NS3 or NS3/NS4A HCV protease, under physiologic (cell culture) conditions;   (ii) a transmembrane domain;   (iii) a signal sequence or any amino acid motif that places the scaffold protein on the extracellular surface of the cell; and   (iv) a detectable moiety (e.g., a Green Fluorescent Protein (GFP) or a luciferase, or any compound that can be directly or indirectly detected),   wherein the amino acid motif or subsequence susceptible to cleavage by the enzyme, e.g., the protease, e.g., HIV protease, e.g., HIV-1 protease (PR), or an NS2/NS3 or NS3/NS4A HCV protease, is positioned within the scaffold protein such that when the detectable moiety is cleaved away from (off from) the scaffold protein by the enzyme, e.g., the protease, e.g., HIV protease, e.g., HIV-1 protease (PR), or an NS2/NS3 or NS3/NS4A HCV protease, the remaining subsequence of scaffold protein on the extracellular surface of the cell lacks the detectable moiety; or   (2) the chimeric polypeptide of (1), wherein the scaffold protein further comprises an endoplasmic reticulum (ER) retention motif or a KDEL motif,   wherein in alternative embodiments the ER retention motif or KDEL motif is positioned in the scaffold protein such that when the enzyme, e.g., the protease, e.g., HIV protease, e.g., HIV-1 protease (PR), or an NS2/NS3 or NS3/NS4A HCV protease, is active the scaffold will be separated into two pieces, leaving the ER retention motif-comprising or KDEL motif-comprising portion of the polypeptide in the ER and freeing the detectable moiety-comprising portion to the cell's extracellular membrane, and if the enzyme, e.g., the protease, e.g., HIV protease, e.g., HIV-1 protease (PR), or an NS2/NS3 or NS3/NS4A HCV protease, is blocked or inactive, the entire scaffold polypeptide will be retained in the ER, and as a consequence will not be detected on the cell's extracellular surface.   
     
     
         71 . A cell-based method for monitoring the activity of any enzyme, e.g., protease or HIV-1 protease (PR), or NS2/NS3 or NS3/NS4A protease of HCV, comprising:
 (1) (a) providing: a nucleic acid encoding a chimeric (hybrid) protein operatively linked to a transcriptional regulatory unit (e.g., a promoter and/or enhancer, e.g., a doxycycline inducible promoter); and, a cell comprising an environment capable of supporting the expression of the chimeric (hybrid) protein by the nucleic acid,   wherein the chimeric (hybrid) protein comprises a chimeric Gal4 expression system comprising (i) an N-terminal Gal4 DNA-binding domain (e.g., DBD: aa 1-147); (ii) an enzyme whose activity is to be monitored, or an enzymatically active fragment thereof; and (iii) a Gal4 C-terminal Transactivation domain (e.g., TAD: aa 768-881),   and the enzyme whose activity is to be monitored or the enzymatically active fragment thereof is positioned in or within the chimeric protein such that an enzymatically active enzyme or enzymatically active fragment thereof is capable of cleaving or physically separating or otherwise functionally separating the N-terminal Gal4 DNA-binding domain from the Gal4 C-terminal Transactivation domain such that the Gal4 can no longer act as a functional transcription factor, and if the enzyme whose activity is to be monitored is inhibited such that it is no longer enzymatically active (or substantially no longer enzymatically active) the Gal4 C-terminal Transactivation domain in conjunction with the N-terminal Gal4 DNA-binding domain can function as a functional transcription factor;   (b) inserting (transfecting) the nucleic acid of (a) into the cell,   wherein optionally the cell does not already express a heterologous or endogenous enzyme, e.g., protease or HIV-1 protease (PR), or NS2/NS3 or NS3/NS4A protease of HCV; and   (c) contacting the cell with a putative (test) enzyme inhibitor,   wherein optionally the enzyme inhibitor comprises a small molecule, a protein, a nucleic acid, a polysaccharide and/or a lipid,   and optionally the enzyme inhibitor is added to the cell before, during and/or after inserting (transfecting) the nucleic acid of (a) into the cell and/or expressing the chimeric protein encoded by the nucleic acid of (a) in the cell,   and optionally cell-based method further comprises a negative control set of cells into which the nucleic acid of (a) also has been inserted and transfected and expresses the chimeric protein encoded by the nucleic acid of (a), but the negative control set of cells is not exposed to the putative (test) enzyme inhibitor or is exposed to a different putative (test) enzyme inhibitor;   (d) determining whether the putative (test) enzyme inhibitor is an effective or sufficient inhibitor of the enzyme or enzymatically active fragment thereof by measuring the ability of the Gal4 C-terminal Transactivation domain in conjunction with the N-terminal Gal4 DNA-binding domain to function as a functional transcription factor,   wherein optionally the ability of the Gal4 C-terminal Transactivation domain in conjunction with the N-terminal Gal4 DNA-binding domain to function as a functional transcription factor is measured by expression of a Fluorescent Protein (FP), e.g., an e-green fluorescent protein, or eGFP (excited with the 488 nm blue laser, an e-cyan fluorescent protein (or eCFP, using a 405 nm violet laser), and/or an mOrange or an mCherry (561 nm yellow laser), where the FP or GFP coding sequence is operably linked to or dependent (for its transcription) on the transcription factor;   (2) the method of (1), wherein the enzyme is a protease or an HIV-1 protease (PR), or a NS2/NS3 or NS3/NS4A protease of HCV; or   (3) the method of (1), wherein the cell is a lymphocyte or a T cell, or a CD4+ T cell, or a mammalian cell or a human cell.   
     
     
         72 . The method of  claim 71 , further comprising:
 (a) running a negative control comprising dividing the plurality of the cells co-expressing the nucleic acid of (a) in the cell and not adding the compound to be screened (the putative (test) enzyme inhibitor) as an inhibitor to one of the divided cell samples; or   (b) running a positive control comprising dividing the plurality of the cells co-expressing the nucleic acid of (a) in the cell and adding a known inhibitor of the enzyme, e.g., a known inhibitor of a protease or an HIV-1 protease (PR), or a NS2/NS3 or NS3/NS4A protease of HCV, to one of the divided cell samples.   
     
     
         73 . The method of  claim 71 , wherein:
 (a) the transcriptional regulatory unit comprises a promoter, an inducible promoter or a constitutive promoter;   (b) the cell is a mammalian cell, a monkey cell or a human cell;   (c) the positive activity of the Gal4 C-terminal Transactivation domain in conjunction with the N-terminal Gal4 DNA-binding domain to function as a functional transcription factor is detected or measured by a high throughput screen, a flow cytometry or microscope visualization;   (d) the compound to be screened as an inhibitor of the enzyme, e.g., protease or HIV-1 protease (PR), or NS2/NS3 or NS3/NS4A protease of HCV, comprises a small molecule, a nucleic acid, a polypeptide or peptide, a peptidomimetic, a polysaccharide or a lipid;   (e) the compound to be screened as an inhibitor of the enzyme, e.g., protease or HIV-1 protease (PR), or NS2/NS3 or NS3/NS4A protease of HCV, is a member of a library of compounds to be screened, or a member of a random peptide library or a chemical compound library;   (f) the transcriptional regulatory unit comprises a promoter, or the transcriptional regulatory unit comprises an inducible promoter or a constitutive promoter;   (g) the cell is a mammalian cell, a monkey cell or a human cell; or a lymphocyte, or a T cell, or a CD4- or CD8-expressing cell; or   (h) the enzyme is a viral protease, a microbial protease or a mammalian protease.   
     
     
         74 . An isolated, recombinant or synthetic nucleic acid encoding a chimeric (hybrid) protein operatively linked to a transcriptional regulatory unit (e.g., a promoter and/or enhancer, e.g., a doxycycline inducible promoter),
 wherein the chimeric (hybrid) protein comprises a chimeric Gal4 expression system comprising (i) an N-terminal Gal4 DNA-binding domain (e.g., DBD: aa 1-147); (ii) an enzyme whose activity is to be monitored, or an enzymatically active fragment thereof; and (iii) a Gal4 C-terminal Transactivation domain (e.g., TAD: aa 768-881),   and the enzyme whose activity is to be monitored or the enzymatically active fragment thereof is positioned in or within the chimeric protein such that an enzymatically active enzyme or enzymatically active fragment thereof is capable of cleaving or physically separating or otherwise functionally separating the N-terminal Gal4 DNA-binding domain from the Gal4 C-terminal Transactivation domain such that the Gal4 can no longer act as a functional transcription factor, and if the enzyme whose activity is to be monitored is inhibited such that it is no longer enzymatically active (or substantially no longer enzymatically active) the Gal4 C-terminal Transactivation domain in conjunction with the N-terminal Gal4 DNA-binding domain can function as a functional transcription factor,   wherein optionally the ability of the Gal4 C-terminal Transactivation domain in conjunction with the N-terminal Gal4 DNA-binding domain to function as a functional transcription factor is measured by expression of a Fluorescent Protein (FP), e.g., an e-green fluorescent protein, or eGFP (excited with the 488 nm blue laser, an e-cyan fluorescent protein (or eCFP, using a 405 nm violet laser), and/or an mOrange or an mCherry (561 nm yellow laser), where the FP or GFP coding sequence is operably linked to or dependent (for its transcription) on the transcription factor.   
     
     
         75 . A cell comprising the isolated, recombinant or synthetic nucleic acid of  claim 74 , wherein optionally the cell is a lymphocyte or a T cell, or a CD4+ T cell, or a mammalian cell or a human cell. 
     
     
         76 . A vector, expression cassette, cosmid or plasmid comprising or having contained therein the isolated, recombinant or synthetic nucleic acid of  claim 74 . 
     
     
         77 . A cell comprising the vector, expression cassette, cosmid or plasmid of  claim 76 , wherein optionally the cell is a lymphocyte or a T cell, or a CD4+ T cell, or a mammalian cell or a human cell. 
     
     
         78 . A chimeric Gal4 expression system comprising (i) an N-terminal Gal4 DNA-binding domain (e.g., DBD: aa 1-147); (ii) an enzyme whose activity is to be monitored, or an enzymatically active fragment thereof; and (iii) a Gal4 C-terminal Transactivation domain (e.g., TAD: aa 768-881), and the enzyme whose activity is to be monitored or the enzymatically active fragment thereof is positioned in or within the chimeric protein such that an enzymatically active enzyme or enzymatically active fragment thereof is capable of cleaving or physically separating or otherwise functionally separating the N-terminal Gal4 DNA-binding domain from the Gal4 C-terminal Transactivation domain such that the Gal4 can no longer act as a functional transcription factor, and if the enzyme whose activity is to be monitored is inhibited such that it is no longer enzymatically active (or substantially no longer enzymatically active) the Gal4 C-terminal Transactivation domain in conjunction with the N-terminal Gal4 DNA-binding domain can function as a functional transcription factor,
 wherein optionally transcription/expression of the chimeric Gal4 expression system is operably linked to a promoter and/or an enhancer, e.g., a doxycycline inducible promoter, 
 wherein optionally the ability of the Gal4 C-terminal Transactivation domain in conjunction with the N-terminal Gal4 DNA-binding domain to function as a functional transcription factor is measured by expression of a Fluorescent Protein (FP), e.g., an e-green fluorescent protein, or eGFP (excited with the 488 nm blue laser, an e-cyan fluorescent protein (or eCFP, using a 405 nm violet laser), and/or an mOrange or an mCherry (561 nm yellow laser), where the FP or GFP coding sequence is operably linked to or dependent (for its transcription) on the transcription factor. 
 
     
     
         79 . The chimeric Gal4 expression system of  claim 78 , wherein: the enzyme is a viral protease, a microbial protease or a mammalian protease; or the enzyme is an HIV-1 protease (PR), or a NS2/NS3 or a NS3/NS4A protease of HCV. 
     
     
         80 . A cell comprising the chimeric Gal4 expression system of any of  claim 78  to  claim 79 , wherein optionally the cell is a lymphocyte or a T cell, or a CD4+ T cell, or a mammalian cell or a human cell. 
     
     
         81 . A multiplexed system adapted for multiplexed analysis of a plurality of enzyme (more than one enzyme) inhibitors or modulators, comprising: a chimeric Gal4 expression system of any of  claim 78  to  claim 79 , or a cell of  claim 76 , or the cell-based method for monitoring the activity of any enzyme of  claim 71 , wherein inhibition of different enzymes is monitored by the expression of a different detectable moiety, e.g., a different Fluorescent Protein (FP), e.g., an e-green fluorescent protein, or eGFP (excited with the 488 nm blue laser, an e-cyan fluorescent protein (or eCFP, using a 405 nm violet laser), and/or an mOrange or an mCherry (561 nm yellow laser). 
     
     
         82 . An isolated, recombinant or synthetic nucleic acid encoding a chimeric (hybrid) protein, wherein the chimeric (hybrid) protein comprises (or consists of) from N- to C-terminus:
 (a) (i) a signal sequence (motif) for Endoplasmic Reticulum (ER) targeting,   (ii) a tag or detection moiety, or “scaffold”, capable of being recognized on a cell surface,   (iii) at least two transmembrane domains that span the ER membrane, with an extra loop at the ER luminal face,   (iv) an enzyme recognition/cleavage site spanning a segment of a gp120/41 boundary, facing the ER lumen, and   (v) an ER retention sequence or motif;   (b) the chimeric (hybrid) protein of (a), wherein the tag or detection moiety, or “scaffold”, comprises a tag for an antibody or an antigen binding fragment thereof (the antibody binding specifically to the tag or detection moiety, or “scaffold”), or the tag or detection moiety, or “scaffold”, comprises a ligand, or the tag or detection moiety, or “scaffold”, comprises a FLAG molecule or equivalent thereof;   (c) the chimeric (hybrid) protein of (a), wherein enzyme recognition/cleavage site comprises a furin enzyme recognition/cleavage site, a calcium-dependent protein convertase enzyme recognition/cleavage site, prohormone convertase-1 (PC1) enzyme recognition/cleavage site, or an enzyme recognition/cleavage site derived from a member of the subtilisin/kexin family of proprotein convertases;   (d) the chimeric (hybrid) protein of (a), wherein enzyme recognition/cleavage site comprises an enzyme recognition/cleavage site within the V3 loop of gp120;   (e) the chimeric (hybrid) protein of (a), wherein the ER retention sequence or motif comprises a KDEL (SEQ ID NO:1) sequence or equivalent thereof;   (f) the chimeric (hybrid) protein of (a), wherein the gp120/41 is an HIV-1 gp120/41;   (g) the chimeric (hybrid) protein of (a), wherein the at least two transmembrane domains that span the ER membrane consist of two transmembrane domains;   (h) the chimeric (hybrid) protein of (a), wherein the at least two transmembrane (TM) domains that span the ER membrane comprise at least one TM of a CRR5;   (i) the chimeric (hybrid) protein of (h), wherein the at least two transmembrane (TM) domains that span the ER membrane comprise TM1 and TM2 from the CRR5;   (j) the chimeric (hybrid) protein of (a), wherein the gp120/gp41 boundary comprises the recognition/cleavage site REKRA (SEQ ID NO:2);   (k) the chimeric (hybrid) protein of (a), wherein the gp120/gp41 boundary further comprises a restriction enzyme recognition site acids at both sides, or comprises additional amino-acids at both sides comprising AKRRVVQREKR (SEQ ID NO:15) and AVGIGALF (SEQ ID NO:16); or   (l) the isolated, recombinant or synthetic nucleic acid encoding the chimeric (hybrid) protein is operatively linked to a transcriptional regulatory unit, or a promoter such as an inducible or constitutive promoter.   
     
     
         83 . A vector, recombinant virus, cloning vehicle, expression cassette, cosmid or plasmid comprising (or consisting of) or having contained therein the isolated, recombinant or synthetic nucleic acid of  claim 82 . 
     
     
         84 . A chimeric or hybrid polypeptide comprising (or consisting of): (a) the polypeptide encoded by the nucleic acid of  claim 82 ; or (b) the chimeric (hybrid) protein of (a), wherein the protein comprises a synthetic protein or peptide, recombinant protein or peptide, a peptidomimetic or a combination thereof. 
     
     
         85 . A chimeric or hybrid protein comprising (or consisting of) from N- to C-terminus:
 (a)) (i) a signal sequence (motif) for Endoplasmic Reticulum (ER) targeting,   (ii) a tag or detection moiety, or “scaffold”, capable of being recognized on a cell surface,   (iii) at least two transmembrane domains that span the ER membrane, with an extra loop at the ER luminal face,   (iv) an enzyme recognition/cleavage site spanning a segment of a gp120/41 boundary, facing the ER lumen, and   (v) an ER retention sequence or motif;   (b) the chimeric (hybrid) protein of (a), wherein the tag or detection moiety, or “scaffold”, comprises a tag for an antibody or an antigen binding fragment thereof (the antibody binding specifically to the tag or detection moiety, or “scaffold”), or the tag or detection moiety, or “scaffold”, comprises a ligand, or the tag or detection moiety comprises a FLAG molecule or equivalent thereof;   (c) the chimeric (hybrid) protein of (a), wherein enzyme recognition/cleavage site comprises a furin enzyme recognition/cleavage site, a calcium-dependent protein convertase enzyme recognition/cleavage site, prohormone convertase-1 (PC1) enzyme recognition/cleavage site, or an enzyme recognition/cleavage site derived from a member of the subtilisin/kexin family of proprotein convertases;   (d) the chimeric (hybrid) protein of (a), wherein enzyme recognition/cleavage site comprises an enzyme recognition/cleavage site within the V3 loop of gp120;   (e) the chimeric (hybrid) protein of (a), wherein the ER retention sequence or motif comprises a KDEL (SEQ ID NO:1) sequence or equivalent thereof;   (f) the chimeric (hybrid) protein of (a), wherein the gp120/41 is an HIV-1 gp120/41;   (g) the chimeric (hybrid) protein of (a), wherein the at least two transmembrane domains that span the ER membrane consist of two transmembrane domains;   (h) the chimeric (hybrid) protein of (a), wherein the at least two transmembrane (TM) domains that span the ER membrane comprise at least one TM of a CRR5;   (i) the chimeric (hybrid) protein of (h), wherein the at least two transmembrane (TM) domains that span the ER membrane comprise TM1 and TM2 from the CRR5;   (j) the chimeric (hybrid) protein of (a), wherein the gp120/gp41 boundary comprises the recognition/cleavage site REKRA (SEQ ID NO:2);   (k) the chimeric (hybrid) protein of (a), wherein the gp120/gp41 boundary further comprises a restriction enzyme recognition site acids at both sides, or comprises additional amino-acids at both sides comprising AKRRVVQREKR (SEQ ID NO:3) and AVGIGALF (SEQ ID NO:4); or   (l) the chimeric (hybrid) protein of (a), wherein the protein comprises (or consists of) a synthetic protein or peptide, recombinant protein or peptide, a peptidomimetic or a combination thereof.   
     
     
         86 . A cell comprising (a) the isolated, recombinant or synthetic nucleic acid of  claim 82 ; (b) the vector, recombinant virus, cloning vehicle, expression cassette, cosmid or plasmid of  claim 83 ; (c) the chimeric or hybrid polypeptide of  claim 84  or  claim 85 ; or, (d) the cell of (a), (b) or (c), wherein the cell is a mammalian or a human cell. 
     
     
         87 . A cell-based method for monitoring the activity of an enzyme, or for screening for an inhibitor of the enzyme, comprising:
 (1) (a) providing: (i) a nucleic acid encoding the chimeric (hybrid) protein of  claim 84  or  claim 85 , or the nucleic acid of  claim 82 , operatively linked to a transcriptional regulatory unit (e.g., a promoter, such as an inducible or constitutive promoter), or (ii) the vector, recombinant virus, cloning vehicle, expression cassette, cosmid or plasmid of  claim 83 ; and, a cell comprising an environment capable of supporting the expression of the chimeric (hybrid) protein by the nucleic acid;   (b) inserting (e.g., transfecting or infecting) the nucleic acid, vector, recombinant virus, cloning vehicle, expression cassette, cosmid or plasmid of (a) into the cell; and   (c) contacting the cell with a putative (test) enzyme inhibitor,   wherein optionally the enzyme inhibitor is added to the cell before, during and/or after inserting (transfecting) the nucleic acid, vector, recombinant virus, cloning vehicle, expression cassette, cosmid or plasmid of (a) into the cell and/or expressing the chimeric protein encoded by a nucleic acid of (a) in the cell,   and optionally the cell-based method further comprises a negative, positive and/or alternative control set of cells into which the nucleic acid, vector, recombinant virus, cloning vehicle, expression cassette, cosmid or plasmid of (a) also has been inserted (or transfected) and expresses the chimeric protein encoded by a nucleic acid of (a), but the negative control set of cells is not exposed to the putative (test) enzyme inhibitor or is exposed to a different putative (test) enzyme inhibitor, or a different amount of putative (test) enzyme inhibitor, or a positive control wherein the cells are exposed to a known inhibitor of the enzyme; and   (d) determining whether the putative (test) enzyme inhibitor is an effective or sufficient inhibitor or modulator of the enzyme or an enzymatically active fragment thereof by measuring the ability of the putative (test) enzyme inhibitor to partially or completely inhibit cleavage of the enzyme recognition/cleavage site;   (2) measuring the ability of the putative (test) enzyme inhibitor to partially or completely inhibit cleavage of the enzyme recognition/cleavage site comprises detecting and/or measuring the amount of tag or detection moiety, or “scaffold”, on the cell surface; or   (3) the method of (1) or (2), wherein the cell is a lymphocyte or a T cell, or a CD4+ T cell, or a human cell.   
     
     
         88 . The method of  claim 87  further comprising running a negative control comprising dividing the plurality of the cells co-expressing a nucleic acid of (a) in the cell and not adding the compound to be screened (the putative (test) enzyme inhibitor) as an inhibitor to one of the divided cell samples. 
     
     
         89 . The method of  claim 87  or  claim 88 , further comprising running a positive control comprising dividing the plurality of the cells co-expressing the nucleic acid of (a) in the cell and adding a known inhibitor of the enzyme, e.g., a known inhibitor of a furin enzyme, a calcium-dependent protein convertase enzyme, prohormone convertase-1 (PC1) enzyme, or an enzyme from a member of the subtilisin/kexin family of proprotein convertases, to one of the divided cell samples. 
     
     
         90 . The method of  claim 87 , wherein:
 (a) the transcriptional regulatory unit comprises a promoter, an inducible promoter or a constitutive promoter;   (b) the cell is a mammalian cell, a monkey cell or a human cell, or the cell is a mammalian cell, a monkey cell or a human cell, or a lymphocyte, or a T cell, or a CD4- or CD8-expressing cell;   (c) the tag or detection moiety, or “scaffold”, is detected or measured on the cell surface by a high throughput screen, a flow cytometry or microscope visualization;   (d) the compound to be screened as an inhibitor of the enzyme comprises a small molecule, a nucleic acid, a polypeptide or peptide, a peptidomimetic, a polysaccharide and/or a lipid; or   (e) the compound to be screened as an inhibitor of the enzyme is a member of a library of compounds to be screened, or a member of a random peptide library or a chemical compound library.