Viral diseases are some of the major scourges of mankind and include such virulent disorders as poliomyelitis, herpes infections and AIDS. In addition, viruses carrying oncogenes are responsible for a number of human tumors and cancers.

Several diagnostic procedures have been developed in effort to improve the detection and diagnosis of viral infections. These procedures involve the detection of viral components in cells of infected individuals or the detection of blood components generated as a response to the presence of a viral infection. Although such methods provide acceptable accuracy in detecting some viral infections, they are oftentimes expensive and time consuming to carry out.

Although accurate and timely diagnosis of some viral infections provides clinicians with better chances of combating viral infection, the high rates of mutations allow quick selection of drug resistant viruses. For HIV, on average every new genome can be expected to carry at least one new mutation, and a single patient will harbor many new viral genomes in various tissues.

As such, for the past decades, universities and pharmaceutical companies have invested considerable resources in efforts to uncover potential anti-viral drug candidates and/or to determine the anti-viral drug resistance of

some viruses.

Present day anti-viral drug screening methods rely on detecting interactions between viral components and molecules having potential anti-viral activity. For example, the identification of inhibitors of virally encoded proteases ("protease inhibitors") relies on the in-vitro screening of purified viral protease with chemical compounds in the presence of synthetic peptide substrates. Initial in-vitro screening is usually followed by a bioassay designed for determining whether a potential protease inhibitor or its derivatives function in virally infected cells prior to additional testing conducted in more complex biological systems.

Screening for drug resistance of certain virus isolates is typically performed by phenotypic testing (plaque reduction assay). This is a labor intensive, time consuming and expensive technique that oftentimes does not correlate well to the clinical response to drug therapy in individual patients.

Nonetheless, because of its derivation from testing for sensitivity to antibacterial agents, this technique is often considered to be the"gold standard".

For example, in HIV infected individuals, phenotypic testing involves the isolation of peripheral blood mononuclear cells (PBMC) from the individuals and culturing these cells with stimulated lymphocytes to produce a viral stock. This stock is tittered down in stimulated PMBCs to a predefined number of infectious units per milliliter following which it is utilized to infect PMBCs in the presence of four concentrations of each of the antiretroviral agents of interest. For example, in the case of azidodeoxythymidine (AZT), concentrations of 0 to 5 micromolar are typically used. Virus stock isolates that exhibit an IC50 (the concentration of drug which inhibits 50% of virus growth compared to the no-drug control wells) at AZT concentrations greater than 1 micromolar are considered resistant to AZT.

Although this assay is easy to implement it is very expensive and time

consuming.

Another alternative drug-resistance assay relies upon the detection of specific genetic mutations in viral genomic sequences. In HIV, for example, such mutations have been highly correlated with phenotypic resistance under laboratory conditions. In general, genotypic assays are more rapid and less expensive than phenotypic testing. However, since they rely upon a direct detection of genetic mutations and since a pattern of mutations can be complex, this technique is less accurate than phenotype testing.

To try and overcome the abovementioned limitations, researchers have attempted to develop recombinant virus assays.

One such recombinant virus assay is the commercially available VIRCO assay which has been used to assess the"phenotypic"sensitivity of reconstructed viruses from well over 2,000 individuals worldwide.

In the VIRCO assay, plasma RNA is reverse transcribed into DNA and a segment of the genome is amplified using PCR. This amplified segment of viral DNA is then combined with a plasmid containing a deleted HIV construct to create a chimeric HIV virus. This plasmid, which contains an HIV provirus, is then introduced into mammalian cells where it replicates and produces a viral stock. The virus can then be grown in the presence of different drugs on standard T-cell lines to perform rapid phenotypic testing.

Recombinant virus assays are inherently limited due to the use of synthesized and thus mutation prone viral genetic material for testing drug resistance. Moreover, the selection of quickly replicating variants occurs during the chimeric HIV stock production.

There is thus a widely recognized need for, and it would be highly advantageous to have, an accurate and rapid method of screening molecules for anti-viral drug candidates and for determining a resistance of a virus to known anti-viral drugs while being devoid of the above limitations.

SUMMARY OF THE INVENTION According to one aspect of the present invention there is provided a nucleic acid construct comprising a polynucleotide cassette encoding a chimeric polypeptide including: (a) a first polypeptide sequence; (b) a second polypeptide sequence being translationally fused to the first polypeptide sequence; and (c) at least one protease recognition site being cleavable by a virally encoded protease, wherein cleavage of the at least one protease recognition site leads to a detectable signal.

According to another aspect of the present invention there is provided a method of uncovering molecules having anti-viral activity, the method comprising the steps of : (a) providing cell infected with a virus encoding a viral protease; (b) introducing into the cell a molecule with potential anti-viral activity and a nucleic acid construct including a polynucleotide cassette encoding a chimeric polypeptide, the chimeric polypeptide including: (i) a first polypeptide sequence; (ii) a second polypeptide sequence being translationally fused to the first polypeptide sequence; and (iii) at least one protease recognition site being cleavable by the viral protease, wherein cleavage of the at least one protease recognition site leads to a detectable signal; and (c) measuring the detectable signal to thereby determine the anti-viral activity of the molecule.

According to further features in preferred embodiments of the invention described below, the above described method further comprising the step of comparing the detectable signal to that from cells not infected with the virus or to cells not including the molecule with potential anti-viral activity to thereby determine the anti-viral activity of the molecule.

According to yet another aspect of the present invention there is provided a method of detecting the presence of a virus in a cell, the method comprising the steps of : (a) introducing into the cell, or incubating with an extract of the cell a polynucleotide cassette encoding a chimeric polypeptide,

the chimeric polypeptide including: (i) a first polypeptide sequence; (ii) a second polypeptide sequence being translationally fused to the first polypeptide sequence; and (iii) at least one protease recognition site being cleavable by protease encoded by the virus, wherein cleavage of the at least one protease recognition site leads to a detectable signal; and (c) measuring the detectable signal to thereby determine the presence of the virus in the cell.

According to still further features in the described preferred embodiments the method described above further comprising the step of comparing the detectable signal to that detected in a cell infected and/or not infected with the virus to thereby determine presence of the virus in the cell.

According to still another aspect of the present invention there is provided a method of determining viral drug resistance, the method comprising the steps of : (a) providing a cell infected with a virus encoding a viral protease; (b) introducing into the cell an anti-viral drug and a nucleic acid construct including a polynucleotide cassette encoding a chimeric polypeptide, the chimeric polypeptide including: (i) a first polypeptide sequence; (ii) a second polypeptide sequence being translationally fused to the first polypeptide sequence; and (iii) at least one protease recognition site being cleavable by the viral protease, wherein cleavage of the at least one protease recognition site leads to a detectable signal; and (c) measuring the detectable signal to thereby determine the resistance of the virus to the anti-viral drug.

According to still further features in the described preferred embodiments the method described above further comprising the step of comparing the detectable signal to that from a cell infected with the virus yet devoid of the anti-viral drug to thereby determine the resistance of the virus to the anti-viral drug.

According to still further features in the described preferred embodiments the at least one protease recognition site is interposed between the first and the second polypeptide sequences.

According to still further features in the described preferred embodiments the chimeric polypeptide further includes a third polypeptide sequence being translationally fused to the second polypeptide sequence.

According to still further features in the described preferred embodiments the nucleic acid construct described above further comprising a promoter sequence being for directing the transcription of the polynucleotide cassette.

According to still further features in the described preferred embodiments the promoter sequence is functional in a eukaryotic cell.

According to still further features in the described preferred embodiments the eukaryotic cell is a mammalian cell.

According to still further features in the described preferred embodiments the nucleic acid construct described above further comprising at least one polynucleotide sequence derived from a non-coding region of a virus genome.

According to still further features in the described preferred embodiments the nucleic acid construct described above further comprising at least one polynucleotide sequence derived from a coding region of a virus genome.

According to still further features in the described preferred embodiments at least one of the first and the second polypeptide sequence encodes protein selected from the group consisting of an enzyme, a substrate protein, a ligand protein and a reporter protein.

According to still further features in the described preferred embodiments the first polypeptide sequence encodes a first fluorophore protein and further wherein the second polypeptide sequence encodes a second fluorophore protein.

According to still further features in the described preferred embodiments the first fluorophore protein is green fluorescence protein and

further wherein the second fluorophore protein is blue fluorescence protein.

According to still further features in the described preferred embodiments there is provided a recombinant virus genome comprising the nucleic acid construct described above.

According to still further features in the described preferred embodiments the virus is an Alphavirus.

According to still further features in the described preferred embodiments there is provided a transformed cell including the nucleic acid construct described above.

According to an additional aspect of the present invention there is provided a nucleic acid construct comprising a polynucleotide cassette encoding a chimeric polypeptide including: (a) a reporter polypeptide sequence; and (b) a protease recognition site polypeptide sequence being integrated within the reporter polypeptide sequence, the protease recognition site being cleavable by a virally encoded protease, wherein cleavage of the protease recognition site leads to abolishment of a reporter function of the reporter polypeptide.

According to still an additional aspect of the present invention there is provided a method of uncovering molecules having anti-viral activity, the method comprising the steps of : (a) providing cells infected with a virus encoding a viral protease; (b) introducing into the cells a molecule with potential anti-viral activity and a nucleic acid construct including a polynucleotide cassette encoding a chimeric polypeptide including: (i) a reporter polypeptide sequence; and (ii) a protease recognition site polypeptide sequence being integrated within the reporter polypeptide sequence, the protease recognition site being cleavable by the viral protease, wherein cleavage of the protease recognition site leads to abolishment of a reporter function of the reporter polypeptide; and (c) measuring the reporter function to thereby determine the anti-viral activity of the molecule.

According to a further aspect of the present invention there is provided a method of detecting the presence of a virus in a cell, the method comprising the steps of : (a) introducing into the cell, or incubating with an extract of the cell, a polynucleotide cassette encoding a chimeric polypeptide, the chimeric polypeptide including: (i) a reporter polypeptide sequence; and (ii) a protease recognition site polypeptide sequence being integrated within the reporter polypeptide sequence, the protease recognition site being cleavable by protease encoded by the virus, wherein cleavage of the protease recognition site leads to abolishment of a reporter function of the reporter polypeptide; and (c) measuring the reporter function to thereby determine the presence of the virus in the cell.

According to still further features in the described preferred embodiments the method described above further comprising the step of comparing the measured reporter function to that from cells non-infected and/or infected with the virus to thereby determine the presence of the active virus in the cell.

According to yet a further aspect of the present invention there is provided a method of determining viral drug resistance, the method comprising the steps of : (a) providing a cell infected with a virus encoding a viral protease; (b) introducing into the cell an anti-viral drug and a nucleic acid construct including a polynucleotide cassette encoding a chimeric polypeptide, the chimeric polypeptide including: (i) a reporter polypeptide sequence; and (ii) a protease recognition site polypeptide sequence being integrated within the reporter polypeptide sequence, the protease recognition site being cleavable by the viral protease, wherein cleavage of the protease recognition site leads to abolishment of a reporter function of the reporter polypeptide; and (c) measuring the reporter function to thereby determine the resistance of the virus to the anti-viral drug.

According to still further features in the described preferred

embodiments the method described above further comprising the step of comparing the measured reporter function to that from cells infected with the virus yet devoid of the anti-viral drug to thereby determine the resistance of the virus to the anti-viral drug.

According to yet an additional aspect of the present invention there is provided a nucleic acid construct comprising at least a portion of a first genome of a first virus, the first genome including at least one polynucleotide sequence encoding a protease recognition site being cleavable by a protease encoded by a second genome of a second virus, wherein the first virus is capable of replicating only in a cell expressing the protease encoded by the second genome of the second virus.

According to still a further aspect of the present invention there is provided a method of uncovering molecules having anti-viral activity, the method comprising the steps of : (a) providing cells infected with a first virus encoding a viral protease; (b) introducing into the cells a molecule with potential anti-viral activity and a nucleic acid construct including at least a portion of a genome of a second virus, the at least a portion of the genome including at least one polynucleotide sequence encoding a protease recognition site being cleavable by the viral protease, wherein the second virus is capable of replicating and lysing the cells upon cleavage of the protease recognition site; and (c) measuring a degree of lysis of the cells to thereby determine the anti-viral activity of the molecule.

According to still further features in the described preferred embodiments the method described above further comprising the step of comparing the degree of lysis to that detected in cells infected with the first and the second virus yet not including the molecule to thereby determine the anti-viral activity of the molecule.

According to still further features in the described preferred embodiments the degree of lysis is measured as a function of time.

According to still a further aspect of the present invention there is provided a method of detecting the presence of a first virus in cells, the method comprising the steps of : (a) introducing into the cells a nucleic acid construct including at least a portion of a genome of a second virus, the at least a portion of the genome including at least one polynucleotide sequence encoding a protease recognition site being cleavable by protease encoded by the first virus, wherein the second virus is capable of replicating and lysing the cells upon cleavage of the protease recognition site; and (c) measuring a degree of lysis of the cells to thereby determine the presence of the first virus.

According to still further features in the described preferred embodiments the method described above further comprising the step of comparing the degree of lysis to that detected in cells infected with the first virus to thereby determine the presence of the first virus.

According to still a further aspect of the present invention there is provided a method of determining viral drug resistance, the method comprising the steps of : (a) providing cells infected with a first virus; (b) introducing into the cells an anti-viral drug and a nucleic acid construct including at least a portion of a genome of a second virus, the at least a portion of the genome including at least one polynucleotide sequence encoding a protease recognition site being cleavable by protease encoded by the first virus, wherein the second virus is capable of replicating and lysing the cells upon cleavage of the protease recognition site; and (c) measuring a degree of lysis of the cells to thereby determine the resistance of the virus to the anti-viral drug.

According to still further features in the described preferred embodiments the method described above further comprising the step of comparing the degree of lysis to that detected in a cell infected with the viruses but not including the molecule to thereby determine the resistance of the virus to the anti-viral drug.

According to still further features in the described preferred embodiments the degree of lysis is measured as a function of time.

According to yet another aspect of the present invention there is provided a chimeric polypeptide comprising: (a) a first polypeptide sequence; (b) a second polypeptide sequence being linked to the first polypeptide sequence; and (c) at least one protease recognition site being cleavable by a virally encoded protease, wherein cleavage of the at least one protease recognition site leads to a detectable signal.

According to still another aspect of the present invention there is provided a method of detecting the presence of an active virus in a cell, the method comprising the steps of : (a) incubating a chimeric polypeptide with an extract of the cell, the chimeric polypeptide including: (i) a first polypeptide sequence ; (ii) a second polypeptide sequence being linked to the first polypeptide sequence; and (iii) at least one protease recognition site being cleavable by protease encoded by the virus, wherein cleavage of the at least one protease recognition site leads to a detectable signal; and (c) measuring the detectable signal to thereby determine the presence of the virus in the cell.

According to yet an additional aspect of the present invention there is provided a method of determining viral drug resistance, the method comprising the steps of : (a) incubating an anti-viral drug and a chimeric polypeptide with an extract of a cell infected with a virus, the chimeric polypeptide including: (i) a first polypeptide sequence; (ii) a second polypeptide sequence being linked to the first polypeptide sequence; and (iii) at least one protease recognition site being cleavable by a protease encoded by the virus, wherein cleavage of the at least one protease recognition site leads to a detectable signal; and (c) measuring the detectable signal to thereby determine the resistance of the virus to the anti-viral drug.

According to yet an additional aspect of the present invention there is provided a method of uncovering molecules having antiviral activity, the

method comprising the steps of : (a) incubating a molecule with potential anti-viral activity and a chimeric polypeptide with an extract of a cell infected with a virus, the chimeric polypeptide including: (i) a first polypeptide sequence; (ii) a second polypeptide sequence being linked to the first polypeptide sequence; and (iii) at least one protease recognition site being cleavable by protease encoded by the virus, wherein cleavage of the at least one protease recognition site leads to a detectable signal; and (c) measuring the detectable signal to thereby determine the anti-viral activity of the molecule.

According to still an additional aspect of the present invention there is provided a method of detecting the presence of a virus in a cell, the method comprising: (a) introducing into a cell, or combining with a cell extract, a chimeric polypeptide including: (i) a first polypeptide sequence encoding an first portion of reporter protein; and (ii) a second polypeptide sequence encoding a second portion of the reporter protein, the second polypeptide being linked to the first polypeptide sequence via at least one protease recognition site being specifically cleavable by the specific protease, wherein cleavage of the at least one protease recognition site leads to separation of the first polypeptide from the second polypeptide, thereby enabling the first and the second polypeptides to self assemble into an active and therefore detectable form of the reporter protein; and (b) detecting the presence or absence of reporter activity to thereby detect the presence of a virus in a cell.

According to still further features in the described preferred embodiments the reporter protein is p-galactosidase.

The present invention successfully addresses the shortcomings of the presently known configurations by providing nucleic acid constructs and method of utilizing same for detecting virus infection, potential anti-viral drug candidates and determining resistance of viral isolates to anti-viral drugs.

BRIEF DESCRIPTION OF THE DRAWINGS The invention is herein described, by way of example only, with reference to the accompanying drawings. With specific reference now to the drawings in detail, it is stressed that the particulars shown are by way of example and for purposes of illustrative discussion of the preferred embodiments of the present invention only, and are presented in the cause of providing what is believed to be the most useful and readily understood description of the principles and conceptual aspects of the invention. In this regard, no attempt is made to show structural details of the invention in more detail than is necessary for a fundamental understanding of the invention, the description taken with the drawings making apparent to those skilled in the art how the several forms of the invention may be embodied in practice.

In the drawings: FIG. 1 is a schematic representation of a Sindbis virus derived expression construct according to the teachings of the present invention.

FIG. 2 is a schematic representation of an expression construct suitable for detecting HIV virus infection according to the teachings of the present invention.

FIG. 3 is a schematic representation of an expression construct including a complementation scheme of the ß-galactosidase (LacZ) reporter polypeptide according to the teachings of the present invention.

FIG. 4. illustrates the construct used to express the Gag-p-galactosidase fusion protein in bacteria according to the teachings of the present invention.

FIG. 5a illustrates the Gag-ß-galactosidase fusion protein of the present invention; HIV-protease cleavage sites are indicated by arrows. Matrix protein (MA), Capsid (CA), P2 and Nuclear capsid (Nc) are cleavage products of the HIV Gag polyprotein. The P-galactosidase protein is fused to the Gag polyprotein via the P2/Nc cleavage site.

FIG. 5b illustrates the predicted proteolytic products of Gag-ß-galactosidase and their detection by polyclonal anti-MA and anti-CA antibodies. Expected size of the cleavage product in kDa is shown at the bottom of the Figure.

FIG. 6 illustrates the expression of HIV-protease in bacteria. M-Blue Ranger Prestained Protein Molecular Weight Marker Mixture (Pierce, Cat.

No. KJ0081) ; Lanes: 1-BL-21 transformed with pET-28a vector; 2-BL-21 cotransformed with Gag-ß-gal/pT5Ap and Pr/pET-28aKl without IPTG; 3-5- BL-21 cotransfonned with Gag-b-gal/pT5Ap and Pr/pET-28ann induced with IPTG for 15', 30'and 60'respectively; 6-like 2, wild type HIV-protease, Pr/pET-28aKI, was replaced by mutant Prive ; 7-9-like 3-5, wild type HIV-protease, Pr/pET-28aK", was replaced by mutant PrIle.

FIG. 7 illustrates proteolytic cleavage of the Gag-p-galactosidase fusion protein by HIV protease.

FIG. 8 illustrates the construct used to express Gag-C-N chimera in bacteria (Gag-C-terminus-a-portion of ß-galactosidase).

FIG. 9 illustrates GagCN activation by HIV-Protease in bacterial cells.

BL-21 cells were cotransformed for each experiment with GagCNAp and Pr/pET-28aKn. Cell lysates of IPTG induced bacteria were prepared by incubation with Reporter Lysis Buffer (Promega). Activity of ß-galactosidase was determined 15', 30'and 60'following IPTG induction, using the (3-galactosidase Enzyme Assay System with Reporter Lysis Buffer (Promega, Cat. No. E2000).

FIG. 10a illustrates the GagCN fusion protein; HIV-protease cleavage sites are indicated by arrows. Matrix protein (MA), Capsid (CA), P2 and Nuclear capsid (Nc) are cleavage products of Gag polyprotein of HIV.

C-terminus of ß-galactosidase is fused to Gag polyprotein through the P2/Nc cleavage site; N-terminus of P-galactosidase is fused to tail of C-terminus through P2/Nc cleavage site.

FIG. 10b illustrates the predicted proteolytic products of Gag-ß-galactosidase and their detection by polyclonal anti-MA and anti-CA antibodies. Expected size of the cleavage product in kDa is shown at the bottom of the Figure.

FIG. 11 illustrates specific proteolytic cleavage of GagCN by HIV-protease and autoproteolysis of aGag-Pr. Bacteria cell lysates were prepared 60 minutes after induction with 0.4mM IPTG, and Western blots were stained with antisera to CA as described in the Examples section. M- Blue Ranger Prestained Protein Molecular Weight Marker Mixture (Pierce, Cat. No. KJ0081) ; Lanes: 1-BL-21 transformed with pET-28a vector; 2- BL-21, transformed with GagCN; 3-specific cleavage of GagCN by HIV-protease in BL-21 cotransformed with GagCN and Pr/pET-28a; 4- autoproteolysis of aGag-Pr ; 5-inefficient proteolysis of aGag-Pr in the presence of C-terminus of (3-galactosidase ; 6-Gag-ß-galactosidase fusion protein.

FIG. 12 illustrates specific proteolytic cleavage of GagCN by HIV-protease as detected by polyclonal anti-MA antibodies. 1-3-as in Figure 11,4-Gag-ß-galactosidase fusion protein.

FIG. 13 illustrates the construct used to express the aGag-Pr chimera in bacteria according to the teachings of the present invention..

FIG. 14a illustrates aGag-Pr autoproteolytic cleavage; HIV-protease cleavage sites are indicated by arrows. a-a portion of ß-galactosidase with myristylation signal and tail of MA, Capsid (CA), P2, Nuclear capsid (Nc), Pl-P6 and Protease are cleavage products of aGag-Pr.

FIG. 14b illustrates the predicted proteolytic products of aGag-Pr and their detection by polyclonal anti-MA and anti-CA antibodies. Expected size of the cleavage product in kDa is shown below.

FIG. 15 illustrates (3-galactosidase activity of the aGag-Pr chimera coexpressed with the C-terminus of ß-galactosidase. Lysates of IPTG induced

BL-21 cells cotransformed with aGag-Pr/pT5Ap and pC-ETKn were used for determining activity.

FIG. 16 illustrates inhibition of auto-proteolytic cleavage of aGag-Pr chimera by HIV-protease inhibitor Saquinavir. M-Blue Ranger Prestained Protein Molecular Weight Marker Mixture (Pierce, Cat. No. KJ0081). Lanes: 1-BL-21 cells transformed with pET-28a vector alone; 2-BL-21/aGag-Pr not induced with IPTG; 3-BL-21/aGag-Pr, induced with 0.4mM IPTG for one hour; 4-BL-21/aGag-Pr, induced with 0.4mM IPTG for one hour in presence of 1mM Saquinavir; 5-same as lane 4, but with 10 mM Saquinavir. aGag-Pr-61 kDa uncleaved polyprotein; a-CA-p2-cleavage product of fast autoproteolysis of aGag-Pr, 36 kDa.

FIG. 17 illustrates the construct used to express the MAaGag-Pr chimera in bacteria according to the teachings of the present invention.

FIGs. 18a-b illustrates the MA. a, Gag-Pr chimeric protein and its potential proteolytic products. Figure 18a schematically illustrates MAaGag-Pr autoproteolytic cleavage; HIV-protease cleavage sites are indicated by arrows. a-a-portion of ß-galactosidase inserted into the MA sequence at the P2/Nc HIV-protease cleavage site and fused with the c-terminus end of MA, Capsid (CA), P2, Nuclear capsid (Nc), Pl-P6 and the Protease enzyme are cleavage products of MAaGag-Pr. Figure 18b illustrates the predicted proteolytic cleavage products of MAaGag-Pr and their detection via polyclonal anti-CA antibodies. Expected size of the cleavage product in kDa is shown at the bottom of the Figure.

FIG. 19 illustrates induction of auto-proteolytic cleavage of MAaGag-Pr chimera in bacteria cells. Comparison of deleted MA peptides from aGag-Pr and MAaGag-Pr chimeras. Detection was effected via polyclonal anti-MA antibodies. Lanes: 1-BL-21/pET-28a vector; 2- BL-21/aGag-Pr without IPTG; 3-5-BL-21/aGag-Pr induced by 0.4mM IPTG for 15', 30'and 60'respectively; 6-BL-21/aGag-Pr+C-ET, incubated with

IPTG for one hour; 7-BL-21/Gag-Pr, induced by IPTG for one hour; 8- BL-21/MAaGag-Pr, induced by IPTG for one hour; 9-11- BL-21/MAaGag-Pr, induced by IPTG for 15', 30'and 60'respectively.

FIG. 20 illustrates the Sindbis virus-based vector used to express GagCN fusion protein in eukaryotic cells according to the teachings of the present invention.. An XbaI/ScaI fragment (5. 5kb) of pGagCN/pT5 was cloned into the XbaI/StuI restriction sites of SinLacZ, replacing the LacZ coding region.

FIG. 21 illustrates (3-galactosidase enzymatic activity in eukaryotic cells expressing the GagCN fusion protein in presence of HIV-protease.

FIG. 22 illustrates the Sindbis virus-based vector used to express gag-Pr fusion protein in eukaryotic cells (pMA-Pr/Sin) according to the teachings of the present invention.. An XbaI/ScaI fragment (1. 8kb) of pGag-Pr/pT5 was cloned into the XbaI/StuI restriction sites of SinLacZ, replacing the LacZ coding region.

FIGs. 23a-d illustrate eukaryotic cells expressing GagCN fusion protein stained for ß-galactosidase enzymatic activity. Cells were stained for p-galactosidase activity according to protocol of Invitrogen supplied with Sindbis Expression System (Cat. No. K750-01). Figure 23a-BHK cells transfected with MA-Pr/Sin (negative control); Figure 23b-cells transfected with GagCN/Sin; Figure 23c-cells cotransfected with GagCN/Sin and MA-Pr/Sin ; Figure 23d-cells transfected with SinLacZ (positive control).

FIG. 24 illustrates inhibition of auto-proteolytic cleavage of the MAaGag-Pr chimera of the present invention by the HIV-protease inhibitor Saquinavir. Lanes: 1-MAaGag-Pr expressed in non-induced BL21 cells ; 2- MAaGag-Pr expressed in BL21 cells induced with 0.4mM IPTG for one hour; 3-5-MAaGag-Pr expressed in BL21 cells induced IPTG in the presence of Saquinavir in concentrations of lmM, 10 mM and 50 mM respectively.

DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention is of nucleic acid constructs and methods utilizing same which can be utilized to detect an active virus infection in an individual, screen molecules for potential anti-viral drugs and to determine a drug resistance of viral isolates. Specifically, the present invention is of nucleic acid constructs, which incorporate a protease cleavage site which when cleaved by a virally encoded protease lead to the generation of a detectable signal.

The principles and operation of the present invention may be better understood with reference to the drawings and accompanying descriptions.

Before explaining at least one embodiment of the invention in detail, it is to be understood that the invention is not limited in its application to the details of construction and the arrangement of the components set forth in the following description or illustrated in the drawings. The invention is capable of other embodiments or of being practiced or carried out in various ways.

Also, it is to be understood that the phraseology and terminology employed herein is for the purpose of description and should not be regarded as limiting.

It is clear that the identification and treatment of viral infection is one of the major goals in the field of virology and certainly the one with the most practical impact for human, animal or plant diseases.

Lack of efficient and cost effective methods for identifying viral infections and for screening molecules for potential anti-viral drugs has proven to be a major hurdle in combating viral infections.

A feature common to most disease causing viruses, such as for example, retro-, herpes-and picornaviruses is the virally encoded protease which functions in processing of the virally encoded polyprotein.

While reducing the present invention to practice, and as shown in the Examples section which follows, the present inventor has shown that chimeric proteins which include various reporter construct schemes can be used to

detect HIV protease activity in both prokaryotic and eukaryotic model systems.

Thus, the constructs and methods of the present invention can be used to detect virally encoded protease found in infected cells or their extracts and thus to detect the presence or absence of viral infection.

Thus, according to one aspect of the present invention there is provided a nucleic acid construct. The nucleic acid construct includes a polynucleotide cassette which encodes a chimeric polypeptide.

As used herein the phrase"chimeric polypeptide"refers to a polypeptide fusion in which sequences from two or more polypeptides are linked via peptide bonds.

The chimeric polypeptide includes a first polypeptide sequence and second polypeptide sequence in translational fusion. Thus, the chimeric polypeptide is generated from a single open reading frame of the nucleic acid construct which is transcribed from a promoter sequence of the construct as a polycistronic message and translated as a single polypeptide.

The chimeric polypeptide further includes at least one protease recognition site which is recognized and cleaved by a virally encoded protease, wherein cleavage of the at least one protease recognition site leads to a detectable signal. As is further described hereinbelow, the amino acid sequence of the protease cleavage site is selected according to a virus of interest. For example, if the virus of interest is the human immunodeficiency virus (HIV) then the protease cleavage site sequence is that which is recognized by the HIV encoded protease.

When the nucleic acid construct is introduced into a cell infected with a virus of interest or is incubated with an extract of such a cell under conditions suitable for effecting transcription and translation of the nucleic acid construct, or when the chimeric polypeptide is incubated with the cell extract under such conditions, the cleavage of this protease cleavage site (s) by a

virally encoded protease leads to a detectable signal.

To generate such a detectable reaction, the first and/or second polynucleotide sequences encode polypeptide (s) which when separated by the cleavage of the protease recognition site produce a detectable signal. For example, the first polynucleotide sequence can encode an enzyme, receptor/ligand, substrate or fluorophore which is activated and thus detectable only following separation from the second polynucleotide sequence. In addition, a polypeptide which is secreted from the cell or imbedded within the cell membrane following separation from the chimeric polypeptide is also suitable for use by the construct according to this aspect of the present invention.

Alternatively, both the first and second polynucleotide sequences can encode different fluorophores which when fused and excited produce a signal of a first wavelength, and when separated and excited produce a signal of a second wavelength. For example, when the amino acid sequence of the green fluorescent protein (GFP) is fused to the amino acid sequence of the blue fluorescent protein (BFP) the resultant chimeric polypeptide emits a first fluorescent color when excited with U. V. radiation. However when separated a second fluorescent color is emitted therefrom when excited with the same U. V. light. This is due to a fluorescence energy transfer (FRET) between BFP and GFP.

It will be appreciated that numerous other types of polypeptides can also be utilized by the construct according to this aspect of the present invention in order to generate a detectable signal following cleavage of the protease cleavage site.

It will further be appreciated that a nucleic acid construct expressing an active reporter polypeptide which includes a protease recognition site within it's amino acid sequence, which reporter polypeptide is inactivated following cleavage of the protease recognition site can also be utilized by the present

invention.

Thus, according to another aspect of the present invention, there is provided a nucleic acid construct having a polynucleotide cassette which encodes a reporter polypeptide sequence including a protease recognition site polypeptide sequence integrated within the reporter polypeptide sequence.

Thus, according to this aspect of the present invention, the nucleic acid construct is capable of expressing a functional reporter polypeptide, for example, GFP, or P-galactosidase which is inactivated upon cleavage of the protease cleavage site. The protease cleavage site can be introduced into the amino acid sequence of, for example, GFP via any one of numerous methods known in the art. For example, mutation or alteration of the GFP encoding polynucleotide sequence can be effected via PCR or other recombinant techniques. In the case of GFP, the protease cleavage site sequence is introduced into the GFP sequence in a position which does not interfere with GFP fluorescence, but which abolishes such fluorescence when cleaved.

The nucleic acid constructs described hereinabove are preferably constructed using commercially available mammalian expression vectors or derivatives thereof. Examples of suitable vectors include, but are not limited to, pcDNA3, pcDNA3. 1 (+/-), pZeoSV2 (+/-), pSecTag2, pDisplay, pEF/myc/cyto, pCMV/myc/cyto, pCR3.1, pSinRep5, DH26S, DHBB which are available from Invitrogen, pCI which is available from Promega, pBK-RSV and pBK-CMV which are available from Stratagene, pTRES which is available from Clontech, and their derivatives and modificants.

Any of the promoter and/or regulatory sequences included in the mammalian expression vectors described above can be utilized to direct the transcription of the polynucleotide cassettes described above. However, since such vectors are readily amenable to sequence modifications via standard recombinant techniques, additional regulatory elements, promoter and/or selection markers can easily be incorporated therein if needed. For example,

since the cap dependent translation of RNA in virally infected cells, within which the above described constructs are expressed, is often downregulated by the infecting virus, the above nucleic acid constructs preferably also include internal ribosome entry site (IRES) sequences for initiation of cap independent translation of the chimeric polypeptide (s).

Thus, the above described nucleic acid constructs express chimeric polypeptides which can be used to detect the presence of a virus in cells or cell extracts by producing a detectable signal or a lack thereof following cleavage by a protease encoded by the virus.

Any of the nucleic acid constructs described above can be included in a construct system. Such a system can include several construct types each including a protease cleavage site of a specific virus and a reporter polypeptide. Thus, such a system enables to determine a virus type in an infected cell according to a specific signal detected, since in an infected cell only the construct type which includes the protease cleavage site recognized by the protease encoded by the virus will produce a detectable signal.

It will be appreciated that detection viruses which encode proteases in a cell can also be effected via a construct which expresses a chimeric polypeptide which alters cellular morphology when cleaved, for example, by lysing the cell in which it expressed following cleavage.

Thus, according to another aspect of the present invention, there is provided a nucleic acid construct which includes at least a portion of a viral genome of a first virus. Such a portion, as is further described in the Examples section which follows, encodes a polyprotein which includes most if not all of the proteins encoded by this virus. Thus, when expressed and processed (cleaved) in a cell, this virally encoded polyprotein leads to a full or partial viral infection cycle. As is further exemplified in the Examples section which follows, the at least a portion of the genome of the first virus is derived from an Alphavirus, such as for example the Sindbis virus (SV).

The construct according to this aspect of the present invention further includes at least one polynucleotide sequence encoding a protease recognition site cleavable by a protease encoded by a second virus. As is further described in the examples section which follows, this polynucleotide sequence replaces the protein recognition site sequence (s) normally found in the sequence of the first viral genome of the construct.

Thus, the construct of this aspect of the present invention encodes a functional or partially functional Alphavirus only in cells which are infected with a second virus encoding a protease capable of cleaving the protease cleavage site of the viral polyprotein expressed from the construct.

Once cleaved, by the protease encoded by the second virus, the polyprotein expressed from the construct follows a viral replication cycle ultimately leading to cell lysis. Thus, according to this aspect of the present invention, cell lysis serves as a detectable signal for the presence of an infecting virus in a cell.

Thus, the above described constructs are useful in detecting the presence of virus in a cell. Detection is effected by introducing any of the above described constructs into a cell suspected of being infected with a virus, or alternatively incubating the reporter expressing constructs (reporter containing polyprotein), with an extract of such a cell, and measuring a detectable signal or a decrease thereof from the cell. Preferably the detectable signal is compared to that measured from cells known to be infected with the virus and/or to cells devoid of the infecting virus to thereby determine the presence of the virus in the cell.

In addition to being useful in detecting the presence of a virus in a cell, the above described nucleic acid constructs according to the various aspects of the present invention can also be utilized to screen and detect molecules which posses anti-viral activities and to determine the drug resistance of a viral isolate.

Thus, according to an additional aspect of the present invention, there is provided a method of uncovering molecules having anti-viral activity, the method is effected by providing a cell infected with a virus which encodes a viral protease and introducing into the cell a molecule with potential anti-viral activity and any of the nucleic acid constructs described hereinabove.

Following a predetermined period of time, the detectable signal or a decrease thereof is measured and compared to that measured from cell infected with the virus but devoid of the molecule, to thereby determine the anti-viral activity of the molecule.

Numerous types of molecules can be screened using this method in an easy and rapid manner provided such molecules can be introduced into the infected cell.

It will be appreciated that in the case of the reporter polypeptide expressing constructs described above, cellular extracts of the infected cell can also be utilized for screening molecules for anti-viral activities.

However, in-situ screening in infected cells is preferred since this method determines anti-viral activity in-situ and as such it is more accurate in predicting future activity of screened molecules in-vivo.

Numerous methods are known in the art for introducing exogenous polynucleotide sequences into mammalian cells. Such methods include, but are not limited to, direct DNA uptake techniques, and virus or liposome mediated transformation (for further detail see, for example,"Methods in Enzymology"Vol. 1-317, Academic Press). Bombardment of cells or cell cultures with nucleic acid coated particles is also envisaged.

According to another aspect of the present invention there is provided a method of determining viral drug resistance.

The method according to this aspect of the present invention is effected by providing a cell infected with a virus encoding a viral protease and introducing into the cell a known anti-viral drug and any one of the nucleic

acid constructs of the present invention.

To determine the resistance of the virus to the drug, the detectable signal is measured and compared to that measured from a cell infected with the virus yet devoid of the anti-viral drug. it will be appreciated that in the case of the reporter polypeptide expressing constructs described hereinabove, drug resistance screening can also be conducted using extracts of the infected cell, although as mentioned above, in-situ methods are inherently advantageous.

Thus, the present invention provides nucleic acid constructs and methods of utilizing same to detect viruses in infected cells, to screen and uncover potential anti-viral drugs and to determine drug resistance of virus isolates.

The chimeric polypeptide described above is preferably expressed from a nucleic acid construct introduced into an infected cell or incubated with a cellular extract of such a cell.

However, as mentioned hereinabove, and according to additional aspects of the present invention, the chimeric polypeptide described above can also be directly utilized for detecting viral presence, for screening anti-viral drug candidates and for determining the resistance of virus strains to anti-viral drugs. In such cases, a collected and preferably purified chimeric polypeptide expressed from a eukaryotic or prokaryotic expression system, or synthesized by methods known in the art can be directly incubated with an extract of an infected cell to thereby produce a detectable reaction when cleaved.

The present invention presents several advantages over prior art methods. It is easily to implementable and executable, and in addition when utilized for uncovering potential viral drugs and for drug resistance screening it can provide results of an accuracy which far exceeds that achieved by presently available in-vitro methods.

Additional objects, advantages, and novel features of the present invention will become apparent to one ordinarily skilled in the art upon examination of the following examples, which are not intended to be limiting.

Additionally, each of the various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below finds experimental support in the following examples.

EXAMPLES Reference is now made to the following examples, which together with the above descriptions, illustrate the invention in a non limiting fashion.

Generally, the nomenclature used herein and the laboratory procedures utilized in the present invention include molecular, biochemical, microbiological and recombinant DNA techniques. Such techniques are thoroughly explained in the literature. See, for example,"Molecular Cloning: A laboratory Manual"Sambrook et al., (1989);"Current Protocols in Molecular Biology"Volumes 1-111 Ausubel, R. M., ed. (1994); Ausubel et al., "Current Protocols in Molecular Biology", John Wiley and Sons, Baltimore, Maryland (1989); Perbal,"A Practical Guide to Molecular Cloning", John Wiley & Sons, New York (1988) ; Watson et al.,"Recombinant DNA", Scientific American Books, New York ; Birren et al. (eds)"Genome Analysis: A Laboratory Manual Series", Vols. 1-4, Cold Spring Harbor Laboratory Press, New York (1998); methodologies as set forth in U. S. Pat. Nos.

4,666,828; 4,683,202; 4,801,531; 5,192,659 and 5,272,057;"Cell Biology: A Laboratory Handbook", Volumes I-III Cellis, J. E., ed. (1994);"Current Protocols in Immunology"Volumes 1-111 Coligan J. E., ed. (1994); Stites et al.

(eds),"Basic and Clinical Immunology" (8th Edition), Appleton & Lange, Norwalk, CT (1994); Mishell and Shiigi (eds),"Selected Methods in Cellular Immunology", W. H. Freeman and Co., New York (1980); available immunoassays are extensively described in the patent and scientific literature, see, for example, U. S. Pat. Nos. 3,791,932; 3, 839, 153; 3,850,752; 3,850,578;

3,853,987; 3,867,517; 3,879,262; 3,901,654; 3,935, 074; 3,984,533; 3,996,345; 4,034,074; 4,098,876; 4,879,219; 5,011,771 and 5,281,521;"Oligonucleotide Synthesis"Gait, M. J., ed. (1984);"Nucleic Acid Hybridization"Hames, B.

D., and Higgins S. J., eds. (1985);"Transcription and Translation"Hames, B.

D., and Higgins S. J., eds. (1984) ;"Animal Cell Culture"Freshney, R. I., ed.

(1986);"Immobilized Cells and Enzymes"IRL Press, (1986);"A Practical Guide to Molecular Cloning"Perbal, B., (1984) and"Methods in Enzymology"Vol. 1-317, Academic Press;"PCR Protocols: A Guide To Methods And Applications", Academic Press, San Diego, CA (1990); Marshal et al.,"Strategies for Protein Purification and Characterization-A Laboratory Course Manual"CSHL Press (1996); all of which are incorporated by reference as if fully set forth herein. Other general references are provided throughout this document. The procedures therein are believed to be well known in the art and are provided for the convenience of the reader. All the information contained therein is incorporated herein by reference.

EXAMPLE I Eukaryotic Constructs wAlich can be used with the present izlvention One nucleic acid construct which can be efficiently utilized by the methods of the present invention is constructed of a recombinant virus genome which is capable of replication in infected cells but which is unable to produce infective viral particles.

Such a pseudovirion can carry a reporter gene, such as for example, P-galactosidase, luciferase or the green fluorescent protein, which is released from the polyprotein expressed by the pseudovirion only in the presence of a protease encoded by a virus of interest. Since such pseudovirions are capable of generating large amounts of the reporter polypeptide when infected into cells, they are ideal for the applications described by the teachings of the present invention.

Alternatively, a recombinant virus which is capable of lysing host cells

infected with a virus of interest can also be utilized. It will be appreciated that in this case, lysis of the host cell can be detected and quantified.

Another nucleic acid construct which can be utilized by the present invention is a mammalian expression vector which expresses a reporter polypeptide translationally fused to another polypeptide via a sequence including a protease cleavage site. Such a construct, which preferably includes a strong constitutive promoter, can be transfected into cells to produce a chimeric protein. In the presence of a virally encoded protease which cleaves the protease cleavage site, the reporter polypeptide is released from the chimeric protein. Such a release either increases or activates the reporter activities of this polypeptide.

In any case, an important feature of any of the nucleic acid constructs described above is the viral protease cleavage site which enables activation of the reporter polypeptide and in the case of recombinant viral vector also enables replication and as such amplification of the virally encoded reporter polypeptide.

Recombiiiaizt viruses One example of a viruses suitable for use with the present invention are members of the Alphavirus family, such as for example, the Sindbis virus (SV).

The life cycle of Alphaviruses allows easy manipulation and usage as expression system. The genomic RNA (49S RNA) of Alphaviruses is an unsegmented plus strand RNA of approximately 11-12 kb in length which contains a 5i cap and 3i polyadenylate tail.

An infectious enveloped Alphavirus particle is assembled on cytoplasmic viral RNA from the viral nucleocapsid proteins which buds through the cell membrane embedded with viral-encoded glycoproteins. Viral entry into cells is probably mediated by endocytosis through coated pits, fusion of the viral membrane with the endosome and release of nucleocapsid and uncoating of viral genome.

During viral replication, the genomic 49S RNA serves as template for synthesis of a complementary negative strand. The negative strand serves as template for full-length genomic RNA and for internally initiated positive-strand 26S subgenomic RNA. The non-structural proteins are translated from the genomic RNA while structural proteins are translated from the subgenomic 26S RNA. All Alphavirus encoded genes are expressed as polyproteins and processed into individual proteins by proteolytic cleavage either during or following translation of the polyprotein. Processing of the structural polyprotein initiates with cleavage of the capsid protein from the structural polyprotein. The translated capsid protein functions as a cis-acting protease to release itself from the nascent polypeptide chain, while cellular host enzymes process the other structural proteins (PE2,6K and E1).

SV is one of the least virulent members of the Alphaviruses, which have been used as a model system for studying the pathogenesis of viral encephalitis. SV can infect cells in either persistent or lytic infection depending on the cell type and the viral strain. Several members of the Alphavirus family, Sindbis virus, Semiliki Forest virus, Venezuelan equine encephalitis virus (VEE) and others are suitable for use as virus-based expression vectors.

Alphavirus vectors are advantageous since the production of the recombinant viruses or pseudovirions takes only one to two days in comparison with two to four months for recombinant adenoviruses, and since the virus (Sindbis virus) is not infectious and not dangerous for use in humans (Strauss J. H., and E. G. Strauss, 1994, Microbiol. Reviews, vol. 58, No3, pp.

491-562). In addition, Alphaviruses are RNA viruses, which do not go through an intermediate DNA stage, as such these viruses do not integrate into the genome of an infected cell. In sharp contrast, retrovirus, herpesviruses and adenoviruses, typically integrate (except of adenovirus) into the host genome prior to initiation of virus activity. Thus Alphaviruses amplify in non-dividing

cells and are likely better suited for the relatively short-term high-level expression levels required by the method of the present invention.

Other advantages of Alphaviruses include high expression, the ability to express reporter polypeptides in non-dividing cells, and a genome which is easily manipulatable via recombinant techniques.

As specifically shown in Figure 1, Recombinant SV can be constructed by replacing the capsid-cleavage site self catalyzed by the capsid protein with a reporter gene sequence, flanked by two protease recognition-site sequences of a virus of interest, such as for example, poliovirus 2APr° and 3CPr°. For example, mutations can be introduced into a sequence corresponding to nucleotides 795-822 of the Sindbis virus 26S RNA sequence (5'-GGGACAGAAGAGTGGTCCGCAGCACCA-3'SEQ ID NO : 1) which code for the amino acid sequence GTEEWSAAP (SEQ ID NO : 2) forming the C/PE2 cleavage site, so as to generate the protease cleavage site of the forming the C/PE2 cleavage site, so as to generate the protease cleavage site of the human poliovirus 1 (Mahoney strain) of the picornavirus family (nucleotide coordinates 3356-3416 of Genbank Accession numbers: V01149 or J02281).

Thus, when the recombinant SV is expressed in cells infected with poliovirus, the reporter polypeptide would be released from the SV polyprotein expressed. Sindbis and Semiliki Forest viruses are neurotrophic and can be used for examination, diagnostics or gene therapy of picornavirus-infected neurons. Picornaviruses are a family of positive-strand RNA viruses the members of which include poliovirus, hepatitis A virus, rhinovirus, foot-and-mouth disease virus and encephalomyocarditis virus. The genetic information contained in the single-stranded, positive sense RNA genome is expressed as a single protein of around 2000 amino acids. This primary product of protein synthesis, designated the polyprotein, is subsequently cleaved in the cytoplasm into the mature viral proteins by a

cis-acting protease sequence. Although such viral proteases typically function in cis, for at least some virally encoded proteases (e. g., polioviruses) catalysis has also been shown to occur in trans.

Since processing of the viral polyprotein, nucleocapsid assembly, and generation of viable SV particles would only occur in cells expressing the poliovirus proteases, only such cells would produce a detectable signal Recombinant SV A Sindbis Expression System, such as that supplied by Invitrogen, can be utilized for constructing the recombinant SV virus described above. This Expression System consists of replicon, coding for four non-structural proteins necessary for SV replication, and defective helper (DH), coding for the structural proteins under subgenomic promoter (sgP). Structural proteins are required for assembly and budding of the virus. Since the replicon, and not the DH, contains the packaging signal, only the RNA of the replicon is packaged in infected cells. Generation of recombinant SV virions is effected by transfecting cells with the recombinant RNA and DH RNA which provides the SV structural proteins in trans. Virus-like particles (pseudovirions) released by the infected cells contain only the recombinant RNA of replicon and are ready to infect new cells and to proliferate but not to form the second generation of pseudovirions.

Expression constructs usefulfor HIV detection : One suitable expression vector for detecting HIV infection includes, for example, an Alphavirus subgenomic promoter directing the expression of a chimeric polynucleotide which includes a reporter gene (e. g. LacZ) placed within the HIV Gag-precursor protein sequence (includes MA, CA, NC and p6 proteins, GenBank Accession number K03455 nucleotide coordinates 789-2290) instead of the HIV NC C-terminal PI and P6 protein coding sequence. (Figure 2). This chimeric polynucleotide includes the HIV MA protein sequence which contains the myristylation signal required for membrane association, the HIV CA protein, which is necessary for virus

assembly and the NC, which promotes Gag-Gag interactions (Ono A., Demirov D, Freed E. O., 2000, J. Virology, V. 74, N11, pp. 5142-5150).

The HIV Gag protein sequence contains five HIV protease cleavage sequences (*-indicates site of cleavage): VSQNY*PIVQN (MA/CA; SEQ ID NO : 3), KARVL*AEAMS (CA/P2; SEQ ID NO : 4), SATIM*MQRGN (P2/NC; SEQ ID NO : 5), ERQAN*FLGKI (NC/P1 ; SEQ ID NO : 6) and RPGNF*LQSRP (P1/P6 ; SEQ ID NO : 7). The reporter gene of this expression construct is preferably translationally fused to nucleotide number 2096 of BglII-site of the NC protein (encoded by nucleotide coordinates 1921-2086 of GenBank Accession number K03455) thus replacing the Pol coding sequence.

Thus, the reporter gene is positioned downstream of the NC/P1 cleavage site represented by SEQ ID NO : 6. This will allow the translocation of the newly synthesized chimera to the sites of HIV virion assembly on the cell membrane and, probably, incorporation into viral particles. There, HIV protease activity will release the reporter polypeptide from the chimeric polypeptide to thereby generate a signal.

An MA-CA-NC-reporter tetrapeptide containing HIV-specific cleavage sites can also be cloned under a subgenomic viral promoter to serve for detecting HIV infection. A chimeric RNA containing non-structural proteins of VEE (Venezuelan equine encephalitis virus), its subgenomic promoter (GenBank Accession number AF075259.1), and nucleotides 789-2096 of Matrix (MA), Capsid (CA) and Nucleocapsid (NC) proteins of HIV1 can be fused to a b-galactosidase gene form pSinLacZ (Invitrogen) to thereby generate an expression construct suitable for the detection of HIV infection.

Co-transfection of BHK cells (baby hamster kidney cells) with this recombinant replicon and a helper construct (e. g., DH26S helper construct) would lead to the generation of a high titer of pseudovirions each expressing a chimeric polypeptide capable of generating (3-galactosidase only in HIV-infected cells.

It will be appreciated that in order to infect lymphocytes, appropriate strains of SV should be selected, such as for example, pTR334, Girdwood S. A. virus, Ockeblo or S. A. AR86 (see, for example, U. S. Pat. No. 6,008,035).

Alternatively lymphocyte-specific pseudovirions can also be formed by using expression constructs based upon the Venezuelan equine encephalitis virus (VEE) (see, for example, U. S. Pat. No. 5,505,947). It will be appreciated that the size of such proposed chimeras is well within the packaging limits of, for example, an Alphavirus pseudovirion.

Various reporter construct schemes can be utilized in order to increase detection and decrease background reporter activity. As specifically illustrated in Figure 3, a p-galactosidase (LacZ) complementation system can be utilized to reduce the probability of background activity of this enzyme. A chimeric, reshuffled p-galactosidase polypeptide which includes the small (a-donor) fragment of p-galactosidase (amino acids 9-214) fused downstream to the large (C-terminus, amino acids 52-998) fragment of this enzyme, can be utilized as a protease activated reporter polypeptide. Such a reporter polypeptide includes the P2/NC protease cleavage site (SEQ ID NO : 5) which when cleaved by a viral protease (e. g. HIV encoded protease) generates the small (a-donor) and large fragments (a-acceptor) of p-galactosidase which can now correctly associate to thereby produce the tetramers with reporter enzymatic activity. Such positioning of the a-portion and C-terminus of p-galactosidase in the expressed chimeric polypeptide should prevent its activity prior to cleavage. Examples 2-3 below detail various complementation construct schemes which can be used to detect protease activity according to the teachings of the present invention.

EXAMPLE 2 Prokaryotic model system Materials and methods Constructionof gag-3-galactosidase The p-galactosidase gene was cloned into the BglII-site (2096) of gag-pr/pT5, replacing PI, P6 and Pr. The gag-pr/pT5 construct has been previously described (Kotler M., et al., J. Virology (1997) V. 71, N. 8, PP. 5774-5781).

To generate this construct, the 5'-terminus of the ß-galactosidase gene encompassing nucleotides 7654-8276, was PCR amplified using SinLacZ as a template (Invitrogen, GenBank accession number 19100) and forward primer 5'-TCTAGAATTCACCATGGAAGATCTTAGCGCTACCATAATG ATGCAGAGGGCAATAACGTTGTTTTACAACGTGAC-3' (SEQ ID NO : 8) which includes the P2/NC HIV-protease cleavage site and a reverse primer, 5'-CGAGACGTCACGGAAAATGCCGC-3' (SEQ ID NO : 9). The resultant PCR product was cloned into pGEM-T-Easy (Promega), and then into SinLacZ (Invitrogen) in order to generate a full length p-galactosidase clone. This full length clone was digested and ligated into the pT5 construct described above.

Gag->galactosidase expression Transformed BL-21 bacteria were induced (IPTG 0.4mM) to express the Gag-ß-galactosidase fusion protein. Approximately 800ml of the growth culture (OD595 0.6) were recovered at 15,30 and 60 minutes following induction. Bacterial cells were spun and the recovered pellet was resuspended in 100 1ll of Leammli sample buffer and 20 1ll of which were loaded onto a 10% SDS-PAGE gel. Following electrophoresis, the gel was blotted onto a nitrocellulose filter which was subjected to a Western blot analysis using polyclonal anti-HIV-protease antibodies (kind gift of Prof. Kotler M.), Deletion of the Agalactosidase portions

An adapter for allowing fusion of p2/Nc HIV-protease cleavage site directly to nucleotide 7794 of FspI-site of p-galactosidase in SinLacZ (LacZ nucleotides 7654-10656) was used to delete the N-terminal 51 amino acids (a portion) of p-galactosidase.

The adapter was generated by annealing two complementary oligonucleotides: 5'-GATCTTAGCGCTACCATAATGATGCAGAGAGGC AATTTGC-3' (SEQ ID NO : 10) and 5'-AATCGCGATGGTATTACTACGT CTCTCCGTTAAACG-3' (SEQ ID NO : 11).

The C-terminus portion of p-galactosidase was also cloned into the EcoRI-site of pET-28aKn to form pC-ET. In addition, this C-terminus portion was also cloned into the XbaIBamHI-sites of pT5Ap, as a fusion to the Gag-protein from gag-pr/pT5. Finally, a 665 bp fragment containing the p2/Nc cleavage site and the N-terminus of P-galactosidase was fused to the C-terminal end of the C-terminus portion of p-galactosidase to generate GagCN.

Agalactosidase fusions The a portion of p-galactosidase (51 N-terminal amino acids) fused to p2/Nc HIV-protease cleavage site was used to replace most of the MA protein of Gag, retaining the MA-myristylation signal.

Forward primer: 5'-AGATCGATGGAGCGCTACCATAATGAT GCAG-3' (SEQ ID NO : 12) containing a Clapi-site and the p2/Nc cleavage site was used along with reverse primer: 5'-GTCAGCTGCTGCGCGCAACTGT TGGGAAGG-3' (SEQ ID NO : 13) which included an AlwNI site to amplify a 178 bp region encompassing the a portion of p-galactosidase. The PCR product was cloned to ClaI site (831) and AlwNI site (1147) of gag-pr/pT5 (coordinates corresponding to the HXB2 HIV strain, GenBank accession number K03455).

Bacterial aGag-Pr expressiosz

Bacterial cells transformed to express an aGag-Pr fusion were induced with IPTG (0.4 mM) or with IPTG and Saquinavir (leim or 10tM) (Fortavase, Rosh). An 800ml sample was recovered from each growth culture (OD595 0. 6) prior to, and 60 minutes following, induction. The recovered cell pellet was resuspended in 100 1ll of Leammli sample buffer and 20 ul were loaded onto a 10% SDS-PAGE gel. Following electrophoresis, the gel was blotted onto a nitrocellulose filter which was subjected to a Western blot analysis using polyclonal anti-CA antibody (kind gift of Prof. Kotler). a-donor-MAproteinfusion A BstEII restriction site (5'-G*GTNACC-3'SEQ ID NO : 14) was inserted into the coding region of MA downstream of nucleotide 1021 (HXB2 strain of HIV-1, GenBank Accession Number K03455). A 190 base pair region of the MA coding sequence, starting with nucleotide number 831 was PCR amplified using forward primer: 5'-GGGGAGAATTAGATCGATGG-3' (SEQ ID NO : 15) and reverse primer: 5'-TTGGTNACCTGATCTAAGTTCTT CTGATCC-3' (SEQ ID NO : 16) and pGag-Pr/pT5 as template DNA. A 178 bp fragment which contained the p2/Nc HIV-protease cleavage site and an a-portion of ß-galactosidase was PCR amplified using forward primer: 5'-TTGGTNACCAGCGCTACCATAATGATGCAG-3' (SEQ ID NO : 17) which included the p2/Nc HIV-protease cleavage site, and reverse primer: 5'-GTCAGCTGCTGCGCGCAACTGTTGGGAAGGG-3' (SEQ ID NO : 18) which included the a-portion of (3-galactosidase (nucleotides 7796-7774, Invitrogen, pSinLacZ, number 19100). These two PCR products were triple ligated into pGag-Pr/pT5 to generate pMAaGag-Pr (Cloned insert: SEQ ID NO : 19).

Results Construction of a reporter fusion activated by protease in E. coli Expression of a reporter protein which can be activated by specific protease was examined in an E. coli model system.

Production of infectious virions from HIV-infected cells depends on expression of HIV protease for cleavage and maturation of structural Gag and Gag-Pol polyproteins (Gottlinger et al., Proc. Natl. Acad. Sci. USA, 86, 5781-5785,1989). During viral infection, generation of new viral particles involves the cleavage of precursor polyprotein by a specific, virally encoded protease. In the case of HIV, the matrix protein (MA), capsid protein (CA) and nucleocapsid protein (Nc) are generated through cleavage of the Gag polyprotein by HIV-protease. The Gag-Pol polyprotein is formed by translational frameshift in the 3'region of the Gag gene, while the HIV-protease is formed by self excision from this precursor at specific sites (Figure 5). The processing of HIV-polyproteins by HIV-protease is thought to be coupled to the budding of viral particles and to occur primarily in the viral particles before or immediately after budding. However, it has also been reported that some polyprotein processing can take place in the cytosole of acutely infected cells and that some cytosolic proteins are subject to cleavage by HIV-protease in acutely infected cells (Vocero-Akbani et al., Nature Med. V. 5, N1, PP. 29-33,1999).

It has been demonstrated that the HIV-protease also recognizes target cleavage sites in context of non-HIV proteins. For example, insertion of P6/protease cleavage site into Thymidylate Synthase, an essential enzyme of DNA metabolism of E. coli, converted Thymidylate Synthase to an HIV-protease substrate (Kupiec et al., J. Biol. Chem., V. 271, N. 31, PP. 18465-18470,1996). The Diphteria toxin is cleaved by HIV-protease when modified to include HIV-protease cleavage sites (Falnes et al., Biochem. J. Oct 1; 343 (Ptl). PP. 199-207,1999).

The HIV-protease can also recognize target sites when introduced into P-galactosidase ; cleavage of this enzyme through HIV-protease specific sites leads to inactivation of ß-galactosidase activity (Baum et al., Proc. Natl. Acad.

Sci. USA, V. 87, pp. 10023-10027, 1990).

Prokaryotic expression vectors pT5 (kind gift of Prof. Kotler M.

Hebrew University, Jerusalem) and pET-28a (Novagen) which include the T7 promoter were used to express HIV protease in BL-21 cells; the Gag-ß-galactosidase fusion protein was used as a substrate.

Most proteases are toxic when over expressed in eukaryotic as well as in prokaryotic cells. Thus, Escherichia coli strain BL-21 (DE3) plysS cells which lack the lon protease, and the ompT outer membrane protease that can degrade proteins during purification were chosen. At least some target proteins are expected to be more stable in BL-21 than in other bacterial strains which do not contain these proteases (Methods in Enzymology, Vol. 185, p 61-88).

The P2/Nc HIV-protease cleavage site was added as a bridge between Gag and (3-galactosidase (Figure 4). HIV-protease cleavage of this Gag protein fusion is illustrated in Figures 5a-b.

As shown in Figure 6, a Western blot probed with anti-HIV-protease antibodies demonstrates that expression of an HIV-protease initiates fifteen minutes following induction with IPTG and steadily increases for one hour following induction.

The activity of HIV-protease was examined by Western blot analysis using anti-CA antibodies and compared with activity of a PrIle mutant (Asp25-Ile25) (kind gift of Prof. Kotler). The appearance of Gag-ß-gal (173 kDa) cleavage products was detected as early as 15 minutes following induction with IPTG and achieved a pronounced level 30 minutes following induction. The Gag polyprotein (55 kDa) appeared as a cleavage product of wild type HIV-protease (HXB2 strain), and at lower levels as a result of activity of Prl"mutant (Figure 7). The MA-CA-P2 cleavage product (43 kDa) is predominant one hour following induction.

Thus, these results indicate that the BL-21/pET host cells/vector system is suitable as a prokaryotic model system for examination of protease

activity. The expression vector Pr/pT5 expresses active HIV-protease, and the cleavage sites introduced into the Gag- (3-galactosidase fusion protein are cleavable by the HIV-protease.

Activation of Gag-C-N via HIV-Protease.

The phenomenon of ß-galactosidase a-complementation is well known in the art. This phenomenon was discovered in extracts of lacZ mutants of E. coli (Ullmann et al., J. Mol. Biol. V. 24, PP. 339-343,1967).

Complementation occurs in vivo in partial diploids and in vitro, when extracts of certain mutant strains are mixed.

In the latter case, ß-galactosidase activity which is absent from each of the initial extracts, is restored upon mixing. It was discovered that one of these strains carried a mutation in the operator-proximal, or a-portion of the gene; while the other strain expressed an intact a-region but displayed a mutation elsewhere in the gene.

Several peptides exhibiting a-donor activity were isolated. The smallest peptide isolated corresponds to residues 3-40 of ß-galactosidase (Zabin et al., Mol Cell. Biochem. V. 49, PP. 87-96, 1982). The nature of the complementation process has been elucidated. The a-acceptors form dimmers, while addition of an a-donor peptide allows the formation of a native-like tetramers. In the free form, a-complementing donor peptides have no obvious structure and apparently do not form a domain. Although a-complementing donor peptides do not form a part of the active site of (3-galactosidase, single amino acid substitutions in a donors can exhibit a profound effect on complementation.

The Gag-C-N fusion protein constructed herein is the first example of a reporter chimeric protein which can be activated by a specific protease (Figure 8). To generate this fusion protein, the N-terminus of ß-galactosidase was ligated to the C-terminus end of ß-galactosidase via an HIV-protease cleavage site (SEQ ID NO : 20,21). In this construct, the C-terminus of

(3-galactosidase is fused in frame to the Gag-protein of HIV. Such a chimera would only display ß-galactosidase activity following cleavage with HIV-protease and formation of active ß-galactosidase tetramers.

The activity of this chimera was examined in BL-21 cells. Cells transformed with the C-terminus portion of ß-galactosidase (amino acids 52-998) exhibited background activity which was similar to that of cells transformed by pET-vector alone even 1 hour post induction with IPTG (Figure 9).

Similarly, very low (3-galactosidase activity was detected in cells expressing the Gag-C-N polypeptide. Under similar conditions and in the presence of an HIV-Protease, activity reaches 80% of that exhibited by the Gag-a-gal fusion protein (positive control), and is 10-folds more then background activity. Gag-C-N activity increases 3-folds in the presence of the protease (Figure 9).

Gag-C-N ß-galactosidase activity observed in the absence of the HIV protease may be explained by interactions between the N-terminus of ß-galactosidase of one molecule and the C-terminus of ß-galactosidase of another chimeric molecule. This trans-complementation of chimeric Gag-C-N is not effective and as such leads to very low (3-galactosidase activity.

Thus, the Gag-C-N fusion protein of the present invention demonstrates that (3-galactosidase reporter activity can be specifically activated by the HIV-protease. Such activity can only arise from cleavage of the Gag-C-N fusion protein by HIV protease and formation of active enzymatic complexes from the C-and N-terminus portions of (3-galactosidase.

Cell lysates from expressing cells were electrophoresed, Western blotted and immunostained with anti-CA or anti-MA antibodies to confirm Gag-C-N cleavage (Figures 10-12). In the absence of the HIV-protease, immunostaining revealed a 191 kDa band which corresponds to Gag-C-N chimera expressed by the BL-21 cells (Figure 10). This band was replaced by

43 and 41 kDa bands (MA-CA-P2 and MA-CA respectively) in cells cotransformed with Gag-C-N and HIV-protease thus indicating cleavage of the Gag polyprotein with HIV-protease. This cleavage leads to the strong P-galactosidase activity observed in BL-21 cells cotransformed with Gag-C-N and HIV-protease.

It should be noted that the 24 kDa band corresponding to CA-protein (Figure 12) resulted from cross reactivity between Anti-MA antibodies and the Capsid protein.

Thus, the HIV-protease is capable of cleaving the Gag-C-N fusion protein to generate detectable P-galactosidase activity.

Activation of aGag via proteolytic cleavage An alternative reporter chimera which can be activated in the presence of the HIV protease, can be constructed from the a-portion of p-galactosidase flanked by sequences of another protein and linked thereto via HIV-protease-cleavage sites.

Thus, proteolytic cleavage would release the a-donor of P-galactosidase and generate a complementation-capable peptide. Addition of the C-terminus portion (co-expression or from extracts) will lead to the formation of an active P-galactosidase.

This type of constructs can be used for determining HIV protease activity. HIV-protease is synthesized in infected cells fused with Gag-polyprotein as a component of Gag-Pol. Embedding of the protease within the polyprotein prevents its cytotoxic effect, but allows effective proteolytic maturation of Gag-Pol and Gag in the budding virions. In contrast to the GagCN construct which might be too large to be efficiently packaged within virions, the oGag chimera is nearly the same size as the Gag polyprotein, and contains all components of Gag, which are necessary for forming of the viral-like particles. The human immunodeficiency virus type 1 Gag precursor in itself is capable of assembling into retrovirus-like particles.

As has been shown recently, the myristylation signal of MA protein is absolutely necessary for particles formation, but the rest of the MA protein can be deleted without affecting HIV particles formation (Accola et al., J. Virology V. 74, Nol2, PP. 5395-5402,2000).

In aGag chimera of the present invention, the a-portion is placed upstream of the Gag protein thus replacing most of the MA-protein (SEQ ID NO : 22). The aGag chimera includes the myristylation signal of the MA-protein which directs transport of the chimera to the cellular membrane where HIV viral particles are formed (Ono et al., J. Virology, V. 74, No. 11, PP. 5142-5150,2000).

A construct including aGag fused to the HIV-protease was generated in order to examine the background activity of the a-portion of ß-galactosidase in absence of C-terminus (Figure 13).

The a-portion includes the N-terminal 51 amino acids of ß-galactosidase (near 1000 amino acids) and appeared to be too small to possess any enzymatic activity. However, to our surprise, the background activity of the aGag-Pr chimera expressed in BL-21 cells in the absence of the C-terminus portion is higher than that observed for Gag-C-N (Figure 15). The cotransformation of BL-21 cells with aGag-Pr/pT5 and C-ET (C-terminus/pET-28a) and induction with IPTG lead to pronounced ß-galactosidase activity in cell lysates ; (3-galactosidase activity of aGag-Pr is at least 2-folds higher in presence of the C-terminus portion (Figure 15).

This increase in activity is less pronounced then that observed following Gag-C-N cleavage by HIV-protease (Figure 9).

Autoproteolytic cleavage of the aGag-Pr chimera (Figures 14a-b) was compared to proteolysis of Gag-C-N in presence of HIV-protease (Figure 11).

Anti-CA antibodies did not detect a 61 kDa in cells expressing the aGag-Pr chimeric protein following 1 hour of induction. Instead a doublet including 36 and 34 kDa bands was observed (a-CA-P2 and a-CA respectively). This

implies that the HIV-protease of aGag-Pr cleaves the aGag-Pr chimera effectively suggesting that the a-portion of ß-galactosidase (N-terminus) is released. Expression of the C-terminus of ß-galactosidase in cells cotransformed with aGag-Pr and C-ET, partially inhibits cleavage of aGag-Pr, since the 61 kDa band representing aGag-Pr remains a major detectable band (Figure 15), while the 36 kDa band (a-CA-P2) appears as a weak band. This partial cleavage may explain the relatively low p-galactosidase activity (only twice higher then aGag-Pr alone) observed in lysates of cells expressing aGag-Pr and the C-terminus portion of P-galactosidase.

To prevent cleavage inhibition, the C-terminus portion of p-galactosidase can be added to the lysates of BL-21/aGag-Pr. In this case, such addition will not inhibit cleavage of aGag-Pr and therefore increase P-galactosidase activity.

HIV-protease inhibitors were used to determine if the fused a-portion reacts with the C-terminal portion in the absence of cleavage.

IPTG induced bacterial cultures which were incubated with Saquinavir (Fortovase, Roche) in concentrations 1 or 10 mM did not exhibit substantial aGag-Pr cleavage (Figure 16) and as such displayed a 60 % reduction in p-galactosidase enzymatic activity as compared to cultures not treated with Saquinavir ( (3-galactosidase enzymatic activity was measured at OD 420 1 hour post induction with IPTG, and background signal, as determined from cells lacking the C-terminus portion was subtracted).

These results indicate that most of the P-galactosidase enzymatic activity results from tetramers formed by non-fused a-donor and the C-terminus portion.

The a-portion of ß-galactosidase was ligated into the middle of the MA-protein (SEQ ID NO : 19) to further prevent the possibility of active complex formation between fused a-donor, and the C-terminus portion

(Figures 17,18a-b). A region of 77 amino acids from the MA protein flanked the N-tenninal end of the a-portion while 13 amino acids of MA protein were fused downstream of the a-portion.

Amino acids 78-119 of MA were replaced by amino acids 9-51 of the a-donor which included, at the N-terminal end, the 10 amino acids comprising the P2/Nc HIV-protease cleavage site (SEQ ID NO : 5). Proteolytic cleavage of this site and the Gag protein releases a fragment which includes the a-donor fused to an additional 13 amino at the C-terminal end (a 55 amino acid peptide).

It should be noted that the estimated rate of cleavage of different HIV-protease cleavage sites within the Gag precursor varies considerably. p2/NC is cleaved rapidly, with a rate of cleavage 14-folds higher then that of MA/CA and 400-folds higher then that of CA/p2 (Pettit et al., J. Virology, V. 68, Nol2, PP. 8017-8027,1994).

This implies that the 12.3 kDa N-terminal peptide of MA (77 amino acids of MA and 5 amino acids of p2/Nc cleavage site) and the a-CA-p2 (36 loba) polypeptide should be released as the first products of autoproteolytic cleavage of MAaGag-Pr.

Electrophoresed and blotted cell lysates immunoprobed with anti-MA polyclonal antibodies detected a band corresponding to the 12.3 kDa peptide, one hour post induction with IPTG (Figure 19, line 11). A similar band was not detected in lysates with Gag-Pr, or aGag-Pr. Treatment of BL21 cells expressing MAaGag-Pr with 10-50 mM of Saquinavir inhibits the proteolytic cleavage of the MAaGag-Pr chimera (Figure 24), further substantiating the fact that MAaGag-Pr is specifically cleaved by the HIV protease.

EXAMPLE 3 Eukaryotic Model system Materials and methods The GagCN was subcloned from the prokaryotic expression vector described above into a Sindbis virus-based vector to generate pGagCN/Sin (Figure 20). In addition, the gag-pr fusion protein was cloned from prokaryotic expression vector gag-pr/pT5 (Kotler et al., J. Virology, V. 71, No.

8, PP. 5774-5781,1997) into SinRepS, to generate the HIV protease construct pMA-Pr/Sin (Figure 22). Expression of the HIV-protease fused to the Gag polyprotein should prevent cytotoxic effect of the protease and more closely mimic proteolytic cleavage of cells infected with HIV.

BHK cells, seeded at a density of 1. 5x105 per well in a 6-well plate were transfected with in-vitro transcribed pGagCN/Sin and pMA-Pr/Sin RNAs, MA-Pr/Sin RNA (negative control) and SinLacZ RNA (positive control).

One ptg of RNA was mixed with 2 pl of Lipofectin (GibcoBRL) in OptiMEM according to manufacture instructions. BHK cells were incubated with the liposomes/RNAs mixture for 6 hours, following which, the transfection medium was exchanged for 3ml of a growth medium (DMEM, containing 5% Fcs, 2mM L-glutamine). Cells were allowed to grow for 18-24 hours and cell lysates were prepared using 400 gel of Reporter Lysis Buffer (Promega). The p-galactosidase assay was performed according to manufacture instructions (Promega, Cat. No. E2000); activity was monitored by observing the change in absorbance at 420 nm using a UV-VIS spectrophotometer.

Results Expression of both MA-Pr/Sin and GagCN/Sin in BHK cells resulted in the appearance of a strong ß-galactosidase activity in cell lysates 24 hours post transfection (Figures 21 and 23c). Activity was 100-fold higher than that

observed for cells separately transfected with MA-Pr/Sin or GagCN/Sin (Figures 23a-b respectively).

In the absence of an HIV-protease, the ß-galactosidase activity of GagCN expressing cells cannot be distinguished from that detected in cells expressing the SinRep5 vector alone confirming fusion of the a-donor downstream of the C-terminus portion of ß-galactosidase prevented enzymatic activity.

Thus, as evident from the results presented hereinabove, the reporter constructs of the present invention can be effectively used for detecting the presence of a viral protease and therefore viral particles within cells. The specificity, sensitivity and lack of background enzymatic activity in the absence of the HIV protease, makes the reporter constructs of the present invention particularly suitable for the detection of specific viral strains isolates even under low viral load conditions.

EXAMPLE 4 Applications Phenotypic Testing of HIVDrug Resistance : The method and constructs of the present invention can be utilized for phenotyping HIV drug resistance.

To test drug resistance, the method of the present invention can be effected as follows: wells of a microtiter plate are coated or filled with anti-HIV drugs in various concentrations and combinations. Several of these wells are left uncoated to be used as controls. Patient peripheral blood mononuclear cells (PBMC) are prepared from 10 ml of anticoagulated whole blood according to known methodology. Approximately 105 of these cells are added to an uncoated well (in triplicate) and serve as untreated control.

Recombinant lymphotrophic pseudovirions carrying a (3-galactosidase reporter construct are added to the remainder of the PBMCs in suitable concentrations.

The treated PBMCs are added to three of the uncoated wells (control) and to the wells including the anti-HIV drugs. The plates are then incubated at 37 °C and 5 % CO2 for 12-24 hours following which a lysis buffer is added to each well. Following lysis, a ß-galactosidase assay buffer is added, the plates are incubated for three hours till overnight, and enzyme activity is determined.

Untreated control cells serve as a background, while treated control cells are considered infected and serve to estimate the titer of HIV in PBMCs. As with standard plaque reduction assays, a 50% inhibitory concentration (IC50) will be reported as the concentration of antiviral drug that reduces the number of active viruses by 50%. IC50 for wild type HIV has to be established for each drug and drug combination and will be used to determine the drug resistance of a virus strain derived from an infected individual. This screening method is easy to implement, eliminates the need to determine the titer of the virus, and can deliver accurate results within 24 hours.

Alternatively, a comparison of ß-galactosidase activity in the presence of different drug cocktails may be also be performed.

Since p-galactosidase activity directly reflects HIV-protease activity, a specific drug cocktail which is most effective in reducing P-galactosidase activity can be determined.

By traversing the need for time consuming IC50 or IC95 testing, a physician can determine with relative ease the most suitable drug cocktail for treating a specific patient.

Although the invention has been described in conjunction with specific embodiments thereof, it is evident that many alternatives, modifications and variations will be apparent to those skilled in the art. Accordingly, it is intended to embrace all such alternatives, modifications and variations that fall within the spirit and broad scope of the appended claims. All publications, patents, patent applications and sequences identified by their accession

numbers mentioned in this specification are herein incorporated in their entirety by reference into the specification, to the same extent as if each individual publication, patent, patent application or sequence identified by their accession number was specifically and individually indicated to be incorporated herein by reference. In addition, citation or identification of any reference in this application shall not be construed as an admission that such reference is available as prior art to the present invention.