Title of Invention	A NOVEL SCREENING METHOD FOR SCREENING OF ANTI-HIV MOLECULES
Abstract	The present invention relates to a novel screening method for screening of anti-HIV therapeutics. In particular, the present invention relates to reporter gene constructs for the detection of the HIV Nef and host PACS-1 protein interaction. Furthermore, the invention relates to a functional interaction for Nef-PACS-1 proteins prepared in a recombinant manner, a method for identifying of Nef-PACS interaction which causes activation of pathway to down regulate MHC class I molecules from cell surface presumably causing immune evasion for HIV in infected cells. The reporter gene construct according to the present invention, after it had been introduced into cells, in the presence of HIV Nef and host PACS-1 proteins result in the expression of reporter luciferase protein which may be used for quantitative/qualitative interaction of HIV Nef and host PACS-1 protein. Furthermore, the invention was used to identify the inhibitor for the interaction of Nef-PACS in the cell.

Title of Invention

A NOVEL SCREENING METHOD FOR SCREENING OF ANTI-HIV MOLECULES

Abstract

The present invention relates to a novel screening method for screening of anti-HIV therapeutics. In particular, the present invention relates to reporter gene constructs for the detection of the HIV Nef and host PACS-1 protein interaction. Furthermore, the invention relates to a functional interaction for Nef-PACS-1 proteins prepared in a recombinant manner, a method for identifying of Nef-PACS interaction which causes activation of pathway to down regulate MHC class I molecules from cell surface presumably causing immune evasion for HIV in infected cells. The reporter gene construct according to the present invention, after it had been introduced into cells, in the presence of HIV Nef and host PACS-1 proteins result in the expression of reporter luciferase protein which may be used for quantitative/qualitative interaction of HIV Nef and host PACS-1 protein. Furthermore, the invention was used to identify the inhibitor for the interaction of Nef-PACS in the cell.

Full Text	Field of Invention The present invention relates to a novel screening method for screening anti-HIV molecules. In particular, the invention relates to mammalian two hybrid system for screening of anti-HIV therapeutics. The present invention further relates to a reporter gene construct for the detection of the Negative factor (Nef) - Phosphofurin acidic cluster sorting protein (PACS-Ifbr) interaction essential for major-histocompatibility-complex (MHC-I) downregulation, resulting immune evasion. Furthermore, the invention relates to a screening model based on Nef and PACS-I interaction in HEK-293 cells using a mammalian two-hybrid system, a method of screening for those inhibitors related to Nef-PACS interaction, a method of screening for inhibiting a pathway initiated with Nef-PACS-1 interaction which downregulate MHC classl from cell surface to transgolgi causing immune evasion. Background of Invention and Description of Prior Art HIV-I is the most prevalent infectious agent world wide and a leading causative agent of acquired immuno deficiency syndrome (AIDS). HIV is a retrovirus, which is transmitted through human fluids either through natural interactions like sexual intercourse or through artificial methods like blood transfusions, infected needles used in injections. The 9-10 Kb of HIV-1 genome express viral proteins (categorized in structural, enzymatic and accessory proteins functions), whose function ranges from the successful viral infection in the cell to the complete life cycle for new virions, has been extensively studied. Every viral protein is considered to be a potential target for developing anti-HIV-1 therapy (Greene and Peterlin, 2002; Stevenson 2003). Further, it was characterized that accessory protein Nef is responsible for HIV-1 pathogenesis (Mellors et. al, 1996) by the experiment where, nef deleted SIVmac 239 infection in rhesus monkey showed low viral load with normal CD4 counts. These monkeys were found healthy with delayed progression to AIDS (Kestier et. al, 1991). Also, the HIV-1 patients who were long-term non-progressor, with no clinical sign of disease progression and normal CD4 counts, upon characterization of virus revealed that majority of them had nef gene deleted from their genome (Daniel et. al, 1992, Deacon et. al, 1995, Kirchhoff et. al, 1995, Mariani et. al, 1996, Salvi et. al, 1996, Switzer et. al, 1998). Interestingly, only expression of nef gene in mice led to the development of AIDS like phenotype suggesting that it harbors major disease determinant (Hanna et. al, 1998). Nef is deludged with functions involving interaction with different host proteins, however, the individual function required for pathogenesis has not been identified till date. Nef is a 27kDa protein, expressed in HIV-1 infected cells even before the formation of provirus suggesting that it initiate functions soon after entry of virus in cell. In vitro and in vivo studies show that Nef protein enhances viral infectivity and replication respectively in infected cells. Nef downregulates the MHC classl from cell surface, which primarily presents viral proteins to activate cytotoxic T lymphocytes. Moreover, Nef functions include activation of Pak kinase, activates cascade of events leading to the activation of T cells without engaging T-cell receptor [TCR] and upregulation of Fas/FasL ligand for killing bystander cells, as well as activates anti-apoptotic pathways for the survival of infected cells. All these functions of Nef are carried out in cell after interaction with host proteins. The molecular pathway for MHC-I downregulation is initiated by functional interaction of Nef and PACS-I in a AP-1 dependent manner in the cell (Piguet et al 2000; Crump et al., 2001; Blagoveshchenskaya et al., 2002). Recently, PACS-2 is also implicated in MHC-I downregulation pathway (Atkins et al., 2008). The underlying cellular pathway for MHC-I downregulation to trans-Golgi network (TGN), requires primarily binding of acidic domain of Nef (62EEEE65) to PACS-I which enables PXXP domain to interact and activate a trans-Golgi network-localized Src family tyrosine kinase (SFK). The Nef/SFK complex then recruits and phosphorylates the tyrosine kinase ZAP-70, which binds class I PI3K in primary CD4+ T cells and in promonocytic cells the complex recruits ZAP-70 homolog Syk to trigger MHC-I down regulation respectively. The PI3K inhibitors repress MHC-I downregulation, identifying them as potential therapeutic agent to combat HIV-1. The PI3K pathways present in multiple HIV-1 reservoirs suggest that the recruitment of Nef-SFK-ZAP-70/Syk-PI3K signaling pathway is a common mechanism for MHC-I downregulation for immune evasion and any PI3K inhibitor will affect globally (Hung et al., 2007). Understanding that the Nef-SFK-ZAP-70/Syk-PI3K signaling pathway is recruited and activated by action of Nef EEEE65 site with PACS-1&2, the Nef-PACS interaction could become a potential target to identify inhibitors as potential therapeutic agent to combat HIV-1 (Fig4&5). Mutational studies have shown that conserved domains of Nef are responsible for regulating function, but the specific role in HIV-1 pathogenesis was not directly understood. In an indirect effort to understand the role of nef gene in the progression to AIDS, the nef gene was isolated from two HIV-1 patients over period of time and observed that the complexity of nef sequence heterogeneity was increased during the progression of HIV-1 infection. However, the scattered regions responsible for downregulation of CD4 and MHC were conserved. Similarly, to understand the role of Nef in HIV-1 pathogenesis efforts were made to characterize nef gene from different stages of HIV-1 disease. One of the studies showed that nefgene isolated from different stages of the disease exhibit mutation in nef which was further related to specific stages of HIV-1 pathogenesis (Krichhoff et. al, 1999). Later on it was shown that these nef alleles isolated from asymptomatic patients are more active in MHC-I down regulation whereas; after progression to AIDS nef was mainly involved in down regulation of CD4 and enhancement of virion infectivity (Carl et. al, 2001). However, other groups could not find any gross abnormality in nef alleles isolated from different stages of disease (Ratner et. al, 1996). The downregulation of MHC-I is considered to be the most important function of Nef because this event causes immune evasion from cytotoxic T lymphocytes (CTL) and is thus considered as a key factor for AIDS development. Macaques with deleted CD8+ T cells exhibited faster progression to AIDS; complementation with CD8+ T cells inhibited virus replication and delayed AIDS progression (Schmitz et. al, 1999). This suggests that the primary CTL response against virus is important to delay the AIDS progression in host. Understanding the molecular Nef pathways responsible for functions involved with viral persistence, replication and infectivity can be used as target for anti HIV-1 therapy. This can enable us to focus on future therapies where the task of interrupting key interaction between viral and host proteins will serve as potential target. Nef-PACS interaction represents a potential target for this approach. Recent approaches towards the development of an alternative model for characterizing specific interactions of Nef with host proteins are either yeast (Rossi et. al, 1996) or mammalian-two-hybrid system (Murakami et. al, 2002). Nef is structurally conserved and the structure function analysis revealed that with maintaining the structure, the accessible nef conserved domain to host proteins is responsible for the function interaction in the cell (Geyer et. al., 2001). In the present study, we developed a mammalian-two hybrid model and studied Nef-PACS-I interaction in HEK-293 cells, an early event which activates ZAP70/PI3K/Syk/AF6 pathway for MHC-I down regulation (Hung et. al, 2007). Further, this interaction was characterized structurally and functionally for primary Nef isolates isolated from patients. The inhibitors designed were screened in this model and it was found that Nef-PACS-I interaction was inhibited, perhaps, which can be used to develop an alternative therapy. The Nef protein is structurally conserved and the structure function analysis revealed that with maintaining the structure, the accessible Nef conserved domain is responsible for the functional interactions in the cell. Therefore, reporter gene construct was developed for maintaining Nef-PACS-Ifbr interaction in HEK-293 cells and screened novel peptide inhibitors for inhibiting Nef-PACS interaction. Mammalian two hybrid models have been used in scientific research in a routine manner (Colas and Brent., 1998). The present invention uses this technique to enable the user to establish the Nef-PACS-I interactions in HEK-293 cells. No other group has developed such a model for the evaluation of Nef-PACS-I interactions earlier and the present invention is the first of it's kind. The present invention serves as a novel tool to identify inhibitors of the aforesaid interactions. Inhibitors, which are so identified, will have property to inhibit MHC-1 down-regulation pathway. There are no known drawbacks to the technique and it is amenable to high-throughput screening. The invention will efficiently allow for the screening of a large number of compounds in a relatively short time period of upto 48 hours. This will also allow for the search of structural analogs of inhibitors identified using the method of the present invention. Inhibitors, which are identified using the present invention, will act through a novel mechanism of inhibiting Nef-PACS-I and 2. This will be an alternate approach for anti-retroviral therapies including but not restricted to the Human immuno deficiency virus (HIV), which causes AIDS. Objects of the Invention The main objective of the present invention is to develop mammalian two-hybrid model for assessing Nef-PACS-Ifbr interaction in HEK-293 cells similar to HIV-I infected cells. Another object of the invention is to provide a novel screening method to identify the molecules useful for inhibition of Nef-PACS-1 interaction in eukaryotic cells. Still another object of the invention is to provide transfecting vectors pACT and pBIND (with and without Nef and PACS-1 gene) with luciferase gene in vector G5Luc as a reporter, in mammalian two hybrid model system where these vectors are co-transfected using cationic polymer. Yet another object of the invention is to define the specific interaction of Nef and PACS-1 by mutating acidic conserved domain in Nef gene, truncated Nef gene and making truncated PACS- lfbr. A further object of the invention is to provide two hybrid model system to maintain the interaction of Nef variants isolated from HIV-1 patients with PACS-1 necessary for immune evasion. Another object of the invention is to screen a molecule 'EEEEE5 which inhibits the interaction of Nef and PACS-1 by showing the reduced luciferase activity with respect to negative control. Still another object of the invention is to provide the mammalian two hybrid model as a novel screening/assay system to identify novel molecules for inhibiting viral-host protein interaction. Summary of the Invention The invention enables the detection and quantification of the function of HIV Nef and host PACS protein interaction in living cells similar to HIV infected cells. The test is based on the qualitative and quantitative evaluation of the expression of reporter luciferase gene which is mediated by the interaction of Nef-PACS protein, cloned in the mammalian-two hybrid vectors. The mutation as well truncations in nef gene inhibits the interaction and no expression of reporter gene is detected. The truncated PACS-lfbr gene, PACS-1 115, the PACS-1 [154] showed interaction with Nef, however, PACS-1 [179] does not interact with Nef suggesting Nef interaction is in AP-1 dependent manner. Furthermore, the Nef-PACS 1 interaction was inhibited by the presence of pentapeptide (EEEEE). Further more, the Nef gene isolated from patients also showed inhibition of interaction. In general the methods of this invention utilize a two-hybrid system, more preferably a mammalian two-hybrid system. A cell is provided containing constructs forming a two-hybrid system. The first construct comprises a DNA binding domain such as GAL4 attached to PACS-I. A second construct is provided that contains a transactivation domain such as VP16 attached to nef. A third construct in the cell is a nucleic acid comprising a DNA binding sequence and a luciferase gene downstream of the DNA binding sequence, wherein the transcription and/or translation of the luciferase gene is to be regulated by this two-hybrid system [transcription of the luciferase gene is regulated by the transactivation domain of the second construct. The reporter construct according to the present invention has the following elements in functional association: a) a reporter plasmid pACT having Vpl6 activation domain, modified by changing polyadenylation region called pACT; b) nef gene fused with activation domain Vpl6; c) PACS-1 gene fused with Gal 4 DNA binding domain. The reporter construct according to the invention can be transferred into cells (for example by transfection) and the recipient cells are analyzed after a defined time period. This time period depends on the amount of Nef and PACS-1 protein produced in the cell for interaction. Since the presence of Nef in the cell could be considered similar to as it is expressed in HIV-1 infected cells, interacting with host proteins, the reasonable amount of the reporter mRNA will produce functional Nef. In the further course, among the host proteins functionally over expressed PACS-1 protein will interact with Nef protein which will mediate expression of luciferase gene expression. The reporter gene constructs without Nef and PACS-1 gene, present inside the cell in a functional form (as in case of e.g. transfection of a respective plasmid) do not mediate the expression of luciferase plasmid. This may be documented and observed in a cell population by analyzing luciferase activity. An advantage of the newly developed reporter gene construct is its uncomplicated and cost-effective use in screening the inhibitors which could be used to develop a novel anti-HIV therapy. Another advantage is the fact that this reporter gene construct could be used in high through put screening for identifying the inhibitors. By means of the invention it is possible for the first time to measure the Nef-PACS interaction in the live cell in a quick and cost effective manner without infection of HIV. The present reporter gene construct causing Nef-PACS interaction is very specific, which is confirmed by the mutation in the acidic domain of the nefgene. The constructs bearing the aforesaid mutated nef gene do not interact with the PACS-1 counterpart. Similarly, the truncated PACS in which the AP-1 binding site is deleted showed loss of interaction with nef. The mammalian two-hybrid system is chosen for making a reporter gene construct of Nef-PACS interaction because HIV infection occurs in mammalian cells and Nef interaction is structure based interaction. Nef structure based interaction depends upon the attained conformational structure in the cell. The post translational modification for attaining conformational structural to attain biological activity is achieved in eukaryotic cells as seen in HIV infected cell. The Nef-PACS protein interaction is structure based interaction also with the involvement of other host proteins, which could be maintained in eukaryotic cells supplementing all proteins required for the interaction. PACS-I fbr region interacts with Nef, when compared with PACS-2fbr showed 76.26% homology, which makes decent hypothesis that PACS-2 will also show tendency to interact with Nef in our mammalian two-hybrid model. Accordingly, the present invention provides a novel screening method for screening of anti-HIV therapeutics, wherein the steps comprising: a) transfecting constructs of SEQ ID No. 18, 25 and 24 respectively into a mammalian cell using 30 to 50 microM of a transfection reagent to obtain a transfected cell; b) analyzing the transfected cell as obtained in step (a) for luciferase activity after incubating for a period of 24 to 48 hours for demonstrating Nef-PACS-1 interaction using dual luciferase kit and luminometer; c) adding the molecule to be screened in the transfected cell of step [a] and incubating for 6 to 24 hours followed by repeating step [b] to assess its inhibitory activity on the Nef-PACS-1 interaction; d) selecting the molecules which are inhibiting the Nef-PACS-1 interaction resulting in no additional luciferase activity with respect to the control as a potential anti-HIV therapeutic. Brief description of the accompanying drawings Figure 1: Nefwt-PACS-Ifbr interaction mediated by AP-1 in reporter gene construct. A) nefwt acidic domain sequence aligned with HIV-1 subtype C nefgene. B) truncated PACS-I fbr gene showing AP-1 binding site. C) Expression of Nefwt/Nef EEmut-PACS-Ifbr, Nefwt/NefEEmut with truncated PACS-I 115, 154 and 179 in HEK-293 cells. The luciferase activity was detected in cell where Nefwt and PACS-Ifbr, 115 and 154 were present. No luciferase activity was found in Nefwt- PACS-I 179 which had truncated AP-1 binding site, establishing that Nefwt-PACS-Ifbr interaction is AP-1 dependant. Also, no luciferase activity was observed in NefEEmut with all PACS-I and truncated PACS-I, single gene vector and control vectors transfected cells. D) Positive control showing protein-protein interaction in reporter gene construct. The MyoD and ID genes were transfected in HEK-293 cells and showed high luciferase activity. The values represent luciferase-renilla ratio for standardizing transfection efficiency. Figure 2: Screening of the inhibitors of Nef-PACS-lfbr interaction in reporter gene construct: Specific inhibition of A)Nefwt-PACS-lfbr, B) Pat-RPOl Nef-PACS-lfbr, C) Pat-RP14 Nef-PACS-lfbr interactions were shown at OuM, 5uM, 2.5uM and 1.25uM concentration of inhibitor D) MyoD/ID interaction was not affected at OuM, 5uM, 2.5uM and 1.25uM concentration of inhibitor. Figure 3 : HIV-1 Nef variants isolated from patients and their mutants interaction with PACS-I fbr in mammalian two hybrid system: A) Pat Nef variants RP01 and RP14, and Nefwt when transfected with PACS-Ifbr showed expression of luciferase activity in HEK-293 cells. The control vectors and single gene vectors transfected showed no luciferase activity in HEK-293 cells. B) MyoD,Id as positive protein-protein interaction Figure 4: Full length protein is responsible for interaction with PACS-Ifbr. A) Digrammatic representation of Nef truncations used in the study. B) In silico modeling of patRPOl Nef protein. C) Nefwt, RP01400, RP01300, RP01200 and NefEEmut were transfected with PACS-Ifbr and showed luciferase activity in HEK-293 cells. D) MyoD-Id interaction for positive protein-protein interaction. Figure 5: Schematic representation of construction of mammalian expression vector VP16pcDNA (called VP16pcDNA+pACT). Detailed Description of the Invention Definitions A "fusion protein" refers to a polypeptide formed by the joining of two or more polypeptides through a peptide bond formed between the amino terminus of one polypeptide and the carboxyl terminus of another polypeptide. The fusion protein may be formed by the chemical coupling of the constituent polypeptides or it may be expressed as a single polypeptide from nucleic acid sequence encoding the single contiguous fusion protein. A single chain fusion protein is a fusion protein halving a single contiguous polypeptide backbone. The term "two-hybrid system" refers to a system comprising two chimeric molecules one of which bears a nucleic acid binding region, the other of which bears an expression control element (e.g. a transactivation or repressor domain). The molecules further a cognate binding pair such that one chimeric molecule is capable of specifically binding to the other chimeric molecule. The two-hybrid system further comprises a nucleic acid encoding a protein binding site that is specifically bound by the protein binding domain on the chimeric molecule thereby anchoring the chimeric molecule to the nucleic acid. The domain of the chimeric molecule recognizes and binds to its cognate binding partner on the second chimeric molecule thereby recruiting that molecule to the nucleic acid whereby the expression control element alters (e.g. activates) expression of a gene or cDNA comprising the underlying nucleic acid. "Transfection" is used herein to mean the delivery of expressible nucleic acid to a target cell, such that the target cell is rendered capable of expressing said nucleic acid. It will be understood that the term "nucleic acid" includes both DNA and RNA without regard to molecular weight, and the term "expression" means any manifestation of the functional presence of the nucleic acid within the cell, including without limitation, both transient expression and stable expression. The term "transactivator" refers to a molecule that induces transcription and/or upregulates transcription of a gene or cDNA. The transactivator may be a complete "native" molecule or a domain of a molecule that is capable of inducing and/or upregulating transcription of a gene or cDNA. "Reporter genes" are genes or cDNAs that express an easily assayable (detectable and/or quantifiable) product. Detection of the assayable product indicates the expression and/or level of expression of the reporter gene. Reporter genes are well known to those of skill in the art. They include, but are not limited to, genes expressing bacterial chloramphenicol acetyl transferase (CAT), beta-galactosidase (.beta.-gal), green fluorescent protein (GFP) and other fluorescent protein, various bacterial luciferase genes, e.g., the luciferase genes encoded by Vibrio harveyi, Vibrio fischeri, and Xenorhabdus luminescens, the firefly luciferase gene FFlux, and the like. The term "transcriptional activators" refers to proteins, which activate transcription in yeast, plant, insect and mammalian cells. These proteins contain two parts: one directs DNA binding and the other, called the activating region, presumably interacts with some component of the transcriptional machinery. Activating regions are typically acidic and require some poorly-understood aspect of structure, probably at least in part an alpha-helix. Here in the present invention, the "transcriptional activator system" utilized is the one, which is formed by fusing a DNA-binding fragment of the yeast activator GAL4 to a highly acidic portion of the herpes simplex virus protein VP16. VP16 activates transcription of immediate early viral genes by using its amino-terminal sequences to attach to one or more host-encoded proteins that recognise DNA sequences in their promoters. Preparation of HIV-1 Nef wt, RP01 and RP14 Nef variants- The nef genes amplified from the randomly selected patients RP01 and RP14 were reamplified with the specific primers as given in table 1 for in frame cloning in the mammalian two-hybrid vector. For generating nefwt, RP01 was used as template using the gene specific primers of SEQ ID No.3 and 4, as given in table 1. The nefwt was made similar to the subtype C reference sequence [refer to figure 1 A] and later on cloned in the mammalian two-hybrid vector for interaction studies. Amplification of PACS-1 gene from rat brain mRNA- mRNA was isolated from rat brain and PACS-1 was amplified using gene specific primers as given in table 1. Rat brain was homogenized in Tri reagent (w/v) and mRNA was isolated by extracting the homogenate with chloroform (1:1). The mRNA was precipitated using equal volume of isopropanol and incubating at minus 20°C for 2hrs. The mRNA pellet was washed with cold 70% alcohol and resuspended in 0.2% diethyl phosphorocyanidate [DEPC] water. Following DNase digestion the mRNA was used for cDNA preparation using random primer pdN6.The PACS-l fbr fragment was amplified using the gene specific primers of seq ID No. 13 and 14 as given in table 1. Using PACS-l fbr fragment as template, truncations of PACS-l were generated and cloned in pDrive vector and confirmed by sequencing. Plasmid Constructs— nef gem was cloned in VP16pcDNA plasmid constructed in lab using pcDNA 3 (Invitrogen) as vector backbone, while the fragment containing the CMV promoter, intron, T7 promoter, nuclear localization signal and multiple cloning region was taken from the mammalian two-hybrid assay vector pACT. The resulting construct thus containing the cloned nef gene was called VP16pCDNA+pACT. The sequence of the construct containing the cloned nefwt gene, is represented by SEQ ID No. 18. Two sets of constructs were generated from the two vectors, pBIND and VP16pCDNA+pACT: • pBIND with PACS-l fbr or PACS-l[115] orPACS-l[154] or PACS-1[179] and • VP16pCDNA+pACT with nejwt or nef/RP01 400 or nef/RP01 300 or nef/RP01 200 or nef RP14. PCR was performed for each gene using the appropriate 5'and 3' primers (as given in Table 1). All of the primers had BamRl and Xbal restriction sites, and the genes were digested with BamHl and subcloned into pBIND and VP16pCDNA+pACT vectors. All constructs were verified by Sanger sequencing in an ABI Automatic Sequencing System (PerkinElmer Applied Biosystems Inc, Foster City, CA). The primers were custom-synthesized by Sigma, Operon and IDT. Table 1: Primers used for «e/mutation and PACS truncations (Formula Removed) Tissue Culture and Transfections—HEK 293 cells were grown in Minimal Essential medium (low glucose, Sigma) supplemented with 10% fetal bovine serum, penicillin (100 units/ml), streptomycin (100 ug/ml) and Gentamycin (50 ug/ml) at 37°C with 5% C02. 1X105 cells were seeded per well in twenty four well plates, one day before transfection. Using commercially available ExGen 500 transfection reagent, all the required plasmid constructs were cotransfected into HEK 293 cells. 0.222 microgram of each plasmid and 5.3 microgram per liter of ExGen 500 reagent were used per well. 48 h post transfection the cells were harvested and Luciferase activity was monitored using the Dual-Luciferase® Reporter assay kit (PROMEGA). The data for basal control were used for the conversion of Luciferase activity to fold activation. As a positive control, the protein-protein interaction vectors pACTMyoD and pBIND-ID encoding the MyoD and ID control proteins, provided with the kit were used. Luciferase Assay—The transfected cells were lysed in IX Passive Lysis Buffer (PLB) provided with the kit, and the cell extracts (containing Luciferase enzyme) were added to the luminometer tubes and the Luciferase and Renilla RLU were measured on Berthold luminometer using the respective substrates for 10s along with 2s delay time. Screening inhibitors which inhibitedNef-PACS interaction in HEK-293 cells: The peptide designed for inhibiting Nef-PACS-1 interaction was screened in the developed mammalian-two hybrid model. The peptide was used at 0, 5, 2.5 and 1.25uM concentration incubated with HEK-293 cells for 4 hours, transfected with nefwt-VP16pcDNA+pACT construct, PACS-KibO-pBIND construct and reporter construct pG5Luc of SEQ ID No. 18, 25 and 24 respectively. Similarly, the cells were also transfected with different constructs of nef variants and PACS variants. The peptides inhibited Nefwt-PACS, RP01-PACS and RP14-PACS interaction at all concentrations. The inhibitor completely inhibited the interaction and was close to control. However, the inhibitor did not affect the MyoD-ID interaction at any concentration (Fig 1D). These results clearly demonstrate that the inhibitor effectively and specifically inhibited the Nef-PACS interaction in HEK-293 cells. In an embodiment of the present invention, fusion with mutated nef and truncated PACS1 have been made to demonstrate the specificity of interaction and that the interaction is AP-1 mediated. In another embodiment of the present invention, the eukaryotic cells used for the preparation of mammalian two-hybrid system may be selected from the group consisting of HEK-293 cells, T cell lines, monocytic cell lines and fibroblast cells wherein Nef-PACS-1 interaction is established. In yet another embodiment, the present invention showed 29.45 times higher ratio of luciferase/renilla domonstrating Nef-PACS-1 interaction in comparison to negative control wherein Nef and PACS-1 were not cloned in vectors. In still another embodiment of the present invention, the model used established the specific interaction of Nef and PACS-1 by mutating acidic conserved domain in Nef gene. In a further embodiment of the present invention, the reporter gene construct mimics the HIV-1 infected cells in terms of interaction by deleting AP-1 binding site in PACS-1. In yet another embodiment of the invention, the Nef variants-PACS-1 interaction is exhibited by luciferase activity in HEK-293 cells. In still another embodiment of the invention, the mammalian two hybrid model is used for screening of a molecule 1EEEE5, which inhibits the interaction of Nef and PACS-1, which is measured by reduced luciferase activity with respect to negative control. In yet another embodiment of the invention is provided the reporter gene construct as a novel screening/assay system to identify novel molecules for inhibiting viral-host protein invention. The following examples are given by way of illustration and therefore should not be construed to limit the scope of the invention. Examplel Reporter gene constructs / mammalian two hybrid model to demonstrate Nef and PACS-Ifbr interaction in HEK-293 cells: The primary interaction of Nef-PACS-I fbr was studied in HEK-293 cells using reporter gene construct. In this experiment, HIV-1 nef variant isolated from patient was modified to match the HIV-1 subtype C nef conserved domain with respect to the acidic domain (EEEEE) and proteolytic domain (WL) (Figla) referred as nefwt (wild type). The conserved acidic domain 62EEEEE66 was mutated to 62AAAAA66 {nefEEmut) for establishing specificity of interaction with PACS-I fbr (Figla). The PACS-I fbr and N-terminal truncated PACS-I 115, PACS-I 154 and PACS-I 179 were made to show API dependent interaction (Figlb). For reporter gene construct, nefwt and nefEEmut gene were cloned in frame to VP16 transactivation domain vector. Similarly the PACS-I fbr gene and truncated PACS-I 115, 154 and 179 were cloned in frame to GAL4 DNA binding domain vector. The VP16 newt, GAL4 PACS-I fbr with luciferase gene vectors when transfected in HEK-293 cells showed 29.45 fold luciferase activity in HEK-293 cell'after 48 hrs post transfection compared to control vectors without Nef and PACS-I fbr genes. However, the nefEEmut, PACS-I with luciferase gene vectors when transfected in HEK-293 cells showed no luciferase activity which correspond to control vector (fig lc). Further, when nefwt was transfected with different truncated PACS-I 115, 154, and 179 it showed 27 fold luciferase activity with PACS-I 115, and 29.45 fold with PACS-I 154 as compared to control. However, the nefwt and truncated PACS-I179 which does not have API binding site does not show luciferase activity. The specificity of interaction with acidic domain was demonstrated by showing no luciferase activity with nefEEmut with PACS-I fbr, 115 and 154 transfected cells (Fig lc). There was no nonspecific luciferase activity found in either of control vectors or with Nef or PACS-Ifbr gene alone in HEK-293 cells (Figlc). MyoD and ID were used as a positive control for protein-protein interaction in mammalian two hybrid system in HEK-293 cells and showed 307 fold luciferase activity (Fig Id). These results show that Nef-PACS-Ifbr interaction in HEK-293 cells is mediated which was similar to the interaction shown in A7 melanomas cells and primary T cells (Piguet et. al, 2000). Example-2 To analyze nef variant isolated from patients for interaction with PACS-I required for MHC-I down regulation: Using the mammalian two-hybrid system of example 1, we showed native interaction of Nef-PACS-I in HEK-293 cells, now the same system was used to understand the functionality of nef variants isolated from patients in terms of Nef-PACS-I interaction. Analysis of nef variants amplified from 7 patients showed that they had variation in acidic domain and neighboring proteolytic cleavage region. The RPOl had mutation in acidic domain in which one E at position 65 is mutated to N where as in RP14 the entire proteolytic cleavage region 64CAWLEAQ69 is deleted and addition of 9 residues were found inserted in the variable region . Here the situation is that in RPOl the acidic domain and proteolytic region is same as nefwt except E mutated to N in acidic domain and in RP14 the complete acidic domain (64CAWLEAQ69) is deleted. The interaction of these nef variants with PACS-I was studied in the said mammalian two-hybrid model. The nef variants RP01 and RP14 were cloned in VP16pcDNA+pACT vector. Both nef variants and nefwt with PACS-I fbr were transfected in HEK-293 cells, the RPQ1 and RP14 variant showed 22.56 and 44.43 fold luciferase activity compared to control vector. RP01 which is close to nefwt showed almost similar fold induction in luciferase activity of 22.56 as was observed for nefwt (23.6 fold luciferase activity) (fig 3A). However, RP14 was 1.88 times higher than Patl luciferase activity inspite of WL region deleted. For showing specificity of RP01 and RP14 interaction with PACS-I, the acidic domain of patl and patl4 were mutated to nefEEmut (RP01) and RP14EEmut (RP14) which when transfected with PACS-I fbr showed no luciferase activity and were similar to control (fig3A). The luciferase activity for MyoD, Id positive control is 304 times compared to control vector (Fig3B). These studies suggest that the Nef variants isolated from patients having said mutations clearly demonstrate functional interaction with PACS-I in HEK-293 cell. Example-3 Both protein conformational structures are responsible for interaction of Nef acidic domain and PACS-I: In a mammalian-two hybrid system, the intriguing feature is that proteins expressed in cell attains native conformational structure and interact with each other in their physiological state. In the structure based interaction of two proteins, change in structure of one protein may lead to perturbation in environment for protein-protein interaction. In nef, the structure based interaction with host protein depends upon the conformational structure of Nef attained in the cell. The fact that the interaction of unstructured N-terminus of Nef with PACS-I is a structure based interaction was shown using this model. To study this hypothesis, the RP01 Nef variant was truncated from C-terminus, thereby, shortening the protein and perturbing the structure as shown in fig 4a. The full length RPOlNef used in the study had all the conserved domain except the myristoylation region (1-40 amino acid residues) (fig 4a). RP01-300, and RP01-200 are subvariants which are truncated from C-terminus (fig4a). The predicted in silico structure of truncated nef subvariants are shown in fig4b. All the subvariants were cloned in VP16pcDNA+pACT vector and their interaction with PACS-I fbr was studied. Only nefwt and RP01 nef full length variant showed 20.13 and 19.0 fold luciferase activities respectively. However, RPOl-300 and RPOl-200 showed no luciferase activity and were same as nefEEmut and control. The positive control interaction of MyoD-Id showed 258 fold luciferase activity. Therefore it was shown that the Nef-PACS-I interaction is typically structure based interaction in HEK-293 cells. Perturbation of nef structure even after keeping the acidic domain intact, does not maintain its interaction with PACS-I in the developed model. Example-4 To study interaction of Patl nef and Patl4 nef variants with PACS-I fbr is API mediated: As shown in fig 1, the nef wt interaction with PACS-I is mediated by API which finally activates the MHC-I downregulation pathway. Similarly to understand whether the Patl and RP14 nef variants, selected naturally in patients, have property for API mediated PACS-I interaction. All PACS-I variants, as described in fig la, were transfected with RPOl and RP14 nef variants. RPOl showed luciferase activity with PACS-Ifbr, PACS-I115 and PACS-I154 of 25.68, 23.3 and 23.86 respectively compared to control, but showed no luciferase activity with PACS-I 179 which lacks the API site (fig4C). Similarly RP14 showed luciferase activity with PACS-Ifbr, PACS-I115 and PACS-I 154 of 42.39, 25.4, and 22.39 respectively compared to control, again no luciferase activity was seen in PACS-I 179 (fig 4D). RP14 nef variant showed decreasing trend of luciferase activity with the decreasing size of PACS-I fragment and finally decreased with deletion of AP-1 site. These results suggest that the Nef variants isolated from HIV-1 infected patients can bind to PACS-I in API dependent manner which presumably have tendency to downregulate MHC-I in patients. Example-5 Novel inhibitors identified for disrupting Nef-PACS interaction in HEK-293 cells: To screen a new class of peptide inhibitor which inhibits the Nef-PACS-Ifbr interaction in Nefwt and primary Nef variants isolated from patients. The peptide designed for inhibiting Nef-PACS-Ifbr interaction was screened in the mammalian-two hybrid model described in example 1. The peptide was used at 0, 5, 2.5 and 1.25 µM concentration, incubated with HEK-293 cells for 4 hours, transfected with nef and PACS-I fbr and MyoD-Id constructs. The peptides inhibited Nefwt, patl and patl4-PACS-Ifbr interaction at all concentrations. The inhibitor completely inhibited the interaction and was close to control. However, the inhibitor did not affect the MyoD-Id interaction at any concentration (Fig2A, B, C &D). These results clearly demonstrate that the inhibitor effectively and specifically inhibited the Nef-PACS-Ifbr interaction in HEK-293 cells and does not interact non-specifically with other proteins. Table 2: Effect of the inhibitor in reporter gene construct in inhibiting Nefwt-PACS interaction at different µM concentration (Formula Removed) The experiment performed using wild-type Nef shows that -100 % inhibition of the Nef-PACS-I interactions are achieved even on addition of 1.25µM of the inhibitor. Table 3: Effect of the inhibitor in reporter gene construct in inhibiting PatlNef-PACS-I interaction at different µM concentration (Formula Removed) The experiment performed using Nef from sample patient-1 shows that -100 % inhibition of the Nef-PACS-I interactions are achieved even on addition of 1.25µM of the inhibitor. Table 4: Effect of the inhibitor in reporter gene construct in inhibiting Patl4Nef-PACS-I interaction at different µM concentration (Formula Removed) The experiment performed using Nef from sample patient-14 shows that ~100 % inhibition of the Nef-PACS-I interactions are achieved even on addition of 1.25uM of the inhibitor. Table 5: Effect of the inhibitor in reporter gene construct in inhibiting MyoD-Id interaction at different µMconcentration (Formula Removed) The above results show 100% inhibition of Nef-PACS-1 (wt and patient samples) interaction even at 1.25µM concentration. Advantages of the Invention: • The reporter gene construct is simple to use and does not involve human or animal models of testing. • This method is amenable to screening a large number of compounds in a relatively short time when coupled with high-throughput technologies or as a stand-alone assay procedure. • Rapid inhibitor optimization is facilitated by quick screening procedure. • The existing anti-HIV therapeutics does not target the Nef-PACS-I interaction and associated pathway. • The present inhibitor(s) will be a novel therapeutic and will overcome HIV-I resistance issues. References: Atkins, K.M., Thomas,L., Youker, T.T., Harriff, M.J., Pissani,F., You,H., and Thomas,G. (2008). HIV-1 Nef binds PACS-2 to assemble a multi-kinase cascade that triggers MHC-I downregulation: Analysis using siRNA and knockout mice. JBC,283, 11772-11784. Blagoveshchenskaya, A.D., Thomas, L., Feliciangeli, S.F., Hung, C.H. and Thomas, G. (2002). HIV-1 Nef downregulates MHC-I by a PACS-1- and PI3K-regulated ARF6 endocytic pathway. Cell 111: 853-66. Colas,P and Brent,R. (1998). The impact of two-hybrid and related methods on biotechnology. TIBTECH 16, 355-363. Crump, CM, Xiang, Y., Thomas, L, Gu, F, Austin, C, Tooze, S.A. and Thomas G. (2001). PACS-1 binding to adaptors is required for acidic cluster motif-mediated protein traffic. EMBO J. 20: 2191-2201. Fackler, O, Moris, A, Tibroni, N, Giese, S, Glass, B, Schwartz, O. and Krausslich, H.G. (2006). Functional characterization of HIV-1 Nef mutants in the context of viral infection. Virology 351:332-339. Greene, W.C and Peterlin,B.M. (2002). Charting HIV's remarkable voyage through the cell: Basic science as a passport for future therapy. Nature medicine 8, 673-680. Hung, C-H, Thomas, L, Ruby, CE, Atkins, K.M, Morris, N.P, Knight, Z.A, Scholz, I, Barklis, E, Weinberg, A.D, Shokat, K.M. and Thomas, G. (2007). HIV-1 Nef assembles a Src family kinase-ZAP-70/Syk-PI3K cascade to downregulate cell-surface MHC-I. Cell host and microbes 1: 121-133. Kestier, H.W, Ringler, D.J, Mori, K, Panicali, D.L, Sehgal, P.K, Daniel, M.D. and Desrosiers, R.C. (1991). Importance of the nef gene for maintenance of high virus loads and for development of AIDS. Cell 65:651-662. Malin, H.M and Emerman, M. (2008). HIV-1 accessory Protein-Ensuring Viral Survival in a Hostile Environment. Cell Host and Microbes 3, 388-398. Piguet V, Wan, L, Borel, C, Mangasarian, A, Demaurex, N, Thomas, G. and Trono, D. (2000). HIV-1 Nef protein binds to the cellular protein PACS-1 to downregulate class I major histocompatibility complexes. Nat. Cell. Biol. 2:163-167. Schmitz, J.E, Kuroda, M.J, Santra, S, Sasseville, V.G, Simon, M.A, Lifton, M.A, Racz, P, Tenner-Racz, K, Dalesandro, M, Scallon, B.J, Ghrayeb, J, Forman, M.A, Montefiori, D.C, Rieber, E.P, Letvin, N.L. and Reimann. K.A.(1999). Control of viremia in Simian Immunodeficiency virus infection by CD8+ lymphocytes. Science 283: 857-860. Stevenson, M. (2003). HIV-1 pathogenesis. Nature Medicine 9, 853-860. We claim: 1. A novel screening method for screening of anti-HIV molecules, wherein the steps comprising: [a] transfecting constructs of SEQ ID No. 18, 25 and 24 into a mammalian cell using 30 to 50 microM of a transfection reagent to obtain a transfected cell; [b] analyzing the transfected cell as obtained in step (a) for luciferase activity after incubating for a period of 24 to 48 hours for demonstrating Nef-PACS-1 interaction using dual luciferase kit and luminometer; [c] adding the molecule to be screened in the transfected cell of step [a] and incubating for 6 to 24 hours followed by repeating step [b] to assess its inhibitory activity on the Nef-PACS-1 interaction; [d] selecting the molecules which are inhibiting the Nef-PACS-1 interaction resulting in no additional luciferase activity with respect to the control as a potential anti-HIV therapeutic. 2. A method as claimed in claim 1, wherein the construct of SEQ ID No. 18 represents nefwt gene cloned in VP16pCDNA+pACT vector. 3. A method as claimed in claim 1, wherein the construct of SEQ ID No. 25 represents PACS-1 gene cloned in pBIND vector. 4. A method as claimed in claim 1, wherein the construct of SEQ ID No. 24 represents the reporter gene construct pG5Luc. 5. A method as claimed in claim 1, wherein the transfection reagent is a commercially available ExGen 500 in vitro Transfection Reagent. 6. A method as claimed in claim 1, wherein the mammalian cell is selected from the group consisting of HEK-293 cells, T cell lines, monocytic cell lines and fibroblast cells wherein Nef-PACS-1 interaction is established. 7. A method as claimed in claim 1, wherein the molecule to be screened is added at a concentration of 5 to 1.25 microM. 8. A method as claimed in claim 1, wherein the control represents a transfected mammalian cell with VP16pCDNA+pACT vector without nef and pBIND vector without PACS-1 along with the reporter construct pG5Luc. 9. A method as claimed in claim 1, wherein the molecule screened as a potential anti-HIV therapeutic is a pentapeptide 'EEEEE'. 10. A novel screening method for screening of anti-HIV therapeutics substantially as herein described with reference to the foregoing examples.

Full Text

Field of Invention
The present invention relates to a novel screening method for screening anti-HIV molecules. In particular, the invention relates to mammalian two hybrid system for screening of anti-HIV therapeutics. The present invention further relates to a reporter gene construct for the detection of the Negative factor (Nef) - Phosphofurin acidic cluster sorting protein (PACS-Ifbr) interaction essential for major-histocompatibility-complex (MHC-I) downregulation, resulting immune evasion. Furthermore, the invention relates to a screening model based on Nef and PACS-I interaction in HEK-293 cells using a mammalian two-hybrid system, a method of screening for those inhibitors related to Nef-PACS interaction, a method of screening for inhibiting a pathway initiated with Nef-PACS-1 interaction which downregulate MHC classl from cell surface to transgolgi causing immune evasion.
Background of Invention and Description of Prior Art
HIV-I is the most prevalent infectious agent world wide and a leading causative agent of acquired immuno deficiency syndrome (AIDS). HIV is a retrovirus, which is transmitted through human fluids either through natural interactions like sexual intercourse or through artificial methods like blood transfusions, infected needles used in injections. The 9-10 Kb of HIV-1 genome express viral proteins (categorized in structural, enzymatic and accessory proteins functions), whose function ranges from the successful viral infection in the cell to the complete life cycle for new virions, has been extensively studied. Every viral protein is considered to be a potential target for developing anti-HIV-1 therapy (Greene and Peterlin, 2002; Stevenson 2003).
Further, it was characterized that accessory protein Nef is responsible for HIV-1 pathogenesis (Mellors et. al, 1996) by the experiment where, nef deleted SIVmac 239 infection in rhesus monkey showed low viral load with normal CD4 counts. These monkeys were found healthy with delayed progression to AIDS (Kestier et. al, 1991). Also, the HIV-1 patients who were long-term non-progressor, with no clinical sign of disease progression and normal CD4 counts, upon characterization of virus revealed that majority of them had nef gene deleted from their genome (Daniel et. al, 1992, Deacon et. al, 1995, Kirchhoff et. al, 1995, Mariani et. al, 1996, Salvi et. al, 1996, Switzer et. al, 1998). Interestingly, only expression of nef gene in mice led to the development of AIDS like phenotype suggesting that it harbors major disease determinant (Hanna et. al, 1998). Nef is deludged with functions involving interaction with different host proteins, however, the individual function required for pathogenesis has not been identified till date.

Nef is a 27kDa protein, expressed in HIV-1 infected cells even before the formation of provirus suggesting that it initiate functions soon after entry of virus in cell. In vitro and in vivo studies show that Nef protein enhances viral infectivity and replication respectively in infected cells. Nef downregulates the MHC classl from cell surface, which primarily presents viral proteins to activate cytotoxic T lymphocytes. Moreover, Nef functions include activation of Pak kinase, activates cascade of events leading to the activation of T cells without engaging T-cell receptor [TCR] and upregulation of Fas/FasL ligand for killing bystander cells, as well as activates anti-apoptotic pathways for the survival of infected cells. All these functions of Nef are carried out in cell after interaction with host proteins. The molecular pathway for MHC-I downregulation is initiated by functional interaction of Nef and PACS-I in a AP-1 dependent manner in the cell (Piguet et al 2000; Crump et al., 2001; Blagoveshchenskaya et al., 2002). Recently, PACS-2 is also implicated in MHC-I downregulation pathway (Atkins et al., 2008).
The underlying cellular pathway for MHC-I downregulation to trans-Golgi network (TGN), requires primarily binding of acidic domain of Nef (62EEEE65) to PACS-I which enables PXXP domain to interact and activate a trans-Golgi network-localized Src family tyrosine kinase (SFK). The Nef/SFK complex then recruits and phosphorylates the tyrosine kinase ZAP-70, which binds class I PI3K in primary CD4+ T cells and in promonocytic cells the complex recruits ZAP-70 homolog Syk to trigger MHC-I down regulation respectively.
The PI3K inhibitors repress MHC-I downregulation, identifying them as potential therapeutic agent to combat HIV-1. The PI3K pathways present in multiple HIV-1 reservoirs suggest that the recruitment of Nef-SFK-ZAP-70/Syk-PI3K signaling pathway is a common mechanism for MHC-I downregulation for immune evasion and any PI3K inhibitor will affect globally (Hung et al., 2007). Understanding that the Nef-SFK-ZAP-70/Syk-PI3K signaling pathway is recruited and activated by action of Nef EEEE65 site with PACS-1&2, the Nef-PACS interaction could become a potential target to identify inhibitors as potential therapeutic agent to combat HIV-1 (Fig4&5).
Mutational studies have shown that conserved domains of Nef are responsible for regulating function, but the specific role in HIV-1 pathogenesis was not directly understood. In an indirect effort to understand the role of nef gene in the progression to AIDS, the nef gene was isolated from two HIV-1 patients over period of time and observed that the complexity of nef sequence heterogeneity was increased during the progression of HIV-1 infection. However, the scattered

regions responsible for downregulation of CD4 and MHC were conserved. Similarly, to understand the role of Nef in HIV-1 pathogenesis efforts were made to characterize nef gene from different stages of HIV-1 disease. One of the studies showed that nefgene isolated from different stages of the disease exhibit mutation in nef which was further related to specific stages of HIV-1 pathogenesis (Krichhoff et. al, 1999). Later on it was shown that these nef alleles isolated from asymptomatic patients are more active in MHC-I down regulation whereas; after progression to AIDS nef was mainly involved in down regulation of CD4 and enhancement of virion infectivity (Carl et. al, 2001). However, other groups could not find any gross abnormality in nef alleles isolated from different stages of disease (Ratner et. al, 1996).
The downregulation of MHC-I is considered to be the most important function of Nef because this event causes immune evasion from cytotoxic T lymphocytes (CTL) and is thus considered as a key factor for AIDS development. Macaques with deleted CD8+ T cells exhibited faster progression to AIDS; complementation with CD8+ T cells inhibited virus replication and delayed AIDS progression (Schmitz et. al, 1999). This suggests that the primary CTL response against virus is important to delay the AIDS progression in host.
Understanding the molecular Nef pathways responsible for functions involved with viral persistence, replication and infectivity can be used as target for anti HIV-1 therapy. This can enable us to focus on future therapies where the task of interrupting key interaction between viral and host proteins will serve as potential target. Nef-PACS interaction represents a potential target for this approach.
Recent approaches towards the development of an alternative model for characterizing specific interactions of Nef with host proteins are either yeast (Rossi et. al, 1996) or mammalian-two-hybrid system (Murakami et. al, 2002). Nef is structurally conserved and the structure function analysis revealed that with maintaining the structure, the accessible nef conserved domain to host proteins is responsible for the function interaction in the cell (Geyer et. al., 2001).
In the present study, we developed a mammalian-two hybrid model and studied Nef-PACS-I interaction in HEK-293 cells, an early event which activates ZAP70/PI3K/Syk/AF6 pathway for MHC-I down regulation (Hung et. al, 2007). Further, this interaction was characterized structurally and functionally for primary Nef isolates isolated from patients. The inhibitors designed were

screened in this model and it was found that Nef-PACS-I interaction was inhibited, perhaps, which can be used to develop an alternative therapy.
The Nef protein is structurally conserved and the structure function analysis revealed that with maintaining the structure, the accessible Nef conserved domain is responsible for the functional interactions in the cell. Therefore, reporter gene construct was developed for maintaining Nef-PACS-Ifbr interaction in HEK-293 cells and screened novel peptide inhibitors for inhibiting Nef-PACS interaction.
Mammalian two hybrid models have been used in scientific research in a routine manner (Colas and Brent., 1998). The present invention uses this technique to enable the user to establish the Nef-PACS-I interactions in HEK-293 cells. No other group has developed such a model for the evaluation of Nef-PACS-I interactions earlier and the present invention is the first of it's kind. The present invention serves as a novel tool to identify inhibitors of the aforesaid interactions. Inhibitors, which are so identified, will have property to inhibit MHC-1 down-regulation pathway. There are no known drawbacks to the technique and it is amenable to high-throughput screening. The invention will efficiently allow for the screening of a large number of compounds in a relatively short time period of upto 48 hours. This will also allow for the search of structural analogs of inhibitors identified using the method of the present invention. Inhibitors, which are identified using the present invention, will act through a novel mechanism of inhibiting Nef-PACS-I and 2. This will be an alternate approach for anti-retroviral therapies including but not restricted to the Human immuno deficiency virus (HIV), which causes AIDS.
Objects of the Invention
The main objective of the present invention is to develop mammalian two-hybrid model for assessing Nef-PACS-Ifbr interaction in HEK-293 cells similar to HIV-I infected cells.
Another object of the invention is to provide a novel screening method to identify the molecules useful for inhibition of Nef-PACS-1 interaction in eukaryotic cells.
Still another object of the invention is to provide transfecting vectors pACT and pBIND (with and without Nef and PACS-1 gene) with luciferase gene in vector G5Luc as a reporter, in mammalian two hybrid model system where these vectors are co-transfected using cationic polymer.

Yet another object of the invention is to define the specific interaction of Nef and PACS-1 by
mutating acidic conserved domain in Nef gene, truncated Nef gene and making truncated PACS-
lfbr.
A further object of the invention is to provide two hybrid model system to maintain the interaction
of Nef variants isolated from HIV-1 patients with PACS-1 necessary for immune evasion.
Another object of the invention is to screen a molecule 'EEEEE5 which inhibits the interaction of Nef and PACS-1 by showing the reduced luciferase activity with respect to negative control.
Still another object of the invention is to provide the mammalian two hybrid model as a novel screening/assay system to identify novel molecules for inhibiting viral-host protein interaction.
Summary of the Invention
The invention enables the detection and quantification of the function of HIV Nef and host PACS protein interaction in living cells similar to HIV infected cells. The test is based on the qualitative and quantitative evaluation of the expression of reporter luciferase gene which is mediated by the interaction of Nef-PACS protein, cloned in the mammalian-two hybrid vectors. The mutation as well truncations in nef gene inhibits the interaction and no expression of reporter gene is detected. The truncated PACS-lfbr gene, PACS-1 115, the PACS-1 [154] showed interaction with Nef, however, PACS-1 [179] does not interact with Nef suggesting Nef interaction is in AP-1 dependent manner. Furthermore, the Nef-PACS 1 interaction was inhibited by the presence of pentapeptide (EEEEE). Further more, the Nef gene isolated from patients also showed inhibition of interaction.
In general the methods of this invention utilize a two-hybrid system, more preferably a mammalian two-hybrid system. A cell is provided containing constructs forming a two-hybrid system. The first construct comprises a DNA binding domain such as GAL4 attached to PACS-I. A second construct is provided that contains a transactivation domain such as VP16 attached to nef. A third construct in the cell is a nucleic acid comprising a DNA binding sequence and a luciferase gene downstream of the DNA binding sequence, wherein the transcription and/or translation of the luciferase gene is to be regulated by this two-hybrid system [transcription of the luciferase gene is regulated by the transactivation domain of the second construct.

The reporter construct according to the present invention has the following elements in functional association:
a) a reporter plasmid pACT having Vpl6 activation domain, modified by changing polyadenylation
region called pACT;
b) nef gene fused with activation domain Vpl6;
c) PACS-1 gene fused with Gal 4 DNA binding domain.
The reporter construct according to the invention can be transferred into cells (for example by transfection) and the recipient cells are analyzed after a defined time period. This time period depends on the amount of Nef and PACS-1 protein produced in the cell for interaction.
Since the presence of Nef in the cell could be considered similar to as it is expressed in HIV-1 infected cells, interacting with host proteins, the reasonable amount of the reporter mRNA will produce functional Nef. In the further course, among the host proteins functionally over expressed PACS-1 protein will interact with Nef protein which will mediate expression of luciferase gene expression.
The reporter gene constructs without Nef and PACS-1 gene, present inside the cell in a functional form (as in case of e.g. transfection of a respective plasmid) do not mediate the expression of luciferase plasmid. This may be documented and observed in a cell population by analyzing luciferase activity.
An advantage of the newly developed reporter gene construct is its uncomplicated and cost-effective use in screening the inhibitors which could be used to develop a novel anti-HIV therapy. Another advantage is the fact that this reporter gene construct could be used in high through put screening for identifying the inhibitors.
By means of the invention it is possible for the first time to measure the Nef-PACS interaction in the live cell in a quick and cost effective manner without infection of HIV.
The present reporter gene construct causing Nef-PACS interaction is very specific, which is confirmed by the mutation in the acidic domain of the nefgene. The constructs bearing the aforesaid

mutated nef gene do not interact with the PACS-1 counterpart. Similarly, the truncated PACS in which the AP-1 binding site is deleted showed loss of interaction with nef.
The mammalian two-hybrid system is chosen for making a reporter gene construct of Nef-PACS interaction because HIV infection occurs in mammalian cells and Nef interaction is structure based interaction. Nef structure based interaction depends upon the attained conformational structure in the cell. The post translational modification for attaining conformational structural to attain biological activity is achieved in eukaryotic cells as seen in HIV infected cell.
The Nef-PACS protein interaction is structure based interaction also with the involvement of other host proteins, which could be maintained in eukaryotic cells supplementing all proteins required for the interaction.
PACS-I fbr region interacts with Nef, when compared with PACS-2fbr showed 76.26% homology, which makes decent hypothesis that PACS-2 will also show tendency to interact with Nef in our mammalian two-hybrid model.
Accordingly, the present invention provides a novel screening method for screening of anti-HIV therapeutics, wherein the steps comprising:
a) transfecting constructs of SEQ ID No. 18, 25 and 24 respectively into a mammalian cell using 30 to 50 microM of a transfection reagent to obtain a transfected cell;
b) analyzing the transfected cell as obtained in step (a) for luciferase activity after incubating for a period of 24 to 48 hours for demonstrating Nef-PACS-1 interaction using dual luciferase kit and luminometer;
c) adding the molecule to be screened in the transfected cell of step [a] and incubating for 6 to 24 hours followed by repeating step [b] to assess its inhibitory activity on the Nef-PACS-1 interaction;
d) selecting the molecules which are inhibiting the Nef-PACS-1 interaction resulting in no additional luciferase activity with respect to the control as a potential anti-HIV therapeutic.
Brief description of the accompanying drawings
Figure 1: Nefwt-PACS-Ifbr interaction mediated by AP-1 in reporter gene construct. A) nefwt acidic domain sequence aligned with HIV-1 subtype C nefgene.

B) truncated PACS-I fbr gene showing AP-1 binding site.
C) Expression of Nefwt/Nef EEmut-PACS-Ifbr, Nefwt/NefEEmut with truncated PACS-I 115, 154
and 179 in HEK-293 cells. The luciferase activity was detected in cell where Nefwt and PACS-Ifbr, 115 and 154 were present. No luciferase activity was found in Nefwt- PACS-I 179 which had truncated AP-1 binding site, establishing that Nefwt-PACS-Ifbr interaction is AP-1 dependant. Also, no luciferase activity was observed in NefEEmut with all PACS-I and truncated PACS-I, single gene vector and control vectors transfected cells.
D) Positive control showing protein-protein interaction in reporter gene construct. The MyoD and
ID genes were transfected in HEK-293 cells and showed high luciferase activity. The values
represent luciferase-renilla ratio for standardizing transfection efficiency.
Figure 2: Screening of the inhibitors of Nef-PACS-lfbr interaction in reporter gene construct:
Specific inhibition of
A)Nefwt-PACS-lfbr,
B) Pat-RPOl Nef-PACS-lfbr,
C) Pat-RP14 Nef-PACS-lfbr interactions were shown at OuM, 5uM, 2.5uM and 1.25uM concentration of inhibitor
D) MyoD/ID interaction was not affected at OuM, 5uM, 2.5uM and 1.25uM concentration of inhibitor.
Figure 3 : HIV-1 Nef variants isolated from patients and their mutants interaction with PACS-I fbr in mammalian two hybrid system: A) Pat Nef variants RP01 and RP14, and Nefwt when transfected with PACS-Ifbr showed expression of luciferase activity in HEK-293 cells. The control vectors and single gene vectors transfected showed no luciferase activity in HEK-293 cells. B) MyoD,Id as positive protein-protein interaction
Figure 4: Full length protein is responsible for interaction with PACS-Ifbr. A) Digrammatic representation of Nef truncations used in the study. B) In silico modeling of patRPOl Nef protein. C) Nefwt, RP01400, RP01300, RP01200 and NefEEmut were transfected with PACS-Ifbr and showed luciferase activity in HEK-293 cells. D) MyoD-Id interaction for positive protein-protein interaction. Figure 5: Schematic representation of construction of mammalian expression vector VP16pcDNA (called VP16pcDNA+pACT).

Detailed Description of the Invention
Definitions
A "fusion protein" refers to a polypeptide formed by the joining of two or more polypeptides through a peptide bond formed between the amino terminus of one polypeptide and the carboxyl terminus of another polypeptide. The fusion protein may be formed by the chemical coupling of the constituent polypeptides or it may be expressed as a single polypeptide from nucleic acid sequence encoding the single contiguous fusion protein. A single chain fusion protein is a fusion protein halving a single contiguous polypeptide backbone.
The term "two-hybrid system" refers to a system comprising two chimeric molecules one of which bears a nucleic acid binding region, the other of which bears an expression control element (e.g. a transactivation or repressor domain). The molecules further a cognate binding pair such that one chimeric molecule is capable of specifically binding to the other chimeric molecule. The two-hybrid system further comprises a nucleic acid encoding a protein binding site that is specifically bound by the protein binding domain on the chimeric molecule thereby anchoring the chimeric molecule to the nucleic acid. The domain of the chimeric molecule recognizes and binds to its cognate binding partner on the second chimeric molecule thereby recruiting that molecule to the nucleic acid whereby the expression control element alters (e.g. activates) expression of a gene or cDNA comprising the underlying nucleic acid.
"Transfection" is used herein to mean the delivery of expressible nucleic acid to a target cell, such that the target cell is rendered capable of expressing said nucleic acid. It will be understood that the term "nucleic acid" includes both DNA and RNA without regard to molecular weight, and the term "expression" means any manifestation of the functional presence of the nucleic acid within the cell, including without limitation, both transient expression and stable expression.
The term "transactivator" refers to a molecule that induces transcription and/or upregulates transcription of a gene or cDNA. The transactivator may be a complete "native" molecule or a domain of a molecule that is capable of inducing and/or upregulating transcription of a gene or cDNA.

"Reporter genes" are genes or cDNAs that express an easily assayable (detectable and/or quantifiable) product. Detection of the assayable product indicates the expression and/or level of expression of the reporter gene. Reporter genes are well known to those of skill in the art. They include, but are not limited to, genes expressing bacterial chloramphenicol acetyl transferase (CAT), beta-galactosidase (.beta.-gal), green fluorescent protein (GFP) and other fluorescent protein, various bacterial luciferase genes, e.g., the luciferase genes encoded by Vibrio harveyi, Vibrio fischeri, and Xenorhabdus luminescens, the firefly luciferase gene FFlux, and the like.
The term "transcriptional activators" refers to proteins, which activate transcription in yeast, plant, insect and mammalian cells. These proteins contain two parts: one directs DNA binding and the other, called the activating region, presumably interacts with some component of the transcriptional machinery. Activating regions are typically acidic and require some poorly-understood aspect of structure, probably at least in part an alpha-helix.
Here in the present invention, the "transcriptional activator system" utilized is the one, which is formed by fusing a DNA-binding fragment of the yeast activator GAL4 to a highly acidic portion of the herpes simplex virus protein VP16. VP16 activates transcription of immediate early viral genes by using its amino-terminal sequences to attach to one or more host-encoded proteins that recognise DNA sequences in their promoters.
Preparation of HIV-1 Nef wt, RP01 and RP14 Nef variants- The nef genes amplified from the randomly selected patients RP01 and RP14 were reamplified with the specific primers as given in table 1 for in frame cloning in the mammalian two-hybrid vector. For generating nefwt, RP01 was used as template using the gene specific primers of SEQ ID No.3 and 4, as given in table 1. The nefwt was made similar to the subtype C reference sequence [refer to figure 1 A] and later on cloned in the mammalian two-hybrid vector for interaction studies.
Amplification of PACS-1 gene from rat brain mRNA- mRNA was isolated from rat brain and PACS-1 was amplified using gene specific primers as given in table 1. Rat brain was homogenized in Tri reagent (w/v) and mRNA was isolated by extracting the homogenate with chloroform (1:1). The mRNA was precipitated using equal volume of isopropanol and incubating at minus 20°C for 2hrs. The mRNA pellet was washed with cold 70% alcohol and resuspended in 0.2% diethyl

phosphorocyanidate [DEPC] water. Following DNase digestion the mRNA was used for cDNA preparation using random primer pdN6.The PACS-l fbr fragment was amplified using the gene specific primers of seq ID No. 13 and 14 as given in table 1. Using PACS-l fbr fragment as template, truncations of PACS-l were generated and cloned in pDrive vector and confirmed by sequencing.
Plasmid Constructs— nef gem was cloned in VP16pcDNA plasmid constructed in lab using pcDNA 3 (Invitrogen) as vector backbone, while the fragment containing the CMV promoter, intron, T7 promoter, nuclear localization signal and multiple cloning region was taken from the mammalian two-hybrid assay vector pACT. The resulting construct thus containing the cloned nef gene was called VP16pCDNA+pACT. The sequence of the construct containing the cloned nefwt gene, is represented by SEQ ID No. 18.
Two sets of constructs were generated from the two vectors, pBIND and VP16pCDNA+pACT:
• pBIND with PACS-l fbr or PACS-l[115] orPACS-l[154] or PACS-1[179] and
• VP16pCDNA+pACT with nejwt or nef/RP01 400 or nef/RP01 300 or nef/RP01 200 or nef RP14.
PCR was performed for each gene using the appropriate 5'and 3' primers (as given in Table 1). All of the primers had BamRl and Xbal restriction sites, and the genes were digested with BamHl and subcloned into pBIND and VP16pCDNA+pACT vectors. All constructs were verified by Sanger sequencing in an ABI Automatic Sequencing System (PerkinElmer Applied Biosystems Inc, Foster City, CA). The primers were custom-synthesized by Sigma, Operon and IDT.
Table 1: Primers used for «e/mutation and PACS truncations
(Formula Removed)
Tissue Culture and Transfections—HEK 293 cells were grown in Minimal Essential medium (low glucose, Sigma) supplemented with 10% fetal bovine serum, penicillin (100 units/ml), streptomycin (100 ug/ml) and Gentamycin (50 ug/ml) at 37°C with 5% C02. 1X105 cells were seeded per well in twenty four well plates, one day before transfection. Using commercially available ExGen 500 transfection reagent, all the required plasmid constructs were cotransfected into HEK 293 cells. 0.222 microgram of each plasmid and 5.3 microgram per liter of ExGen 500 reagent were used per well. 48 h post transfection the cells were harvested and Luciferase activity was monitored using the Dual-Luciferase® Reporter assay kit (PROMEGA). The data for basal control were used for the conversion of Luciferase activity to fold activation. As a positive control, the protein-protein interaction vectors pACTMyoD and pBIND-ID encoding the MyoD and ID control proteins, provided with the kit were used.
Luciferase Assay—The transfected cells were lysed in IX Passive Lysis Buffer (PLB) provided with the kit, and the cell extracts (containing Luciferase enzyme) were added to the luminometer tubes and the Luciferase and Renilla RLU were measured on Berthold luminometer using the respective substrates for 10s along with 2s delay time.
Screening inhibitors which inhibitedNef-PACS interaction in HEK-293 cells:
The peptide designed for inhibiting Nef-PACS-1 interaction was screened in the developed mammalian-two hybrid model. The peptide was used at 0, 5, 2.5 and 1.25uM concentration incubated with HEK-293 cells for 4 hours, transfected with nefwt-VP16pcDNA+pACT construct, PACS-KibO-pBIND construct and reporter construct pG5Luc of SEQ ID No. 18, 25 and 24 respectively. Similarly, the cells were also transfected with different constructs of nef variants and PACS variants. The peptides inhibited Nefwt-PACS, RP01-PACS and RP14-PACS interaction at all concentrations. The inhibitor completely inhibited the interaction and was close to control. However, the inhibitor did not affect the MyoD-ID interaction at any concentration (Fig 1D). These results clearly demonstrate that the inhibitor effectively and specifically inhibited the Nef-PACS interaction in HEK-293 cells.
In an embodiment of the present invention, fusion with mutated nef and truncated PACS1 have been made to demonstrate the specificity of interaction and that the interaction is AP-1 mediated.

In another embodiment of the present invention, the eukaryotic cells used for the preparation of
mammalian two-hybrid system may be selected from the group consisting of HEK-293 cells, T cell
lines, monocytic cell lines and fibroblast cells wherein Nef-PACS-1 interaction is established.
In yet another embodiment, the present invention showed 29.45 times higher ratio of
luciferase/renilla domonstrating Nef-PACS-1 interaction in comparison to negative control wherein
Nef and PACS-1 were not cloned in vectors.
In still another embodiment of the present invention, the model used established the specific
interaction of Nef and PACS-1 by mutating acidic conserved domain in Nef gene.
In a further embodiment of the present invention, the reporter gene construct mimics the HIV-1
infected cells in terms of interaction by deleting AP-1 binding site in PACS-1.
In yet another embodiment of the invention, the Nef variants-PACS-1 interaction is exhibited by
luciferase activity in HEK-293 cells.
In still another embodiment of the invention, the mammalian two hybrid model is used for screening
of a molecule 1EEEE5, which inhibits the interaction of Nef and PACS-1, which is measured by
reduced luciferase activity with respect to negative control.
In yet another embodiment of the invention is provided the reporter gene construct as a novel
screening/assay system to identify novel molecules for inhibiting viral-host protein invention.
The following examples are given by way of illustration and therefore should not be construed to limit the scope of the invention.
Examplel
Reporter gene constructs / mammalian two hybrid model to demonstrate Nef and PACS-Ifbr interaction in HEK-293 cells:
The primary interaction of Nef-PACS-I fbr was studied in HEK-293 cells using reporter gene construct. In this experiment, HIV-1 nef variant isolated from patient was modified to match the HIV-1 subtype C nef conserved domain with respect to the acidic domain (EEEEE) and proteolytic domain (WL) (Figla) referred as nefwt (wild type). The conserved acidic domain 62EEEEE66 was mutated to 62AAAAA66 {nefEEmut) for establishing specificity of interaction with PACS-I fbr (Figla). The PACS-I fbr and N-terminal truncated PACS-I 115, PACS-I 154 and PACS-I 179 were made to show API dependent interaction (Figlb). For reporter gene construct, nefwt and nefEEmut gene were cloned in frame to VP16 transactivation domain vector. Similarly the PACS-I fbr gene and truncated PACS-I 115, 154 and 179 were cloned in frame to GAL4 DNA binding domain vector.

The VP16 newt, GAL4 PACS-I fbr with luciferase gene vectors when transfected in HEK-293 cells showed 29.45 fold luciferase activity in HEK-293 cell'after 48 hrs post transfection compared to control vectors without Nef and PACS-I fbr genes. However, the nefEEmut, PACS-I with luciferase gene vectors when transfected in HEK-293 cells showed no luciferase activity which correspond to control vector (fig lc).
Further, when nefwt was transfected with different truncated PACS-I 115, 154, and 179 it showed 27 fold luciferase activity with PACS-I 115, and 29.45 fold with PACS-I 154 as compared to control. However, the nefwt and truncated PACS-I179 which does not have API binding site does not show luciferase activity. The specificity of interaction with acidic domain was demonstrated by showing no luciferase activity with nefEEmut with PACS-I fbr, 115 and 154 transfected cells (Fig lc). There was no nonspecific luciferase activity found in either of control vectors or with Nef or PACS-Ifbr gene alone in HEK-293 cells (Figlc). MyoD and ID were used as a positive control for protein-protein interaction in mammalian two hybrid system in HEK-293 cells and showed 307 fold luciferase activity (Fig Id). These results show that Nef-PACS-Ifbr interaction in HEK-293 cells is mediated which was similar to the interaction shown in A7 melanomas cells and primary T cells (Piguet et. al, 2000).
Example-2
To analyze nef variant isolated from patients for interaction with PACS-I required for MHC-I down regulation:
Using the mammalian two-hybrid system of example 1, we showed native interaction of Nef-PACS-I in HEK-293 cells, now the same system was used to understand the functionality of nef variants isolated from patients in terms of Nef-PACS-I interaction.
Analysis of nef variants amplified from 7 patients showed that they had variation in acidic domain and neighboring proteolytic cleavage region. The RPOl had mutation in acidic domain in which one E at position 65 is mutated to N where as in RP14 the entire proteolytic cleavage region 64CAWLEAQ69 is deleted and addition of 9 residues were found inserted in the variable region . Here the situation is that in RPOl the acidic domain and proteolytic region is same as nefwt except E mutated to N in acidic domain and in RP14 the complete acidic domain (64CAWLEAQ69) is deleted. The interaction of these nef variants with PACS-I was studied in the said mammalian two-hybrid model.

The nef variants RP01 and RP14 were cloned in VP16pcDNA+pACT vector. Both nef variants and nefwt with PACS-I fbr were transfected in HEK-293 cells, the RPQ1 and RP14 variant showed 22.56 and 44.43 fold luciferase activity compared to control vector. RP01 which is close to nefwt showed almost similar fold induction in luciferase activity of 22.56 as was observed for nefwt (23.6 fold luciferase activity) (fig 3A). However, RP14 was 1.88 times higher than Patl luciferase activity inspite of WL region deleted. For showing specificity of RP01 and RP14 interaction with PACS-I, the acidic domain of patl and patl4 were mutated to nefEEmut (RP01) and RP14EEmut (RP14) which when transfected with PACS-I fbr showed no luciferase activity and were similar to control (fig3A). The luciferase activity for MyoD, Id positive control is 304 times compared to control vector (Fig3B). These studies suggest that the Nef variants isolated from patients having said mutations clearly demonstrate functional interaction with PACS-I in HEK-293 cell.
Example-3
Both protein conformational structures are responsible for interaction of Nef acidic domain and PACS-I:
In a mammalian-two hybrid system, the intriguing feature is that proteins expressed in cell attains native conformational structure and interact with each other in their physiological state. In the structure based interaction of two proteins, change in structure of one protein may lead to perturbation in environment for protein-protein interaction. In nef, the structure based interaction with host protein depends upon the conformational structure of Nef attained in the cell. The fact that the interaction of unstructured N-terminus of Nef with PACS-I is a structure based interaction was shown using this model.
To study this hypothesis, the RP01 Nef variant was truncated from C-terminus, thereby, shortening the protein and perturbing the structure as shown in fig 4a. The full length RPOlNef used in the study had all the conserved domain except the myristoylation region (1-40 amino acid residues) (fig 4a). RP01-300, and RP01-200 are subvariants which are truncated from C-terminus (fig4a). The predicted in silico structure of truncated nef subvariants are shown in fig4b. All the subvariants were cloned in VP16pcDNA+pACT vector and their interaction with PACS-I fbr was studied. Only nefwt and RP01 nef full length variant showed 20.13 and 19.0 fold luciferase activities respectively.

However, RPOl-300 and RPOl-200 showed no luciferase activity and were same as nefEEmut and control. The positive control interaction of MyoD-Id showed 258 fold luciferase activity. Therefore it was shown that the Nef-PACS-I interaction is typically structure based interaction in HEK-293 cells. Perturbation of nef structure even after keeping the acidic domain intact, does not maintain its interaction with PACS-I in the developed model.
Example-4 To study interaction of Patl nef and Patl4 nef variants with PACS-I fbr is API mediated:
As shown in fig 1, the nef wt interaction with PACS-I is mediated by API which finally activates the MHC-I downregulation pathway. Similarly to understand whether the Patl and RP14 nef variants, selected naturally in patients, have property for API mediated PACS-I interaction. All PACS-I variants, as described in fig la, were transfected with RPOl and RP14 nef variants. RPOl showed luciferase activity with PACS-Ifbr, PACS-I115 and PACS-I154 of 25.68, 23.3 and 23.86 respectively compared to control, but showed no luciferase activity with PACS-I 179 which lacks the API site (fig4C). Similarly RP14 showed luciferase activity with PACS-Ifbr, PACS-I115 and PACS-I 154 of 42.39, 25.4, and 22.39 respectively compared to control, again no luciferase activity was seen in PACS-I 179 (fig 4D). RP14 nef variant showed decreasing trend of luciferase activity with the decreasing size of PACS-I fragment and finally decreased with deletion of AP-1 site. These results suggest that the Nef variants isolated from HIV-1 infected patients can bind to PACS-I in API dependent manner which presumably have tendency to downregulate MHC-I in patients.
Example-5
Novel inhibitors identified for disrupting Nef-PACS interaction in HEK-293 cells:
To screen a new class of peptide inhibitor which inhibits the Nef-PACS-Ifbr interaction in Nefwt and primary Nef variants isolated from patients.
The peptide designed for inhibiting Nef-PACS-Ifbr interaction was screened in the mammalian-two hybrid model described in example 1. The peptide was used at 0, 5, 2.5 and 1.25 µM concentration, incubated with HEK-293 cells for 4 hours, transfected with nef and PACS-I fbr and MyoD-Id constructs. The peptides inhibited Nefwt, patl and patl4-PACS-Ifbr interaction at all concentrations. The inhibitor completely inhibited the interaction and was close to control. However, the inhibitor

did not affect the MyoD-Id interaction at any concentration (Fig2A, B, C &D). These results clearly demonstrate that the inhibitor effectively and specifically inhibited the Nef-PACS-Ifbr interaction in HEK-293 cells and does not interact non-specifically with other proteins.
Table 2: Effect of the inhibitor in reporter gene construct in inhibiting Nefwt-PACS interaction at different µM concentration
(Formula Removed)

The experiment performed using wild-type Nef shows that -100 % inhibition of the Nef-PACS-I interactions are achieved even on addition of 1.25µM of the inhibitor.
Table 3: Effect of the inhibitor in reporter gene construct in inhibiting PatlNef-PACS-I interaction at different µM concentration
(Formula Removed)

The experiment performed using Nef from sample patient-1 shows that -100 % inhibition of the Nef-PACS-I interactions are achieved even on addition of 1.25µM of the inhibitor.
Table 4: Effect of the inhibitor in reporter gene construct in inhibiting Patl4Nef-PACS-I interaction at different µM concentration
(Formula Removed)

The experiment performed using Nef from sample patient-14 shows that ~100 % inhibition of the Nef-PACS-I interactions are achieved even on addition of 1.25uM of the inhibitor.
Table 5: Effect of the inhibitor in reporter gene construct in inhibiting MyoD-Id interaction at different µMconcentration
(Formula Removed)

The above results show 100% inhibition of Nef-PACS-1 (wt and patient samples) interaction even at 1.25µM concentration.
Advantages of the Invention:
• The reporter gene construct is simple to use and does not involve human or animal models of testing.
• This method is amenable to screening a large number of compounds in a relatively short time when coupled with high-throughput technologies or as a stand-alone assay procedure.
• Rapid inhibitor optimization is facilitated by quick screening procedure.
• The existing anti-HIV therapeutics does not target the Nef-PACS-I interaction and associated pathway.
• The present inhibitor(s) will be a novel therapeutic and will overcome HIV-I resistance issues.

References:
Atkins, K.M., Thomas,L., Youker, T.T., Harriff, M.J., Pissani,F., You,H., and Thomas,G. (2008). HIV-1 Nef binds PACS-2 to assemble a multi-kinase cascade that triggers MHC-I downregulation: Analysis using siRNA and knockout mice. JBC,283, 11772-11784.
Blagoveshchenskaya, A.D., Thomas, L., Feliciangeli, S.F., Hung, C.H. and Thomas, G. (2002). HIV-1 Nef downregulates MHC-I by a PACS-1- and PI3K-regulated ARF6 endocytic pathway. Cell 111: 853-66.
Colas,P and Brent,R. (1998). The impact of two-hybrid and related methods on biotechnology. TIBTECH 16, 355-363.
Crump, CM, Xiang, Y., Thomas, L, Gu, F, Austin, C, Tooze, S.A. and Thomas G. (2001). PACS-1 binding to adaptors is required for acidic cluster motif-mediated protein traffic. EMBO J. 20: 2191-2201.
Fackler, O, Moris, A, Tibroni, N, Giese, S, Glass, B, Schwartz, O. and Krausslich, H.G. (2006). Functional characterization of HIV-1 Nef mutants in the context of viral infection. Virology 351:332-339.
Greene, W.C and Peterlin,B.M. (2002). Charting HIV's remarkable voyage through the cell: Basic science as a passport for future therapy. Nature medicine 8, 673-680.
Hung, C-H, Thomas, L, Ruby, CE, Atkins, K.M, Morris, N.P, Knight, Z.A, Scholz, I, Barklis, E, Weinberg, A.D, Shokat, K.M. and Thomas, G. (2007). HIV-1 Nef assembles a Src family kinase-ZAP-70/Syk-PI3K cascade to downregulate cell-surface MHC-I. Cell host and microbes 1: 121-133.
Kestier, H.W, Ringler, D.J, Mori, K, Panicali, D.L, Sehgal, P.K, Daniel, M.D. and Desrosiers, R.C. (1991). Importance of the nef gene for maintenance of high virus loads and for development of AIDS. Cell 65:651-662.
Malin, H.M and Emerman, M. (2008). HIV-1 accessory Protein-Ensuring Viral Survival in a Hostile Environment. Cell Host and Microbes 3, 388-398.
Piguet V, Wan, L, Borel, C, Mangasarian, A, Demaurex, N, Thomas, G. and Trono, D. (2000). HIV-1 Nef protein binds to the cellular protein PACS-1 to downregulate class I major histocompatibility complexes. Nat. Cell. Biol. 2:163-167.
Schmitz, J.E, Kuroda, M.J, Santra, S, Sasseville, V.G, Simon, M.A, Lifton, M.A, Racz, P, Tenner-Racz, K, Dalesandro, M, Scallon, B.J, Ghrayeb, J, Forman, M.A, Montefiori, D.C, Rieber, E.P, Letvin, N.L. and Reimann. K.A.(1999). Control of viremia in Simian Immunodeficiency virus infection by CD8+ lymphocytes. Science 283: 857-860.
Stevenson, M. (2003). HIV-1 pathogenesis. Nature Medicine 9, 853-860.

We claim:
1. A novel screening method for screening of anti-HIV molecules, wherein the steps comprising:
[a] transfecting constructs of SEQ ID No. 18, 25 and 24 into a mammalian cell using 30 to 50 microM of a transfection reagent to obtain a transfected cell;
[b] analyzing the transfected cell as obtained in step (a) for luciferase activity after incubating for a period of 24 to 48 hours for demonstrating Nef-PACS-1 interaction using dual luciferase kit and luminometer;
[c] adding the molecule to be screened in the transfected cell of step [a] and incubating for 6 to 24 hours followed by repeating step [b] to assess its inhibitory activity on the Nef-PACS-1 interaction;
[d] selecting the molecules which are inhibiting the Nef-PACS-1 interaction resulting in no additional luciferase activity with respect to the control as a potential anti-HIV therapeutic.

2. A method as claimed in claim 1, wherein the construct of SEQ ID No. 18 represents nefwt gene cloned in VP16pCDNA+pACT vector.
3. A method as claimed in claim 1, wherein the construct of SEQ ID No. 25 represents PACS-1 gene cloned in pBIND vector.
4. A method as claimed in claim 1, wherein the construct of SEQ ID No. 24 represents the reporter gene construct pG5Luc.
5. A method as claimed in claim 1, wherein the transfection reagent is a commercially available ExGen 500 in vitro Transfection Reagent.
6. A method as claimed in claim 1, wherein the mammalian cell is selected from the group consisting of HEK-293 cells, T cell lines, monocytic cell lines and fibroblast cells wherein Nef-PACS-1 interaction is established.

7. A method as claimed in claim 1, wherein the molecule to be screened is added at a concentration of 5 to 1.25 microM.
8. A method as claimed in claim 1, wherein the control represents a transfected mammalian cell with VP16pCDNA+pACT vector without nef and pBIND vector without PACS-1 along with the reporter construct pG5Luc.
9. A method as claimed in claim 1, wherein the molecule screened as a potential anti-HIV therapeutic is a pentapeptide 'EEEEE'.
10. A novel screening method for screening of anti-HIV therapeutics substantially as herein described with reference to the foregoing examples.

Documents:

http://ipindiaonline.gov.in/patentsearch/GrantedSearch/viewdoc.aspx?id=eWlyVLuLZGRR/OmCBwZZJg==&loc=+mN2fYxnTC4l0fUd8W4CAA==

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Patent Number

278079

Indian Patent Application Number

1709/DEL/2008

PG Journal Number

52/2016

Publication Date

16-Dec-2016

Grant Date

13-Dec-2016

Date of Filing

18-Jul-2008

Name of Patentee

COUNCIL OF SCIENTIFIC & INDUSTRIAL RESEARCH

Applicant Address

ANUSANDHAN BHAWAN, RAFI MARG, NEW DELHI-110001,INDIA.

Inventors:

#	Inventor's Name	Inventor's Address
1	RAJ KAMAL TRIPATHI	CENTRAL DRUG RESEARCH INSTITUTE, CHATTAR MANZIL PALACE, POST BOX NO 173, LUCKNOW 226 001, INDIA.
2	DHARAMSHEELA	CENTRAL DRUG RESEARCH INSTITUTE, CHATTAR MANZIL PALACE, POST BOX NO 173, LUCKNOW 226 001, INDIA.
3	PANKAJ SINGH	CENTRAL DRUG RESEARCH INSTITUTE, CHATTAR MANZIL PALACE, POST BOX NO 173, LUCKNOW 226 001, INDIA.
4	NA	NA
5	RICHA VERMA	CENTRAL DRUG RESEARCH INSTITUTE, CHATTAR MANZIL PALACE, POST BOX NO 173, LUCKNOW 226 001, INDIA.
6	JIMUT KANTI GHOSH	CENTRAL DRUG RESEARCH INSTITUTE, CHATTAR MANZIL PALACE, POST BOX NO 173, LUCKNOW 226 001, INDIA.
7	RAVISHANKAR RAMACHANDRAN	CENTRAL DRUG RESEARCH INSTITUTE, CHATTAR MANZIL PALACE, POST BOX NO 173, LUCKNOW 226 001, INDIA.

PCT International Classification Number

C07K 14/00

PCT International Application Number

N/A

PCT International Filing date

PCT Conventions:

#	PCT Application Number	Date of Convention	Priority Country
1			NA