JHU/CCR Fellowship in Molecular Targets and Drug Discovery Technologies Project Details

Project Sponsor/Mentor:	Vinay K. Pathak
Title:	Principal Investigator
Address:	NCI-Frederick,CCR 1050 Boyle St. Building 535, Room 334, Frederick, Maryland 21702
Telephone:	301-846-1710
Fax:	301-846-6013
Email:	pathakv@mail.nih.gov
Sponsoring Laboratory/Branch:	Retroviral Replication Laboratory

Project Title:	Inhibitors of Vif-A3G/A3F Interactions
Target(s) of Interest:	APOBEC3G and APOBEC3F

Project Synopsis:
Introduction. Identification of host restriction factors APOBEC3G (A3G) and APOBEC3F (A3F) has revealed important new targets for anti-HIV-1 drug development. A3G and A3F are cytidine deaminases that are packaged into HIV-1 virions in the absence of the virally encoded Vif protein and deaminate deoxycytidines in viral DNA to deoxyuridines, resulting in extensive G-to-A hypermutation and abrogation of viral replication. HIV-1 Vif binds to A3G and A3F, and induces their proteasomal degradation in the virus producer cells, suppressing their virion incorporation and restoring viral replication. A3G and A3F are believed to be components of a potent innate antiviral immune mechanism and HIV-1 has evolved to express the Vif protein as a counterdefense to these host restriction factors. The antiviral activities of A3G and A3F strongly inhibit replication of Vif-deficient HIV-1 in patients. Thus, compounds that inhibit the ability of Vif to induce proteasomal degradation of A3G and/or A3F could form the basis of a new class of antiviral drugs that augment the host innate immunity to block HIV-1 replication. Preliminary Studies. We have carried out extensive mutational analysis of HIV-1 Vif, A3G and A3F to elucidate the structural determinants of the Vif-A3G and Vif-A3F interactions (1, 2). These studies have shown that these two interactions are distinct. Amino acids 40-44 of Vif form a major determinant that interacts with amino acids 126-132 in A3G, whereas amino acid 14-17 of Vif interact with the C-terminal amino acids 283-300 of A3F. These studies have identified two distinct molecular targets for antiviral drug development; small molecule inhibitors that block either interaction are expected to allow either A3G or A3F to potently inhibit HIV-1 replication. We have developed an in vivo bimolecular fluorescence complementation (BiFC) assay to analyze interactions between A3G molecules and between A3G and viral proteins (3). In this assay, the enhanced yellow fluorescent protein gene is split into an N-terminal (NY) and a C-terminal (CY) fragment; these fragments do not exhibit fluorescence, and are used to generate fusion proteins with target proteins of interest; interactions between the target proteins can result in the association of NY and CY, resulting in reconstitution of fluorescence. Thus, fluorescence complementation is an indicator of an interaction between the two proteins of interest. A recent study used an in vivo assay to identify a small molecule inhibitor that interferes with Vif-induced proteasomal degradation of A3G (4). In this study, an A3G-eYFP expression plasmid and either a wild-type HIV-1 or HIV-1Vif were cotransfected into 293T cells; cells cotranfected with HIV-1Vif exhibited a higher level of fluorescence than cells cotransfected with wild-type HIV-1. A screen of 30,000 compounds resulted in the identification of one candidate inhibitor named RN-18, which inhibited HIV-1 replication with a 50% inhibitory concentration of 3 M. The potential cytotoxiciy of this compound, and the mechanism by which it inhibits HIV-1 replication, have not been fully elucidated. Development of in vivo assays for identification of inhibitors of A3G-Vif interactions. We are developing in vivo and in vitro assays for identification of small molecules that interfere with the ability of Vif to induce degradation of A3G and A3F. The first assay is similar to that described by Nathans and coworkers (4), except that we have generated 293T cells that stably express A3G-eYFP; in order to obtain an optimal ratio of A3G and Vif expression in the cells, we have generated cells that express high, medium and low levels of A3G as determined by flow cytometry. We will infect these cells with either wild-type HIV-1 or HIV-1Vif and characterize the levels of eYFP fluorescence. We will use the wild-type HIV-1 infected cells to identify small molecule inhibitors that increase eYFP fluorescence; these candidate inhibitors, which increased the steady-state levels of expression of A3G-eYFP, may have blocked Vif-induced degradation of A3G. To facilitate high-throughput screening of small molecule inhibitors, we would like to establish a stable cell line that expresses both A3G-eYFP and Vif. To accomplish this goal, we are developing another cell line in which the Vif protein is expressed along with the red fluorescent protein mCherry by using in internal ribosomal entry site. We will transfect this plasmid into cells that stably express A3G-eYFP, and isolate cells that are mCherry-positive but express low or no eYFP fluorescence. In these cells, Vif expression presumably resulted in proteasomal degradation of A3G-eYFP. We will verify this hypothesis by incubating these cells with a proteasomal inhibitor, MG132; an increase in eYFP fluorescence in the presence of MG132 will verify that A3G was being degraded through the proteasomal pathway. The two assays described above have the disadvantage that any molecules that interfere with the proteasomal degradation pathway would be identified as a potential inhibitor; however, because long-term inhibition of the proteasomal degradation pathway is likely to be toxic to cells, it would be desirable to identify molecules that specifically interfere with the Vif-A3G and Vif-A3F interactions. To accomplish this goal, we have developed a novel assay in which A3G and Vif fusion proteins are tagged at the N- or C-terminus with the NY or CY fragments of eYFP. Interactions between A3G and Vif allow the YFP N- and C-terminal fragments to associate with each other in a manner that allows complementation and reconstitution of fluorescence in cotransfected cells. Our studies indicate that expression of Vif-NY, Vif-CY, and NY-Vif results is about fivefold reduction in the mean fluorescence intensity of eYFP positive cells; using these A3G and Vif expression plasmids, we will establish a stable cell line that expresses both proteins. We will identify cell lines that increase the expression of eYFP fluorescence in the presence of MG132 to verify that both proteins are expressed. Experimental Plan. We will generate these three in vivo assays by establishing stable cell lines that express the A3G and Vif expression plasmids as described above. We will validate these assays by characterization of the A3G protein expression by FACS analysis as well as western blotting analysis. We will carry out a screen of a library of 50,000 compounds (DiverSet, ChemBridge Corp.) that are available from the manufacturer. We will determine whether the candidate inhibitors are cytotoxic using a standard XTT assay. We will carry out secondary screens by analyzing the effects of candidate inhibitors on HIV-1 replication and in vitro cytidine deaminase activity assays to determine the enzymatic activity of A3G and A3F. Future Directions. We will develop in vitro assays for analysis of A3G-Vif and A3F-Vif interactions. In collaboration with the Protein Expression Laboratory, we have overexpressed wild-type A3G in the bacculovirus system and have obtained several milligrams of enzymatically active A3G. We are also currently in the process of expressing wild type Vif using a similar approach, and will express A3G-NY and Vif-CY proteins. We will also collaborate with Dr. Terence Burke, Jr. (Laboratory of Medicinal Chemistry) to carry out structure-activity studies to improve the therapeutic index of promising candidate inhibitors. REFERENCES: 1. Russell, R. A. & Pathak, V. K. (2007) Journal of virology 81, 8201-8210. 2. Russell, R. A., Smith, J., Barr, R., Bhattacharyya, D., & Pathak, V. K. (2009) Journal of virology 83, 1992-2003. 3. Friew, Y. N., Boyko, V., Hu, W. S., & Pathak, V. K. (2009) Retrovirology 6, 56. 4. Nathans, R., Cao, H., Sharova, N., Ali, A., Sharkey, M., Stranska, R., Stevenson, M., & Rana, T. M. (2008) Nature biotechnology 26, 1187-1192.
Fellow Research Plan:
The fellow will develop in vivo assays for identification small molecule inhibitors of the Vif-A3G and Vif-A3F interaction. We are developing fluorescence based in vivo assays for identification small molecule inhibitors that interfere with these interactions. These studies will include learning molecular biology techniques, such as manipulation and construction of plasmids, and cell culture techniques that involve transfection, infection, and characterization of infected cells using flow cytometry, p24 ELISA, and western blotting analysis. In addition, our laboratory is in the process of purchasing a High Content ImageXpress System from Molecular Devices, which will be used for quantification of fluorescence in mammalian cells. The ImageXpress system consists of an inverted epifluorescent microscope that is automated for scanning live cells in standard multi-well plates (6- through 1536-well plates) and fixed cells on slides. The system will be configured with: an environmental control chamber for maintenance of cells at the desired temperature, CO2, and humidity; a robotic arm for automated loading of plates, allowing the analysis of up to 200 plates/day; a liquid handler for automation of assays that include addition of specific reagents to the wells at desired time points. The system is designed for validation of high-throughput cell-based assays, and for secondary and tertiary screens of potential inhibitors identified in larger screens. The system includes five software modules that are designed to quantitatively analyze intracellular events, such as intracellular trafficking and translocation of fluorescently labeled proteins, nuclear import, and punctate structures such as P bodies. The system can also be used for quantifying cell viability and cell proliferation. The fluorescence-based assays will be used to identify small molecule inhibitors that interfere with the Vif-A3G and Vif-A3F interactions and prevent the degradation of the APOBEC3 proteins. We will use this system validate these assays and carry out small-scale screens for potential inhibitors.