Final Report Summary - HCV PATHOLOGY (Mapping of Hepatitis C virus NS4B protein interactions with its host network) Over 150 million individuals are infected with hepatitis C virus (HCV) worldwide. Current therapies are inadequate for the majority of these patients (Liang T.J. et al., 2000, Ann. Intern. Med.). Our long-term goals are to better understand the life cycle of HCV and translate this knowledge into novel antiviral strategies. HCV is believed to replicate its genome in association with cytoplasmic membranes, similarly to other positive strand RNA viruses. Nevertheless, the mechanisms of assembly and maintenance of the RNA replication complex remain largely unknown. HCV non-structural (NS) proteins NS3A, NS4B and NS5A are implicated in these processes. Mapping their interaction with the host proteins may shed light on the viral life cycle. Moreover, these new interactions may serve as drug targets, which may help create new therapeutics against an important human pathogen.Mapping viral interactions is difficult. Current methodologies lack sensitivity or are incompatible with viral proteins. The latter is mainly because many viral processes occur near, on or within membranes. My expertise is in bioengineering microfluidic devices for use in proteomics. In this project, our aim is to create a microfluidic based protein array that will allow us to address the issue of mapping viral protein interactions. Project objectives1. Establish a microfluidic fab at Bar Ilan University and integrate the technology. 2. Fabricate and establish the microfluidic-based protein arrays.3. Create a Human proteome synthetic library.4. Express Human proteins on chip.5. Establish and characterize known protein interaction controls. 6. Screen HCV non-structural proteins ResultsWe have established photolithography processes such as SU-8 and SPR220 in the new core facility calss 100 clean room at the BINA Institute. These processes fully support our needs for mold fabrication and allow us to improve and create new designs of microfluidic devices. In addition, we have a class 10,000 clean room in which now supports a full fabrication process for integrated microfluidic devices as well as microarray printing. The above facility makes us the top microfluidic lab in Israel in terms of fabrication capacity.Using the above mentioned facilities we have established a microfluidic-based protein array platform (Glick et al., 2012). The platform combines microarrays with a microfluidic device and in vitro protein expression to create fresh and active proteins. In parallel, we have created a synthetic gene library using assembly PCR that encompasses over 15,000 human ORFs. Success rate for this stage was at 85% providing us with over 13,000 synthetic genes. Expression experiments demonstrate that over 80% of these genes fully express in our microfluidic platform. These 10,000 human proteins can now serve as targets for binding assays. Next we used NS5A to characterize the sensitivity of our microfluidic platform and found it to be between 60-70%. (Much higher than other screening platforms like yeast two hybrid.) We further tested the compatibility of our platform with NS4B via the only validated interactor ATF6b. We found that we can clearly detect this interaction with our platform. Making it the only high throughput platform that can detect this specific interaction. These experiments demonstrated the compatibility of the microfluidic protein arrays with viral proteins (Ben-Ari et. al., 2013). Next, we screened the HCV NS3 against human proteins and discovered a battery of new binding partners. WE are now in the process of validating the biological relevance of these partners. However, NS3 is a protease. As such one of its viral functions is protein cleavage. There are only 2 known host factors cleaved by NS3 and both are important players in apoptosis. We tested the top hits from the screen for cleavage by NS3 and found new cleave targets. This results is extremely important since blocking proteases is one of the established routes for viral therapeutics including for HCV treatment. A significant part of this project is to disseminate the microfluidic technology into the scientific community. We have confronted this objective on four fronts. First, we published a methods paper demonstrating how the platform works. The paper was accepted for publication in JOVE and a movie clip demonstrating the process is under production (Glick et al., 2012). Next, we published a paper in Lab on chip on using a large scale integrated microfluidic platform to characterize viral protein interaction with host (Ben-Ari et al., 2013).Our second approach was to established several collaborations: - In collaboration with Professor Haim Breitbart of Bar Ilan University we have helped discover new sperm co-receptors that are important in the initiation of fertilization (Jaldety et al., 2012). - In collaboration with Professor Shirit Einav of Stanford University we helped characterize new HCV core -host interactions (Neveu et al., 2012). - In collaboration with professor Ronit Sarid of Bar Ilan University we characterized the oligomeric state of PICT-1 using our microfluidic platform (Borodianskiy-Shteinberg et al., 2014). - In collaboration with Professor Ariella Openheim of the Hebrew University we helped characterize new receptors for SV40 (Drayman et al., 2014).The third approach was to establish a lab on a chip course. In this course, I disseminate my knowledge in microfluidics and lab on a chip field in general to students. During the project period, I have taught the course to over a hundred students.The fourth approach was to present our work in conferences such as Molecular viral Hepatitis conference, ISBMB annual meeting, Israeli biophysical annual meeting, FISB meeting, American biophysical society meetings, Affinity proteomic conference, EMBL microfluidics, etc. The results are very positive. We have numerous requests for collaboration in a wide range of scientific questions. Clearly, the microfluidic platform we have established in this project has made a significant impact on the community.
