Grant: $299,998 - National Science Foundation - Jun. 9, 2009
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Award Description: The overall goal of this project is to elucidate and quantify the dynamics of the fusion machine of the HIV-host cell virological synapse. Based on the dynamics, methods will be engineered to alter time-dependent molecular processes controlling virus-cell fusion at the synapse. Our approach relies on creating fluorescent constructs to track receptor diffusion and assembly, on triggering assembly of receptors by pseudoparticles (virus-like particles, or VLPs). Assembly is controlled on the host cell side with nanoparticle tagged antibodies and on the viral side with nanoparticle tagged protein inhibitors. The three specific objectives that will be addressed in order to achieve the overall goal of the project are: 1. Quantify the dynamics of host cell receptor diffusion and assembly triggered by viral spike. Host cell receptor diffusion rates will be correlated with infection rates of the virus. 2. Determine the effects of nanoparticle tagging on host receptor diffusion and assembly dynamics. Nanoparticle size will be correlated with diffusion rates of receptors. 3. Modulate the dynamics of the virus-host encounter by envelope inhibitors, unmodified and modified with NPs. Nanoparticle size will be correlated with infection rates of the virus.
Project Description: a) First Deliverable: A stable cell line expressing CD4-YFP has been created. We have observed stable expression of YFP using fluorescence microscopy over 5 passages. Sub-cloning techniques were used to excise the CCR5-CFP gene from a vector expressing the same resistance gene (Neomycin) as pCD4-YFP. This DNA sequence was inserted into another vector expressing resistance to Zeocin antibiotic. This allows for a stable co-transfection to take place. These stably transfected cells will now be used to introduce CCR5-CFP. A sample of these CD4-YFP transfected cells will be used as a control for FRET and infectivity assays. b) Second Deliverable: Functional (single-round infection) virus-like-particles (VLPs) have also been created by co-transfection. Single round infection has been verified using luciferase activity in infected cells. We also possess DNA plasmids to make virus-like particles with spikes that represent strains from different parts of the world. In the upcoming year, we will use these different strains to compare synapse dynamics of globally important HIV-1 forms. c) Third Aim-Deliverable: On the nanoparticle-antibody conjugation aim (Aim 3), progress has been made through identification of an appropriate non-neutralizing antibody producing cell line OKT4. These cells are being grown up in culture currently and will soon be used to harvest the antibody OKT4a. d) We have successfully obtained apFRET and FRAP experimental data. apFRET will be used to determine the amount of receptor co-localization after exposure to gp120 monomer and gp140 trimer at varying concentrations. FRAP experiments will be used to determine lateral receptor mobility. In conclusion, we have made important advancements into the project in the last 4 months that will enable us to meet our goals and probe these new concepts.
Jobs Summary: The jobs created include retention through partial salary support for a highly skilled postdoctoral fellow (Mark Contarino), full support for a Ph.D. candidate (Venkat Ramalingasamy), and summer support for an undergraduate researcher from another Institution (Gene.. from Duke University). Partial summer salary support for the PIs (2 persons) allowed focused activities during the summer that enabled the excellent progress described below. Retention of Dr. Mark Contarino, postdoctoral fellow, allowed fast training of the graduate researcher, and continued mentoring of an outstanding young researcher with academic aspirations. The undergraduate visiting scholar (Gene ) was a valuable asset to our project whom we hope to attract also next summer. We feel that this training experience was a critical point for Gene’s career exposing him to research early (right after his freshman year) and influencing his choices. The connections and collaborations already stemming from this project are impressive. Drs. Papazoglou and Chaiken spearheaded an internal competition for Tobacco funds based on discussions and brainstorming for this project. (Total jobs reported: 5)
Project Status: Less Than 50% Completed
This award's data was last updated on Jun. 9, 2009. Help expand these official descriptions using the wiki below.