Research Projects

1. Mechanisms of HIV cell-cell transmission
   Recent studies have revealed that the efficiency of HIV dissemination is greatly facilitated by cell contact between infected and uninfected T cells. Infected T cells have been found to form adhesive contacts with uninfected CD4+ T cells. These contacts are called virological synapses (VS) because of some similarity to other adhesive structures in the immune system call immunological synapses. These structures require viral Env proteins to be expressed on the cell surface where they interact with CD4 on target cells. Using infectious fluorescent virus systems we are able to quantify and visualize the amount of viral transfer that occurs through VS. In vitro we find that these can be over 10,000-fold more efficient at transferring viral antigen from cell to cell. High resolution, real-time confocal microscopy allows us to directly visualize the changes in cellular distribution of viral protein that occur during VS formation. We find that the VS causes the massive transfer of viral particles into target T cells through an endocytic route that is still largely uncharacterized. The VS-mediated viral transfer can be resistant to patient antibodies that are capable of neutralizing homologous cell free virus. Our ongoing studies are directed at understanding the cellular mechanisms that regulate VS transfer. We are also working to understand how transfer may provide an important mechanism to evade humoral immune responses. The work will help us understand how this mode of efficient viral dissemination may allow HIV to spread efficiently in vivo.
2. Examining the role of cell-cell transmission of HIV in vivo
   HIV researchers have long sought a genetically tractable, economical alternative to the costly primate models for HIV infection. In a recent breakthrough in the field, investigators are using mice with humanized immune systems as in vivo models for HIV infection. These mouse systems transplant human hematopoietic stem cells into immunodeficient mice and allow diverse lineages of human immunocytes to develop. Importantly, the human immune systems are highly susceptible to HIV and can support sustained HIV viral loads in animals that are challenged. In humanized mouse systems, we plan to study the T cell dynamics that allow HIV to spread within a living organism. To understand the dynamics of T cells in an organism we will investigate the trafficking, interactions and viral propagation of HIV infected T cells. Our aim is to understand how T cell migration and interactions contribute to HIV dissemination in vivo.
3. Role of host factors in regulating HIV assembly and virological synapse function
   We are interested in understanding how the viral Gag protein is targeted to specific membranes where viral assembly occurs. Viral genetic studies have revealed that determinants in the MA domain of Gag interact with species-specific host factors to regulate assembly. Using genetic and biochemical approaches, we have been working to identify host cofactors that participate in regulation of assembly. We have identified factors that interact the matrix domain and are testing their role in assembly and virological synapse formation. Current studies are exploring the roles of small G proteins of the Rab family and ubiquitin in modulating HIV assembly, budding and cell-cell transfer. Understanding the cellular pathways that regulate assembly will allow us to design novel inhibitors to block viral assembly.
4. Chemical genetics of HIV assembly
   To monitor the process of assembly with direct imaging, we have developed infectious proviral clones of HIV that carry spectral variants of the green fluorescent protein that engage efficiently in fluorescence resonance energy transfer (FRET). Energy transfer between cyan and yellow fuorescent proteins can serve as a quantitative measure for viral oligomerization which drives viral assembly at the plasma membrane. Recent data suggest that viral assembly is dependent upon cellular factors that help to target the viral proteins to appropriate cellular locations. Current studies are testing high throughput FRET-based assays to identify small molecules that may block HIV assembly. Antagonists of viral assembly may be useful probes to identify key steps in the process. These molecules may also serve as possible therapeutic agents.