Dr. Markus Thali
Overall we are interested in understanding how (genetic, biochemical) information can be transmitted from cell to cell and how such information flow determines the fate of cells, tissues, and ultimately organisms. The main object of our current studies is the retrovirus HIV-1, though we also support investigations by colleagues who focus on other viruses, including influenza virus and endogenous retroviruses. Importantly, in all of our projects we collaborate more or less extensively with other scientists, both on campus and at other institutions, thus forming transient alliances to tackle emerging questions.
Multiscale analyses of HIV-1 cell-to-cell transmission
Successful dissemination of HIV-1 in infected individuals depends on efficient transmission of viral particles from infected (producer) to uninfected (target) cells. In vitro propagation studies have established that HIV-1 particles are most effectively transmitted to target cells if they bud at the so-called virological synapse (VS), a transient multiform (spatially and temporally) adhesion structure at the interface between producer and target cells. Such synaptic virus transmission is thought to also contribute to viral spread in vivo, e.g. when HIV-1 is transmitted to uninfected cells in secondary lymphoid organs of infected individuals. The events leading to the formation, maintenance, and disassembly of the VS are poorly understood, however. It remains also unclear why infected cells, which express the viral envelope glycoprotein (Env), do not always fuse with uninfected cells that carry the viral receptor, thus forming a syncytium.
Focusing on the molecular landscape at the surface of the producer cell, our laboratory, and others, have shown that HIV-1 exits from infected T lymphocytes at membrane segments that are enriched in tetraspanins, cellular scaffold proteins that are known to regulate various cell-cell fusion processes. Not unexpectedly, then, we found that their presence at the virological presynapse, together with other cellular and viral factors, facilitates fusion-less encounters between producer and target cells. While small, T cell-based syncytia do form in lymph nodes of infected individuals, it is clear that in the majority of cases transient alignments of infected and uninfected cells do not result in fusion. Because central features of HIV-1 pathogenesis, including not only virus transmission but also e.g. the establishment of latently infected cells, depend on such fusion-less encounters, we continue to investigate how various viral and cellular factors, together, regulate cell-cell fusion.
Approaches and Innovation
a) quantitative imaging, biophysical techniques
For our investigations we are using various virological and cell biological methods. Particular emphasis has been placed on applying quantitative imaging methods, including e.g. restoration fluorescence microscopy. We have also teamed up with Pierre-Emmanuel Milhiet at the Centre de Biochimie Structurale in Montpelier, France, who does single molecule analyses (SPT) of membrane proteins, and here at UVM we helped to implement super resolution microscopy. Using this new tool, we have already successfully visualized individual budding HIV-1 particles (e.g. see Roy et al., JVI 2013).
b) 3D tissue culture systems – analyses of virus spread at the cell population level
While we will continue to adapt sophisticated techniques that allow us to achieve nanometer resolutions with our imaging approaches, we are also moving in the opposite direction, scale-wise. It is becoming more and more evident that likely all cellular (and by extension also virological) processes are influenced by cues received from the extracellular milieu. Therefore, in collaboration with Alan Howe (at the UVM Department of Pharmacology) we are moving our quantitative imaging analyses from 2D into 3D cell culture systems, as they better recapitulate the microenvironment of living tissue. We already found, for example, that small HIV-1-induced syncytia, as they were observed in lymphoid tissue, using intravital fluorescence microscopy, by our collaborator Thorsten Mempel (at MGH/HMS in Boston), can be recapitulated and thus further analyzed in such 3D in vitro systems e.g. by time lapse imaging and tracking of infected cells over several days.
Thinking beyond viral pathogenesis
Last but not least, while our research endeavor aims at characterizing steps in the replication cycle of HIV-1 that may serve as targets for the development of anti-viral strategies, we try not to lose sight of the big picture. The traditional definition of viruses as obligate intracellular parasites clearly does not render justice to these genetic entities, whose ancestors, self-replicating RNA-based genetic elements, are thought to have predated cellular life. Irrespective of whether or not the host immune system controls their levels of replication, relatively few viruses cause disease, and it is now clear that viruses and virus-like elements played important roles not only at early stages but throughout the evolution of life. Understanding these positive, even essential functions of viruses and virus-like elements will not only be interesting per se, it will also lead to a better understanding of how some of them, under certain circumstances, can inflict harm.
Weng J, Krementsov DN, Khurana S, Roy NH, Thali M. Formation of syncytia is repressed by tetraspanins in human immunodeficiency virus type 1-producing cells. J Virol. 2009 Aug;83(15):7467-74.
Krementsov DN, Weng J, Lambele M, Roy NH, Thali M. Tetraspanins regulate cell-to-cell transmission of HIV-1. Retrovirology. 2009 Jul 14;6:64.
Krementsov DN, Rassam P, Margeat E, Roy NH, Schneider-Schaulies J, Milhiet PE, Thali M. HIV-1 assembly differentially alters dynamics and partitioning of tetraspanins and raft components. Traffic. 2010 Nov;11(11):1401-14.
Roy NH, Chan J, Lambele M, Thali M. Clustering and mobility of HIV-1 Env at viral assembly sites predict its propensity to induce cell-cell fusion. J Virol. 2013 Jul;87(13):7516-25
Symeonides M, Lambele M, Roy NH, Thali M Evidence showing that tetraspanins inhibit HIV-1-induced cell-cell fusion at a post-hemifusion stage. Viruses. 2014 Mar 7;6(3):1078-90
Roy NH, Lambele M, Chan J, Symeonides M, Thali M Ezrin Is a Component of the HIV-1 Virological Presynapse and Contributes to the Inhibition of Cell-Cell Fusion. J Virol. 2013 Jul;87(13):7516-25