Molecular regulation of HIV-1 and influenza virus spread
Current activities – perspectives
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 studies is the retrovirus HIV-1, though recently we have also started to investigate how other viruses, particularly influenza virus, replicate, and we are in the pilot experiment phase for research that aims at elucidating how information transfer from cell to cell is regulating developmental processes. Importantly, in all of our projects we collaborate more or less extensively with other scientists, both on campus and at other institutions.
Multiscale analyses of HIV-1 assembly, release and 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) that forms between producer and target cells. Such synaptic virus transmission is thought to be prevalent also in vivo, when HIV-1 spreads in secondary lymphoid organs of infected individuals. The events leading to the formation, maintenance, and disassembly of the VS are poorly understood.
Focusing on the molecular landscape at the surface of the producer cell, our laboratory is investigating mechanisms that control the synaptic transmission of HIV-1. Because we and others have shown that this virus exits from infected cells at membrane segments that are enriched in tetraspanins, we are trying to define what roles these cellular scaffold proteins play during HIV-1 transmission from cell-to-cell. So far we found that individual members of the tetraspanin family, though present at viral budding sites, (indeed even recruited to those sites,) are dispensable for particle release. In fact, and as also documented by others (e.g. see Sato et al., 2008, J Virol 82:1021), these proteins, when acquired by newly formed viral particles, reduce the infectivity of virions. However, while their presence at HIV-1 exit sites thus appears to restrict viral spread, we also found that tetraspanins prevent the formation of syncytia that can form upon an interaction of the viral envelope glycoprotein expressed at the surface of producer cells and the viral receptors present on the target cells. The virus may benefit from such fusion prevention, as this guarantees prolonged survival of newly infected cells. For this reason, but also because we found that tetraspanins overall are downregulated in infected cells, we are further testing the hypothesis that HIV-1 evolved to temporarily and spatially adjust the levels of tetraspanins such that they allow for sustained virus propagation.
Role of tetraspanins in influenza virus replication
Recently, we have also started to compare (at the molecular level) the assembly and release process of HIV-1 with that of influenza virus and we are examining the characteristics of the two exit sites. In collaboration with Dr. Megan Shaw at Mount Sinai Medical School, whose recent proteomic and genetic analyses identified CD81 as a host factor that positively regulates influenza virus replication, we have started scrutinizing how exactly this tetraspanin, through its presence in producer and target cells, promotes transmission of this virus. Together with the HIV studies we expect that these analyses will provide further insight into the molecular mechanisms underlying the spread of pathogenic viruses. They may, however, also contribute to a better understanding of how members of the tetraspanin family of proteins regulate cellular processes such as adhesion and membrane fusion that are required for normal physiology and/or various non-viral pathologies.
Approaches – 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 restoration fluorescence microscopy and Fluorescence Recovery after Photobleaching (FRAP). We have also teamed up with Dr. 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 have recently embarked on Atomic Force Microscopy (AFM) studies and proteomic analyses of viral particles. Further, in our quest to use the most advanced techniques available, a couple years ago we have teamed up with Dr. David Warshaw and Dr. Douglas Taatjes, the chair of the Molecular Physiology and Biophysics Department and the Director of the Microscopy Imaging Center here at UVM’s College of Medicine, respectively, and helped implementing super resolution microscopy (also referred to as nanoscopy). Using this new tool, we have now already successfully visualized individual budding HIV-1 particles (e.g. see Roy et al).
b) 3D tissue culture systems
While we attempt to adapt sophisticated techniques that will 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 many cellular (and by extension also virological) processes are triggered, or at least are influenced, by cues received from the extracellular milieu. Therefore, in collaboration with Dr. Alan Howe (at UVM’s Department of Pharmacology) we are currently also attempting to move our quantitative imaging analyses from 2D- into 3D-cell culture systems, as they better recapitulate the microenvironment of living tissue.
Thinking beyond viral pathogenesis
Last but not least, while our research endeavor primarily aims at characterizing steps in the replication cycle of HIV-1 and influenza virus that may serve as targets for the development of anti-viral strategies, we try not to lose sight of the big picture. The classical 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 can control 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 the early stages of life but throughout evolution. 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.
Roy NH, Chan J, Lambelé 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, doi: 10.1128/JVI.00790-13
Wesley CS, Guo H, Chaudhry KA, Thali M, Yin JC-P, Clason T, Wesley UV. Loss of PTB or negative regulation of Notch mRNA reveals distinct zones of Notch and Actin protein accumulation in Drosophila embryo. PLoS One. 2011;6(7):e21876.
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.
Krementsov DN, Weng J, Lambelé M, Roy NH, Thali M. Tetraspanins regulate cell-to-cell transmission of HIV-1. Retrovirology. 2009 Jul 14;6:64.
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.
Nydegger, S, Khurana, S, Krementsov, DK, Foti, M, Thali, M. Mapping of tetraspanin-enriched microdomains that can function as gateways for HIV-1. J Cell Biol. 2006 Jun 5;173(5):795-807.
All Thali publications