Molecular Mechanisms of Viral Entry
All enveloped viruses enter target cells by fusing the viral membrane with a cell membrane, which releases the viral genetic information into the cell. This process is catalyzed by envelope glycoproteins found on the virion surface (Fig 1). The goal of my laboratory is to elucidate the mechanism by which enveloped viruses enter target cells in terms of the conformational dynamics of the viral envelope glycoproteins. Although the details of the entry pathways and the structures of the glycoproteins among different viruses can vary significantly, viruses have evolved common mechanisms of surmounting the energetic barrier to fusing two membranes. The energy needed to cross this barrier on a physiologically relevant timescale is contained entirely within the metastable conformation of the glycoprotein. This energy is released through transition from the metastable pre-fusion conformation to a highly stable post-fusion conformation, which occurs through multiple intermediate steps. We aim to characterize this dynamic process in molecular detail, with a focus on common mechanistic strategies utilized by distinct viruses.
Figure 1. Envelope glycoproteins from HIV-1, Ebola virus, influenza A, and respiratory syncytial virus. While the structures depict single heterodimeric protomers, all of these glycoproteins are present as trimers of heterodimers on the viral surface. The homologous receptor-binding domains are in blue; membrane fusion domains are in red.
In addition to their role in viral entry, envelope glycoproteins are also the primary target for attack by antibodies. As such, viruses have evolved multiple strategies for escaping neutralization by antibodies. Of most interest to us is the idea that envelope glycoproteins conceal their functional centers, such as receptor-binding sites, which form antigenic hotspots, through conformational changes. We seek to understand the diverse and conserved mechanisms of conformational masking in immune evasion. We are also interested in understanding how some antibodies successfully neutralize viruses, despite their protective abilities.
Our highly interdisciplinary approach centers on the application of single-molecule fluorescence microscopy and spectroscopy, and involves kinetic and thermodynamic analysis of single-molecule data, and structural modeling (Fig 2). Because single-molecule imaging is an emerging technology, we are also highly interested in the continued development of new fluorescence-based methods for interrogating the function of individual macromolecules, as well as improved means of acquiring, processing, and analyzing single-molecule fluorescence data.
Figure 2. Experimental design for imaging the conformational dynamics of single Env glycoproteins on the surface on native HIV-1 virions using total internal reflection fluorescence microscopy.
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