Tijana Ivanovic, Ph.D.

Viral outbreaks and pandemics arise when animal viruses adapt so that they can recognize and use human cells as their host. Viral persistence in human circulation is subsequently driven by rapid acquisition of mutations in viral proteins that allow evasion of the adaptive human immune response.

Recent research in the lab has shown that viral ability to recognize and invade host cells in the presence of immune system antibodies is determined by the viral particle structure. For example, the same antibodies binding to the same target viral protein may or may not neutralize viral infection depending on the overall structural organization of the virus particle displaying these targets.

We are further working to define how the structural organization of virus particles might enable entry into cells not displaying fitting receptors. Viral ability to establish new infections under suboptimal conditions is probed as a possible mechanism of adaptation to new hosts with the potential to lead to new pandemics. Complementing our research on virus cell entry is our effort to define the mechanisms of virus particle assembly and regulation of the assembly outcomes (i.e., structural features of virus particles such as shape, size, or viral protein incorporation). We have developed novel quantitative and high-throughput approaches for studying virus particle assembly, and detailed functional characterizations are underway.

Trainees learn how to think deeply as virologists and acquire a unique combination of interdisciplinary skills that together permit them to seek answers to sophisticated questions. We combine molecular virology, in vitro reconstitution, biophysics, and quantitative cell biology to answer difficult-to-tackle mechanistic questions in virology and build upon those discoveries to ultimately enable prediction and prevention of undesirable viral adaptation (e.g., drug resistance, immune escape, or pandemic adaptation). Our work spans diverse viral pathogens and/or their model systems, including but not limited to influenza virus and reovirus.

Viral cell-entry mechanisms

Viruses initiate infection by delivering the genomes from within the viral capsid or a membrane envelope to the target cell. Enveloped viruses accomplish this by fusing/merging their membrane with a cellular membrane. Membrane fusion is mediated by the viral surface glycoproteins, which undergo a series of large-scale conformational changes that allow them first to insert in the target membrane, then to bring the viral and cellular membranes together. By measuring the fusion kinetics of individual virus particles and their mutants in the presence or absence of antibodies or inhibitors, we have uncovered the detailed mechanism of membrane fusion by influenza virus.

Influenza cell-entry glycoprotein hemagglutinin (HA) is present at a high density on virus particles. Three to five HAs that insert next to each other cooperate to bring about membrane fusion. However, the steps leading to HA insertion are stochastic (noncooperative, random), so substoichiometric (nonsaturating) antibody or inhibitor amounts prevent fusion by the majority of particles.

A further consequence of the mechanism of membrane fusion we uncovered is that very large particles are virtually impossible to inhibit. Tens of micrometers-long viral filaments require more than 90% of HAs to be inactivated for any reduction in the fusion yield. This insight led to a model where the pre-existing diversity in virus particle size in pleomorphic (mixed-shape) viruses enables viral adaptation to cell-entry pressure in a way that does not require an initiating genetic change.

We are asking:

1. How does virus particle shape influence viral adaptation to cell-entry pressure?
2. What is the mechanism of membrane fusion by other pleomorphic viruses (e.g., Ebola virus)?
3. What role do other viral structural features (e.g., glycoprotein density) play in the mechanism of membrane fusion?
4. What role do the interactions between viral glycoproteins and cellular receptors play in membrane fusion?

Left: Electron micrograph revealing pleomorphic influenza A virions. A filamentous and a spherical virion are indicated. Pleomorphic virus structure is employed by other circulating (e.g., measles and RSV) or emerging (e.g., highly pathogenic avian influenza, Ebola, Nipah, and Hendra viruses) devastating viral pathogens. Middle: Home-built TIRF microscope for single-virion and single-molecule imaging. As our research evolves, we update the design of our microscopes to meet the changing experimental needs. Right: Longest filamentous virions are effectively resistant to inhibition by HA-targeting antibodies.

Credit: NIAID

Viral assembly mechanisms

We have established a total internal reflection fluorescence (TIRF)-based single-particle platform for quantitative real-time imaging of reovirus assembly, disassembly, and transcription.

Reovirus virion consists of two concentric icosahedral protein shells. The inner, core particle encloses the genome and serves as the transcriptional unit in the infected cell cytoplasm. The outer capsid delivers the core across the cellular membrane to initiate infection. Disassembly of the outer capsid activates cores for transcription, and its assembly around cores represses transcription.

In our experiment, fluorescently labeled cores are immobilized on microscope coverslips. Fluorescently tagged oligonucleotides detect nascent viral transcripts. Fluorescently tagged outer-capsid components assemble around immobilized cores and repress transcription. We are working toward a quantitative model of reovirus outer-capsid assembly and transcriptional regulation.

In addition to studying the assembly of regularly shaped, icosahedral virions, we are interested in mechanisms of assembly of irregularly shaped pleomorphic viruses. For this, we have developed a novel, high-throughput assay enabling quantification of virus particle size distributions directly in the medium around infected cells.

We are asking:

1. What is the mechanism of pleomorphic virion assembly?
2. How is the shape of pleomorphic virions regulated?
3. What are the viral and cellular determinants of viral assembly outcomes?
4. How do viruses with segmented genomes ensure efficient packaging of the full genome complement?

Left: Electron micrograph of Reovirus virions, cores, and recoated cores in vitro. Reovirus virions display a regular, icosahedral particle structure. Recoated cores are indistinguishable from virions in structure and function. Right: Single particle platform for study of reovirus assembly, disassembly, and transcription. Schematic of the experiment design and illustration of what transcription/assembly might look like under the microscope. Red square: dots correspond to viral cores (all particles). Blue and green squares: cores that have incorporated the outer-capsid proteins sigma1 (blue) and mu1/sigma3 (green). Orange square: actively transcribing cores.

Credit: NIAID