Ivanovic Lab Brandeis University, Biochemistry Department
Our research integrates molecular virology, cell biology, single-molecule biophysics, and computational modeling. As a result, trainees in the Ivanovic lab learn not only how to think deeply as virologists, but also acquire an unusual combination of interdisciplinary skills that together permit one to seek answers to sophisticated questions. Opportunities for learning are illustrated below and include how to manipulate viral genomes to generate ‘designer’ viruses, how to use viruses to learn new cell biology, how to design and build from components specialized microscopes, and how to write computer codes simulating single-molecule events in virus system models.
A combination of viral segments from a H1N1 (blue) and a H3N2 (yellow) virus allowing foreign gene expression without attenuation of virus replication. Left - small (A) and large (B) plaque morphologies (yellow circles) of viruses with different gene combinations. Foreign genes can be inserted in the PA gene segment of the 'large-plaquer' virus without attenuation of infectivity.
The ability to introduce a foreign-gene cassette into a nonattenuated influenza virus is being exploited in different projects in the lab as illustrated below:
GFP-expressing influenza virus is used to set up a CRISPRi screen for host factors uniquely permitting cell entry by the filamentous virus particles. The requirements for large-cargo internalization are likely shared among the diverse filamentous human pathogens including Ebola virus, measles, and RSV. This line of research might thus lead to novel, wide-spectrum treatments directly targeting viral ability to adapt to external pressure.
Left - under HA pressure, filamentous particles from a mixed-sized virus population can enter, while cell entry by short particles is inhibited. A macropinocytosis inhibitor EIPA eliminates the selective advantage of filamentous particles. A phenotype analogous to that observed in the presence to EIPA is expected from inhibition of host factors required for filamentous virus-internalization in our CRISPRi screen.
A virus is designed that expresses an ER-retained HA-antibody fragment to generate virus particles with reduced HA incorporation. An elegant consequence of this approach is that the level of scFv expression is proportional to the level of infection, thus achieving uniform HA knockdown among produced virus particles. This tool will set the stage for assessing the contribution of natural HA density to HA functions.
Home-built Total Internal Reflection Fluorescence (TIRF) microscope, version 1 of 3. As our research evolves, we update the design of our microscopes to meet the changing experimental needs.
Molecular events at the interface between the virus and the target membrane. HAs at the contact interface are shown in green.
Influenza virus membrane fusion viewed under the TIRF microscope. The diffraction limited fluorescent dots are individual virus particles. Fluorescent dye in the viral membrane gets brighter when the viral and target membranes merge, then diffuses away.
Stochastic simulations of the molecular events at the interface between individual virus particles and the target membrane. The hexagonal lattice represents HAs at this interface. Fusion cluster (FC) is a group of neighboring HAs that successfully engage the target membrane and combine forces to mediate fusion. The depicted stochastic model reproduces the measured effect of inhibitors on membrane fusion.