Person:

Piccinotti, Silvia

Rabies virus (RABV) subverts host neuronal circuitry to gain access to the brain where it causes generally incurable, lethal encephalitis. The single glycoprotein (G) dictates two defining steps for infection and neuroinvasion: receptor-mediated endocytosis and transport of virus. We generate two recombinant VSV (rVSV) clones that genetically incorporate G (rVSV RABV G) from the fixed RABV strains, SAD B19 and CVS, to study internalization into epithelial cells and compartmentalized primary cultures of peripheral neurons. Through the use of chemical inhibitors and markers for specific endocytic routes, we demonstrate that the predominant RABV entry route in both epithelial and neuronal cells is dynamin- and clathrin- dependent. Viral endocytosis is mediated by actin-dependent, partially coated clathrin pits as evidenced by live high resolution confocal microscopy of envelopment in epithelial cells and transmission electron micrographs in neuronal and non-neuronal cells. Thus, we corroborate the hypothesis that particle size is the sole viral determinant of actin-dependence of coated pits. Through a combination of high resolution microscopy and infectivity-based approaches, we link molecular mechanisms of viral uptake at the single particle level to productive infection. Targeted pharmacological disruption of endosomal acidification at the neurites or cell bodies of peripheral neurons demonstrates that fusion and viral genome release at the cell body, the site of replication, is a prerequisite for infection. This work extends the current understanding of RABV entry by providing a detailed characterization of endocytosis from the plasma membrane to the site of fusion and correlating it with establishment of infection into neuronal populations relevant for pathogenesis in vivo.

Background: Most odors are perceived to have the same quality over a large concentration range, but the neural mechanisms that permit concentration-invariant olfactory perception are unknown. In larvae of the vinegar fly Drosophila melanogaster, odors are sensed by an array of 25 odorant receptors expressed in 21 olfactory sensory neurons (OSNs). We investigated how subsets of larval OSNs with overlapping but distinct response properties cooperate to mediate perception of a given odorant across a range of concentrations. Results: Using calcium imaging, we found that ethyl butyrate, an ester perceived by humans as fruity, activated three OSNs with response thresholds that varied across three orders of magnitude. Whereas wild-type larvae were strongly attracted by this odor across a 500-fold range of concentration, individuals with only a single functional OSN showed attraction across a narrower concentration range corresponding to the sensitivity of each ethyl butyrate-tuned OSN. To clarify how the information carried by different OSNs is integrated by the olfactory system, we characterized the response properties of local inhibitory interneurons and projection neurons in the antennal lobe. Local interneurons only responded to high ethyl butyrate concentrations upon summed activation of at least two OSNs. Projection neurons showed a reduced response to odors when summed input from two OSNs impinged on the circuit compared to when there was only a single functional OSN. Conclusions: Our results show that increasing odor concentrations induce progressive activation of concentration-tuned olfactory sensory neurons and concomitant recruitment of inhibitory local interneurons. We propose that the interplay of combinatorial OSN input and local interneuron activation allows animals to remain sensitive to odors across a large range of stimulus intensities.

The single glycoprotein (G) of rabies virus (RABV) dictates all viral entry steps from receptor engagement to membrane fusion. To study the uptake of RABV into primary neuronal cells in culture, we generated a recombinant vesicular stomatitis virus in which the G protein was replaced with that of the neurotropic RABV CVS-11 strain (rVSV CVS G). Using microfluidic compartmentalized culture, we examined the uptake of single virions into the termini of primary neurons of the dorsal root ganglion and ventral spinal cord. By pharmacologically disrupting endocytosis at the distal neurites, we demonstrate that rVSV CVS G uptake and infection are dependent on dynamin. Imaging of single virion uptake with fluorescent endocytic markers further identifies endocytosis via clathrin-coated pits as the predominant internalization mechanism. Transmission electron micrographs also reveal the presence of viral particles in vesicular structures consistent with incompletely coated clathrin pits. This work extends our previous findings of clathrin-mediated uptake of RABV into epithelial cells to two neuronal subtypes involved in rabies infection in vivo. Chemical perturbation of endosomal acidification in the neurite or somal compartment further shows that establishment of infection requires pH-dependent fusion of virions at the cell body. These findings correlate infectivity to existing single particle evidence of long-range endosomal transport of RABV and clathrin dependent uptake at the plasma membrane.