Matthew Taylor, Ph.D.
The Taylor Lab is interested in the intercellular spread of neuroinvasive viruses and how spread impacts pathogen evolution and pathogenesis. Neuroinvasive viruses, including Herpes Simplex virus and West Nile virus, preferentially infect the cells of the nervous system, transmitting viral particles between neurons. Using these connections, viral infection invades the central nervous system resulting in severe disease and death. We study the events of virion transmission within neurons utilizing live-cell imaging of primary neuronal cultures to capture the events of virion transport, egress and infection. Combining this technique with an ever-expanding cohort of fluorescent fusions to viral and cellular proteins in conjunction with loss-of-function mutants allows the dissection of specific steps in virion transmission that have never been visualized or fully understood.
There are two main projects that are being developed in the laboratory related to the spread of Alphaherpes virus and West Nile virus. A central aspect to both projects is the well-characterized in vitro model of compartmentalized neuronal cultures (Figure 1). In these cultures, dissociated superior cervical ganglia neurons are plated in one compartment and extend axons to a far compartment. Epithelial cells can be cultured on these isolated axons, simulating the peripheral cellular environment. This system physically separates primary infection of neuronal cell bodies from axon-to-cell spread.
Understanding the restriction on Alphaherpes virus spread - We recently published that neuron-to-cell anterograde spread of infection involves a limited number of viral particles for two alphaherpes viruses: HSV-1 and pseudorabies virus (PRV) (Taylor et al, PNAS 2012).
Taking advantage of the compartmentalized neuronal cultures, I developed two methodologies to quantify viral infection after anterograde transport (Figure 2). In the first method, the average number of viral genomes in newly infected cells is quantified using a three-color fluorescence diversity assay (Figure 2A). The second method to quantifying anterograde spread involved live cell imaging of fluorescent capsids infecting isolated epithelial cells (Figure 2C-E). Both methods quantified axon-to-cell anterograde spread, and the resulting data allowed us to conclude that a limited number of virions initiate infection following spread.
To follow up on these results, research in my laboratory will focus on the mechanism behind the bottleneck on viral spread. I hypothesize the bottleneck is due, in part, to a function of viral and cellular proteins, which restricts the number of viral particles transmitted from the axon to the susceptible cell. This hypothesis will be tested with three experimental directions:
- Characterizing the viral proteins that alter the number of virions transmitted during anterograde spread. This project will utilize targeted and random mutagenesis of viral genomes with subsequent testing on the capacity of viral mutants to effect anterograde spread.
- Identifying the cellular immune responses triggered by virion entry. A combination of live cell imaging of fluorescent reporters and genetic knock-out cell lines will be used to determine the impact of innate immunity on limiting virion transmission.
- Understanding restrictions during intra-host viral spread. Utilizing a well-characterized animal model for anterograde neuronal spread will correlate findings from compartmentalized neurons to in vivo spread of viral infection.
By characterizing the factors involved in restricting the number of virions transmitted, we can begin to understand not only how axon-to-cell spread is regulated but also what impact the restriction has on viral spread between cells and within an infected host.
Characterizing the neuronal spread of West Nile virus. – West Nile virus is an emergent zoonotic infection that has caused regional epidemics of viral encephalitis across the United States since early 2000. Like alphaherpes viruses, West Nile virus spreads within infected hosts utilizing the connections of the nervous system. Unlike herpes, this positive strand RNA virus has not been characterized for the number of viral particles transmitted between neurons. Research into the intracellular transmission of West Nile virus will initially mirror the work done with HSV and PRV.
Anterograde-directed viral spread may represent a potent target for treating a broad range of diseases that are caused by viral spread in the nervous system. The population bottlenecks restrict the size and genetic properties of spreading viral populations. As a single genome initiates viral replication in susceptible cells, that genome cannot contain mutations that would negatively impact viral replication or spread. Thus, the population bottleneck could act as a selection pressure, enriching the population of viral genomes for enhanced kinetics of anterograde transport, genome replication, and virion assembly. In essence, anterograde spread could result in a viral population with a reduced number of defective virions, allowing the most productive viral genomes to propagate within and between hosts. If we can experimentally modify the number of virions transmitted from axons, we can determine the relative fitness of viral populations following anterograde spread.
B.S. Microbiology/Biochemistry University of Washington, 2000
Ph.D. Microbiology & Immunology Stanford University, 2008
Post-doctoral Studies Princeton University
Articles of Significance
- Taylor, M.P., Kobiler, O., and Enquist, L.W. (2012) Alphaherpesvirus axon-to-cell spread involves limited virion transmission. Proc. Natl. Acad. Sci.,109(42): 17046–51. PMCID: 3479527
- Taylor, M.P., Kratchmarov, R., and Enquist, L.W. (2013) Live cell imaging of alphaherpes virus anterograde transport and spread. Journal of Visualized Experiments. 2013; (78). PMCID: 23978901
- Kratchmarov, R., Taylor, M.P., and Enquist, L.W. (2013) Role of Us9 phosphorylation in axonal sorting and anterograde transport of Pseudorabies Virus. PLoS One, 8(3): e58776. PMCID: 3602541
- Taylor, M. P., Lyman, M.G., Kramer, T., Kratchmarov, R., and Enquist L.W. (2012) Visualization of an alphaherpesvirus membrane protein that is essential for anterograde axonal spread of infection in neurons. mBio,3(2): e00063. PMCID: 3315705
- R Kratchmarov, MP Taylor, LW Enquist (2012). Making the case: Married versus Separate models of alphaherpes virus anterograde transport in axons. Reviews in Medical Virology, 1-12