1995 — 2020 |
Enquist, Lynn W. |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. R37Activity Code Description: To provide long-term grant support to investigators whose research competence and productivity are distinctly superior and who are highly likely to continue to perform in an outstanding manner. Investigators may not apply for a MERIT award. Program staff and/or members of the cognizant National Advisory Council/Board will identify candidates for the MERIT award during the course of review of competing research grant applications prepared and submitted in accordance with regular PHS requirements. |
Genetic Analysis of Herpesvirus Tropism and Virulence
With few exceptions, viral infections of the central nervous system (CNS) begin in peripheral tissues and then reach the CNS either by hematogenous routes or transport in nerves. This proposal focuses on the first step in the nerve route pathway ? invasion of the peripheral nervous system (PNS). The primary emphasis is on alpha herpesviruses that have evolved to enter the PNS efficiently. The overarching questions are why PNS invasion by alpha herpesviruses is so efficient and how do peripheral axons defend themselves against viral invaders. Our recent studies revealed that axonal entry by alpha herpesvirus particles rapidly stimulated new, local protein synthesis to increase efficiency of virus particle retrograde transport to cell bodies. We suggested that axonal infection induces an immediate damage response that increases efficiency of axonal transport. Axons also sense both type I and II interferons (IFN) produced in infected peripheral tissues, resulting in a novel antiviral response (local axonal production of phosphorylated STAT1 after IFNß exposure resulting in reduction of virus particle transport toward the cell bodies). A hallmark of the axonal response to viral infection and IFN exposure is rapid translation of new axonal proteins from repressed axonal mRNAs. Remarkably, infection by herpesviruses, as well as IFN exposure rapidly changes the total axonal proteome and does so before new viral proteins are produced. Our data suggests that the PNS axons not only sense peripheral virus infection. We are interested in the mechanisms of these axonal responses and their virus specificity. We will explore the interplay and specificity between axonal detection and local defensive action. The unique focus of this proposal is on axon biology immediately after virion entry and subsequent transport of distinctive viral/cellular cargos in PNS axons with or without cytokine exposure before viral gene products are made. Our hypotheses are (1) that axons are front-line sensors and responders to diverse viral PNS invasion; (2) they locally sense and respond to entry of different virus particles; and (3) they respond to IFN produced by infected non-neuronal tissues and mount distinct, local antiviral responses. We have developed three technologies to explore these ideas: tri-compartment Campenot chambers to physically isolate axons from their cell bodies; optical imaging technology to follow entry events and subsequent axonal transport of individual virus particles in the presence or absence of IFN; and bioorthogonal noncanonical amino acid tagging (BONCAT) or Click chemistry and SILAC mass spectrometry approaches to label and quantitate new proteins synthesized immediately in axons after infection or IFN treatment. Understanding the fundamentals of the immediate and local response of PNS axons to incoming virus particles and inflammatory cytokines before any new viral gene products are expressed will lead to a deeper understanding of how the PNS and ultimately the CNS, is protected from infection.
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2000 — 2004 |
Enquist, Lynn W. |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Genertic Analysis of Herpesvirus Tropism and Virulence
The long term goal is to understand in molecular terms how a neurotropic herpes virus invades and spreads in the mammalian nervous system. Most alpha herpesviruses (e.g., herpes simplex virus HSV; varicella-zoster virus, VZV; and pseudorabies virus, PRV, display a striking neurotropism infecting both the peripheral and central nervous system, and do so in every animal species that can be infected. In PRV, two genes (gE, gI) are required for anterograde, but not retrograde spread in the nervous system of living animals. A new gene (Us9) was recently discovered that also is required for anterograde spread of PRV. All three gene products also influence virulence. Research in this proposal will continue a focused genetic and biochemical attack to determine the functions of the gE, gI and Us9 gene products in invasion, spread and pathogenesis in the nervous system. Four specific aims are proposed to test a general model of directional spread of virus in the nervous system: In aim 1, the localization and trafficking of gE, gI and Us9 proteins in cultured neurons and infected nervous system tissue will be determined. In aim 2 alanine substitution mutations in the gE ectodomain will be analyzed to define sites of interaction with putative cellular receptors. In addition, the role of the gI cytoplasmic tail on gE function will be determined. In aim 3, a genetic and biochemical analysis of Us9 will be initiated to determine its role in anterograde trans-neuronal spread. Finally, in aim 4: the role of gD, a viral ligand essential for virion infection, will be analyzed to determine if gD is involved in gE/gI/Us9-dependent and -independent spread of virus in defined neuronal circuits. The knowledge to be learned from completion of this work is important because the direction taken by the virus in a neuron after primary infection or reactivation from latency, as well the extent of spread in a neuronal circuit can be the difference between a minor peripheral infection or a lethal viral encephalitis.
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2003 |
Enquist, Lynn W. |
R13Activity Code Description: To support recipient sponsored and directed international, national or regional meetings, conferences and workshops. |
Viruses and Cells Gordon Conference @ Gordon Research Conferences
DESCRIPTION (provided by applicant): The Gordon Conference, Viruses and Cells, is a premier meeting in the field of Virology, emphasizing the dynamic interaction between viruses and the cells and organisms they infect. The 2003 meeting will be held at II Ciocco, Lucca, Italy, from May 18-23. The invited speakers will discuss recent ground-breaking findings on the mechanisms of viral replication and pathogenesis, as well as the host antiviral and immune responses to viral infection. The most recent developments will be highlighted both by selecting several short talks from abstracts submitted at the time of registration and through the use of prominently featured poster sessions. Participants will include a mixture of principal investigators from well-established and newly established laboratories, postdoctoral fellows, graduate students, and investigators from industry. This conference is one of the few smaller meetings in virology that encompasses a broad view of the field. Funding is requested to support the registration and travel of graduate students and postdoctoral fellows to this meeting.
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2008 — 2017 |
Enquist, Lynn W. |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Neuronal Spread of Herpesvirus Infection
NIH NS060699 renewal Neuronal spread of herpesvirus infections Project Summary One of the most exciting areas in biology is the nervous system and how it works. Viral infections of the nervous system have provided exceptional insight at many levels, from pathogenesis to basic biology. The mechanism(s) by which alpha herpesvirus infections spread into, within, and out of the nervous system are understood in principle, but not in any detail despite considerable effort. This unique biology leads to efficient host-to-host transmission and establishment of these viruses in their natural host populations with minimal pathogenesis. A long-term goal of my laboratory is to determine the molecular mechanisms by which neuroinvasive alpha-herpesviruses move in and out of the mammalian nervous system. These mechanisms will provide targets for manipulation that could substantially expand our understanding of infection transmission. Work in this renewal proposal continues to build on powerful imaging technology developed in the past funding period to reveal how herpes virion components move inside neurons and from neurons to non-neuronal cells in vitro and in vivo. We seek to identify and quantify critical events and potential bottlenecks in long distance transmission of infection from the peripheral nervous system (PNS) to peripheral epithelial cells. Experiments are divided among three aims all featuring light and video microscopy: Imaging individual virion egress events using multi-color TIRF microscopy; assaying axon-cell egress and spread events with chambered neurons, three color virus technology, and fast epifluorescence imaging; and imaging in vivo/ex vivo PRV invasion of the PNS at the single cell and single particle level. The technology and knowledge obtained from these studies have broad application. They enable a better understanding of herpesvirus cell biology at the single cell and particle level, provide insight into potential bottlenecks during host-host transmission and have implications for intervention strategies. This technology and knowledge also will provide opportunities to develop enhanced viral tracers for understanding the organization and functional architecture of the nervous system.
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2009 — 2010 |
Card, John Patrick (co-PI) [⬀] Enquist, Lynn W. Wang, Samuel Sheng-Hung (co-PI) [⬀] |
RC1Activity Code Description: NIH Challenge Grants in Health and Science Research |
Viral Brainbow: Tracing Brain Circuits With Connection Order Specificity
DESCRIPTION (provided by applicant): This application addresses broad Challenge Area (06) Enabling Technologies, 06-NS-106: Validating new methods to study brain connectivity. We propose to test a new method that provides substantial improvement over previous Cre-conditional viral tracers. The technology combines the Brainbow multicolor cell marking technology with the retrograde, circuit tracing properties of pseudorabies virus (PRV), a neuroinvasive alpha herpesvirus. We have constructed a prototype PRV Brainbow virus called PRV263 that we propose will enable simultaneous identification of distinct chains of neurons projecting to a phenotypically defined population of neurons, and promises to provide predictive data on the strength of different connections among those neurons. Importantly, these PRV Brainbow tracers will have distinct advantages over present tracers. Our concept takes advantage of conditional, site-specific recombination of the genome of a DNA virus to produce multiple reporters so that neurons upstream (presynaptic) of a Cre recombinase (Cre) expressing neuron will be a different color from the Cre-expressing neuron. This novel concept will be expanded to produce second generation prototypes of PRV Brainbow tracers that do not rely on Cre-transgenic mice and can be used in the many mammalian species susceptible to PRV infection. A third generation prototype will be constructed that not only marks circuits, but also reports on neuronal activity. In this latter concept, the PRV Brainbow virus also will include a genetically encoded calcium indicator that fluoresces when calcium is bound. As viral tracing of neural circuitry has become an essential tool in the neuroscience community, our new tracers will be immediately applicable for many ongoing fundamental research projects in neuroscience in a variety of animals. These new tools have promise to reveal detailed functional insights into neural circuit organization that have not been possible to achieve in the past. Viral tracing of neural circuitry has become an essential tool in the neuroscience community. The new, robust viral tracers that will result from our work have promise to reveal detailed functional insights into trans-neuronal spread of herpesviruses, as well as neural circuit organization that have not been possible to achieve in the past. These neural tracers would be powerful tools to elucidate brain micro-circuitry, providing a better understanding of nervous system functions.
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