Lynn Enquist
Systems and Circuits
Systems and Circuits

Henry L. Hillman Professor in Molecular Biology

Professor of molecular biology and the Princeton Neuroscience Institute

Research: Neurovirology
Areas of Research: Cell Biology, Genomics, Microbiology & Virology, Neuroscience
lenquist@princeton.edu
Research Lab
609-258-2415
314 Schultz Laboratory
(609) 258-4990
Faculty Assistant:
Matt Montondo
montondo@princeton.edu
(609) 258-266

Research Focus

Neurovirology

My research focuses on mechanisms of herpesvirus pathogenesis. Most alpha-herpesviruses (e.g., herpes simplex virus HSV; varicella-zoster virus, VZV; and pseudorabies virus, PRV) are parasites of the peripheral nervous system (PNS) in their natural hosts. This life style is remarkable because of all the neurotropic viruses that also enter the PNS (e.g., rabies), only the alpha-herpesviruses routinely STOP in the PNS of their natural hosts and establish a reactivatable, latent infection. Normally, this PNS infection is relatively benign, promoting survival of both virus and host. However, aberrations in this pathway give rise to the set of common diseases caused by these viruses. For example, invasion of the CNS is a rare, but exceedingly serious possibility. The directional spread of a herpes infection from epithelial surfaces to PNS neurons (where latency is established), and then, upon reactivation, spread back to epithelial surfaces constitutes the virus survival strategy. Research in my laboratory is directed toward answering several basic questions: What are the virus- and cell-encoded mechanisms that direct virus into and back out of the PNS? Why does the virus only occasionally enter the CNS in its natural host? What are the molecular roadblocks that make CNS infection rare in the natural host and frequent in the non-natural host? What are the viral gene products that promote disease and how do they work? How do the PNS and CNS respond to viral infection? These basic questions continue to lead us into exciting (and sometimes unexpected) areas. Two major areas of work are outlined below.

Pseudorabies Virus (PRV) as a Model System

PRV is a broad host range herpesvirus that causes fatal encephalitis in a wide variety of animal species except its natural host, the adult pig. PRV is not a human pathogen and grows well in the lab. Using rodent and chick embryo models and defined mutations in specific PRV genes, we are studying mechanisms of spread from site of primary infection, evasion of innate and acquired immune defense, and cell/tissue damage. We have identified several PRV gene products that affect the extent of PRV pathogenesis in both model systems affecting cell to cell spread, direction of spread, and host response to infection. We have constructed infectious bacterial artificial chromosomes carrying the entire PRV genome and are using E. coli mutagenesis and recombination techniques for genetic analysis of PRV. cDNA analyses coupled with gene array technology are being developed to understand the role of virus and host genes in virus replications in various cell types and tissues of infected animals.

Genetics of Directional Spread of Virus In and Between Neurons

Our work with PRV infections in rats, mice, and chicken embryos demonstrates that directional spread of virus in the nervous system is regulated, in part, by several viral genes. We have developed technology to culture primary embryonic sensory and autonomic PNS neurons from mice, rats, and chickens. Based on these systems, we have defined two distinct processes involved in directional spread: (1) movement of virion components into axons and long distance movement of these components to axon terminals; and (2) interactions between the infected axon terminals and connected cells that promote assembly, virus release, or both. The small type II membrane protein called Us9 affects the first process: it promotes the movement of virion envelope (but not tegument or capsid) components into axons. The type I viral membrane proteins gE and gI also are required for directional spread in the nervous system, but the mechanism for their action is distinct from that of Us9. gE and gI affect the entry of all viral components into axons. gE expression also exacerbates symptoms and speeds time to death in vivo, but this phenotype is not correlated with virus spread. Green and red fluorescent protein fusions to Us9, gE, gI, and other membrane proteins, as well as capsid and tegument proteins enable us to follow synthesis, transport, and assembly of virions in cultured neurons as well as in tissue. The genetics and biochemistry of these proteins and others that affect endocytosis, targeting, virus assembly, egress, spread, and virulence are a major focus of our work.

Tracing the Hardwiring of the Nervous System with a Virus

Unlike infections of the natural host (the adult pig), PRV infections of all other permissive species (e.g., baby pigs, chicken embryos, rodents, dogs, cats, and cows, to name a few) are lethal and quickly transmitted through peripheral nerves to the brain and spinal cord. These infections enable us to study not only mechanisms of virulence and how the nervous system responds to infection, but also how the virus spreads in the nervous system. Amazingly enough, in all permissive species, the virus spreads only in chains of synaptically connected neurons (trans-neuronal spread). Therefore, PRV can be used as a self-amplifying tracer of neuronal circuits. The ability to reveal a neural circuit (neuron A is wired to neuron B is wired to neuron C etc) is powerful technology, but demands attention to many details. Tracing studies are rewarding because they require close collaboration between neurobiologists and virologists. For example, circuit tracing requires use of mutant viruses that are much less virulent than wild type PRV. Understanding why these mutants are good tracing strains has led us to a better understanding of virulence and mechanisms by which virus spreads between neurons. In addition, we capitalize on our ability to manipulate PRV to construct less virulent, innovative tracing viruses: e.g., viruses expressing beta-galactosidase and variants of green fluorescent protein, as well as viruses that replicate only in certain neurons and not others. These viruses provide powerful tools to study neural circuits and how the nervous system responds to infection.


Selected Publications

  • Granstedt AE, Bosse JB, Thiberge SY, Enquist LW. (2013) In vivo imaging of alphaherpesvirus infection reveals synchronized activity dependent on axonal sorting of viral proteins. Proc Natl Acad Sci. 110: E3516-25.  Pubmed
  • Sun XR, Badura A, Pacheco DA, Lynch LA, Schneider ER, Taylor MP, Hogue IB, Enquist LW, Murthy M, Wang SS. (2013) Fast GCaMPs for improved tracking of neuronal activity. Nat Commun. 4: 2170. Pubmed
  • Granstedt AE, Brunton BW, Enquist LW. (2013) Imaging the transport dynamics of single alphaherpesvirus particles in intact peripheral nervous system explants from infected mice. MBio. 4(3). pii: e00358-13. Pubmed
  • Koyuncu OO, Hogue IB, Enquist LW. (2013) Virus infections in the nervous system. Cell Host Microbe. 13: 379-93. Pubmed
  • Kratchmarov R, Kramer T, Greco TM, Taylor MP, Ch'ng TH, Cristea IM, Enquist LW. (2013) Glycoproteins gE and gI are required for efficient KIF1A-dependent anterograde axonal transport of alphaherpesvirus particles in neurons. J Virol. 87: 9431-40. Pubmed
  • Kratchmarov R, Taylor MP, Enquist LW. (2013) Role of us9 phosphorylation in axonal sorting and anterograde transport of pseudorabies virus. PLoS One. 8: e58776. Pubmed
  • Kramer T, Enquist LW. (2013) Directional spread of alphaherpesviruses in the nervous system. Viruses 5: 678-707. Pubmed
  • Koyuncu OO, Perlman DH, Enquist LW. (2013) Efficient retrograde transport of pseudorabies virus within neurons requires local protein synthesis in axons. Cell Host Microbe. 13: 54-66. Pubmed
  • Kramer T, Greco TM, Taylor MP, Ambrosini AE, Cristea IM, Enquist LW. (2012) Kinesin-3 mediates axonal sorting and directional transport of alphaherpesvirus particles in neurons. Cell Host Microbe. 12: 806-14. Pubmed
  • Taylor MP, Kobiler O, Enquist LW. (2012) Alphaherpesvirus axon-to-cell spread involves limited virion transmission. Proc Natl Acad Sci. 109: 17046-51. Pubmed
  • Card JP, Enquist LW. (2012) Use and visualization of neuroanatomical viral transneuronal tracers. Visualization Techniques: From Immunohistochemistry to Magnetic Resonance Imaging. Neuromethods 70: 225-268. Journal link
  • Kratchmarov R, Taylor MP, Enquist LW. (2012) Making the case: married versus separate models of alphaherpes virus anterograde transport in axons. Rev Med Virol. 22: 378-91. Pubmed
  • Enquist LW (2012) Ten Years under the JVI Flag. J. Virol. 86: 7025-7026. Journal link
  • Kramer T, Enquist LW. (2012) Alphaherpesvirus infection disrupts mitochondrial transport in neurons. Cell Host Microbe. 11: 504-14. Pubmed
  • Taylor MP, Kramer T, Lyman MG, Kratchmarov R, Enquist LW. (2012) Visualization of an alphaherpesvirus membrane protein that is essential for anterograde axonal spread of infection in neurons. MBio. 3: e00063-12. Pubmed
  • Enquist LW. (2012) Five questions about viral trafficking in neurons. PLoS Pathog. 8: e1002472. Pubmed
  • Kobiler O, Brodersen P, Taylor MP, Ludmir EB, Enquist LW. (2011) Herpesvirus replication compartments originate with single incoming viral genomes. MBio. 2: e00278-11. Pubmed
  • Szpara ML, Tafuri YR, Parsons L, Shamim SR, Verstrepen KJ, Legendre M, Enquist LW. (2011) A wide extent of inter-strain diversity in virulent and vaccine strains of alphaherpesviruses. PLoS Pathog. 7: e1002282. Pubmed
  • Szpara ML, Tafuri YR, Enquist LW. (2011) Preparation of viral DNA from nucleocapsids. J Vis Exp. pii: 3151. doi: 10.3791/3151. Pubmed
  • Card JP, Kobiler O, Ludmir EB, Desai V, Sved AF, Enquist LW. (2011) A dual infection pseudorabies virus conditional reporter approach to identify projections to collateralized neurons in complex neural circuits. PLoS One. 6: e21141. Pubmed
  • Haugo AC, Szpara ML, Parsons L, Enquist LW, Roller RJ. (2011) Herpes simplex virus 1 pUL34 plays a critical role in cell-to-cell spread of virus in addition to its role in virus replication. J Virol. 85: 7203-15. Pubmed
  • Kramer T, Greco TM, Enquist LW, Cristea IM. (2011) Proteomic characterization of Pseudorabies virus extracellular virions. J Virol. 85: 6427-6441. Pubmed
  • Taylor MP, Koyuncu OO, Enquist LW. (2011) Subversion of the actin cytoskeleton during viral infection. Nat Rev Microbiol. 9: 427-439. Pubmed
  • Szpara ML, Parson L, Enquist LW (2010). Sequence variability in clinical and lab isolates of Herpes Simplex Virus 1 reveals new mutations. J Virol. 84: 5303-5313. PubMed
  • Szpara ML, Kobiler O, Enquist LW. (2010) A common neuronal response to alphaherpesvirus infection.. J Neuroimmune Pharmacol 5: 418-427. PubMed
  • Granstedt AE, Kuhn B, Wang SS, Enquist LW (2010). Calcium imaging of neuronal circuits in vivo using a circuit-tracing pseudorabies virus. Cold Spring Harb Protoc. 2010: pdb.prot5410. PubMed
  • Curanovic D, Enquist L. (2009) Directional transneuronal spread of alpha-herpesvirus infection. Future Virol. 4: 591-603. PubMed
  • McCarthy KM, Tank DW, Enquist LW. (2009) Pseudorabies virus infection alters neuronal activity and connectivity in vitro. PLoS Pathog. 5: e1000640. PubMed
  • Granstedt AE, Szpara ML, Kuhn B, Wang SS, Enquist LW. (2009) Fluorescence-based monitoring of in vivo neural activity using a circuit-tracing pseudorabies virus. PLoS One. 4: e6923. PubMed
  • Ludmir EB, Enquist LW. (2009) Viral genomes are part of the phylogenetic tree of life. Nat Rev Microbiol. 7: 615. PubMed
  • Curanovic D, Enquist LW. (2009) Virion-incorporated glycoprotein B mediates transneuronal spread of pseudorabies virus. J Virol. 83: 7796-4804. PubMed
  • Curanovic D, Ch'ng TH, Szpara M, Enquist L. (2009) Compartmented neuron cultures for directional infection by alpha herpesviruses. Curr Protoc Cell Biol. Chapter 26: Unit 26.4. PubMed
  • Lyman MG, Kemp CD, Taylor MP, Enquist LW. (2009) Comparison of the pseudorabies virus Us9 protein with homologs from other veterinary and human alphaherpesviruses. J Virol. 83: 6978-6986. PubMed
  • Enquist, L.W. for the Editors of the Journal of Virology. (2009) Virology in the 21st century. J Virol. 83: 5296-5308. PubMed
  • Lyman MG, Enquist LW. (2009) Herpesvirus interactions with the host cytoskeleton. J Virol. 83: 2058-2066. PubMed
  • Curanović D, Lyman MG, Bou-Abboud C, Card JP, Enquist LW. (2009) Repair of the UL21 locus in pseudorabies virus Bartha enhances the kinetics of retrograde, transneuronal infection in vitro and in vivo. J Virol. 83: 1173-1183. PubMed
  • Lyman MG, Curanovic D, Brideau AD, Enquist LW. (2008) Fusion of enhanced green fluorescent protein to the pseudorabies virus axonal sorting protein Us9 blocks anterograde spread of infection in mammalian neurons. J Virol. 82: 10308-10311. PubMed
  • Liu WW, Goodhouse J, Jeon NL, Enquist LW. (2008) A microfluidic chamber for analysis of neuron-to-cell spread and axonal transport of an alpha-herpesvirus. PLoS One. 3: e2382. PubMed
  • Lyman MG, Curanovic D, Enquist LW. (2008) Targeting of pseudorabies virus structural proteins to axons requires association of the viral Us9 protein with lipid rafts. PLoS Pathog. 4: e1000065. PubMed
  • Ekstrand MI, Enquist LW, Pomeranz LE. (2008) The alpha-herpesviruses: molecular pathfinders in nervous system circuits. Trends Mol Med. 14: 134-140. PubMed
  • Lyman MG, Feierbach B, Curanovic D, Bisher M, Enquist LW. (2007) Pseudorabies virus Us9 directs axonal sorting of viral capsids. J Virol. 81: 11363-11371. PubMed
  • Favoreel HW, Enquist LW, Feierbach B. (2007) Actin and Rho GTPases in herpesvirus biology. Trends Microbiol. 15: 426-433. PubMed
  • Ch'ng TH, Spear PG, Struyf F, Enquist LW. (2007) Glycoprotein D-independent spread of pseudorabies virus infection in cultured peripheral nervous system neurons in a compartmented system. J Virol. 81: 10742-10757. PubMed
  • Feierbach B, Bisher M, Goodhouse J, Enquist LW. (2007) In vitro analysis of transneuronal spread of an alphaherpesvirus infection in peripheral nervous system neurons. J Virol. 81: 6846-6857. PubMed
  • Viney TJ, Balint K, Hillier D, Siegert S, Boldogkoi Z, Enquist LW, Meister M, Cepko CL, Roska B. (2007) Local retinal circuits of melanopsin-containing ganglion cells identified by transsynaptic viral tracing. 17: 981-988. PubMed
  • Reynolds AE, Enquist LW (2006). Biological interactions between herpesviruses and cyclooxygenase enzymes. Rev Med Virol. 16: 393-403. PubMed
  • Feierbach B, Piccinotti S, Bisher M, Denk W, Enquist LW (2006). Alpha-herpesvirus infection induces the formation of nuclear actin filaments. PLoS Pathog 2: e85. PubMed
  • Paulus C, Sollars PJ, Pickard GE and Enquist LW (2006). Transcriptome signature of virulent and attenuated pseudorabies virus-infected rodent brain. J Virol 80: 1773-1786. PubMed
  • Song CK, Enquist LW and Bartness TJ (2005). New developments in tracing neural circuits with herpesviruses. Virus Res 111: 235-249. PubMed
  • del Rio T, DeCoste CJ and Enquist LW (2005). Actin is a component of the compensation mechanism in pseudorabies virus virions lacking the major tegument protein VP22. J Virol 79: 8614-8619. PubMed
  • Ch'ng TH and Enquist LW (2005). Neuron-to-cell spread of pseudorabies virus in a compartmented neuronal culture system. J Virol 79: 10875-10889. PubMed
  • Ch'ng TH and Enquist LW (2005). Efficient axonal localization of alphaherpesvirus structural proteins in cultured sympathetic neurons requires viral glycoprotein E. J Virol 79: 8835-8846. PubMed
  • Smith GA, Pomeranz L, Gross SP and Enquist LW (2004). Local modulation of plus-end transport targets herpesvirus entry and egress in sensory axons. Proc Natl Acad Sci. 101: 16034-16039. PubMed
  • Smeraski CA, Sollars PJ, Ogilvie MD, Enquist LW and Pickard GE (2004). Suprachiasmatic nucleus input to autonomic circuits identified by retrograde transsynaptic transport of pseudorabies virus from the eye. J Comp Neurol 471: 298-313. PubMed
  • Ray N and Enquist LW (2004). Transcriptional response of a common permissive cell type to infection by two diverse alphaherpesviruses. J Virol 78: 3489-3501. PubMed
  • Klupp BG, Hengartner CJ, Mettenleiter TC and Enquist LW (2004). Complete, annotated sequence of the pseudorabies virus genome. J Virol 78: 424-440. PubMed
  • Brittle EE, Reynolds AE and Enquist LW (2004). Two modes of pseudorabies virus neuroinvasion and lethality in mice. J Virol 78: 12951-12963. PubMed
  • Enquist LW and Card JP (2003). Recent advances in the use of neurotropic viruses for circuit analysis. Curr Opin Neurobiol 13: 603-606. PubMed