Jennifer H. LaVail, Ph.D. - US grants

Important nutrients or effector molecules, e.g., hormones, growth factors, toxins, enxymes or pathogens, are taken in by the neuronal plasma membrane and must be sorted to specific sites in the cell. Uptake and sorting of membrane compartments by epithelial cells is a research field of intense current investigation. However, relatively little attention is being focused on neurons, despite their highly elongated shape and vital interdependence on one another. In this grant the identity of the membrane compartments that transport macromolecules from the surface of the perikaryon to the axon terminal will be examined. We hypothesize that this path will involve perikaryal membrane that is fated for exocytosis by nerve terminals, i.e., neurons are capable of transcellular transport. Wheat germ agglutinin (WGA) will be used as a selective probe for the glycoproteins of neuronal membranes, due to its ability to bind selectively to N-acetyglucosamine and sialic acid residues. Iodiated WGA and EM autoradiography as well as immunocytochemical techniques will be the prime methods of procedure used to localize the WGA in various cellular compartments in the neuron cell body or axon. Evidence will also be sought for the intercellular transfer of WGA from nerve to muscle at the neuromuscular junction. Understanding the intracellular path followed by WGA through the neuron will provide us with new insight about the circulation of membrane within neurons. The information will also increase our understanding not only of axonal transport mechanisms, in general, but also of the cellular basis for many pathological conditions, including pathological intoxications with environmental toxins. We need to know the normal patterns of organelle and membrane sorting if we are to understand fully abnormal situations, either peripherally (e.g., peripheral neuropathies) or centrally (e.g., optic nerve compression in glaucoma). Moreover, information gained about the transfer of macromolecules between neurons should clarify the mechanisms by which trophic factors from neurons influence muscle cell development or neurons influence other neurons during development. Lastly, understanding the cellular basis for the transport of probes is expected to lead to the improved application of these probes in neuroanatomical tracing studies.

DESCRIPTION (provided by applicant): Understanding the mechanisms of axonal transport of neurotropic viruses is the key to understanding and controlling their spread in the nervous system. To multiply and spread, to move initially from the site of entry and later to the site of release, Herpes simplex virus (HSV) must use neuronal host cell proteins and mechanisms. Our understanding of the interplay of specific viral proteins that piggyback on the mechanisms and of the neuronal proteins that are exploited, is critical but almost nonexistent. In this application we continue to focus on the anterograde transport of HSV. Based on our previous results, our hypothesis is that the nucleocapsid and envelope components of HSV are independently transported in the axon and that the components require specific kinesin related proteins. 1) We have begun to examine the transport of the nucleocapsid and envelope components. To do this we have developed two viral mutant strains and revertants that will facilitate our research into these transport mechanisms. 2) In this proposal we shall continue these studies and carry out co-immunoprecipitation assays to identify the motor proteins associated with the nucleocapsid component. 3) We shall also determine whether or not the virus egresses from the cell by budding, after the envelope proteins are delivered to the axon membrane and the nucleocapsids cluster near that region of membrane. These results will provide important new cell biological information about the recognition signals of particular organelles. They will also have significant clinical benefits. The anterograde transport of HSV to the cornea in human herpetic keratitis results in severe consequences, including corneal scars, glaucoma and possibly encephalitis. Our results will provide new insight into the identification of viral and host proteins necessary for viral envelope and nucleocapsid transport and a rational basis for the design of innovative antiviral drugs for prevention and intervention. Furthermore, the genome of HSV can be altered to serve as a vector for introduction of novel genes into the nervous system. Our results will elucidate the mechanisms that target the vector to particular neuron types and to particular regions of infected neurons.

The ability of a virus to usurp the normal cell biology of a host cell for viral replication and spread depends on both viral gene expression and host cell responses. The key to controlling viral invasion of an epithelium lies in understanding the mechanisms by which virus spreads within and between host cells. We shall focus on mechanisms by which Herpes simplex virus type 1 (HSV) invades and usurps the machinery of corneal epithelial cells. The available information about the regulation of HSV infection and spread in the cornea is limited. However, it is clear that at least three mechanisms are involved. 1) Free virus can enter the cornea from a distance, attach and fuse with a host cell plasma membrane and enter the cell (viral fusion mechanism). 2) A different mechanism is used for transfer of virions from an infected cell to coupled adjacent cells (cell-to-cell spread mechanism). 3) Finally, a less widely recognized mechanism involves the movement of virus in an infected cell as the cell moves from one location to another (translational mechanism). We hypothesize that viral envelope glycoproteins and host cell surface receptors play key roles in the spread of HSV in cornea in the first and second mechanisms, but not in the translational mechanism. We will use two mouse model systems: first, primary infection with virus delivered to the corneal surface injury; and second, a model of recurrent herpetic infection by viral delivery to the basal surface of the intact cornea via infected trigeminal ganglion cell axons. Using genetic, pharmacological and immunocytochemical tools, we shall define the contributions of viral fusion and cell-to-cell spread by their different sensitivity to mutations in the HSV envelope glycoproteins. The role of translocation of virally infected cells will be determined by delivering HSV as a pulse, using Valacyclovir to eliminate secondary cell infection. These in vivo experiments will provide fundamental, cell biological information about the transfer of HSV between squamous epithelial cells in vivo and the response of host cell junctions to injury. In addition, the results will also have significant clinical benefits. A better understanding of the differences between spread of virus after corneal injury and of the spread that results from reactivated HSV delivered to the cornea in human herpetic keratitis will focus attention on whether different strategies are required for treatment of initial as opposed to recurrent infections. Moreover, our results will lead to identification of viral and host proteins that are necessary for viral spread and a rational basis for the design of innovative antiviral drugs.

Affinity; Agarose; Antibodies; Axon Terminals; Axonal Transport; Axoplasmic Transport; Biochemical; CRISP; Capsid; Capsid Proteins; Cell Body; Cell model; Cell/Tissue, Immunohistochemistry; Cellular model; Coat Proteins; Computer Retrieval of Information on Scientific Projects Database; Coupled; Cytoplasm; DNA; DNA, Viral; Deoxyribonucleic Acid; Funding; Ganglion Cells (Retina); Gel; Grant; HHV-1; HSV-1; HSV1; Herpes Simplex Virus 1; Herpes Simplex Virus Type 1; Herpesvirus 1 (alpha), Human; Herpesvirus 1, Human; Human herpes simplex virus type 1; Human herpesvirus 1; Human herpesvirus type 1; IHC; Immunohistochemistry; Immunohistochemistry Staining Method; Institution; Investigators; Label; Mammals, Mice; Mass Spectrum; Mass Spectrum Analysis; Mice; Modeling; Motor Cell; Motor Neurons; Murine; Mus; NIH; National Institutes of Health; National Institutes of Health (U.S.); Nerve Cells; Nerve Endings, Presynaptic; Nerve Unit; Neural Cell; Neurocyte; Neurons; Nucleocapsid; Photometry/Spectrum Analysis, Mass; Presynaptic Terminals; Process; Proteins; Research; Research Personnel; Research Resources; Researchers; Resources; Retinal Ganglion Cells; Sepharose; Silver Staining; Source; Spectrometry, Mass; Spectroscopy, Mass; Spectrum Analyses, Mass; Spectrum Analysis, Mass; Synaptic Boutons; Synaptic Terminals; Transmission; United States National Institutes of Health; Viral; Viral Coat Proteins; Viral Envelope Proteins; Viral Gene Products; Viral Gene Proteins; Viral Outer Coat Protein; Viral Proteins; Virus; Viruses, General; Visual System; Visual system structure; anterograde transport; cell body (neuron); coat (nonenveloped virus); gene product; herpes simplex i; herpes virus 1, human; human alphaherpesvirus 1; motoneuron; mutant; neural cell body; neuronal; neuronal cell body; retinal ganglion; silver impregnation; soma; synaptobrevin; therapeutic target; tool; transmission process; vesicle-associated membrane protein; viral DNA; virus DNA; virus protein

DESCRIPTION (provided by applicant): Herpes simplex virus (HSV) infections are responsible for recurrent corneal herpetic keratitis, as well as sporatic acute encephalitis. Even with antiviral treatment, the incidence of mortality or severe neurological deficits after HSV encephalitis remains high. Our long-term goal is to identify the viral and infected host proteins that promote the spread of HSV into the central nervous system. Using a novel in vivo mouse retinal ganglion cell model, we have found that the HSV viral protein, Us9, is necessary for the long distance spread specifically of viral nucleocapsid and viral DNA from an infected neuron cell body toward the axon terminal. Understanding the mechanisms underlying targeted delivery of virus leads directly to the identification of vulnerable steps in HSV and other neurotropic viral infections, such as CMV and VZV. We now propose to define the regions of the Us9 that are necessary for efficient nucleocapsid sorting. We shall also define the viral proteins and host proteins that associate with Us9 in infected axons. Lastly, we shall test the function of virus and host motor proteins in the delivery of nucleocapsids within an infected axon using a novel, microscale culture system. Understanding the pathophysiology of the virus in mature neurons is important, because the mechanisms of HSV transmission between neurons is essential for development of new antiviral drugs that block viral encephalitic spread. In addition, what we learn about HSV transmission will be relevant for fighting other virus infections. PUBLIC HEALTH RELEVANCE: Herpes simplex type 1 (HSV) and type 2 are responsible for the majority of herpetic encephalitis cases. HSV is also the pathogen responsible for recurrent scarring of the corneal epithelium in ocular herpetic keratitis, a common cause of blindness. HSV can become latent in infected neurons and can become resistant to treatment with acyclovir and acyclovir-derived drugs. Thus, exploration of the mechanisms used by the virus to infect sensory neurons and travel to the central nervous system is of value in that it will provide insight into potential vulnerabilities of the pathogen that can be exploited therapeutically.