Individual herpes simplex virus 1 (HSV-1) particles exit by exocytosis and accumulate at preferential egress sites.

The human pathogen herpes simplex virus 1 (HSV-1) produces a lifelong infection in the majority of the world's population. While the generalities of alpha herpesvirus assembly and egress pathways are known, the precise molecular and spatiotemporal details remain unclear. In order to study this aspect of HSV-1 infection, we engineered a recombinant HSV-1 strain expressing a pH-sensitive reporter, gM-pHluorin. Using a variety of fluorescent microscopy modalities, we can detect individual virus particles undergoing intracellular transport and exocytosis at the plasma membrane. We show that particles exit from epithelial cells individually, not bulk release of many particles at once, as has been reported for other viruses. In multiple cell types, HSV-1 particles accumulate over time at the cell periphery and cell-cell contacts. We show that this accumulation effect is the result of individual particles undergoing exocytosis at preferential sites and that these egress sites can contribute to cell-cell spread. We also show that the viral membrane proteins gE, gI, and US9, which have important functions in intracellular transport in neurons, are not required for preferential egress and clustering in non-neuronal cells. Importantly, by comparing HSV-1 to a related alpha herpesvirus, pseudorabies virus, we show that this preferential exocytosis and clustering effect are cell type dependent, not virus dependent. This preferential egress and clustering appear to be the result of the arrangement of the microtubule cytoskeleton, as virus particles co-accumulate at the same cell protrusions as an exogenous plus end-directed kinesin motor.IMPORTANCEAlpha herpesviruses produce lifelong infections in their human and animal hosts. The majority of people in the world are infected with herpes simplex virus 1 (HSV-1), which typically causes recurrent oral or genital lesions. However, HSV-1 can also spread to the central nervous system, causing severe encephalitis, and might also contribute to the development of neurodegenerative diseases. Many of the steps of how these viruses infect and replicate inside host cells are known in depth, but the final step, exiting from the infected cell, is not fully understood. In this study, we engineered a novel variant of HSV-1 that allows us to visualize how individual virus particles exit from infected cells. With this imaging assay, we investigated preferential egress site formation in certain cell types and their contribution to the cell-cell spread of HSV-1.

LIVE-CELL FLUORESCENCE MICROSCOPY OF HSV-1 CELLULAR EGRESS BY EXOCYTOSIS.

The human pathogen Herpes Simplex Virus 1 (HSV-1) produces a lifelong infection in the majority of the world's population. While the generalities of alpha herpesvirus assembly and egress pathways are known, the precise molecular and spatiotemporal details remain unclear. In order to study this aspect of HSV-1 infection, we engineered a recombinant HSV-1 strain expressing a pH-sensitive reporter, gM-pHluorin. Using a variety of fluorescent microscopy modalities, we can detect individual virus particles undergoing intracellular transport and exocytosis at the plasma membrane. We show that particles exit from epithelial cells individually, not bulk release of many particles at once, as has been reported for other viruses. In multiple cell types, HSV-1 particles accumulate over time at the cell periphery and cell-cell contacts. We show that this accumulation effect is the result of individual particles undergoing exocytosis at preferential sites and that these egress sites can contribute to cell-cell spread. We also show that the viral membrane proteins gE, gI, and US9, which have important functions in intracellular transport in neurons, are not required for preferential egress and clustering in non-neuronal cells. Importantly, by comparing HSV-1 to a related alpha herpesvirus, pseudorabies virus, we show that this preferential exocytosis and clustering effect is cell type-dependent, not virus dependent. This preferential egress and clustering appears to be the result of the arrangement of the microtubule cytoskeleton, as virus particles co-accumulate at the same cell protrusions as an exogenous plus end-directed kinesin motor.

The Use of Campenot Trichambers for the Study of Peripheral Neuronal Growth and Survival in Presence of Thrombotic Factors and Serpins.

The mechanisms underlying nervous system injury, such as spinal cord injury (SCI), traumatic brain injury (TBI), and peripheral nerve injury are complex and not well understood. Following acute tissue damage and cell death, inflammatory processes cause ongoing damage. Many factors regulate this inflammation, including factors that modulate chemokine expression. Serine proteases, including those of the thrombotic and thrombolytic pathways (e.g., thrombin, tPA, uPA) are upregulated during nervous system damage and can modulate the release and bioavailability of many chemokines. Virus-derived immunomodulators, such as Serp-1, a serine protease inhibitor (serpin), have protective effects by reducing inflammation and tissue damage. However, the precise mechanisms of Serp-1 neuroprotection are still being studied. Compartmentalized in vitro neuron culture systems, such as the Campenot trichamber, are useful for such mechanistic studies. This chapter provides a protocol for assembling and culturing rodent embryonic superior cervical ganglion (SCG) and dorsal root ganglion (DRG) neurons in Campenot trichambers, as well as instructive examples of the types of experiments enabled by these methods.

Methods and Applications of Campenot Trichamber Neuronal Cultures for the Study of Neuroinvasive Viruses.

The development of compartmentalized neuron culture systems has been invaluable in the study of neuroinvasive viruses, including the alpha herpesviruses Herpes Simplex Virus 1 (HSV-1) and Pseudorabies Virus (PRV). This chapter provides updated protocols for assembling and culturing rodent embryonic superior cervical ganglion (SCG) and dorsal root ganglion (DRG) neurons in Campenot trichamber cultures. In addition, we provide several illustrative examples of the types of experiments that are enabled by Campenot cultures: (1) Using fluorescence microscopy to investigate axonal outgrowth/extension through the chambers, and alpha herpesvirus infection, intracellular trafficking, and cell-cell spread via axons. (2) Using correlative fluorescence microscopy and cryo electron tomography to investigate the ultrastructure of virus particles trafficking in axons.

Serine Proteases and Chemokines in Neurotrauma: New Targets for Immune Modulating Therapeutics in Spinal Cord Injury.

Progressive neurological damage after brain or spinal cord trauma causes loss of motor function and treatment is very limited. Clotting and hemorrhage occur early after spinal cord (SCI) and traumatic brain injury (TBI), inducing aggressive immune cell activation and progressive neuronal damage. Thrombotic and thrombolytic proteases have direct effects on neurons and glia, both healing and also damaging bidirectional immune cell interactions. Serine proteases in the thrombolytic cascade, tissue- and urokinase-type plasminogen activators (tPA and uPA), as well as the clotting factor thrombin, have varied effects, increasing neuron and glial cell growth and migration (tPA), or conversely causing apoptosis (thrombin) and activating inflammatory cell responses. tPA and uPA activate plasmin and matrix metalloproteinases (MMPs) that break down connective tissue allowing immune cell invasion, promoting neurite outgrowth. Serine proteases also activate chemokines. Chemokines are small proteins that direct immune cell invasion but also mediate neuron and glial cell communication. We are investigating a new class of therapeutics, virus-derived immune modulators; One that targets coagulation pathway serine proteases and a second that inhibits chemokines. We have demonstrated that local infusion of these biologics after SCI reduces inflammation providing early improved motor function. Serp-1 is a Myxomavirus-derived serine protease inhibitor, a serpin, that inhibits both thrombotic and thrombolytic proteases. M-T7 is a virus-derived chemokine modulator. Here we review the roles of thrombotic and thrombolytic serine proteases and chemoattractant proteins, chemokines, as potential therapeutic targets for SCI. We discuss virus-derived immune modulators as treatments to reduce progressive inflammation and ongoing nerve damage after SCI.

Transplanted Human Neural Progenitor Cells Attenuate Motor Dysfunction and Lengthen Longevity in a Rat Model of Ataxia.

The spastic Han Wistar (sHW) rat serves as a model for human ataxia presenting symptoms of motor deterioration, weight loss, shortened lifespan, and Purkinje neuron loss. Past studies revealed that human neural progenitor cells (NPCs) improved ataxic symptoms at 20 d posttransplantation in sHW rats. In this study, we investigated the fate and longer-term effectiveness of these transplanted NPCs. Rats were placed into four treatment groups: an untreated normal control group (n = 10), an untreated mutant rat control (n = 10), a mutant group that received an injection of dead NPCs (n = 9), and a mutant group that received live NPCs (n = 10). Bilateral cerebellar injections containing 500,000 of either live or dead NPCs were performed on mutant sHW rats at 40 d of age. Motor activity for all mutant rats started to decline in open field testing around day 35. However, at day 45, the live NPC-treated mutants exhibited significant improvements in open field activity. Similar improvements were observed during rotarod testing and weight gain through the completion of the experiments (100 d). Immunohistochemistry revealed few surviving human NPCs in the cerebella of 80- and 100-d-old NPC-treated mutants; while cresyl violet staining revealed that live NPC-treated mutants had significantly more surviving Purkinje neurons compared to mutants that were untreated or received dead NPCs. Direct stereotactic implantation of NPCs alleviated the symptoms of ataxia, acting as a neuroprotectant, supporting future clinical applications of these NPCs in the areas of ataxia as well as other neurodegenerative diseases.

Transplantation of Human Neural Progenitor Cells Reveals Structural and Functional Improvements in the Spastic Han-Wistar Rat Model of Ataxia.

The use of regenerative medicine to treat nervous system disorders like ataxia has been proposed to either replace or support degenerating neurons. In this study, we assessed the ability of human neural progenitor cells (hNPCs) to repair and restore the function of dying neurons within the spastic Han-Wistar rat (sHW), a model of ataxia. The sHW rat suffers from neurodegeneration of specific neurons, including cerebellar Purkinje cells and hippocampal CA3 pyramidal cells leading to the observed symptoms of forelimb tremor, hind-leg rigidity, gait abnormality, motor incoordination, and a shortened life span. To alleviate the symptoms of neurodegeneration and to replace or augment dying neurons, neuronal human progenitor cells were implanted into the sHW rats. At 30 d of age, male sHW mutant rats underwent subcutaneous implantation of an Alzet osmotic pump that infused cyclosporine (15 mg/kg/d) used to suppress the rat's immune system. At 40 d, sHW rats received bilateral injections (500,000 cells in 5 µL media) of live hNPCs, dead hNPCs, live human embryonic kidney cells, or growth media either into the cerebellar cortex or into the hippocampus. To monitor results, motor activity scores (open-field testing) and weights of the animals were recorded weekly. The sHW rats that received hNPC transplantation into the cerebellum, at 60 d of age, displayed significantly higher motor activity scores and sustained greater weights and longevities than control-treated sHW rats or any hippocampal treatment group. In addition, cerebellar histology revealed that the transplanted hNPCs displayed signs of migration and signs of neuronal development in the degenerated Purkinje cell layer. This study revealed that implanted human progenitor cells reduced the ataxic symptoms in the sHW rat, identifying a future clinical use of these progenitor cells against ataxia and associated neurodegenerative diseases.