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Alphaherpesvirus tegument assembly, secondary envelopment, and exocytosis processes are understood in broad strokes, but many of the individual steps in this pathway, and their molecular and cell biological details, remain unclear. Viral tegument and membrane proteins form an extensive and robust protein interaction network, such that essentially any structural protein can be deleted, yet particles are still assembled, enveloped, and released from infected cells. We conceptually divide the tegument proteins into three groups: conserved inner and outer teguments that participate in nucleocapsid and membrane contacts, respectively, and 'middle' tegument proteins, consisting of some of the most abundant tegument proteins that serve as central hubs in the protein interaction network, yet which are unique to the alphaherpesviruses. We then discuss secondary envelopment, reviewing the tegument-membrane contacts and cellular factors that drive this process. We place this viral process in the context of cell biological processes, including the endocytic pathway, ESCRT machinery, autophagy, secretory pathway, intracellular transport, and exocytosis mechanisms. Finally, we speculate about potential relationships between cellular defenses against oligomerizing or aggregating membrane proteins and the envelopment and egress of viruses.

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 in clusters at the corners, edges, and cell-cell contacts. We show that this clustering effect is the result of individual particles undergoing exocytosis at preferential sites. We also show that the viral membrane proteins gE, gI, and US9, which have important functions in intracellular transport in neurons, have a subtle effect on 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.ImportanceAlpha herpesviruses produce lifelong infections of 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.

Antivirals are indispensable tools that can be targeted at viral domains directly or indirectly at cellular domains to obstruct viral infections and reduce pathogenicity. Despite their transformative use in healthcare, antivirals have been clinically approved to treat only 10 of the more than 200 known pathogenic human viruses. Additionally, many virus functions are intimately coupled with host cellular processes, which present challenges in antiviral development due to the limited number of clear targets per virus, necessitating an extensive insight into these molecular processes. Compounding this challenge, many viral pathogens have evolved to evade effective antivirals. We hypothesize that a Viral Attachment Blocking Chimera (VirABloC) composed of a viral binder and a bulky scaffold that sterically blocks interactions between a viral particle and a host cell may be suitable for the development of antivirals agnostic to the extravirion epitope that is being bound. We test this hypothesis by modifying a nanobody that specifically recognizes a non-essential epitope presented on the extra virion surface of Pseudorabies virus strain 486 with a 3-dimensional wireframe DNA origami structure ∼100 nm in diameter. The nanobody switches from having no inhibitory properties (tested up to 50 µM) to 4.2 ± 0.9 nM IC50when conjugated with the DNA origami scaffold. Mechanistic studies support that inhibition is mediated by the non-covalent attachment of the DNA origami scaffold to the virus particle, which obstructs the attachment of the viruses onto host cells. These results support the potential of VirABloC as a generalizable approach to developing antivirals.

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.

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.

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.

The central nervous system has long been thought to regulate insulin secretion, an essential process in the maintenance of blood glucose levels. However, the anatomical and functional connections between the brain and insulin-producing pancreatic β-cells remain undefined. Here, we describe a functional transneuronal circuit connecting the hypothalamus to β-cells in mice. This circuit originates from a subpopulation of oxytocin neurons in the paraventricular hypothalamic nucleus (PVN(OXT)) and via the sympathetic autonomic branch it reaches the islets of the endocrine pancreas to innervate β-cells. Stimulation of PVN(OXT) neurons rapidly suppresses insulin secretion and causes hyperglycemia. Conversely, silencing of these neurons elevates insulin levels by dysregulating neuronal signaling and secretory pathways in β-cells and induces hypoglycemia. PVN(OXT) neuronal activity is triggered by glucoprivation. Our findings thus reveal that a subset of PVN(OXT) neurons form functional multisynaptic circuits with β-cells in mice to regulate insulin secretion and their function is necessary for the β-cell response to hypoglycemia.

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.

Axonal sorting, the controlled passage of specific cargoes from the cell soma into the axon compartment, is critical for establishing and maintaining the polarity of mature neurons. To delineate axonal sorting events, we took advantage of two neuroinvasive alpha-herpesviruses. Human herpes simplex virus 1 (HSV-1) and pseudorabies virus of swine (PRV; suid herpesvirus 1) have evolved as robust cargo of axonal sorting and transport mechanisms. For efficient axonal sorting and subsequent egress from axons and presynaptic termini, progeny capsids depend on three viral membrane proteins (Us7 (gI), Us8 (gE), and Us9), which engage axon-directed kinesin motors. We present evidence that Us7-9 of the veterinary pathogen pseudorabies virus (PRV) form a tripartite complex to recruit Kif1a, a kinesin-3 motor. Based on multi-channel super-resolution and live TIRF microscopy, complex formation and motor recruitment occurs at the trans-Golgi network. Subsequently, progeny virus particles enter axons as enveloped capsids in a transport vesicle. Artificial recruitment of Kif1a using a drug-inducible heterodimerization system was sufficient to rescue axonal sorting and anterograde spread of PRV mutants devoid of Us7-9. Importantly, biophysical evidence suggests that Us9 is able to increase the velocity of Kif1a, a previously undescribed phenomenon. In addition to elucidating mechanisms governing axonal sorting, our results provide further insight into the composition of neuronal transport systems used by alpha-herpesviruses, which will be critical for both inhibiting the spread of infection and the safety of herpesvirus-based oncolytic therapies., Author summary Alpha-herpesviruses represent a group of large, enveloped DNA viruses that are capable to establish a quiescent (also called latent) but reactivatable form of infection in the peripheral nervous system of their hosts. Following reactivation of latent genomes, virus progeny is formed in the soma of neuronal cells and depend on sorting into the axon for anterograde spread of infection to mucosal sites and potentially new host. We studied two alpha-herpesviruses (the veterinary pathogen pseudorabies virus (PRV) and human herpes simplex virus 1 (HSV-1)) and found viral membrane proteins Us7, Us8, and Us9 form a complex, which is able to recruit kinsin-3 motors. Motor recruitment facilitates axonal sorting and subsequent transport to distal egress sites. Complex formation occurs at the trans-Golgi network and mediates efficiency of axonal sorting and motility characteristics of egressing capsids. We also used an artificial kinesin-3 recruitment system, which allows controlled induction of axonal sorting and transport of virus mutants lacking Us7, Us8, and Us9. Overall, these data contribute to our understanding of anterograde alpha-herpesvirus spread and kinesin-mediated sorting of vesicular axonal cargoes.

Alpha herpesviruses naturally infect the peripheral nervous system, and can spread to the central nervous system causing severe deadly or debilitating disease. Because alpha herpesviruses spread along synaptic circuits, and infected neurons exhibit altered electrophysiology and increased spontaneous firing, we hypothesized that alpha herpesviruses use activity-dependent synaptic vesicle-like regulated secretory mechanisms for egress and spread from neurons. To address this hypothesis, we used a compartmentalized primary neuron culture system to measure egress and spread of pseudorabies virus (PRV), pharmacological and optogenetics approaches to modulate neuronal firing activity, and a live-cell fluorescence microscopy assay to directly visualize the exocytosis of individual virus particles from infected neurons. Using tetrodotoxin to silence neuronal activity, we observed no inhibition of virus spread, and using potassium chloride or optogenetics to elevate neuronal activity, we also show no increase in virus spread. Using a live-cell fluorescence microscopy method to directly measure virus egress from infected neurons, we observed no association between virus particle exocytosis and intracellular Ca2+ signaling. Finally, we observed virus particle exocytosis occurs in association with constitutive secretory Rab GTPases, Rab6a and Rab8a, not Rab proteins that are associated with the Ca2+-regulated secretory pathway in neurons, Rab3a and Rab11a. Therefore, we conclude that alpha herpesvirus egress and spread is independent of neuronal activity and Ca2+ signaling because virus particle exocytosis uses constitutive secretory mechanisms in neurons.Author SummaryAlpha herpesviruses, including important human pathogens Herpes Simplex Virus 1 and 2, and Varicella-Zoster Virus, are among the very few viruses that naturally infect the nervous system. These viruses cause recurrent herpetic and zosteriform lesions, peripheral neuropathies, and deadly or debilitating central nervous system diseases. Many of the molecular and cellular mechanisms of viral egress and spread remain unknown, particularly in the context of specialized neuronal cell biology. Our results indicate that elevated firing activity of infected neurons is not functionally or mechanistically linked to virus egress and spread; therefore, therapies targeting peripheral neuropathic symptoms, elevated neuronal activity, and synaptic vesicle secretory mechanisms are unlikely to affect virus spread in the nervous system.

Nicotine self-administration is associated with decreased expression of the glial glutamate transporter (GLT-1) and the cystine-glutamate exchange protein xCT within the nucleus accumbens core (NAcore). N-acetylcysteine (NAC) has been shown to restore these proteins in a rodent model of drug addiction and relapse. However, the specific molecular mechanisms driving its inhibitory effects on cue-induced nicotine reinstatement are unknown. Here, we confirm that extinction of nicotine-seeking behavior is associated with impaired NAcore GLT-1 function and expression and demonstrate that reinstatement of nicotine seeking rapidly enhances membrane fraction GLT-1 expression. Extinction and cue-induced reinstatement of nicotine seeking was also associated with increased tumor necrosis factor alpha (TNFα) and decreased glial fibrillary acidic protein (GFAP) expression in the NAcore. NAC treatment (100 mg/kg/day, i.p., for 5 days) inhibited cue-induced nicotine seeking and suppressed AMPA to NMDA current ratios, suggesting that NAC reduces NAcore post-synaptic excitability. In separate experiments, rats received NAC and an antisense vivo-morpholino to selectively suppress GLT-1 expression in the NAcore during extinction and were subsequently tested for cue-induced reinstatement of nicotine seeking. NAC treatment rescued NAcore GLT-1 expression and attenuated cue-induced nicotine seeking, which was blocked by GLT-1 antisense. NAC also reduced TNFα expression in the NAcore. Viral manipulation of the NF-κB pathway, which is downstream of TNFα, revealed that cue-induced nicotine seeking is regulated by NF-κB pathway signaling in the NAcore independent of GLT-1 expression. Ultimately, these results are the first to show that immunomodulatory mechanisms may regulate known nicotine-induced alterations in glutamatergic plasticity that mediate cue-induced nicotine-seeking behavior.

Inflammatory bowel disease (IBD) and Clostridium difficile infection cause gastrointestinal (GI) distension and, in severe cases, toxic megacolon with risk of perforation and death. Herpesviruses have been linked to severe GI dilatation. MHV-68 is a model for human gamma herpesvirus infection inducing GI dilatation in interleukin-10 (IL-10)-deficient mice but is benign in wildtype mice. MHV-68 also causes lethal vasculitis and pulmonary hemorrhage in interferon gamma receptor-deficient (IFN&gamma, R&minus, /&minus, ) mice, but GI dilatation has not been reported. In prior work the Myxomavirus-derived anti-inflammatory serpin, Serp-1, improved survival, reducing vasculitis and pulmonary hemorrhage in MHV-68-infected IFN&gamma, mice with significantly increased IL-10. IL-10 has been investigated as treatment for GI dilatation with variable efficacy. We report here that MHV-68 infection produces severe GI dilatation with inflammation and gut wall degradation in 28% of INF&gamma, R-/- mice. Macrophage invasion and smooth muscle degradation were accompanied by decreased concentrations of T helper (Th2), B, monocyte, and dendritic cells. Plasma and spleen IL-10 were significantly reduced in mice with GI dilatation, while interleukin-1 beta (IL-1&beta, ), IL-6, tumor necrosis factor alpha (TNF&alpha, ) and INF&gamma, increased. Treatment of gamma herpesvirus-infected mice with exogenous IL-10 prevents severe GI inflammation and dilatation, suggesting benefit for herpesvirus-induced dilatation.

Background Viral transneuronal tracing has become a well established technology used to define the synaptic architecture of polysynaptic neural networks. New Method In this report we define the neuroinvasive profile and reporter expression of a new recombinant of the Bartha strain of pseudorabies virus (PRV). The new recombinant, PRV-290, expresses the mTurquoise2 fluorophor and is designed to complement other isogenic recombinants of Bartha that express different reporters of infection. Results & Comparison with Existing Methods: PRV-290 was injected either alone or in combination with isogenic recombinants of PRV that express enhanced green fluorescent protein (EGFP; PRV-152) or monomeric red fluorescent protein (mRFP; PRV-614). Circuits previously defined using PRV-152 and PRV-614 were used for the analysis. The data demonstrate that PRV-290 is a retrograde transneuronal tracer with temporal kinetics similar to those of its isogenic recombinants. Stable expression of the diffusible mTurquoise2 reporter filled infected neurons, with the extent and intensity of labeling increasing with advancing post inoculation survival. In multiple injection experiments, PRV-290 established productive infections in neurons also replicating PRV-152 and/or PRV-614. This novel demonstration of three recombinants infecting individual neurons represents an important advance in the technology. Conclusion Collectively, these data demonstrate that PRV-290 is a valuable addition to the viral tracer toolbox for transneuronal tracing of neural circuitry.

In the nearly two decades since the popularization of green fluorescent protein (GFP), fluorescent protein-based methodologies have revolutionized molecular and cell biology, allowing us to literally see biological processes as never before. Naturally, this revolution has extended to virology in general, and to the study of alpha herpesviruses in particular. In this review, we provide a compendium of reported fluorescent protein fusions to herpes simplex virus 1 (HSV-1) and pseudorabies virus (PRV) structural proteins, discuss the underappreciated challenges of fluorescent protein-based approaches in the context of a replicating virus, and describe general strategies and best practices for creating new fluorescent fusions. We compare fluorescent protein methods to alternative approaches, and review two instructive examples of the caveats associated with fluorescent protein fusions, including describing several improved fluorescent capsid fusions in PRV. Finally, we present our future perspectives on the types of powerful experiments these tools now offer.

Fluorescent protein fusions to herpesvirus capsids have proven to be a valuable method to study virus particle transport in living cells. Fluorescent protein fusions to the amino terminus of small capsid protein VP26 are the most widely used method to visualize pseudorabies virus (PRV) and herpes simplex virus (HSV) particles in living cells. However, these fusion proteins do not incorporate to full occupancy and have modest effects on virus replication and pathogenesis. Recent cryoelectron microscopy studies have revealed that herpesvirus small capsid proteins bind to capsids via their amino terminus, whereas the carboxy terminus is unstructured and therefore may better tolerate fluorescent protein fusions. Here, we describe a new recombinant PRV expressing a carboxy-terminal VP26-mCherry fusion. Compared to previously characterized viruses expressing amino-terminal fusions, this virus expresses more VP26 fusion protein in infected cells and incorporates more VP26 fusion protein into virus particles, and individual virus particles exhibit brighter red fluorescence. We performed single-particle tracking of fluorescent virus particles in primary neurons to measure anterograde and retrograde axonal transport, demonstrating the usefulness of this novel VP26-mCherry fusion for the study of viral intracellular transport. IMPORTANCE Alphaherpesviruses are among the very few viruses that are adapted to invade the mammalian nervous system. Intracellular transport of virus particles in neurons is important, as this process underlies both mild peripheral nervous system infection and severe spread to the central nervous system. VP26, the small capsid protein of HSV and PRV, was one of the first herpesvirus proteins to be fused to a fluorescent protein. Since then, these capsid-tagged virus mutants have become a powerful tool to visualize and track individual virus particles. Improved capsid tags will facilitate fluorescence microscopy studies of virus particle intracellular transport, as a brighter particle will improve localization accuracy of individual particles and allow for shorter exposure times, reducing phototoxicity and improving the time resolution of particle tracking in live cells.

Many molecular and cell biological details of the alphaherpesvirus assembly and egress pathway remain unclear. Recently we developed a live-cell fluorescence microscopy assay of pseudorabies virus (PRV) exocytosis, based on total internal reflection fluorescence (TIRF) microscopy and a virus-encoded pH-sensitive fluorescent probe. Here, we use this assay to distinguish three classes of viral exocytosis in a nonpolarized cell type: (i) trafficking of viral glycoproteins to the plasma membrane, (ii) exocytosis of viral light particles, and (iii) exocytosis of virions. We find that viral glycoproteins traffic to the cell surface in association with constitutive secretory Rab GTPases and exhibit free diffusion into the plasma membrane after exocytosis. Similarly, both virions and light particles use these same constitutive secretory mechanisms for egress from infected cells. Furthermore, we show that viral light particles are distinct from cellular exosomes. Together, these observations shed light on viral glycoprotein trafficking steps that precede virus particle assembly and reinforce the idea that virions and light particles share a biogenesis and trafficking pathway., IMPORTANCE The alphaherpesviruses, including the important human pathogens herpes simplex virus 1 (HSV-1), HSV-2, and varicella-zoster virus (VZV), are among the few viruses that have evolved to exploit the mammalian nervous system. These viruses typically cause mild recurrent herpetic or zosteriform lesions but can also cause debilitating herpes encephalitis, more frequently in very young, old, immunocompromised, or nonnatural hosts. Importantly, many of the molecular and cellular mechanisms of viral assembly and egress remain unclear. This study addresses the trafficking of viral glycoproteins to the plasma membrane, exocytosis of light particles, and exocytosis of virions. Trafficking of glycoproteins affects immune evasion and pathogenesis and may precede virus particle assembly. The release of light particles may also contribute to immune evasion and pathogenesis. Finally, exocytosis of virions is important to understand, as this final step in the virus replication cycle produces infectious extracellular particles capable of spreading to the next round of host cells.

Bioinspired organ-level in vitro platforms are emerging as effective technologies for fundamental research, drug discovery, and personalized healthcare. In particular, models for nervous system research are especially important, due to the complexity of neurological phenomena and challenges associated with developing targeted treatment of neurological disorders. Here we introduce an additive manufacturing-based approach in the form of a bioinspired, customizable 3D printed nervous system on a chip (3DNSC) for the study of viral infection in the nervous system. Micro-extrusion 3D printing strategies enabled the assembly of biomimetic scaffold components (microchannels and compartmented chambers) for the alignment of axonal networks and spatial organization of cellular components. Physiologically relevant studies of nervous system infection using the multiscale biomimetic device demonstrated the functionality of the in vitro platform. We found that Schwann cells participate in axon-to-cell viral spread but appear refractory to infection, exhibiting a multiplicity of infection (MOI) of 1.4 genomes per cell. These results suggest that 3D printing is a valuable approach for the prototyping of a customized model nervous system on a chip technology.

The nuclear chromatin structure confines the movement of large macromolecular complexes to interchromatin corrals. Herpesvirus capsids of approximately 125 nm assemble in the nucleoplasm and must reach the nuclear membranes for egress. Previous studies concluded that nuclear herpesvirus capsid motility is active, directed, and based on nuclear filamentous actin, suggesting that large nuclear complexes need metabolic energy to escape nuclear entrapment. However, this hypothesis has recently been challenged. Commonly used microscopy techniques do not allow the imaging of rapid nuclear particle motility with sufficient spatiotemporal resolution. Here, we use a rotating, oblique light sheet, which we dubbed a ring-sheet, to image and track viral capsids with high temporal and spatial resolution. We do not find any evidence for directed transport. Instead, infection with different herpesviruses induced an enlargement of interchromatin domains and allowed particles to diffuse unrestricted over longer distances, thereby facilitating nuclear egress for a larger fraction of capsids.

Dual-color live cell fluorescence microscopy of fast intracellular trafficking processes, such as axonal transport, requires rapid switching of illumination channels. Typical broad-spectrum sources necessitate the use of mechanical filter switching, which introduces delays between acquisition of different fluorescence channels, impeding the interpretation and quantification of highly dynamic processes. Light Emitting Diodes (LEDs), however, allow modulation of excitation light in microseconds. Here we provide a step-by-step protocol to enable any scientist to build a research-grade LED illuminator for live cell microscopy, even without prior experience with electronics or optics. We quantify and compare components, discuss our design considerations, and demonstrate the performance of our LED illuminator by imaging axonal transport of herpes virus particles with high temporal resolution.

Egress of newly assembled herpesvirus particles from infected cells is a highly dynamic process involving the host secretory pathway working in concert with viral components. To elucidate the location, dynamics, and molecular mechanisms of alpha herpesvirus egress, we developed a live-cell fluorescence microscopy method to visualize the final transport and exocytosis of pseudorabies virus (PRV) particles in non-polarized epithelial cells. This method is based on total internal reflection fluorescence (TIRF) microscopy to selectively image fluorescent virus particles near the plasma membrane, and takes advantage of a virus-encoded pH-sensitive probe to visualize the precise moment and location of particle exocytosis. We performed single-particle tracking and mean squared displacement analysis to characterize particle motion, and imaged a panel of cellular proteins to identify those spatially and dynamically associated with viral exocytosis. Based on our data, individual virus particles travel to the plasma membrane inside small, acidified secretory vesicles. Rab GTPases, Rab6a, Rab8a, and Rab11a, key regulators of the plasma membrane-directed secretory pathway, are present on the virus secretory vesicle. These vesicles undergo fast, directional transport directly to the site of exocytosis, which is most frequently near patches of LL5β, part of a complex that anchors microtubules to the plasma membrane. Vesicles are tightly docked at the site of exocytosis for several seconds, and membrane fusion occurs, displacing the virion a small distance across the plasma membrane. After exocytosis, particles remain tightly confined on the outer cell surface. Based on recent reports in the cell biological and alpha herpesvirus literature, combined with our spatial and dynamic data on viral egress, we propose an integrated model that links together the intracellular transport pathways and exocytosis mechanisms that mediate alpha herpesvirus egress., Author Summary Pseudorabies virus, an alpha herpesvirus, is an important veterinary pathogen, and related to human varicella-zoster virus and herpes simplex viruses. New alpha herpesvirus particles are assembled inside an infected cell, and must exit from the infected cell by taking advantage of cellular mechanisms. How these virus particles are transported inside the infected cell and secreted at the cell surface is not understood in great detail. In particular, how this process unfolds over time is not easily observed using previous methods. In this study, we developed a new method to observe this egress process. Using this method, we described how virus particles move on their way out: individual virus particles travel to the cell surface, directly to the exit site, where they pause for several seconds before crossing out of the cell. We identified several cellular proteins that are involved in this process. After exiting, virus particles remained stuck to the outer cell surface. Finally, we draw connections between our observations and other recent studies to propose an integrated model of how alpha herpesvirus particles exit from infected cells.

Alpha herpesvirus genomes encode the capacity to establish quiescent infections (i.e. latency) in the peripheral nervous system for the life of their hosts. Multiple times during latency, viral genomes can reactivate to start a productive infection, enabling spread of progeny virions to other hosts. Replication of alpha herpesviruses is well studied in cultured cells and many aspects of productive replication have been identified. However, many questions remain concerning how a productive or a quiescent infection is established. While infections in vivo often result in latency, infections of dissociated neuronal cultures in vitro result in a productive infection unless lytic viral replication is suppressed by DNA polymerase inhibitors or interferon. Using primary peripheral nervous system neurons cultured in modified Campenot tri-chambers, we previously reported that reactivateable, quiescent infections by pseudorabies virus (PRV) can be established in the absence of any inhibitor. Such infections were established in cell bodies only when physically isolated axons were infected at a very low multiplicity of infection (MOI). In this report, we developed a complementation assay in compartmented neuronal cultures to investigate host and viral factors in cell bodies that prevent establishment of quiescent infection and promote productive replication of axonally delivered genomes (i.e. escape from silencing). Stimulating protein kinase A (PKA) signaling pathways in isolated cell bodies, or superinfecting cell bodies with either UV-inactivated PRV or viral light particles (LP) promoted escape from genome silencing and prevented establishment of quiescent infection but with different molecular mechanisms. Activation of PKA in cell bodies triggers a slow escape from silencing in a cJun N-terminal kinase (JNK) dependent manner. However, escape from silencing is induced rapidly by infection with UVPRV or LP in a PKA- and JNK-independent manner. We suggest that viral tegument proteins delivered to cell bodies engage multiple signaling pathways that block silencing of viral genomes delivered by low MOI axonal infection., Author summary Alpha herpesvirus infections stay life-long in infected human and animal hosts`nervous systems in a silent state ready to reactivate upon various stress signals. Remarkably, infection of epithelial cells with these viruses results in productive infection whereas infection of peripheral nervous system neurons results in non-productive silent infection (i.e. latency) in the natural hosts. More interestingly, infection of dissociated peripheral neurons in culture also results in productive infection unless DNA replication inhibitors are used. To study the molecular mechanisms of escape from latency, we used primary neurons cultured in compartmented tri-chambers. By this way, we recapitulated the natural route of infection by infecting axons with low dose of virus which resulted in a silent infection in a small number of neuronal cell bodies without the use of any inhibitors. Using these cultures, we developed a new complementation assay to investigate the molecular signals leading to escape from latency and establishment of productive infection. We found two different mechanisms to escape from latency: Cellular stress-mediated slow route and viral tegument mediated-fast route. Furthermore, we showed that the stress-mediated pathway requires protein kinase A and c-Jun N-terminal kinase activity while the viral tegument-mediated fast escape does not require these host cell kinase activities. We also concluded that a general response to DNA virus infection or presence of excess herpesviral genomes in the nucleus to saturate silencing complexes is not enough to escape from latency. Induction of a productive infection requires presence of tegument proteins or activation of PKA and JNK pathway.

Correction for ‘3D printed nervous system on a chip’ by Blake N. Johnson et al., Lab Chip, 2016, 16, 1393–1400.