A hallmark property of the neurotropic alpha-herpesvirinae is the dissemination of infection to sensory and autonomic ganglia of the peripheral nervous system following an initial exposure at mucosal surfaces. The peripheral ganglia serve as the latent virus reservoir and the source of recurrent infections such as cold sores (herpes simplex virus type I) and shingles (varicella zoster virus). However, the means by which these viruses routinely invade the nervous system is not fully understood. We report that an internal virion component, the pUL37 tegument protein, has a surface region that is an essential neuroinvasion effector. Mutation of this region rendered herpes simplex virus type 1 (HSV-1) and pseudorabies virus (PRV) incapable of spreading by retrograde axonal transport to peripheral ganglia both in culture and animals. By monitoring the axonal transport of individual viral particles by time-lapse fluorescence microscopy, the mutant viruses were determined to lack the characteristic sustained intracellular capsid motion along microtubules that normally traffics capsids to the neural soma. Consistent with the axonal transport deficit, the mutant viruses did not reach sites of latency in peripheral ganglia, and were avirulent. Despite this, viral propagation in peripheral tissues and in cultured epithelial cell lines remained robust. Selective elimination of retrograde delivery to the nervous system has long been sought after as a means to develop vaccines against these ubiquitous, and sometimes devastating viruses. In support of this potential, we find that HSV-1 and PRV mutated in the effector region of pUL37 evoked effective vaccination against subsequent nervous system challenges and encephalitic disease. These findings demonstrate that retrograde axonal transport of the herpesviruses occurs by a virus-directed mechanism that operates by coordinating opposing microtubule motors to favor sustained retrograde delivery of the virus to the peripheral ganglia. The ability to selectively eliminate the retrograde axonal transport mechanism from these viruses will be useful in trans-synaptic mapping studies of the mammalian nervous system, and affords a new vaccination paradigm for human and veterinary neurotropic herpesviruses.

CD95/Fas ligand binds to the death receptor CD95 to induce apoptosis in sensitive cells. We previously reported that CD95L mRNA is enriched in sequences that, when converted to si/shRNAs, kill all cancer cells by targeting critical survival genes (Putzbach et al., 2017). We now report expression of full-length CD95L mRNA itself is highly toxic to cells and induces a similar form of cell death. We demonstrate that small (s)RNAs derived from CD95L are loaded into the RNA induced silencing complex (RISC) which is required for the toxicity and processing of CD95L mRNA into sRNAs is independent of both Dicer and Drosha. We provide evidence that in addition to the CD95L transgene a number of endogenous protein coding genes involved in regulating protein translation, particularly under low miRNA conditions, can be processed to sRNAs and loaded into the RISC suggesting a new level of cell fate regulation involving RNAi.

CD95/Fas ligand binds to the death receptor CD95 to induce apoptosis in sensitive cells. We previously reported the CD95L mRNA is enriched in sequences that, when converted to si/shRNAs, kill all cancer cells by targeting critical survival genes (Putzbach et al., 2017). We now report expression of full-length CD95L mRNA, itself, is highly toxic to cells and induces a similar form of cell death. We demonstrate that small (s)RNAs derived from CD95L are loaded into the RNA induced silencing complex (RISC) RISC which is required for the toxicity and that processing of CD95L mRNA into sRNAs is independent of both Dicer and Drosha. We provide evidence that in addition to the CD95L transgene a number of endogenous protein coding genes involved in regulating protein translation, particularly under low miRNA conditions, can be processed to sRNAs and loaded into the RISC suggesting a new level of cell fate regulation involving RNAi.

Sensory-guided limb control relies on communication across sensorimotor loops. For active touch with the hand, the longest loop is the transcortical continuation of ascending pathways, particularly the lemnisco-cortical and corticocortical pathways carrying tactile signals via the cuneate nucleus, ventral posterior lateral (VPL) thalamus, and primary somatosensory (S1) and motor (M1) cortices to reach corticospinal neurons and influence descending activity. We characterized excitatory connectivity along this pathway in the mouse. In the lemnisco-cortical leg, disynaptic cuneate→VPL→S1 connections excited mainly layer (L) 4 neurons. In the corticocortical leg, S1→M1 connections from L2/3 and L5A neurons mainly excited downstream L2/3 neurons, which excite corticospinal neurons. The findings provide a detailed new wiring diagram for the hand/forelimb-related transcortical circuit, delineating a basic but complex set of cell-type-specific feedforward excitatory connections that selectively and extensively engage diverse intratelencephalic projection neurons, thereby polysynaptically linking subcortical somatosensory input to cortical motor output to spinal cord.

CD95/Fas is an apoptosis inducing death receptor. However, it also has multiple nonapoptotic activities that are tumorigenic. Chronic stimulation of CD95 on breast cancer cells can increase their cancer initiating capacity through activation of a type I interferon (IFN-I)/STAT1 pathway when caspases are inhibited. We now show that this activity relies on the canonical components of the CD95 death-inducing signaling complex, FADD and caspase-8, and on the activation of NF-κB. We identified caspase-2 as the antagonistic caspase that downregulates IFN-I production. Once produced, IFN-Is bind to their receptors activating both STAT1 and STAT2 resulting in upregulation of the double stranded (ds)RNA sensor proteins RIG-I and MDA5, and a release of a subset of endogenous retroviruses. Thus, CD95 is part of a complex cell autonomous regulatory network that involves activation of innate immune components that drive cancer stemness and contribute to therapy resistance.

Upon replication in mucosal epithelia and transmission to nerve endings, capsids of herpes simplex virus 1 (HSV-1) travel retrogradely within axons to peripheral ganglia, where life-long latent infections are established. A capsid-bound tegument protein, pUL37, is an essential effector of retrograde axonal transport and also houses a deamidase activity that antagonizes innate immune signaling. In this report, we examined whether the deamidase of HSV-1 pUL37 contributes to the neuroinvasive retrograde axonal transport mechanism. We conclude that neuroinvasion is enhanced by the deamidase, but the critical contribution of pUL37 to retrograde axonal transport functions independently of this activity. IMPORTANCE Herpes simplex virus 1 invades the nervous system by entering nerve endings and sustaining long-distance retrograde axonal transport to reach neuronal nuclei in ganglia of the peripheral nervous system. The incoming viral particle carries a deamidase activity on its surface that antagonizes antiviral responses. We examined the contribution of the deamidase to the hallmark neuroinvasive property of this virus.

Sensory-guided limb control relies on communication across sensorimotor loops. For active touch with the hand, the longest loop is the transcortical continuation of ascending pathways, particularly the lemnisco-cortical and corticocortical pathways carrying tactile signals via the cuneate nucleus, ventral posterior lateral (VPL) thalamus, and primary somatosensory (S1) and motor (M1) cortices to reach corticospinal neurons and influence descending activity. We characterized excitatory connectivity along this pathway in the mouse. In the lemnisco-cortical leg, disynaptic cuneate→VPL→S1 connections excited mainly layer (L) 4 neurons. In the corticocortical leg, S1→M1 connections from L2/3 and L5A neurons mainly excited downstream L2/3 neurons, which excite corticospinal neurons. The findings provide a detailed new wiring diagram for the hand/forelimb-related transcortical circuit, delineating a basic but complex set of cell-type-specific feedforward excitatory connections that selectively and extensively engage diverse intratelencephalic projection neurons, thereby polysynaptically linking subcortical somatosensory input to cortical motor output to spinal cord.

Upon replication in mucosal epithelia and transmission to nerve endings, capsids of herpes simplex virus type I (HSV-1) travel retrograde within axons to peripheral ganglia where life-long latent infections are established. A capsid-bound tegument protein, pUL37, is an essential effector of retrograde axonal transport and also houses a deamidase activity that antagonizes innate immune signaling. In this report, we examined whether the deamidase of HSV-1 pUL37 contributes to the neuroinvasive retrograde axonal transport mechanism. We conclude that neuroinvasion is enhanced by the deamidase, but the critical contribution of pUL37 to retrograde axonal transport functions independently of this activity.IMPORTANCEHerpes simplex virus type 1 invades the nervous system by entering nerve endings and sustaining long-distance retrograde axonal transport to reach neuronal nuclei in ganglia of the peripheral nervous system. The incoming viral particle carries a deamidase activity on its surface that antagonizes antiviral responses. We examined the contribution of the deamidase to the hallmark neuroinvasive property of this virus.