scholarly journals CRISPR/Cas9-constructed pseudorabies virus mutants reveal the importance of UL13 in alphaherpesvirus escape from genome silencing

2020 ◽  
Author(s):  
Jolien Van Cleemput ◽  
Orkide O. Koyuncu ◽  
Kathlyn Laval ◽  
Esteban A. Engel ◽  
Lynn W. Enquist
Keyword(s):  
Nervous System ◽  
Peripheral Nervous System ◽  
Pseudorabies Virus ◽  
Neuronal Cell ◽  
Gene Replacement ◽  
Productive Infection ◽  
Tegument Protein ◽  
Neuronal Cultures ◽  
Tegument Proteins

Latent and recurrent productive infection of long-living cells, such as neurons, enables alphaherpesviruses to persist in their host populations. Still, the viral factors involved in these events remain largely obscure. Using a complementation assay in compartmented primary peripheral nervous system (PNS) neuronal cultures, we previously reported that productive replication of axonally-delivered genomes is facilitated by PRV tegument proteins. Here, we sought to unravel the role of tegument protein UL13 in this escape from silencing. We first constructed four new PRV mutants in the virulent Becker strain using CRISPR/Cas9-mediated gene replacement: (i) PRV Becker defective for UL13 expression (PRV ΔUL13), (ii) PRV where UL13 is fused to eGFP (PRV UL13-eGFP) and two control viruses (iii and iv) PRV where VP16 is fused with mTurquoise at either the N-terminus (PRV mTurq-VP16) or C-terminus (PRV VP16-mTurq). Live cell imaging of PRV capsids showed efficient retrograde transport after axonal infection with PRV UL13-eGFP, although we did not detect dual-color particles. Surprisingly, immunofluorescence staining of particles in mid-axons indicated that UL13 might be co-transported with PRV capsids in PNS axons. Superinfecting nerve cell bodies with UV-inactivated PRV ΔUL13 failed to efficiently promote escape from genome silencing when compared to UV-PRV wild type and UV-PRV UL13-eGFP superinfection. However, UL13 does not act directly in the escape from genome silencing, as AAV-mediated UL13 expression in neuronal cell bodies was not sufficient to provoke escape from genome silencing. Based on this, we suggest that UL13 may contribute to initiation of productive infection through phosphorylation of other tegument proteins. Importance Alphaherpesviruses have mastered various strategies to persist in an immunocompetent host, including the induction of latency and reactivation in peripheral nervous system (PNS) ganglia. We recently discovered that the molecular mechanism underlying escape from latency by the alphaherpesvirus pseudorabies virus (PRV) relies on a structural viral tegument protein. This study aimed at unravelling the role of tegument protein UL13 in PRV escape from latency. First, we confirmed the use of CRISPR/Cas9-mediated gene replacement as a versatile tool to modify the PRV genome. Next, we used our new set of viral mutants and AAV vectors to conclude on the indirect role of UL13 in PRV escape from latency in primary neurons and on its spatial localization during retrograde capsid transport in axons. Based on these findings, we speculate that UL13 phosphorylates one or more tegument proteins, thereby priming these putative proteins to induce escape from genome silencing.

scholarly journals Neuron-to-Cell Spread of Pseudorabies Virus in a Compartmented Neuronal Culture System

2005 ◽  
Vol 79 (17) ◽  
pp. 10875-10889 ◽  
Author(s):  
T. H. Ch'ng ◽  
L.W. Enquist
Keyword(s):  
Nervous System ◽  
Peripheral Nervous System ◽  
Culture System ◽  
Pseudorabies Virus ◽  
Neuronal Cell ◽  
Long Distance ◽  
Peripheral Tissues ◽  
Natural Hosts ◽  
Mucosal Cells ◽  
Cell Spread

ABSTRACT Alphaherpesviruses are parasites of the peripheral nervous system in their natural hosts. After the initial infection of peripheral tissues such as mucosal cells, these neurotropic viruses will invade the peripheral nervous system that innervates the site of infection via long-distance axonal transport of the viral genome. In natural hosts, a latent and a nonproductive infection is usually established in the neuronal cell bodies. Upon reactivation, the newly replicated genome will be assembled into capsids and transported back to the site of entry, where a localized infection of the epithelial or mucosal cells will produce infectious virions that can infect naïve hosts. In this paper, we describe an in vitro method for studying neuron-to-cell spread of alphaherpesviruses using a compartmented culture system. Using pseudorabies virus as a model, we infected neuron cell bodies grown in Teflon chambers and observed spread of infection to nonneuronal cells plated in a different compartment. The cells are in contact with the neurons via axons that penetrate the Teflon barrier. We demonstrate that wild-type neuron-to-cell spread requires intact axons and the presence of gE, gI, and Us9 proteins, but does not require gD. We also provide ultrastructural evidence showing that capsids enclosed within vesicles can be found along the entire length of the axon during viral egress.

scholarly journals Pseudorabies Virus Infection Accelerates Degradation of the Kinesin-3 Motor KIF1A

2020 ◽  
Vol 94 (9) ◽  
Author(s):  
Hao Huang ◽  
Orkide O. Koyuncu ◽  
Lynn W. Enquist
Keyword(s):  
Nervous System ◽  
Peripheral Nervous System ◽  
Pseudorabies Virus ◽  
Time Window ◽  
Proteasomal Degradation ◽  
Neuronal Cultures ◽  
Human Pathogens ◽  
Herpes Simplex Viruses ◽  
Varicella Zoster ◽  
Axonal Sorting

ABSTRACT Alphaherpesviruses, including pseudorabies virus (PRV), are neuroinvasive pathogens that establish lifelong latency in peripheral ganglia following the initial infection at mucosal surfaces. The establishment of latent infection and subsequent reactivations, during which newly assembled virions are sorted into and transported anterogradely inside axons to the initial mucosal site of infection, rely on axonal bidirectional transport mediated by microtubule-based motors. Previous studies using cultured peripheral nervous system (PNS) neurons have demonstrated that KIF1A, a kinesin-3 motor, mediates the efficient axonal sorting and transport of newly assembled PRV virions. Here we report that KIF1A, unlike other axonal kinesins, is an intrinsically unstable protein prone to proteasomal degradation. Interestingly, PRV infection of neuronal cells leads not only to a nonspecific depletion of KIF1A mRNA but also to an accelerated proteasomal degradation of KIF1A proteins, leading to a near depletion of KIF1A protein late in infection. Using a series of PRV mutants deficient in axonal sorting and anterograde spread, we identified the PRV US9/gE/gI protein complex as a viral factor facilitating the proteasomal degradation of KIF1A proteins. Moreover, by using compartmented neuronal cultures that fluidically and physically separate axons from cell bodies, we found that the proteasomal degradation of KIF1A occurs in axons during infection. We propose that the PRV anterograde sorting complex, gE/gI/US9, recruits KIF1A to viral transport vesicles for axonal sorting and transport and eventually accelerates the proteasomal degradation of KIF1A in axons. IMPORTANCE Pseudorabies virus (PRV) is an alphaherpesvirus related to human pathogens herpes simplex viruses 1 and 2 and varicella-zoster virus. Alphaherpesviruses are neuroinvasive pathogens that establish lifelong latent infections in the host peripheral nervous system (PNS). Following reactivation from latency, infection spreads from the PNS back via axons to the peripheral mucosal tissues, a process mediated by kinesin motors. Here, we unveil and characterize the underlying mechanisms for a PRV-induced, accelerated degradation of KIF1A, a kinesin-3 motor promoting the sorting and transport of PRV virions in axons. We show that PRV infection disrupts the synthesis of KIF1A and simultaneously promotes the degradation of intrinsically unstable KIF1A proteins by proteasomes in axons. Our work implies that the timing of motor reduction after reactivation would be critical because progeny particles would have a limited time window for sorting into and transport in axons for further host-to-host spread.

scholarly journals Pseudorabies Virus Infection Accelerates Degradation of the Kinesin-3 Motor KIF1A

2019 ◽  
Author(s):  
Hao Huang ◽  
Orkide O. Koyuncu ◽  
Lynn W. Enquist
Keyword(s):  
Nervous System ◽  
Peripheral Nervous System ◽  
Pseudorabies Virus ◽  
Time Window ◽  
Proteasomal Degradation ◽  
Neuronal Cultures ◽  
Human Pathogens ◽  
Herpes Simplex Virus 1 ◽  
Varicella Zoster ◽  
Axonal Sorting

AbstractAlphaherpesviruses, including pseudorabies virus (PRV), are neuroinvasive pathogens that establish life-long latency in peripheral ganglia following the initial infection at mucosal surfaces. The establishment of latent infection and the subsequent reactivations during which newly-assembled virions are sorted into and transported anterogradely inside axons to the initial mucosal site of infection, rely on axonal bidirectional transport mediated by microtubule-based motors. Previous studies using cultured peripheral nervous system (PNS) neurons have demonstrated that KIF1A, a kinesin-3 motor, mediates the efficient axonal sorting and transport of newly-assembled PRV virions. In this study, we report that KIF1A, unlike other axonal kinesins, is an intrinsically unstable protein prone to proteasomal degradation. Interestingly, PRV infection of neuronal cells leads not only to a non-specific depletion of KIF1A mRNA, but also to an accelerated proteasomal degradation of KIF1A proteins, leading to a near depletion of KIF1A protein late in infection. Using a series of PRV mutants deficient in axonal sorting and anterograde spread, we identified the PRV US9/gE/gI protein complex as a viral factor facilitating the proteasomal degradation of KIF1A proteins. Moreover, by using compartmented neuronal cultures that fluidically and physically separate axons from cell bodies, we found that the proteasomal degradation of KIF1A occurs in axons during infection. We propose that PRV anterograde sorting complex, gE/gI/US9, recruits KIF1A to viral transport vesicles for axonal sorting and transport, and eventually accelerates the proteasomal degradation of KIF1A in axons.ImportancePseudorabies virus (PRV) is an alphaherpesvirus related to human pathogens herpes simplex virus −1, −2 and varicella zoster virus. Alphaherpesviruses are neuroinvasive pathogens that establish life-long latent infections in the host peripheral nervous system (PNS). Following reactivation from latency, infection spreads from the PNS back via axons to the peripheral mucosal tissues, a process mediated by kinesin motors. Here, we unveil and characterize the underlying mechanisms for a PRV-induced, accelerated degradation of KIF1A, a kinesin-3 motor promoting the sorting and transport of PRV virions in axons. We show that PRV infection disrupts the synthesis of KIF1A, and simultaneously promotes the degradation of intrinsically unstable KIF1A proteins by proteasomes in axons. Our work implies that the timing of motor reduction after reactivation would be critical because progeny particles would have a limited time window for sorting into and transport in axons for further host-to-host spread.

scholarly journals Changes in peripheral nervous system at participants of accident consequences elimination in Chernobyl APP and population of the Tomsk northern industrial center

2004 ◽  
Vol 3 (4) ◽  
pp. 71-80
Author(s):  
Ya. V. Porovsky ◽  
V. I. Zhankova ◽  
A. I. Ryzhov ◽  
Ye. V. Kalyanov ◽  
F. F. Tetenev
Keyword(s):  
Nervous System ◽  
Immune System ◽  
Peripheral Nervous System ◽  
Radiochemical Plant ◽  
Chemical Complex ◽  
Tomsk Region ◽  
Industrial Center ◽  
Sensory Polyneuropathy ◽  
Siberian Chemical

A clinical, electroneuromyographic (ENMG) and pathomorphological investigation of 19 eliminators of accident consequences (EAC) in Chernobyl APP in 1986 and 27 Tomsk region inhabitants living in the accident area that has taken place at radiochemical plant of Siberian Chemical Complex in 1993 has been made with the aim of the influence study of low ionizing radiation levels on the peripheral nervous system. Symptoms of sensory polyneuropathy prevailed in both groups clinically. Mixed affection type has been found at EAC by ENGM method, affection of myelinic nerve fibre membrane has been found at people living in accident trace area. Morphofunctional changes in skin allow considering the role of immune system in mechanisms of neuroglial and neuronal damages, distant by time.

scholarly journals Nervous system injury in alcoholic disease

2019 ◽  
Vol 11 (2S) ◽  
pp. 83-88
Author(s):  
O. E. Zinovyeva ◽  
N. V. Vashchenko ◽  
O. E. Mozgovaya ◽  
T. A. Yanakaeva ◽  
A. Yu. Emelyanova
Keyword(s):  
Nervous System ◽  
Peripheral Nervous System ◽  
Alcohol Intoxication ◽  
Clinical Manifestations ◽  
Symptomatic Treatment ◽  
Chronic Alcohol ◽  
Neurological Manifestations ◽  
Chronic Alcohol Intoxication ◽  
Alcoholic Disease

The paper considers various variants of nervous system injury in alcoholic disease. It discusses the epidemiology, pathogenesis, diagnosis, and clinical manifestations of central and peripheral nervous system lesions in the presence of acute and chronic alcohol intoxication. Attention is paid to the issues of etiotropic, pathogenetic, and symptomatic treatment for neurological manifestations of alcoholic disease and to the role of neurotropic B vitamins in the treatment of alcohol-induced deficiency and non-deficiency states.

The role of NGF in the peripheral nervous system

1995 ◽  
Vol 8 (1,2) ◽  
pp. 97-98
Author(s):  
Jack Diamond ◽  
Sandra Lourenssen ◽  
Yvonne Kril ◽  
Jim Fawcett ◽  
Andrew Gloster
Keyword(s):  
Nervous System ◽  
Peripheral Nervous System
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