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 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 Identification and Visualization of Functionally Important Domains and Residues in Herpes Simplex Virus Glycoprotein K(gK) Using a Combination of Phylogenetics and Protein Modeling

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Paul J. F. Rider ◽  
Lyndon M. Coghill ◽  
Misagh Naderi ◽  
Jeremy M. Brown ◽  
Michal Brylinski ◽  
...  
Keyword(s):  
Herpes Simplex ◽  
Evolutionary Rate ◽  
Quantitative Measure ◽  
Rate Variation ◽  
Human Pathogens ◽  
Herpes Simplex Viruses ◽  
Varicella Zoster ◽  
Conserved Sequence ◽  
Glycoprotein K ◽  
Herpes Simplex Virus Glycoprotein

Abstract Alphaherpesviruses are a subfamily of herpesviruses that include the significant human pathogens herpes simplex viruses (HSV) and varicella zoster virus (VZV). Glycoprotein K (gK), conserved in all alphaherpesviruses, is a multi-membrane spanning virion glycoprotein essential for virus entry into neuronal axons, virion assembly, and pathogenesis. Despite these critical functions, little is known about which gK domains and residues are most important for maintaining these functions across all alphaherpesviruses. Herein, we employed phylogenetic and structural analyses including the use of a novel model for evolutionary rate variation across residues to predict conserved gK functional domains. We found marked heterogeneity in the evolutionary rate at the level of both individual residues and domains, presumably as a result of varying selective constraints. To clarify the potential role of conserved sequence features, we predicted the structures of several gK orthologs. Congruent with our phylogenetic analysis, slowly evolving residues were identified at potentially structurally significant positions across domains. We found that using a quantitative measure of amino acid rate variation combined with molecular modeling we were able to identify amino acids predicted to be critical for gK protein structure/function. This analysis yields targets for the design of anti-herpesvirus therapeutic strategies across all alphaherpesvirus species that would be absent from more traditional analyses of conservation.

scholarly journals Suppression of the Interferon-Mediated Innate Immune Response by Pseudorabies Virus

2006 ◽  
Vol 80 (13) ◽  
pp. 6345-6356 ◽  
Author(s):  
Alla Brukman ◽  
L. W. Enquist
Keyword(s):  
Immune Response ◽  
Cell Activation ◽  
Pseudorabies Virus ◽  
Nk Cell ◽  
Innate Immune ◽  
Human Pathogens ◽  
Wild Type ◽  
Varicella Zoster ◽  
Simplex Virus ◽  
Hsv 1

ABSTRACT Pseudorabies virus (PRV) is an alphaherpesvirus related to the human pathogens herpes simplex virus type 1 (HSV-1) and varicella-zoster virus. PRV is capable of infecting and killing a wide variety of mammals. How it avoids innate immune defenses in so many hosts is not understood. While the anti-interferon (IFN) strategies of HSV-1 have been studied, little is known about how PRV evades the IFN-mediated immune response. In this study, we determined if wild-type PRV infection can overcome the establishment of a beta interferon (IFN-β)-induced antiviral state in primary rat fibroblasts. Using microarray technology, we found that the expression of a subset of genes normally induced by IFN-β in these cells was not induced when the cells were simultaneously infected with a wild-type PRV strain. Expression of transcripts associated with major histocompatibility complex class I antigen presentation and NK cell activation was reduced, while transcripts associated with inflammation either were unaffected or were induced by viral infection. This suppression of IFN-stimulated gene expression occurred because IFN signal transduction, in particular the phosphorylation of STAT1, became less effective in PRV-infected cells. At least one virion-associated protein is involved in inhibition of STAT1 tyrosine phosphorylation. This ability to disarm the IFN-β response offers an explanation for the uniform lethality of virulent PRV infection of nonnatural hosts.

scholarly journals In Vitro Analysis of Transneuronal Spread of an Alphaherpesvirus Infection in Peripheral Nervous System Neurons

2007 ◽  
Vol 81 (13) ◽  
pp. 6846-6857 ◽  
Author(s):  
B. Feierbach ◽  
M. Bisher ◽  
J. Goodhouse ◽  
L. W. Enquist
Keyword(s):  
Nervous System ◽  
Peripheral Nervous System ◽  
Pseudorabies Virus ◽  
Viral Glycoprotein ◽  
In Vitro System ◽  
Vitro Analysis ◽  
In Vitro Analysis ◽  
Postsynaptic Target

ABSTRACT The neurotropic alphaherpesviruses invade and spread in the nervous system in a directional manner between synaptically connected neurons. Until now, this property has been studied only in living animals and has not been accessible to in vitro analysis. In this study, we describe an in vitro system in which cultured peripheral nervous system neurons are separated from their neuron targets by an isolator chamber ring. Using pseudorabies virus (PRV), an alphaherpesvirus capable of transneuronal spread in neural circuits of many animals, we have recapitulated in vitro all known genetic requirements for retrograde and anterograde transneuronal spread as determined previously in vivo. We show that in vitro transneuronal spread requires intact axons and the presence of the viral proteins gE, gI, and Us9. We also show that transneuronal spread is dependent on the viral glycoprotein gB, which is required for membrane fusion, but not on gD, which is required for extracellular spread. We demonstrate ultrastructural differences between anterograde- and retrograde-traveling virions. Finally, we show live imaging of dynamic fluorescent virion components in axons and postsynaptic target neurons.

Varicella-Zoster Virus Infections of the Nervous System

2001 ◽  
Vol 125 (6) ◽  
pp. 770-780 ◽  
Author(s):  
B. K. Kleinschmidt-DeMasters ◽  
Donald H. Gilden
Keyword(s):  
Nervous System ◽  
Varicella Zoster Virus ◽  
Peripheral Nervous System ◽  
Cerebral Arteries ◽  
Infectious Agent ◽  
Spinal Ganglia ◽  
Ependymal Cells ◽  
Pcr Analysis ◽  
Neurologic Complications ◽  
Varicella Zoster

Abstract Background.—Diseases that present with protean manifestations are the diseases most likely to pose diagnostic challenges for both clinicians and pathologists. Among the most diverse disorders caused by a single known toxic, metabolic, neoplastic, or infectious agent are the central and peripheral nervous system complications of varicella-zoster virus (VZV). Methods.—The pathologic correlates of the neurologic complications of VZV infection, as well as current methods for detecting viral infections, are discussed and presented in pictorial format for the practicing pathologist. Results.—Varicella-zoster virus causes chickenpox (varicella), usually in childhood; most children manifest only mild neurologic sequelae. After chickenpox resolves, the virus becomes latent in neurons of cranial and spinal ganglia of nearly all individuals. In elderly and immunocompromised individuals, the virus may reactivate to produce shingles (zoster). After zoster resolves, many elderly patients experience postherpetic neuralgia. Uncommonly, VZV can spread to large cerebral arteries to cause a spectrum of large-vessel vascular damage, ranging from vasculopathy to vasculitis, with stroke. In immunocompromised individuals, especially those with cancer or acquired immunodeficiency syndrome, deeper tissue penetration of the virus may occur (as compared with immunocompetent individuals), with resultant myelitis, small-vessel vasculopathy, ventriculitis, and meningoencephalitis. Detection of the virus in neurons, oligodendrocytes, meningeal cells, ependymal cells, or the blood vessel wall often requires a combination of morphologic, immunohistochemical, in situ hybridization, and polymerase chain reaction (PCR) methods. The PCR analysis of cerebrospinal fluid remains the mainstay for diagnosing the neurologic complications of VZV during life. Conclusions.—Varicella-zoster virus infects a wide variety of cell types in the central and peripheral nervous system, explaining the diversity of clinical disorders associated with the virus.

scholarly journals Defensive Perimeter in the Central Nervous System: Predominance of Astrocytes and Astrogliosis during Recovery from Varicella-Zoster Virus Encephalitis

2015 ◽  
Vol 90 (1) ◽  
pp. 379-391 ◽  
Author(s):  
John E. Carpenter ◽  
Amy C. Clayton ◽  
Kevin C. Halling ◽  
Daniel J. Bonthius ◽  
Erin M. Buckingham ◽  
...  
Keyword(s):  
Central Nervous System ◽  
Nervous System ◽  
Human Brain ◽  
Varicella Zoster Virus ◽  
Peripheral Nervous System ◽  
Cellular Response ◽  
Cell Types ◽  
Varicella Zoster ◽  
Human Host ◽  
The Central Nervous System

ABSTRACTVaricella-zoster virus (VZV) is a highly neurotropic virus that can cause infections in both the peripheral nervous system and the central nervous system. Several studies of VZV reactivation in the peripheral nervous system (herpes zoster) have been published, while exceedingly few investigations have been carried out in a human brain. Notably, there is no animal model for VZV infection of the central nervous system. In this report, we characterized the cellular environment in the temporal lobe of a human subject who recovered from focal VZV encephalitis. The approach included not only VZV DNA/RNA analyses but also a delineation of infected cell types (neurons, microglia, oligodendrocytes, and astrocytes). The average VZV genome copy number per cell was 5. Several VZV regulatory and structural gene transcripts and products were detected. When colocalization studies were performed to determine which cell types harbored the viral proteins, the majority of infected cells were astrocytes, including aggregates of astrocytes. Evidence of syncytium formation within the aggregates included the continuity of cytoplasm positive for the VZV glycoprotein H (gH) fusion-complex protein within a cellular profile with as many as 80 distinct nuclei. As with other causes of brain injury, these results suggested that astrocytes likely formed a defensive perimeter around foci of VZV infection (astrogliosis). Because of the rarity of brain samples from living humans with VZV encephalitis, we compared our VZV results with those found in a rat encephalitis model following infection with the closely related pseudorabies virus and observed similar perimeters of gliosis.IMPORTANCEInvestigations of VZV-infected human brain from living immunocompetent human subjects are exceedingly rare. Therefore, much of our knowledge of VZV neuropathogenesis is gained from studies of VZV-infected brains obtained at autopsy from immunocompromised patients. These are not optimal samples with which to investigate a response by a human host to VZV infection. In this report, we examined both flash-frozen and paraffin-embedded formalin-fixed brain tissue of an otherwise healthy young male with focal VZV encephalitis, most likely acquired from VZV reactivation in the trigeminal ganglion. Of note, the cellular response to VZV infection mimicked the response to other causes of trauma to the brain, namely, an ingress of astrocytes and astrogliosis around an infectious focus. Many of the astrocytes themselves were infected; astrocytes aggregated in clusters. We postulate that astrogliosis represents a successful defense mechanism by an immunocompetent human host to eliminate VZV reactivation within neurons.

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 Comparison of the Pseudorabies Virus Us9 Protein with Homologs from Other Veterinary and Human Alphaherpesviruses

2009 ◽  
Vol 83 (14) ◽  
pp. 6978-6986 ◽  
Author(s):  
M. G. Lyman ◽  
C. D. Kemp ◽  
M. P. Taylor ◽  
L. W. Enquist
Keyword(s):  
Pseudorabies Virus ◽  
Fluorescent Protein ◽  
Membrane Topology ◽  
Human Pathogens ◽  
Herpesvirus Type ◽  
Varicella Zoster ◽  
Green Fluorescent ◽  
Simplex Virus ◽  
Hsv 1

ABSTRACT Pseudorabies virus (PRV) Us9 is a small, tail-anchored (TA) membrane protein that is essential for axonal sorting of viral structural proteins and is highly conserved among other members of the alphaherpesvirus subfamily. We cloned the Us9 homologs from two human pathogens, varicella-zoster virus (VZV) and herpes simplex virus type 1 (HSV-1), as well as two veterinary pathogens, equine herpesvirus type 1 (EHV-1) and bovine herpesvirus type 1 (BHV-1), and fused them to enhanced green fluorescent protein to examine their subcellular localization and membrane topology. Akin to PRV Us9, all of the Us9 homologs localized to the trans-Golgi network and had a type II membrane topology (typical of TA proteins). Furthermore, we examined whether any of the Us9 homologs could compensate for the loss of PRV Us9 in anterograde, neuron-to-cell spread of infection in a compartmented chamber system. EHV-1 and BHV-1 Us9 were able to fully compensate for the loss of PRV Us9, whereas VZV and HSV-1 Us9 proteins were unable to functionally replace PRV Us9 when they were expressed in a PRV background.

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