scholarly journals The Number of Alphaherpesvirus Particles Infecting Axons and the Axonal Protein Repertoire Determines the Outcome of Neuronal Infection

mBio ◽  
2015 ◽  
Vol 6 (2) ◽  
Author(s):  
Orkide O. Koyuncu ◽  
Ren Song ◽  
Todd M. Greco ◽  
Ileana M. Cristea ◽  
Lynn W. Enquist
Keyword(s):  
Nervous System ◽  
Peripheral Nervous System ◽  
Viral Entry ◽  
Retrograde Transport ◽  
Productive Infection ◽  
Long Distance ◽  
Viral Genomes ◽  
Neuronal Nuclei ◽  
Viral Particles ◽  
The Impact

ABSTRACTInfection by alphaherpesviruses invariably results in invasion of the peripheral nervous system (PNS) and establishment of either a latent or productive infection. Infection begins with long-distance retrograde transport of viral capsids and tegument proteins in axons toward the neuronal nuclei. Initial steps of axonal entry, retrograde transport, and replication in neuronal nuclei are poorly understood. To better understand how the mode of infection in the PNS is determined, we utilized a compartmented neuron culturing system where distal axons of PNS neurons are physically separated from cell bodies. We infected isolated axons with fluorescent-protein-tagged pseudorabies virus (PRV) particles and monitored viral entry and transport in axons and replication in cell bodies during low and high multiplicities of infection (MOIs of 0.01 to 100). We found a threshold for efficient retrograde transport in axons between MOIs of 1 and 10 and a threshold for productive infection in the neuronal cell bodies between MOIs of 1 and 0.1. Below an MOI of 0.1, the viral genomes that moved to neuronal nuclei were silenced. These genomes can be reactivated after superinfection by a nonreplicating virus, but not by a replicating virus. We further showed that viral particles at high-MOI infections compete for axonal proteins and that this competition determines the number of viral particles reaching the nuclei. Using mass spectrometry, we identified axonal proteins that are differentially regulated by PRV infection. Our results demonstrate the impact of the multiplicity of infection and the axonal milieu on the establishment of neuronal infection initiated from axons.IMPORTANCEAlphaherpesvirus genomes may remain silent in peripheral nervous system (PNS) neurons for the lives of their hosts. These genomes occasionally reactivate to produce infectious virus that can reinfect peripheral tissues and spread to other hosts. Here, we use a neuronal culture system to investigate the outcome of axonal infection using different numbers of viral particles and coinfection assays. We found that the dynamics of viral entry, transport, and replication change dramatically depending on the number of virus particles that infect axons. We demonstrate that viral genomes are silenced when the infecting particle number is low and that these genomes can be reactivated by superinfection with UV-inactivated virus, but not with replicating virus. We further show that viral invasion rapidly changes the profiles of axonal proteins and that some of these axonal proteins are rate limiting for efficient infection. Our study provides new insights into the establishment of silent versus productive alphaherpesvirus infections in the PNS.

scholarly journals Building a mechanistic mathematical model of hepatitis C virus entry

2018 ◽  
Author(s):  
Mphatso Kalemera ◽  
Dilyana Mincheva ◽  
Joe Grove ◽  
Christopher J. R. Illingworth
Keyword(s):  
Mathematical Model ◽  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Viral Entry ◽  
Productive Infection ◽  
Virus Particles ◽  
Quantitative Studies ◽  
Receptor Dynamics ◽  
Viral Lifecycle ◽  
The Impact

AbstractThe mechanism by which hepatitis C virus (HCV) gains entry into cells is a complex one, involving a broad range of host proteins. Entry is a critical phase of the viral lifecycle, and a potential target for therapeutic or vaccine-mediated intervention. However, the mechanics of HCV entry remain poorly understood. Here we describe a novel computational model of viral entry, encompassing the relationship between HCV and the key host receptors CD81 and SR-B1. We conduct experiments to thoroughly quantify the influence of an increase or decrease in receptor availability upon the extent of viral entry. We use these data to build and parameterise a mathematical model, which we then validate by further experiments. Our results are consistent with sequential HCV-receptor interactions, whereby initial interaction between the HCV E2 glycoprotein and SR-B1 facilitates the accumulation CD81 receptors, leading to viral entry. However, we also demonstrate that a small minority of virus can achieve entry in the absence of SR-B1. Our model estimates the impact of the different obstacles that viruses must surmount to achieve entry; among virus particles attaching to the cell surface, 20-35% accumulate sufficient CD81 receptors, of these 4-8% then complete the subsequent steps to achieve productive infection. Furthermore, we make estimates of receptor stoichiometry; between 3 and 6 CD81 receptors are likely to be required to achieve viral entry. Our model provides a tool to investigate the entry characteristics of HCV variants and outlines a framework for future quantitative studies of the multi-receptor dynamics of HCV entry.

scholarly journals RNA Structures and Their Role in Selective Genome Packaging

Viruses ◽  
2021 ◽  
Vol 13 (9) ◽  
pp. 1788
Author(s):  
Liqing Ye ◽  
Uddhav B. Ambi ◽  
Marco Olguin-Nava ◽  
Anne-Sophie Gribling-Burrer ◽  
Shazeb Ahmad ◽  
...  
Keyword(s):  
Viral Evolution ◽  
Complex Mixture ◽  
Rna Structures ◽  
Rna Packaging ◽  
Genome Packaging ◽  
Viral Genomes ◽  
Viral Particles ◽  
The Impact ◽  
Packaging Signals

To generate infectious viral particles, viruses must specifically select their genomic RNA from milieu that contains a complex mixture of cellular or non-genomic viral RNAs. In this review, we focus on the role of viral encoded RNA structures in genome packaging. We first discuss how packaging signals are constructed from local and long-range base pairings within viral genomes, as well as inter-molecular interactions between viral and host RNAs. Then, how genome packaging is regulated by the biophysical properties of RNA. Finally, we examine the impact of RNA packaging signals on viral evolution.

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.

Evidence for axonal ‘decision regions’ in the axolotl peripheral nervous system

Development ◽  
1988 ◽  
Vol 102 (4) ◽  
pp. 823-836
Author(s):  
S. Wilson ◽  
N. Holder
Keyword(s):  
Nervous System ◽  
Peripheral Nervous System ◽  
Spatial Organization ◽  
Retrograde Transport ◽  
Branch Points ◽  
Spinal Nerves ◽  
Lateral Motor Column ◽  
Decision Region ◽  
Muscle Nerve ◽  
Axial Musculature

Horseradish peroxidase (HRP) was employed to analyse the spatial organization of axons within nerves of the axolotl peripheral nervous system. HRP applications to the lateral motor column, spinal nerves and muscle nerve branches were examined after orthograde or retrograde transport. Axons change relative positions at particular limb regions, notably at the limb plexi, but also at branch points at other limb levels. Such areas of axon reorganization (termed ‘decision regions’ in line with Tosney & Landmesser (1985) J. Neurosci. 5, 2345) are interspersed by lengths of nerve in which axons run parallel to one another. A decision region is also described which involves only axons destined for axial musculature. The detailed anatomy of axon groups is discussed in terms of the likely mechanisms responsible for its formation during development. We conclude that, despite considerable variation in nerve pattern not seen in higher vertebrates, neuromuscular specificity in the axolotl limb is established largely by local pathway cues guiding axons to their appropriate targets.

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 A pseudorabies virus serine/threonine kinase, US3, promotes retrograde transport in axons via Akt/mToRC1

2021 ◽  
Author(s):  
Andrew D. Esteves ◽  
Orkide O Koyuncu ◽  
Lynn W. Enquist
Keyword(s):  
Pseudorabies Virus ◽  
Current Model ◽  
Retrograde Transport ◽  
Productive Infection ◽  
Local Translation ◽  
Serine Threonine Kinase ◽  
Long Distance ◽  
Threonine Kinase ◽  
Tegument Proteins ◽  
Long Distance Movement

Infection of peripheral axons by alpha herpesviruses (AHVs) is a critical stage in establishing a life-long infection in the host. Upon entering the cytoplasm of axons, AHV nucleocapsids and associated inner-tegument proteins must engage the cellular retrograde transport machinery to promote the long-distance movement of virion components to the nucleus. The current model outlining this process is incomplete and further investigation is required to discover all viral and cellular determinants involved as well as the temporality of the events. Using a modified tri-chamber system, we have discovered a novel role of the pseudorabies virus (PRV) serine/threonine kinase, US3, in promoting efficient retrograde transport of nucleocapsids. We discovered that transporting nucleocapsids move at similar velocities both in the presence and absence of a functional US3 kinase; however fewer nucleocapsids are moving when US3 is absent and move for shorter periods of time before stopping, suggesting US3 is required for efficient nucleocapsid engagement with the retrograde transport machinery. This led to fewer nucleocapsids reaching the cell bodies to produce a productive infection 12hr later. Furthermore, US3 was responsible for the induction of local translation in axons as early as 1hpi through the stimulation of a PI3K/Akt-mToRC1. These data describe a novel role for US3 in the induction of local translation in axons during AHV infection, a critical step in transport of nucleocapsids to the cell body.

scholarly journals A Pseudorabies Virus Serine/Threonine Kinase, US3, Promotes Retrograde Transport in Axons via Akt/mToRC1

2022 ◽  
Author(s):  
Andrew D. Esteves ◽  
Orkide O. Koyuncu ◽  
Lynn W. Enquist
Keyword(s):  
Cell Body ◽  
Pseudorabies Virus ◽  
Retrograde Transport ◽  
Productive Infection ◽  
Local Translation ◽  
Serine Threonine Kinase ◽  
Long Distance ◽  
Threonine Kinase ◽  
Mtorc1 Signaling ◽  
Axonal Translation

Infection of peripheral axons by alpha herpesviruses (AHVs) is a critical stage in establishing a life-long infection in the host. Upon entering the cytoplasm of axons, AHV nucleocapsids and associated inner-tegument proteins must engage the cellular retrograde transport machinery to promote the long-distance movement of virion components to the nucleus. The current model outlining this process is incomplete and further investigation is required to discover all viral and cellular determinants involved as well as the temporality of the events. Using a modified tri-chamber system, we have discovered a novel role of the pseudorabies virus (PRV) serine/threonine kinase, US3, in promoting efficient retrograde transport of nucleocapsids. We discovered that transporting nucleocapsids move at similar velocities both in the presence and absence of a functional US3 kinase; however fewer nucleocapsids are moving when US3 is absent and move for shorter periods of time before stopping, suggesting US3 is required for efficient nucleocapsid engagement with the retrograde transport machinery. This led to fewer nucleocapsids reaching the cell bodies to produce a productive infection 12hr later. Furthermore, US3 was responsible for the induction of local translation in axons as early as 1hpi through the stimulation of a PI3K/Akt-mToRC1 pathway. These data describe a novel role for US3 in the induction of local translation in axons during AHV infection, a critical step in transport of nucleocapsids to the cell body. Importance Neurons are highly polarized cells with axons that can reach centimeters in length. Communication between axons at the periphery and the distant cell body is a relatively slow process involving the active transport of chemical messengers. There’s a need for axons to respond rapidly to extracellular stimuli. Translation of repressed mRNAs present within the axon occurs to enable rapid, localized responses independently of the cell body. AHVs have evolved a way to hijack local translation in the axons to promote their transport to the nucleus. We have determined the cellular mechanism and viral components involved in the induction of axonal translation. The US3 serine/threonine kinase of PRV activates Akt-mToRC1 signaling pathways early during infection to promote axonal translation. When US3 is not present, the number of moving nucleocapsids and their processivity are reduced, suggesting that US3 activity is required for efficient engagement of nucleocapsids with the retrograde transport machinery.

Imaging Techniques Combined With Enzyme-Based Procedures That Facilitate the Simultaneous Imaging of Nerves, Neurons, Cartilage And Bones During Development In Gambusia

1999 ◽  
Vol 5 (S2) ◽  
pp. 1074-1075
Author(s):  
E. Rosa-Molinar
Keyword(s):  
Nervous System ◽  
Peripheral Nervous System ◽  
Imaging Techniques ◽  
Retrograde Transport ◽  
Nerve Fibers ◽  
Staining Procedure ◽  
Alizarin Red ◽  
Persistent Problem ◽  
Simultaneous Visualization ◽  
And Cartilage

A persistent problem in elucidating the anatomy of the peripheral nervous system has been an inability to stain both myelinated and unmyelinated nerve fibers. To overcome this problem, our laboratory developed two workmg protocols for reliably and differentially labeling and staining the peripheral nervous system and combined them with an enzyme clearing and staining procedure for the simultaneous visualization of bone and cartilage.One protocol uses anti-acetylated α-tubulin immunohistochemistry to follow the course, peripheral branching, and origin of the ventral spinal nerve innervating the axial musculature and a second uses anterograde and retrograde transport of selectively applied 3000 molecular weight (MW) biotin dextran amines and/or biocytin to identify specific afferent and efferent projections and their cell bodies. Both procedures can be combined with an enzyme clearing and staining procedure for the simultaneous visualization of bone (alizarin red S) and cartilage (alcian blue) in whole-mount preparations.

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