scholarly journals Dual-Color Herpesvirus Capsids Discriminate Inoculum from Progeny and Reveal Axonal Transport Dynamics

2016 ◽  
Vol 90 (21) ◽  
pp. 9997-10006 ◽  
Author(s):  
Julian Scherer ◽  
Zachary A. Yaffe ◽  
Michael Vershinin ◽  
Lynn W. Enquist
Keyword(s):  
Central Nervous System ◽  
Nervous System ◽  
Pseudorabies Virus ◽  
Fluorescent Protein ◽  
Cytoplasmic Dynein ◽  
Virus Preparation ◽  
Viral Capsids ◽  
Transport Dynamics ◽  
Dual Color ◽  
The Central Nervous System

ABSTRACT Alphaherpesviruses such as herpes simplex virus and pseudorabies virus (PRV) are neuroinvasive double-stranded DNA (dsDNA) viruses that establish lifelong latency in peripheral nervous system (PNS) neurons of their native hosts. Following reactivation, infection can spread back to the initial mucosal site of infection or, in rare cases, to the central nervous system, with usually serious outcomes. During entry and egress, viral capsids depend on microtubule-based molecular motors for efficient and fast transport. In axons of PNS neurons, cytoplasmic dynein provides force for retrograde movements toward the soma, and kinesins move cargo in the opposite, anterograde direction. The dynamic properties of virus particles in cells can be imaged by fluorescent protein fusions to the small capsid protein VP26, which are incorporated into capsids. However, single-color fluorescent protein tags fail to distinguish the virus inoculum from progeny. Therefore, we established a dual-color system by growing a recombinant PRV expressing a red fluorescent VP26 fusion (PRV180) on a stable cell line expressing a green VP26 fusion (PK15-mNG-VP26). The resulting dual-color virus preparation (PRV180G) contains capsids tagged with both red and green fluorescent proteins, and 97% of particles contain detectable levels of mNeonGreen (mNG)-tagged VP26. After replication in neuronal cells, all PRV180G progeny exclusively contain monomeric red fluorescent protein (mRFP)-VP26-tagged capsids. We used PRV180G for an analysis of axonal capsid transport dynamics in PNS neurons. Fast dual-color total internal reflection fluorescence (TIRF) microscopy, single-particle tracking, and motility analyses reveal robust, bidirectional capsid motility mediated by cytoplasmic dynein and kinesin during entry, whereas egressing progeny particles are transported exclusively by kinesins. IMPORTANCE Alphaherpesviruses are neuroinvasive viruses that infect the peripheral nervous system (PNS) of infected hosts as an integral part of their life cycle. Establishment of a quiescent or latent infection in PNS neurons is a hallmark of most alphaherpesviruses. Spread of infection to the central nervous system is surprisingly rare in natural hosts but can be fatal. Pseudorabies virus (PRV) is a broad-host-range swine alphaherpesvirus that enters neuronal cells and utilizes intracellular transport processes to establish infection and to spread between cells. By using a virus preparation with fluorescent viral capsids that change color depending on the stage of the infectious cycle, we find that during entry, axons of PNS neurons support robust, bidirectional capsid motility, similar to cellular cargo, toward the cell body. In contrast, progeny particles appear to be transported unidirectionally by kinesin motors toward distal egress sites.

Targeting gene-modified hematopoietic cells to the central nervous system: Use of green fluorescent protein uncovers microglial engraftment

2001 ◽  
Vol 7 (12) ◽  
pp. 1356-1361 ◽  
Author(s):  
Josef Priller ◽  
Alexander Flügel ◽  
Tim Wehner ◽  
Matthias Boentert ◽  
Carola A. Haas ◽  
...  
Keyword(s):  
Central Nervous System ◽  
Nervous System ◽  
Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Hematopoietic Cells ◽  
System Use ◽  
The Central Nervous System ◽  
Green Fluorescent

Imaging Molecular Targeting In Living Neural Cultures

1999 ◽  
Vol 5 (S2) ◽  
pp. 1228-1229
Author(s):  
Christopher S. Wallace ◽  
Michael A. Silverman ◽  
Michelle A. Burack ◽  
Janis E. Lochner ◽  
Richard G. Allen ◽  
...  
Keyword(s):  
Central Nervous System ◽  
Nervous System ◽  
Hippocampal Neurons ◽  
Protein Targeting ◽  
Fluorescent Protein ◽  
Complex Structure ◽  
Time Lapse ◽  
The Central Nervous System

Recent technical advances in the ability to attach an endogenously fluorescent protein sequence—i.e., green fluorescent protein or GFP and its derivatives--to any protein of experimental interest promises to mark a new era of progress in the study of protein targeting. Bringing these new tools to bear on neurons of the central nervous system has been challenging, however, because they have a very complex structure and are relatively difficult to transfect because they are post-mitotic.We use two cell culture approaches to characterize protein trafficking within neurons of the central nervous system in vitro. The first is a dissociated culture of hippocampal neurons from embryonic (El8) rats which is especially suited to analysis by conventional light microscopy because these neurons are grown on glass coverslips at low density. Neurons cultured in this way develop a morphology comparable to that seen in vivo and permit the establishment of axons and dendrites to be analyzed by time-lapse microscopy.

scholarly journals Anterograde transneuronal viral tract tracing reveals central sensory circuits from white adipose tissue

2009 ◽  
Vol 296 (3) ◽  
pp. R501-R511 ◽  
Author(s):  
C. Kay Song ◽  
Gary J. Schwartz ◽  
Timothy J. Bartness
Keyword(s):  
Central Nervous System ◽  
Nervous System ◽  
Adipose Tissue ◽  
White Adipose Tissue ◽  
Pseudorabies Virus ◽  
Sensory Cells ◽  
Tract Tracing ◽  
Herpes Simplex Virus 1 ◽  
The Central Nervous System ◽  
Hsv 1

The origins of the sympathetic nervous system (SNS) innervation of white adipose tissue (WAT) have been defined using the transneuronal viral retrograde tract tracer, pseudorabies virus. Activation of this SNS innervation is acknowledged as the principal initiator of WAT lipolysis. The central control of WAT lipolysis may require neural feedback to a brain-SNS-WAT circuit via WAT afferents. Indeed, conventional tract tracing studies have demonstrated that peripheral pseudounipolar dorsal root ganglion (DRG) sensory cells innervate WAT. The central nervous system projections of WAT afferents remain uncharted, however, and form the focus of the present study. We used the H129 strain of the herpes simplex virus-1 (HSV-1), an anterograde transneuronal viral tract tracer, to define the afferent circuits projecting from WAT to the central nervous system. Siberian hamster inguinal (IWAT) or epididymal WAT was injected with H129 and the neuraxis processed for HSV-1 immunoreactivity. We found substantial overlap in the pattern of WAT sensory afferent projections with multiple SNS outflow sites along the neuraxis, suggesting the possibility of WAT sensory-SNS circuits that could regulate WAT SNS drive and thereby lipolysis. Previously, we demonstrated that systemic 2-deoxy-d-glucose (2DG) elicited increases in the SNS drive to IWAT. Here, we show that systemic 2DG administration also significantly increases multiunit spike activity arising from decentralized IWAT afferents. Collectively, these data provide structural and functional support for the existence of a sensory WAT pathway to the brain, important in the negative feedback control of lipid mobilization.

scholarly journals STUDIES ON PSEUDORABIES (INFECTIOUS BULBAR PARALYSIS, MAD ITCH)

1934 ◽  
Vol 59 (6) ◽  
pp. 729-749 ◽  
Author(s):  
E. Weston Hurst
Keyword(s):  
Central Nervous System ◽  
Nervous System ◽  
Peripheral Nerves ◽  
Pseudorabies Virus ◽  
Blood Stream ◽  
Reverse Direction ◽  
Bulbar Paralysis ◽  
The Central Nervous System ◽  
Intravenous Inoculation ◽  
Subcutaneous Inoculation

After intramuscular, intradermal and subcutaneous inoculation, the pseudorabies virus reaches the central nervous system by way of the peripheral nerves, although it is circulating in the blood. Centrifugal spread from the infected nervous tissues by the neural route also occurs. After intracerebral inoculation the virus passes in the reverse direction, down the nervous axis. The Aujeszky strain invades the blood stream more readily than does the Iowa strain; but possibly with repeated passage the latter is approximating in this respect more closely the classical Aujeszky strain. After intravenous inoculation, effective with even small doses, virus is rapidly removed from the blood, and multiple infective foci are established in various organs; thence ascent of the virus by the peripheral nerves leads to infection of the central nervous system, the symptomatology differing according to whether the spinal cord or the medulla is first reached. The lack of evidence that the virus can penetrate directly the hemato-encephalic barrier deserves emphasis. When subcutaneous inoculation is practised in an area deprived of its nerve supply, the ability of the virus to invade the blood stream permits it to establish infective foci in the various viscera, and, after a predictable delay, the course of infection resembles that following intravenous injection. The pseudorabies virus is pantropic; i.e., it readily attacks cells derived from any embryonic layer. Lesions in the adrenal gland following intravenous inoculation are very like those due to herpes virus similarly introduced, this being one point of similarity in the pathogenic action of the two organisms. The relation of the pseudorabies virus to other viruses affecting the central nervous system is discussed.

scholarly journals Imaging the Transport Dynamics of Single Alphaherpesvirus Particles in Intact Peripheral Nervous System Explants from Infected Mice

mBio ◽  
2013 ◽  
Vol 4 (3) ◽  
Author(s):  
Andrea E. Granstedt ◽  
Bingni W. Brunton ◽  
Lynn W. Enquist
Keyword(s):  
Nervous System ◽  
Salivary Gland ◽  
Peripheral Nervous System ◽  
Pseudorabies Virus ◽  
Molecular Mechanisms ◽  
Fluorescent Protein ◽  
Virus Particles ◽  
Transport Dynamics ◽  
Ganglion Neurons

ABSTRACT Alphaherpesvirus particles travel long distances in the axons of neurons using host microtubule molecular motors. The transport dynamics of individual virions in neurons have been assessed in cultured neurons, but imaging studies of single particles in tissue from infected mice have not been reported. We developed a protocol to image explanted, infected peripheral nervous system (PNS) ganglia and associated innervated tissue from mice infected with pseudorabies virus (PRV). This ex vivo preparation allowed us to visualize and track individual virions over time as they moved from the salivary gland into submandibular ganglion neurons of the PNS. We imaged and tracked hundreds of virions from multiple mice at different time points. We quantitated the transport velocity, particle stalling, duty cycle, and directionality at various times after infection. Using a PRV recombinant that expressed monomeric red fluorescent protein (mRFP)-VP26 (red capsid) and green fluorescent protein (GFP)-Us9 (green membrane protein), we corroborated that anterograde transport in axons occurs after capsids are enveloped. We addressed the question of whether replication occurs initially in the salivary gland at the site of inoculation or subsequently in the neurons of peripheral innervating ganglia. Our data indicate that significant amplification of infection occurs in the peripheral ganglia after transport from the site of infection and that these newly made particles are transported back to the salivary gland. It is likely that this reseeding of the infected gland contributes to massive invasion of the innervating PNS ganglia. We suggest that this “round-trip” infection process contributes to the characteristic peripheral neuropathy of PRV infection. IMPORTANCE Much of our understanding of molecular mechanisms of alphaherpesvirus infection and spread in neurons comes from studying cultured primary neurons. These techniques enabled significant advances in our understanding of the viral and neuronal components needed for efficient replication and directional spread between cells. However, in vitro systems cannot recapitulate the environment of innervated tissue in vivo with associated defensive properties, such as innate immunity. Therefore, in this report, we describe a system to image the progression of infection by single virus particles in tissue harvested from infected animals. We explanted intact innervated tissue from infected mice and imaged fluorescent virus particles in infected axons of the specific ganglionic neurons. Our measurements of virion transport dynamics are consistent with published in vitro results. Importantly, this system enabled us to address a fundamental biological question about the amplification of a herpesvirus infection in a peripheral nervous system circuit.

scholarly journals Visualizing Sphingosine-1-Phosphate Receptor 1(S1P1) Signaling During Central Nervous System De- and Re-Myelination

2021 ◽  
Author(s):  
Ezzat Hashemi ◽  
Hsing-Chuan Tsai ◽  
Ezra Yoseph ◽  
Monica Moreno ◽  
Li-Hao Yeh ◽  
...  
Keyword(s):  
Central Nervous System ◽  
Nervous System ◽  
Fluorescent Protein ◽  
Demyelinating Disease ◽  
Oligodendrocyte Progenitor Cells ◽  
Reporter Mice ◽  
Sphingosine 1 Phosphate ◽  
The Central Nervous System ◽  
Green Fluorescent

Abstract Multiple sclerosis (MS) is an inflammatory demyelinating disease of the central nervous system (CNS) mediated by aberrant immune responses. The current immune modulatory therapies are unable to protect and repair the brain damage caused by the immune attack. One of the therapeutic targets for MS is the sphingosine-1-phosphate (S1P) pathways, which signals via sphingosine-1-phosphate receptors 1-5 (S1P1-5), in the CNS and immune cells. In light of the potential neuro-protective properties of S1P signaling, we utilized the S1P1-GFP (Green fluorescent protein) reporter mice in the cuprizone-induced-demyelination model, to investigate the in vivo S1P- S1P1 signaling in the CNS. We observed S1P1 signaling in a subset of neural stem cells in the subventricular zone (SVZ) during demyelination. Additionally, oligodendrocyte progenitor cells in the SVZ and mature oligodendrocytes in the medial corpus callosum (MCC) expressed S1P1 signaling during remyelination. We did not observe S1P1 signaling in neurons and astrocytes in the cuprizone model. This approach was unable to determine S1P1-GFP signaling in the myeloid cells because of their aberrant GFP expression in GFP reporter mice. Significant S1P1 signaling was observed in lymphocytes during demyelination and inflammation. Our findings reveal β-arrestin dependent S1P1 signaling in oligodendrocyte lineage cells, indicating a role of S1P1 signaling during remyelination.

Sign in / Sign up

Export Citation Format

Share Document