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.

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.

The Pseudorabies Virus Serine/Threonine Kinase Us3 Contains Mitochondrial, Nuclear and Membrane Localization Signals

Virus Genes ◽  
2004 ◽  
Vol 29 (1) ◽  
pp. 131-145 ◽  
Author(s):  
Christine M. Calton ◽  
Jessica A. Randall ◽  
Melissa W. Adkins ◽  
Bruce W. Banfield
Keyword(s):  
Pseudorabies Virus ◽  
Membrane Localization ◽  
Serine Threonine Kinase ◽  
Threonine Kinase

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 Protein Kinase KIS Localizes to RNA Granules and Enhances Local Translation

2008 ◽  
Vol 29 (3) ◽  
pp. 726-735 ◽  
Author(s):  
Serafí Cambray ◽  
Neus Pedraza ◽  
Marta Rafel ◽  
Eloi Garí ◽  
Martí Aldea ◽  
...  
Keyword(s):  
Cortical Neurons ◽  
Molecular Mechanisms ◽  
Local Translation ◽  
Serine Threonine Kinase ◽  
Threonine Kinase ◽  
Rna Granules ◽  
Axon Development ◽  
Brain Extracts ◽  
Cultured Cortical Neurons ◽  
Actin Mrna

ABSTRACT The regulation of mRNA transport is a fundamental process for cytoplasmic sorting of transcripts and spatially controlled translational derepression once properly localized. There is growing evidence that translation is locally modulated as a result of specific synaptic inputs. However, the underlying molecular mechanisms that regulate this translational process are just emerging. We show that KIS, a serine/threonine kinase functionally related to microtubule dynamics and axon development, interacts with three proteins found in RNA granules: KIF3A, NonO, and eEF1A. KIS localizes to RNA granules and colocalizes with the KIF3A kinesin and the β-actin mRNA in cultured cortical neurons. In addition, KIS is found associated with KIF3A and 10 RNP-transported mRNAs in brain extracts. The results of knockdown experiments indicate that KIS is required for normal neurite outgrowth. More important, the kinase activity of KIS stimulates 3′ untranslated region-dependent local translation in neuritic projections. We propose that KIS is a component of the molecular device that modulates translation in RNA-transporting granules as a result of local signals.

scholarly journals Two Modes of Herpesvirus Trafficking in Neurons: Membrane Acquisition Directs Motion

2006 ◽  
Vol 80 (22) ◽  
pp. 11235-11240 ◽  
Author(s):  
Sarah E. Antinone ◽  
Gregory A. Smith
Keyword(s):  
Cell Body ◽  
Intracellular Transport ◽  
Membrane Lipids ◽  
Neuronal Cell ◽  
Retrograde Transport ◽  
Bound State ◽  
Neuronal Cell Body ◽  
Progeny Virus ◽  
Long Distance ◽  
Viral Particles

ABSTRACT Alphaherpesvirus infection of the mammalian nervous system is dependent upon the long-distance intracellular transport of viral particles in axons. How viral particles are effectively trafficked in axons to either sensory ganglia following initial infection or back out to peripheral sites of innervation following reactivation remains unknown. The mechanism of axonal transport has, in part, been obscured by contradictory findings regarding whether capsids are transported in axons in the absence of membrane components or as enveloped virions. By imaging actively translocated viral structural components in living peripheral neurons, we demonstrate that herpesviruses use two distinct pathways to move in axons. Following entry into cells, exposure of the capsid to the cytosol resulted in efficient retrograde transport to the neuronal cell body. In contrast, progeny virus particles moved in the anterograde direction following acquisition of virion envelope proteins and membrane lipids. Retrograde transport was effectively shut down in this membrane-bound state, allowing for efficient delivery of progeny viral particles to the distal axon. Notably, progeny viral particles that lacked a membrane were misdirected back to the cell body. These findings show that cytosolic capsids are trafficked to the neuronal cell body and that viral egress in axons occurs after capsids are enshrouded in a membrane envelope.

scholarly journals Heterogeneity of a Fluorescent Tegument Component in Single Pseudorabies Virus Virions and Enveloped Axonal Assemblies

2005 ◽  
Vol 79 (7) ◽  
pp. 3903-3919 ◽  
Author(s):  
T. del Rio ◽  
T. H. Ch'ng ◽  
E. A. Flood ◽  
S. P. Gross ◽  
L. W. Enquist
Keyword(s):  
Cell Body ◽  
Secretory Pathway ◽  
Pseudorabies Virus ◽  
Molecular Mechanisms ◽  
Fluorescent Protein ◽  
Brefeldin A ◽  
Cultured Neurons ◽  
Long Distance ◽  
Heterogeneous Distribution ◽  
Green Fluorescent

ABSTRACT The molecular mechanisms responsible for long-distance, directional spread of alphaherpesvirus infections via axons of infected neurons are poorly understood. We describe the use of red and green fluorescent protein (GFP) fusions to capsid and tegument components, respectively, to visualize purified, single extracellular virions and axonal assemblies after pseudorabies virus (PRV) infection of cultured neurons. We observed heterogeneity in GFP fluorescence when GFP was fused to the tegument component VP22 in both single extracellular virions and discrete puncta in infected axons. This heterogeneity was observed in the presence or absence of a capsid structure detected by a fusion of monomeric red fluorescent protein to VP26. The similarity of the heterogeneous distribution of these fluorescent protein fusions in both purified virions and in axons suggested that tegument-capsid assembly and axonal targeting of viral components are linked. One possibility was that the assembly of extracellular and axonal particles containing the dually fluorescent fusion proteins occurred by the same process in the cell body. We tested this hypothesis by treating infected cultured neurons with brefeldin A, a potent inhibitor of herpesvirus maturation and secretion. Brefeldin A treatment disrupted the neuronal secretory pathway, affected fluorescent capsid and tegument transport in the cell body, and blocked subsequent entry into axons of capsid and tegument proteins. Electron microscopy demonstrated that in the absence of brefeldin A treatment, enveloped capsids entered axons, but in the presence of the inhibitor, unenveloped capsids accumulated in the cell body. These results support an assembly process in which PRV capsids acquire a membrane in the cell body prior to axonal entry and subsequent transport.

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