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.

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 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 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.

From sand to sea: tracing the production and trade in glass beads from the 10th-century Cirebon shipwreck in the Java Sea

2020 ◽  
Author(s):  
Carolyn Swan
Keyword(s):  
Glass Production ◽  
Glass Beads ◽  
Rock Crystal ◽  
Long Distance ◽  
Merchant Ship ◽  
The Indian Ocean ◽  
Java Sea ◽  
Indian Ocean World ◽  
Long Distance Movement ◽  
Lapis Lazuli

Around the year 970 CE, a merchant ship carrying an assortment of goods from East Africa, Persia, India, Sri Lanka, Southeast Asia, and China foundered and sank to the bottom of the Java Sea. Thousands of beads made from many different materials—ceramic, jet, coral, banded stone, lapis lazuli, rock crystal, sapphire, ruby, garnet, pearl, gold, and glass—attest to the long-distance movement and trade of these small and often precious objects throughout the Indian Ocean world. The beads made of glass are of particular interest, as closely-dated examples are very rare and there is some debate as to where glass beads were being made and traded during this period of time. This paper examines 18 glass beads from the Cirebon shipwreck that are now in the collection of Qatar Museums, using a comparative typological and chemical perspective within the context of the 10th-century glass production. Although it remains uncertain where some of the beads were made, the composition of the glass beads points to two major production origins for the glass itself: West Asia and South Asia.

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