Rabies Virus Hijacks and Accelerates the p75NTR Retrograde Axonal Transport Machinery

Gluska, Shani; Zahavi, Eitan Erez; Chein, Michael; Gradus, Tal; Bauer, Anja; Finke, Stefan; Perlson, Eran

doi:10.1371/journal.ppat.1004348

Cited by 99 publications

(110 citation statements)

References 57 publications

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“…Recent work on p75NTR receptor and RABV cointernalization at axon endings showed that RABV not only hijacks but also accelerates axonal transport mechanisms (24). Whereas p75NTR-dependent transport manipulation is related to early phases of neuronal infections, we show that also posttranslational modification of microtubules can be induced by the expression of a virus protein.…”

Section: Discussionmentioning

confidence: 53%

A Dynein Light Chain 1 Binding Motif in Rabies Virus Polymerase L Protein Plays a Role in Microtubule Reorganization and Viral Primary Transcription

Bauer

Nolden

Nemitz

et al. 2015

J Virol

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Rabies virus (RABV) polymerase L together with phosphoprotein P forms the PL polymerase complex that is essential for replication and transcription. However, its exact mechanism of action, interactions with cellular factors, and intracellular distribution are yet to be understood. Here by imaging a fluorescently tagged polymerase (mCherry-RABV-L), we show that L accumulates at acetylated and reorganized microtubules (MT). In silico analysis revealed a dynein light chain 1 (DLC1) binding motif in L that could mediate MT binding through dynein motors. As DLC1 binding by polymerase cofactor P is known, we compared the impact of the DLC1-binding motifs in P and L. Viruses with mutations in the respective motifs revealed that both motifs are required for efficient primary transcription, indicating that DLC1 acts as a transcription enhancer by binding to both P and L. Notably, also the levels of cellular DLC1 protein were regulated by both motifs, suggesting regulation of the DLC1 gene expression by both P and L. Finally, disruption of the motif in L resulted in a cell-type-specific loss of MT localization, demonstrating that DLC1 is involved in L-mediated cytoskeleton reorganization. Overall, we conclude that DLC1 acts as a transcription factor that stimulates primary RABV transcription by binding to both P and L. We further conclude that L influences MT organization and posttranslational modification, suggesting a model in which MT manipulation by L contributes to efficient intracellular transport of virus components and thus may serve as an important step in virus replication. IMPORTANCERegulation of rabies virus polymerase complex by viral and cellular factors thus far has not been fully understood. Although cellular dynein light chain 1 (DLC1) has been reported to increase primary transcription by binding to polymerase cofactor phosphoprotein P, the detailed mechanism is unknown, and it is also not known whether the large enzymatic polymerase subunit L is involved. By fluorescence microscopy analysis of fluorescence-tagged rabies virus L, in silico identification of a potential DLC1 binding site in L, and characterization of recombinant rabies virus mutants, we show that a DLC1 binding motif in L is involved in cytoskeleton localization and reorganization, primary transcription regulation by DLC1, and regulation of cellular DLC1 gene expression. By providing evidence for a direct contribution of a DLC1 binding motif in L, our data significantly increase the understanding of rabies virus polymerase regulation and host manipulation by the virus as well. Rhabdoviridae) are neurotropic viruses that enter the central nervous system (CNS) by axonal transport along microtubules (1). The negative-sense genomic viral RNA encodes five proteins, of which the 240-kDa large enzymatic subunit L and the 35-kDa cofactor phosphoprotein P form the PL polymerase complex (2). The viral polymerase is a ribonucleoprotein (RNP)-dependent polymerase that only uses the genomic RNA as a template for RNA synthesis when it is encap...

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Section: Discussionmentioning

confidence: 53%

A Dynein Light Chain 1 Binding Motif in Rabies Virus Polymerase L Protein Plays a Role in Microtubule Reorganization and Viral Primary Transcription

Bauer

Nolden

Nemitz

et al. 2015

J Virol

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“…Recently, Hislop et al showed that pseudotyping lentiviral vector with rabies virus envelope glycoprotein (RVG) mediates axonal retrograde transport through Rab5-positive endosomes and accumulation within the Rab7 compartment (39). Gluska et al also showed that rabies virus not only hijacks the signaling endosome system but also accelerates the p75 receptor retrograde axonal transport machinery, representing a fast and efficient mechanism to gain access to the central nervous system (40). These studies indicate that pathogens have evolved to take advantage of long distance signaling mechanisms employed by neurons.…”

Section: Figmentioning

confidence: 98%

Signaling Over Distances

Saito

Cavalli

2016

Molecular & Cellular Proteomics

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Neurons are extremely polarized cells. Axon lengths often exceed the dimension of the neuronal cell body by several orders of magnitude. These extreme axonal lengths imply that neurons have mastered efficient mechanisms for long distance signaling between soma and synaptic terminal. These elaborate mechanisms are required for neuronal development and maintenance of the nervous system. Neurons can fine-tune long distance signaling through calcium wave propagation and bidirectional transport of proteins, vesicles, and mRNAs along microtubules. The signal transmission over extreme lengths also ensures that information about axon injury is communicated to the soma and allows for repair mechanisms to be engaged. This review focuses on the different mechanisms employed by neurons to signal over long axonal distances and how signals are interpreted in the soma, with an emphasis on proteomic studies. We also discuss how proteomic approaches could help further deciphering the signaling mechanisms operating over long distance in axons. Neurons have unique and highly polarized morphologies. The axon allows intracellular communication between the cell soma and the distantly located synaptic terminal. The extreme length of axons relative to the cell soma diameter poses great challenges for neuronal development, maintenance, and repair. Anterograde and retrograde axonal transport of proteins vesicles and RNA along the microtubule tracks in the axon is crucial to sustain the required signaling events underlying development and maintenance of the nervous system. The retrograde transport system is also used following axon injury to communicate information from the site of injury back to the soma. Communication between distant axon locations and the cell soma is also ensured by a more rapid signal encoded in calcium waves. Incoming signals from distant axon locations are interpreted by the soma to regulate gene expression for survival or repair. The neuronal epigenome is also influenced by incoming signals to appropriately coordinate transcriptional responses. In this review, we discuss the state of the field regarding the different mechanisms employed by neurons to signal intracellularly over long distances in axons and how signals are interpreted in the cell soma, with an emphasis on proteomic studies. We also discuss how the use of proteomics could further help in understanding long distance signaling system in axons. The communication employed by neurons to signal along dendrites has been reviewed in detail elsewhere (1) and will not be discussed here.Calcium Influx, Back Propagation, and Long Distance Effects-Alterations in axonal calcium levels affect multiple and diverse signaling events, including local protein synthesis and degradation. These local signals can have long distance effects. Axon injury provides a powerful system to study the role of calcium in axonal signaling, because injury-induced calcium influx in the axon triggers important downstream events at the injury site and the soma (Fig.

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“…Colocalization of EGFP-P particles with nucleoprotein N. Although EGFP-P has been previously used as a marker for extracellular virions (8) and retrograde axonal transport during virus entry (9,11,12), here we checked colocalization with nucleoprotein N to see whether EGFP-P particles transported in the axons at later phases of DRG infection indeed could represent viral RNPs. Therefore, DRG neurons were infected at the cell soma, and 3 days later the cells were fixed and immunostained with an N-specific antibody.…”

Section: Rabv Infection and Live Imaging Of Drg Neuronsmentioning

confidence: 99%

“…By the combination of GFP-tagged rabies virus RNPs and incorporation of a membrane-anchored red fluorescent protein into virus particles, double-labeled rabies virus particles were further used to show retrograde axonal transport of complete, membrane-enveloped virions (11), suggesting that in neurons, receptor usage by the viral glycoprotein and subsequent receptor-dependent endocytosis are crucial for entering the longdistance transport to the cell soma. In neurons from mouse dorsal root ganglions (DRGs), most of the retrogradely transported RABV was cotransported with low-affinity nerve growth factor receptor (p75NTR) and colocalized with acidified transport vesicles (12), confirming RABV transport in vesicles. This provided direct evidence that one of the three proposed neuronal RABV receptors (13) is indeed a receptor that directs incoming virus particles in the retrograde transport pathway.…”

mentioning

confidence: 96%

“…This provided direct evidence that one of the three proposed neuronal RABV receptors (13) is indeed a receptor that directs incoming virus particles in the retrograde transport pathway. Notably, RABV not only was cotransported with p75NTR but also accelerated the p75NTR retrograde axonal transport machinery, indicating that RABV modulates axonal transport in the course of retrograde infection (12).…”

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confidence: 97%

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Anterograde Glycoprotein-Dependent Transport of Newly Generated Rabies Virus in Dorsal Root Ganglion Neurons

Bauer

Nolden

Schröter

et al. 2014

J Virol

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Rabies virus (RABV) spread is widely accepted to occur only by retrograde axonal transport. However, examples of anterograde RABV spread in peripheral neurons such as dorsal root ganglion (DRG) neurons indicated a possible bidirectional transport by an uncharacterized mechanism. Here, we analyzed the axonal transport of fluorescence-labeled RABV in DRG neurons by livecell microscopy. Both entry-related retrograde transport of RABV after infection at axon endings and postreplicative transport of newly formed virus were visualized in compartmentalized DRG neuron cultures. Whereas entry-related transport at 1.5 m/s occurred only retrogradely, after 2 days of infection, multiple particles were observed in axons moving in both the anterograde and retrograde directions. The dynamics of postreplicative retrograde transport (1.6 m/s) were similar to those of entry-related retrograde transport. In contrast, anterograde particle transport at 3.4 m/s was faster, indicating active particle transport. Interestingly, RABV missing the glycoproteins did not move anterogradely within the axon. Thus, anterograde RABV particle transport depended on the RABV glycoprotein. Moreover, colocalization of green fluorescent protein (GFP)-labeled ribonucleoproteins (RNPs) and glycoprotein in distal axonal regions as well as cotransport of labeled RNPs with membrane-anchored mCherry reporter confirmed that either complete enveloped virus particles or vesicle associated RNPs were transported. Our data show that anterograde RABV movement in peripheral DRG neurons occurs by active motor protein-dependent transport. We propose two models for postreplicative long-distance transport in peripheral neurons: either transport of complete virus particles or cotransport of RNPs and G-containing vesicles through axons to release virus at distal sites of infected DRG neurons. IMPORTANCERabies virus retrograde axonal transport by dynein motors supports virus spread over long distances and lethal infection of the central nervous system. Though active rabies virus transport has been widely accepted to be unidirectional, evidence for anterograde spread in peripheral neurons supports the hypothesis that in some neurons RABV also enters the anterograde pathway by so-far unknown mechanisms. By live microscopy we visualized fast anterograde axonal transport of rabies virus. The velocities exceeded those of retrograde movements, suggesting that active, most likely kinesin-dependent transport machineries are involved. Dependency of anterograde transport on the expression of virus glycoprotein G and cotransport with vesicles further suggest that complete enveloped virus particles or cotransport of virus ribonucleoprotein and G-containing vesicles occurred. These data provide the first insight in the mechanism of anterograde rabies virus transport and substantially contribute to the understanding of RABV replication and spread of newly formed virus in peripheral neurons.

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Rabies Virus Hijacks and Accelerates the p75NTR Retrograde Axonal Transport Machinery

Cited by 99 publications

References 57 publications

A Dynein Light Chain 1 Binding Motif in Rabies Virus Polymerase L Protein Plays a Role in Microtubule Reorganization and Viral Primary Transcription

A Dynein Light Chain 1 Binding Motif in Rabies Virus Polymerase L Protein Plays a Role in Microtubule Reorganization and Viral Primary Transcription

Signaling Over Distances

Anterograde Glycoprotein-Dependent Transport of Newly Generated Rabies Virus in Dorsal Root Ganglion Neurons

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