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Glycoproteins gE and gI are required for efficient KIF1A-dependent anterograde axonal transport of alphaherpesvirus particles in neurons.

ABSTRACT: Alphaherpesviruses, including pseudorabies virus (PRV), spread directionally within the nervous systems of their mammalian hosts. Three viral membrane proteins are required for efficient anterograde-directed spread of infection in neurons, including Us9 and a heterodimer composed of the glycoproteins gE and gI. We previously demonstrated that the kinesin-3 motor KIF1A mediates anterograde-directed transport of viral particles in axons of cultured peripheral nervous system (PNS) neurons. The PRV Us9 protein copurifies with KIF1A, recruiting the motor to transport vesicles, but at least one unidentified additional viral protein is necessary for this interaction. Here we show that gE/gI are required for efficient anterograde transport of viral particles in axons by mediating the interaction between Us9 and KIF1A. In the absence of gE/gI, viral particles containing green fluorescent protein (GFP)-tagged Us9 are assembled in the cell body but are not sorted efficiently into axons. Importantly, we found that gE/gI are necessary for efficient copurification of KIF1A with Us9, especially at early times after infection. We also constructed a PRV recombinant that expresses a functional gE-GFP fusion protein and used affinity purification coupled with mass spectrometry to identify gE-interacting proteins. Several viral and host proteins were found to associate with gE-GFP. Importantly, both gI and Us9, but not KIF1A, copurified with gE-GFP. We propose that gE/gI are required for efficient KIF1A-mediated anterograde transport of viral particles because they indirectly facilitate or stabilize the interaction between Us9 and KIF1A.

SUBMITTER: Kratchmarov R

PROVIDER: S-EPMC3754139 | biostudies-literature | 2013 Sep

REPOSITORIES: biostudies-literature

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Glycoproteins gE and gI are required for efficient KIF1A-dependent anterograde axonal transport of alphaherpesvirus particles in neurons.

Kratchmarov Radomir R Kramer Tal T Greco Todd M TM Taylor Matthew P MP Ch'ng Toh Hean TH Cristea Ileana M IM Enquist Lynn W LW

Journal of virology 20130626 17

Alphaherpesviruses, including pseudorabies virus (PRV), spread directionally within the nervous systems of their mammalian hosts. Three viral membrane proteins are required for efficient anterograde-directed spread of infection in neurons, including Us9 and a heterodimer composed of the glycoproteins gE and gI. We previously demonstrated that the kinesin-3 motor KIF1A mediates anterograde-directed transport of viral particles in axons of cultured peripheral nervous system (PNS) neurons. The PRV ...[more]

PMID: 23804637

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Project description:UNLABELLED:Pseudorabies virus (PRV), an alphaherpesvirus with a broad host range, replicates and spreads in chains of synaptically connected neurons. The PRV protein Us9 is a small membrane protein that is highly conserved among alphaherpesviruses and is essential for anterograde axonal spread in neurons. Specifically, the Us9 protein is required for the sorting of newly assembled PRV particles into axons. However, the molecular details underlying the function of Us9 are poorly understood. Here we constructed PRV strains that express functional green fluorescent protein (GFP)-Us9 fusion proteins in order to visualize axonal transport of viral particles in infected rat superior cervical ganglion neurons. We show that GFP-Us9-labeled structures are transported exclusively in the anterograde direction within axons. Additionally, the vast majority of anterograde-directed capsids (labeled with VP26-monomeric red fluorescent protein) and a viral membrane protein (labeled with glycoprotein M fused to mCherry) are cotransported with GFP-Us9 in the anterograde direction. In contrast, during infection with PRV strains that express nonfunctional mutant GFP-Us9 proteins, cotransport of mutant GFP-Us9 with capsids in axons is abolished. These findings show that axonal sorting of progeny viral particles is dependent upon the association of viral structures with membranes that contain functional Us9 proteins. This association is required for anterograde spread of infection in neurons. IMPORTANCE:Alphaherpesviruses, such as pseudorabies virus (PRV), are parasites of the mammalian nervous system. These viruses spread over long distances in chains of synaptically connected neurons. PRV encodes several proteins that mediate directed virion transport and spread of infection. Us9 is a highly conserved viral membrane protein that is essential for anterograde neuronal spread of infection. In the absence of Us9, newly replicated viral particles are assembled in the cell body but are not sorted into or transported within axons. Here, we constructed and characterized novel PRV strains that express functional green fluorescent protein (GFP)-Us9 fusion proteins in order to visualize its localization in living neurons during infection. This enabled us to better understand the function of Us9 in facilitating the spread of infection. We show that all viral particles moving in the anterograde direction are labeled with GFP-Us9, suggesting that the presence of Us9 determines the capacity for directed transport within axons.

Project description:During infection of neurons by alphaherpesviruses including Pseudorabies virus (PRV) and Herpes simplex virus type 1 (HSV-1) viral nucleocapsids assemble in the cell nucleus, become enveloped in the cell body then traffic into and down axons to nerve termini for spread to adjacent epithelia. The viral membrane protein US9p and the membrane glycoprotein heterodimer gE/gI play critical roles in anterograde spread of both HSV-1 and PRV, and several models exist to explain their function. Biochemical studies suggest that PRV US9p associates with the kinesin-3 motor KIF1A in a gE/gI-stimulated manner, and the gE/gI-US9p complex has been proposed to recruit KIF1A to PRV for microtubule-mediated anterograde trafficking into or along the axon. However, as loss of gE/gI-US9p essentially abolishes delivery of alphaherpesviruses to the axon it is difficult to determine the microtubule-dependent trafficking properties and motor-composition of Δ(gE/gI-US9p) particles. Alternatively, studies in HSV-1 have suggested that gE/gI and US9p are required for the appearance of virions in the axon because they act upstream, to help assemble enveloped virions in the cell body. We prepared Δ(gE/gI-US9p) mutant, and control parental PRV particles from differentiated cultured neuronal or porcine kidney epithelial cells and quantitated the efficiency of virion assembly, the properties of microtubule-dependent transport and the ability of viral particles to recruit kinesin motors. We find that loss of gE/gI-US9p has no significant effect upon PRV particle assembly but leads to greatly diminished plus end-directed traffic, and enhanced minus end-directed and bidirectional movement along microtubules. PRV particles prepared from infected differentiated mouse CAD neurons were found to be associated with either kinesin KIF1A or kinesin KIF5C, but not both. Loss of gE/gI-US9p resulted in failure to recruit KIF1A and KF5C, but did not affect dynein binding. Unexpectedly, while KIF5C was expressed in undifferentiated and differentiated CAD neurons it was only found associated with PRV particles prepared from differentiated cells.

Dataset Information

Glycoproteins gE and gI are required for efficient KIF1A-dependent anterograde axonal transport of alphaherpesvirus particles in neurons.

Publications

Glycoproteins gE and gI are required for efficient KIF1A-dependent anterograde axonal transport of alphaherpesvirus particles in neurons.

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