Directional Transneuronal Spread of α-Herpesvirus Infection

Neurotropic herpesviruses depend on long-distance axon transport for the initial establishment of latency in peripheral ganglia (retrograde transport) and for viral spread in axons to exposed body surfaces following reactivation (anterograde transport). Images of neurons infected with herpes simplex virus type 1 (HSV-1), acquired using electron microscopy, have led to a debate regarding why different types of viral structures are seen in axons and which of these particles are relevant to the axon transport process. In this study, we applied time-lapse fluorescence microscopy to image HSV-1 virion components actively translocating to distal axons in primary neurons and neuronal cell lines. Key to these findings, only a small fraction of viral particles were engaged in anterograde transport during the egress phase of infection at any given time. By selective analysis of the composition of the subpopulation of actively transporting capsids, a link between transport of fully assembled HSV-1 virions and the neuronal secretory pathway was identified. Last, we have evaluated the seemingly opposing findings made in previous studies of HSV-1 axon transport in fixed cells and demonstrate a limitation to assessing the composition of individual HSV-1 particles using antibody detection methods.Several members of the alphaherpesvirus subfamily infect and establish latency within the peripheral nervous system of their natural hosts. The human pathogen herpes simplex virus type 1 (HSV-1) and the veterinary pathogen pseudorabies virus (PRV) are commonly studied models of herpesvirus neural infection. These viruses initially infect exposed skin or mucosal tissue and spread to innervating sensory or sympathetic nerve endings. Neurotropic herpesviruses enter nerve terminals by fusion of the envelope with the plasma membrane (47), resulting in release of the capsid and tegument into the cytosol (36, 37). Following fusion, capsids together with a subset of tegument proteins engage the host microtubule transport machinery and move intracellularly to the neuronal cell body (4,35,58), where the DNA genomes are deposited in nuclei and latency is established. HSV-1 and PRV are highly conserved both in postentry capsid transport dynamics and in the composition of the moving particle in axons during this early stage of infection (4, 35). Upon reactivation, progeny viral particles sort into axons from the neuronal cell body and move anterograde back toward nerve terminals, where infection spreads to exposed tissue and ultimately to new hosts. Reactivated spread of HSV-1 can result in several pathologies that range from mild (herpes labialis) to severe (keratitis). The ability of neurotropic herpesviruses to undergo long-distance directed movement in axons is critical both for the establishment of latency and for recurrent disease resulting from reactivation.Understanding the process by which HSV-1 traffics in axons is fundamental to deciphering the molecular mechanisms of microtubule-based transport and how the virus disseminates in animals ...

“…HSV-1 and PRV infection (14,16). The small amount of HSV-1 axon transport relative to that of PRV is formally documented in a side-by-side experiment as part of this study ( Fig.…”

Section: Discussionsupporting

confidence: 53%

Resolving the Assembly State of Herpes Simplex Virus during Axon Transport by Live-Cell Imaging

Antinone

Zaichick

Smith

2010

“…For example, an axon-targeted viral particle must contain Us9 in the transport vesicle membrane to recruit KIF1A for axonal sorting. The virion envelope inside the transport vesicle must carry the viral fusion glycoprotein complex gB/gH/gL to enable entry into a new cell after release from the axon (52,53). However, other data are not compatible with this model.…”

Section: Discussionmentioning

confidence: 99%

Glycoproteins gE and gI Are Required for Efficient KIF1A-Dependent Anterograde Axonal Transport of Alphaherpesvirus Particles in Neurons

Kratchmarov

Kramer

Greco

et al. 2013

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.

“…For PrV, it has now been largely accepted that enveloped virions within vesicles constitute the most abundant, if not exclusive, cargo for anterograde intraaxonal transport following high-resolution ultrastructural electron microscopi-cal studies (5,13,28), as well as live-cell analysis by video microscopy of fluorescently labeled virions (2,3,25) and reinterpretation of earlier results (5,8,13,41).…”

mentioning

confidence: 83%

Ultrastructural Analysis of Virion Formation and Intraaxonal Transport of Herpes Simplex Virus Type 1 in Primary Rat Neurons

Negatsch

Granzow

Maresch

et al. 2010

After primary replication at the site of entry into the host, alphaherpesviruses infect and establish latency in neurons. To this end, they are transported within axons retrograde from the periphery to the cell body for replication and in an anterograde direction to synapses for infection of higher-order neurons or back to the periphery. Retrograde transport of incoming nucleocapsids is well documented. In contrast, there is still significant controversy on the mode of anterograde transport. By high-resolution transmission electron microscopy of primary neuronal cultures from embryonic rat superior cervical ganglia infected by pseudorabies virus (PrV), we observed the presence of enveloped virions in axons within vesicles supporting the "married model" of anterograde transport of complete virus particles within vesicles (C. Maresch, H. Granzow, A. Negatsch, B.G. Klupp, W. Fuchs, J.P. Teifke, and T.C. Mettenleiter, J. Virol. 84:5528-5539, 2010). We have now extended these analyses to the related human herpes simplex virus type 1 (HSV-1). We have demonstrated that in neurons infected by HSV-1 strains HFEM, 17؉ or SC16, approximately 75% of virus particles observed intraaxonally or in growth cones late after infection constitute enveloped virions within vesicles, whereas approximately 25% present as naked capsids. In general, the number of HSV-1 particles in the axons was significantly less than that observed after PrV infection.Herpesviruses are characterized by a distinct virion morphology and the property to establish latent infections with episodes of spontaneous reactivation. Herpesvirus virions contain a DNA genome enclosed in an icosahedral capsid shell, which is in turn embedded in tegument proteins and surrounded by a lipid envelope containing virally encoded, mostly glycosylated proteins. Within the Herpesviridae, three subfamilies, designated the Alpha-, Beta-, and Gammaherpesvirinae, have been recognized (9). The alphaherpesviruses contain pathogens of humans and animals with neuroinvasive properties resulting in infection of and latency in neurons. The genus Simplexvirus encompasses the ubiquitous human herpes simplex viruses, types 1 and 2 (HSV-1 and HSV-2), whereas varicella-zoster virus and several relevant animal pathogens, e.g., the porcine pseudorabies virus (PrV) (Suid herpesvirus 1 [30]), belong to the genus Varicellovirus.Alphaherpesviruses are pantropic but neuroinvasive, i.e., they infect the nervous system after primary replication in mucosal membranes. Neuroinvasion entails two long-distance transport processes of different directionalities (3). There is general consent that retrograde intraaxonal transport of incoming alphaherpesvirus particles to the neuronal cell body for productive replication or establishment of latent infection is effected by dynein-mediated microtubule-associated transport of nucleocapsids coated with "inner" tegument proteins (1,14,26), as occurs during infection of nonpolarized cultured cells (reviewed in reference 39). After reactivation from latency, anterograde...