Inner tegument proteins of Herpes Simplex Virus are sufficient for intracellular capsid motility in neurons but not for axonal targeting

Upon reactivation from latency and during lytic infections in neurons, alphaherpesviruses assemble cytosolic capsids, capsids associated with enveloping membranes, and transport vesicles harboring fully enveloped capsids. It is debated whether capsid envelopment of herpes simplex virus (HSV) is completed in the soma prior to axonal targeting or later, and whether the mechanisms are the same in neurons derived from embryos or from adult hosts. We used HSV mutants impaired in capsid envelopment to test whether the inner tegument proteins pUL36 or pUL37 necessary for microtubule-mediated capsid transport were sufficient for axonal capsid targeting in neurons derived from the dorsal root ganglia of adult mice. Such neurons were infected with HSV1-ΔUL20 whose capsids recruited pUL36 and pUL37, with HSV1-ΔUL37 whose capsids associate only with pUL36, or with HSV1-ΔUL36 that assembles capsids lacking both proteins. While capsids of HSV1-ΔUL20 were actively transported along microtubules in epithelial cells and in the somata of neurons, those of HSV1-ΔUL36 and -ΔUL37 could only diffuse in the cytoplasm. Employing a novel image analysis algorithm to quantify capsid targeting to axons, we show that only a few capsids of HSV1-ΔUL20 entered axons, while vesicles transporting gD utilized axonal transport efficiently and independently of pUL36, pUL37, or pUL20. Our data indicate that capsid motility in the somata of neurons mediated by pUL36 and pUL37 does not suffice for targeting capsids to axons, and suggest that capsid envelopment needs to be completed in the soma prior to targeting of herpes simplex virus to the axons, and to spreading from neurons to neighboring cells.


Introduction
Many pathogens spread within the nervous system by fast intracellular axonal transport mediated by microtubule motors, and progeny particles are transmitted via synapses from neuron to neuron [1,2]. Such life style allows pathogens to hide from the extracellular complement system, from antibodies, and from immune cells. But it comes with the challenge of finding the tiny exit gates from the neuronal somata, and of specific targeting to dendrites or to axons. It has been estimated that the cross section of the axonal outlet occupies less than 2 thousandth of the surface area of the soma [3,4]. The molecular post codes achieving this remarkable intracellular trafficking control are not well understood; neither for host nor for pathogen cargoes.
Alphaherpesviruses such as herpes simplex viruses (HSV) replicate in the skin, the eyes, and the oral, genital, or nasal mucosa, and then enter local axon endings of sensory or autonomic neurons in which they establish latency with little gene expression. Latent HSV-1 genomes have been detected in human cranial ganglia, especially in the trigeminal ganglia, while dorsal root ganglia (DRG) can harbor both, HSV-1 and HSV-2 [5][6][7]. Viral gene expression can be reactivated upon stress and a decline in the local immune responses, and progeny viruses are transported back to the periphery, where they cause lytic infections. Efficient targeting of alphaherpesviruses to the axons is essential for axonal spread and thus disease. Severe outcomes are potentially blinding herpes keratitis upon spread within the eyes or life-threatening herpes encephalitis upon spread within the brain [reviewed in 8,9].
HSV-1 cytosolic capsids and complete virions within transport vesicles are targeted from the neuronal somata to axons to a varying extent; this has led to different hypotheses on the mode of neuronal alphaherpesvirus assembly [reviewed in 23,40,41]. According to the married model, capsid envelopment occurs exclusively in the soma, and only transport vesicles harboring complete virions enter axons. The separate model refers to different cargo structures being targeted independently of each other to the axons; namely capsids with associated tegument proteins as well as vesicles harboring viral envelope proteins and tegument proteins associated to their cytosolic tails.
Many structural proteins contribute to the neuronal spread of alphaherpesviruses, but the molecular determinants that are required for microtubule motor recruitment and for targeting from the soma to the axon gate have not been fully dissected. Purified HSV-1 capsids with inner tegument proteins can recruit the microtubule motors kinesin-1, kinesin-2, dynein and its cofactor dynactin to their surface, are translocated along microtubules in vitro, and can dock to nuclear pores [11,[42][43][44]. pUL36 of HSV-1 and of the porcine pseudorabies virus (PRV) harbor potential binding motives for kinesin-1 light chains, and PRV-pUL36 has been shown to bind dynein and dynactin [45][46][47]. For these reasons and based on many additional functional studies, pUL36 and pUL37 are considered the most likely candidates for microtubule motor recruitment [reviewed in 23,45,46,48].
We therefore hypothesized that pUL36 and pUL37 might be sufficient for axonal capsid targeting and axonal capsid transport. As capsid envelopment may be a fast process, and since therefore the steady-state concentration of cytosolic capsids might be low, we used HSV-1 mutants to enrich for assembly intermediates. We infected neurons with HSV1-ΔUL20, HSV1-ΔUL37 or HSV1-ΔUL36 that accumulated cytosolic capsids decorated with pUL36 and pUL37, only with pUL36, or lacking both. Our previous data indicate that acquisition of both pUL36 and pUL37 is essential to enable capsids to enlist microtubule transport in epithelial cells [20]. However, while these inner tegument proteins enabled intracellular capsid motility of HSV1-ΔUL20 in epithelial cells and the somata of DRG neurons, they did not suffice to target cytosolic capsids to the axons. Our data indicate that HSV-1 particles have to acquire additional tegument and envelope proteins, and suggest that cytoplasmic envelopment needs to be completed prior to axonal transport which is in accordance with the married model for assembly of alphaherpesviruses in neurons.

HSV-1 infection of mature neurons from dorsal root ganglia of adult mice
To investigate HSV-1 axonal targeting, we cultured primary neurons derived from dissociated DRG of adult mice until they had developed mature neurites. Within 3 to 5 days of culture in vitro (div), the neurons expressed the axonal microtubule-associated protein tau (not shown), phosphorylated neurofilament, un-phosphorylated neurofilament, and ankyrinG. In the somata, there were short β-III-tubulin microtubules and careful analysis often revealed a perinuclear microtubule-organizing center (S1Aii Fig, arrow), but individual microtubules could not be discerned in the neurites (S1A Likewise ankyrinG, another axonal marker was targeted to the neurites (S1D Fig). In situ, peripheral sensory axons also contain ankyrinG along the entire axons up to the dermal-epidermal junction [50].
To evaluate the microtubule polarity in these neurites, we transduced the neurons at 1 div with a lentiviral vector expressing end-binding protein 3 (EB3), which associates with dynamically growing microtubule plus-ends [51]. At 4 div, live-cell imaging as well as time-projections showed that all EB3-GFP comets moved away from the soma, suggesting that most, if not all, microtubule plus-ends faced towards the distal ends of the neurites (not shown). These results indicate that the DRG neurons had regrown neurites with axonal features and with a unipolar microtubule polarity. We have shown previously that such neurons are productively infected, and that they transmit HSV-1 to co-cultured epithelial cells upon infection with the HSV1 (17 + )Lox-Che (c.f. Table 1), which had been cloned into a bacterial artificial chromosome (BAC), and which expresses mCherry as a reporter [52,53].
HSV-1 mutants lacking pUL36, pUL37, or pUL20 assemble different cytosolic capsids To generate cytosolic capsids with different tegument protein composition, and thus to determine whether the requirements for intracellular capsid motility parallel those for axonal targeting, we constructed HSV1(17 + )Lox-CheVP26-ΔUL36 [20], -CheVP26-ΔUL37 [20], and -CheVP26-ΔUL20 (this study) in the same genetic background (c.f. Table 1). We deleted the ATG start codon and a second ATG, and inserted three stop codons into the UL20 gene, since it includes the promotor of UL19 that codes for the major capsid protein VP5. Restriction analyses of pHSV1(17 + )Lox-ΔUL20 and -CheVP26-ΔUL20 indicated the addition of mCherry to VP26 (AscI and BamHI, C in S2A Fig), and that a resistance gene inserted with the UL20 mutations had been removed (XhoI, asterisks in S2A Fig). Further HindIII (S2A Fig), EcoRI, or EcoRV digestions resulted in the expected fragment sizes (not shown). HSV1 (17 + )Lox-ΔUL20 and -CheVP26-ΔUL20 were recovered by transfecting the corresponding BACs into Flp-In-CV-1-cells that express pUL20 in trans [54]. Sequencing of the mutated region confirmed the introduction of the intended changes (not shown). The lack of the ATGs and the introduced stop codons prevented the expression of pUL20, whereas pUL37 expression was unchanged (S2B Fig). The intra-and extracellular titers of HSV1(17 + )Lox-ΔUL20 and -CheVP26-ΔUL20 were about 1,000-fold lower than their parental strains in non-complementing Vero cells, but higher in a pUL20 complementing cell line (S2C and S2D Fig). Similar results have been reported for HSV1-ΔUL20 mutants in other genetic Using conventional electron microscopy, we next analyzed virus morphogenesis. Upon infection of Vero cells with HSV1(17 + )Lox (Fig 1A) or -CheVP26 (not shown), viral particle maturation proceeded as expected with the formation of nuclear capsids, the appearance of cytosolic capsids, partially and completely enveloped cytoplasmic capsids, and extracellular virions associated with the plasma membrane. With secondary envelopment, capsids acquired an electron dense tegument layer, and the electron dense genomes were well preserved (Fig  1Ai and 1Aiii; asterisk). Therefore the morphology of capsids and tegument of virions inside authentic transport vesicles was more similar to that of extracellular virions (Fig 1Aii; black arrow) than to that of cytosolic capsids (Fig 2Aiii; white arrowhead) even if they were partially enveloped (Fig 1Ai and 1Aiii, black arrowhead). Nuclear capsid egress of the mutants HSV1 (17 + )Lox-ΔUL20 (Fig 1Bi and 1Bii) and -CheVP26-ΔUL20 (Fig 1Biii) was unaffected, resulting in many cytosolic capsids, and several of them were closely associated with cytoplasmic membranes (Fig 1Bi and 1Bii; black arrowhead). We detected neither capsids with the characteristic morphology of completed secondary envelopment nor virions bound to the plasma membrane in the absence of pUL20. Furthermore, in contrast to infection with HSV1(17 + )Lox-ΔUL36 or -ΔUL37 [20], we did not detect any cytoplasmic or extracellular L-particles, which are viral envelopes with an electron dense tegument but lacking capsids [58][59][60].

HSV-1-pUL20 is dispensable for capsid motility in epithelial cells and the somata of neurons
To characterize the intracellular capsid motility in the presence of different inner tegument proteins, we infected Vero cells with HSV1(17 + )Lox-CheVP26, -CheVP26-ΔUL36, -CheVP26-ΔUL37, or -CheVP26-ΔUL20 and acquired confocal fluorescence time-lapse movies with a temporal resolution of 5 images per second. For a direct comparison, we analyzed in parallel movies with -CheVP26 and -CheVP26-ΔUL20 recorded in this study as well as movies that we had recorded with -CheVP26-ΔUL36 and -CheVP26-ΔUL37 previously and published in Sandbaumhüter et al. [20]. The time-projections of the tracks show that capsids of Lox-CheVP26 (Fig 4Ai) and -CheVP26-ΔUL20 (Fig 4Aiv) exerted short and long range transport towards the nucleus and towards the cell periphery (S1 and S4 Movies). In contrast, the tracks of -CheVP26-ΔUL36 (Fig 4Aii) and -CheVP26-ΔUL37 (Fig 4Aiii) revealed only random, undirected motility (S2 and S3 Movies).
While the average track length (Fig 4Bi) and the maximum step velocity (Fig 4Bii) were similar for the capsids of -CheVP26, -CheVP26-ΔUL36, -CheVP26-ΔUL37, and -CheVP26-ΔUL20, there were more tracks with a length of more than 5 μm and with a velocity exceeding 1 μm/s for the capsids of the parental Lox-CheVP26 and the -CheVP26-ΔUL20 than for the capsids of -CheVP26-ΔUL36 and -CheVP26-ΔUL37. We furthermore determined the mean square displacement exponent (MSD ex ) for each track (Fig 4Biii). The MSD ex is a characteristic feature for the displacement mode of a single motile particle from its starting position over time. The MSD is defined as the square of the travelled distance from the starting point of the track, which is calculated for each time point, and plotted against the time. The slope of such a curve in a log-log plot defines the MSD ex and indicates whether a particle has undergone active transport (MSD ex > 1), free diffusion (MSD ex = 1), or confined diffusion [MSD ex <1; 61,62,63]. While the MSD ex of the majority of the tracks denoted free or confined diffusion as expected for any intracellular cargo, the MSD ex was greater than 1 for more tracks of Lox-CheVP26 and -CheVP26-ΔUL20 than for -CheVP26-ΔUL36 or -CheVP26-ΔUL37.
We furthermore infected DRG neurons with HSV1(17 + )Lox-CheVP26, -CheVP26-ΔUL36, -CheVP26-ΔUL37, or -CheVP26-ΔUL20 and acquired spinning disk microscopy time lapse movies of the cell bodies with a temporal resolution of 20 images per second. Similar as in the epithelial cells, capsids of the parental (Fig 5Ai; S5 Movie) and -CheVP26-ΔUL20 (Fig 5Aiv; S8 Movie) were transported over short and longer distances as indicated by the time projection lines. In contrast, the capsids of -CheVP26-ΔUL36 (Fig 5Aii; S6 Movie) and -CheVP26-ΔUL37 (Fig 5Aiii, S7 Movie) only moved in an undirected fashion. Quantification revealed that the transport characteristics in DRG neuronal cell bodies were the same as in epithelial cells (Fig 5Bi to 5iii). Thus, the motility features of the cytosolic capsids of -CheVP26-ΔUL36 or -CheVP26-ΔUL37 in epithelial cells and sensory neurons were indicative for intracellular diffusion which is also the mode of motility in the absence of microtubules after nocodazole treatment [62]. In contrast, the capsids of -CheVP26 and -CheVP26-ΔUL20 had transport characteristics typical for directed, active microtubule-dependent transport.
HSV-1 axonal targeting in the absence of pUL36, pUL37, or pUL20 After we had characterized the intracellular motility of capsids in the presence of pUL36 and pUL37 (-ΔUL20), of only pUL36 (-ΔUL37), or lacking both (-ΔUL36), we asked which capsid Axonal targeting of HSV1 particles types could be targeted to the axonal outlet. We infected primary DRG neurons with HSV1 (17 + )Lox, -ΔUL36, -ΔUL37, or ΔUL20 (Fig 6) or the respective HSV1(17 + )Lox-CheVP26 strains (S3 Fig, Fig 7). At 24 to 26 hpi, the cells were fixed, permeabilized and labeled with antibodies directed against the capsid protein VP26 (Fig 6i and 6iv panels, green in 6iii), and the tegument proteins VP22 (Fig 6A-6D, ii and v panels, red in iii and vi), the tegument protein VP13/14 (Fig 7, ii panels, green in iii), the envelope protein gD (Fig 6E-6H, ii and iv panels, red iii and vi), the envelope protein gB (Fig 7, vi panels, green in viii), or the neuron specific beta-3-tubulin (Fig 7vii). Representative images from at least 3 independent experiments for the different strains are shown in Figs 6, S3 and 7.
After infection with HSV1(17 + )Lox (Fig 6A and 6E) or HSV1(17 + )Lox-CheVP26 (Figs S3A and 7A), the nucleoplasm (Figs 6Ai, 6Ei and S3Ai), the cytoplasm (Figs 6Ai, 6Ei and S3Ai) and the axons (Fig 6Aiv, 6Eiv and 7A) were filled with capsids; in the cytoplasm, the capsids were often clustered. Infection with -ΔUL36 (Figs 6B, 6F, S3B and 7B) resulted in a dispersed and more random distribution of capsids in the cytoplasm of the somata (S3B Fig), with fewer capsids being targeted to the axons (Figs 6Biv, 6Fi, 6Fv and 7B). After infection with -ΔUL37 (Figs 6C, 6G, S3C and 7C), capsids clustered within the cytoplasm and the cell morphology was often altered; again very few capsids had been targeted to the axons (Figs 6Civ, 6Giv, 7Ci and 7Cv). The stronger cytopathic effects as indicated by membrane blebbing upon infection upon infection with the HSV1-ΔUL37 strains (S3C Fig) might be due to the lack of pUL37 targeting RIG-I and blocking RNA-induced activation [64], but we did not investigate this further.
Unexpectedly, there were also only very few axonal capsids upon infection with -ΔUL20 (Figs 6Hiv and 7D). The capsid distribution of -ΔUL20 in the somata was similar to that of the parental HSV1(17 + )Lox, but with reduced accumulation at the plasma membrane (Fig 6Div,  S3Di). After infection with -ΔUL36, -ΔUL37, or -ΔUL20, the outer tegument proteins VP22 (Fig 6Bv, 6Cv and 6Dv) and VP13/14 (Fig 7Bii, 7Cii and 7Dii) as well as gD (Fig 6Fv, 6Gv and 6Hv) and the envelope proteins gB (Fig 7Bvi, 7Cvi and 7Dvi) had still been targeted to the axons as for the parental strain (Figs 6A, 6E and 7A), indicating that axonal transport per se had not been impaired. Nevertheless, the deletion mutants -ΔUL36, -ΔUL37, and -ΔUL20 were incapable of efficient axonal capsid transport.
For quantitation, we scaled up two experiments to image random axonal regions and determined the number of different viral structures per axon length using a novel automated image analysis algorithm (S4 Fig). We determined the imaged axon length, the total number of capsids as detected by anti-VP26, and the fraction of these capsids colocalizing with gD or VP22  (c.f. S1 Table). After infection with the parental strain HSV1(17 + )Lox, we counted 20 to 30 capsids per 100 μm axon length ( Fig 8A; total VP26, black bars) of which 75% colocalized with gD ( Fig 8B; VP26 + gD). Furthermore, there was a similar number of membrane structures fixed and processed for immunoelectron microscopy with antibodies against pUL36 (ii and iii) or pUL37 (i and iv). (B) Immunogold labeling of cytoplasmic capsids was quantified after labeling with anti-pUL36 (Bi) or anti-pUL37 (Bii  containing gD and colocalizing with VP26 or not (Fig 8C, gD); the size of these structures varied considerably ( Fig 8D). After infection with Lox-ΔUL36 (Fig 8; light grey columns), or -ΔUL20 (Fig 8; dark grey columns), there were only few capsids targeted to the axons (Fig 8A), which again mostly co-localized with gD ( Fig 8B). However, the number of gD-containing membrane structures was only moderately reduced for the HSV-1 mutants suggesting that axonal transport per se had not been inhibited ( Fig 8C). In uninfected neurons, there was also  some anti-gD background signal (Fig 8C, mock), but these cross-reacting structures were much smaller (Fig 8D). We also quantified the number of capsids detected by anti-VP26 ( Fig  8E), and the fraction of these capsids colocalizing with the outer tegument protein VP22 ( Fig  8F) of a parallel set of samples. After infection with the parental strain HSV1(17 + )Lox, we counted again 20 to 30 capsids per 100 μm axon length ( Fig 8E; total VP26, black bars), of which 60% to 70% colocalized with VP22 ( Fig 8F; VP26 + VP22). Furthermore, there was a similar number of structures containing VP22 colocalizing with VP26 or not (Fig 8G; VP22) which also varied considerably in size ( Fig 8H). After infection with Lox-ΔUL36, or -ΔUL20, again fewer capsids had been targeted to the axons (Fig 8E), of which almost 80% co-localized with VP22 ( Fig 8F). The number of VP22 containing structures was moderately reduced for the HSV-1 mutants supporting the notion that axonal transport per se had not been inhibited ( Fig 8G). In uninfected neurons, there was almost no anti-VP22 background signal (Fig 8G,  mock), and again these cross-reacting structures were much smaller ( Fig 8H). These experiments show that the inner tegument proteins pUL36 and pUL37 on the capsids of -ΔUL20 were not sufficient for axonal targeting. Instead, association with membranes and outer tegument proteins, and thus possibly completion of secondary envelopment seem to be a prerequisite for efficient capsid targeting into the axons.

Discussion
Animal models as well as cultured neurons have contributed tremendously to elucidating the mechanisms of viral neuroinvasion, spread within the nervous system, and peripheral recurrent diseases [1,2,23]. Various aspects of human diseases are mimicked in murine HSV-1 infection models [65]. For example, upon infection of the cornea, HSV-1 reaches the trigeminal ganglia and causes herpetic keratitis; infection of the flank skin establishes latency in the DRG, and results in zosteriform skin lesions upon reactivation [52, [66][67][68][69][70]. To further characterize alphaherpesvirus axonal targeting, we investigated here HSV infection of primary neurons derived from the DRGs of adult mice, and developed novel image processing algorithms   Table. to determine the amount of different axonal assembly intermediates in an unbiased fashion. We infected such neurons with HSV-1 mutants that assemble cytosolic capsids lacking pUL36 and pUL37, capsids associated only with pUL36, or capsids that recruited both, pUL36 and pUL37 to determine whether these inner tegument proteins implicated in microtubule-mediated transport were sufficient for intracellular capsid motility in epithelial cells and the somata of neurons as well as for capsid targeting to axons. Our data indicate that in addition to acquiring pUL36 and pUL37, capsids needed to complete secondary envelopment to be efficiently targeted to axons, to spread from neurons to neighboring cells, and thus to cause recurrent diseases.

HSV infection of primary neurons derived from adult mice
Embryonic and neonatal neurons dissociated from trigeminal ganglia, DRG, or superior cervical ganglia of chicken, mouse, rat, or human have been used to study axonal trafficking and egress of HSV particles [71][72][73][74][75][76][77][78]. However, primary cultures from embryonic tissue do not present optimal models for age-related changes in differentiation, physiology, or late-onset disease [79][80][81]. We and others [82] therefore used neurons from adult mice to complement results obtained by infecting adult animals with HSV-1 [74,83] and to obtain functional insights into the process of axonal targeting of HSV-1 viral structures. The neurons from the DRG of adult mice formed neurites with axonal features which contained ankyrinG and microtubules of uniform polarity with the plus-ends pointing towards the axon endings. Furthermore, we have shown previously that HSV1(17 + )Lox strains that have been cloned into a bacterial artificial chromosome and therefore lack the Ori L [84,85] infect such neurons and spread the infection to neighboring epithelial cells [52,53]. Hence murine adult DRG neurons provide a versatile system to study productive HSV-1 infection and transport in axons with unipolar microtubules in vivo and in vitro.

Dissection of HSV-1 axonal structures
While there is a growing consensus that the swine alphaherpesvirus PRV relies on the married model for axonal transport in all neuron types, the picture is less clear for HSV-1. According to the married model, capsids are enveloped exclusively in the somata, and thus it would be sufficient to expose a host or a viral postal code on the cytosolic membrane surface to target vesicles harboring fully assembled virions to axons [reviewed in 23,40,41]. But for HSV-1 two types of axonal cargo, free cytosolic capsids as well as capsids surrounded by an envelope and a transport vesicle membrane, have been detected to a varying degree [71,73,76,78,[86][87][88].
Complementary methods have been used to image viral assembly intermediates. Antibodies have less access to capsids associated with membranes than to cytosolic capsids [89,90]. While fluorescent protein tags circumvent this challenge, they may shift the relative abundance of different assembly intermediates [57,72,73,84,[91][92][93][94]. Furthermore, the resolution limit of confocal microscopy is larger than a capsid diameter of 125 nm leading to an overestimation of capsid colocalization with membrane markers. On the other hand, conventional electron micrographs provide sufficient contrast to distinguish hexagonal capsids from membrane vesicles or fully enveloped capsids within transport vesicles but only upon sufficient heavy metal deposition and in very thin sections of 50 nm, which results in an underestimation of membrane-associated capsids [47,71,87]. In embryonic rat hippocampal neurons, many cytosolic HSV-1 capsids are indeed not associated with cytoplasmic membranes as shown by 3-dimensional cryoelectron tomography [88]. The latter is in fact the best imaging method for this question, but limited to specialized centers and not amenable to medium-throughput [95]. For these reasons, we developed novel image quantitation algorithms and novel mutants to characterize HSV-1 axonal targeting, and used both, fluorescent tagging and antibodies to detect the capsid protein VP26, in combination with antibodies against the tegument proteins VP22 and VP13/14, and the envelope proteins gD and gB.
The inner tegument proteins pUL36 and pUL37 are not sufficient for axonal targeting The separate model of alphaherpesvirus egress postulates that different subassemblies, namely cytosolic capsids as well as membrane vesicles conveying envelope proteins, are targeted to axons to be assembled in the axons or just prior to trans-synaptic spread or even during budding at the axonal plasma membrane [reviewed in 23,40,41]. Neuronal cargoes enlist the so-called smart motors that utilize stabilized microtubules which lead to the tiny outlets to dendrites or to axons [96,97]. One such smart motor is kinesin-1 that preferentially binds to de-tyrosinated and acetylated microtubules, modifications that are typical for axonal microtubules while others, such as kinesin-5 or kinesin-13 prefer tyrosinated microtubules [97,98]. Based on extensive genetic, biochemical and cell biology experiments, pUL36 and pUL37 are considered the most likely candidates for motor recruitment to cytosolic capsids [reviewed in 45,48]. Alphaherpesviruses lacking pUL36 or pUL37 are impaired in microtubule-mediated intracellular transport [15,16,[18][19][20][30][31][32][99][100][101]. HSV-1 capsids exposing the inner tegument proteins pUL36 and pUL37 but not naked capsids translocate along microtubules in vitro and recruit kinesin-1 and kinesin-2 from a brain cytosolic extract [11,43]. Finally, pUL36 of HSV-1 and PRV contains binding sites for dynein and dynactin, and possibly the light chains of kinesin-1 [46,47].
We therefore generated HSV-1 mutants that assembled un-enveloped cytosolic capsids with different tegument proteins. Consistent with their proposed function to recruit microtubule motors, the capsids of HSV1-ΔUL20 harboring pUL36 and pUL37 but not the ones of HSV1-ΔUL36 or HSV1-ΔUL37 were capable of active transport in epithelial cells or the somata of DRG neurons. The capsids of HSV1-ΔUL20 therefore resemble capsids which recruit the smart motor kinesin-1 from a cytosolic brain extract in vitro [11]. Our data indicate that capsid-associated pUL36 and pUL37 were sufficient for microtubule transport, but not for efficient axonal targeting.
It is possible that the capsids of HSV1-ΔUL20 had been associated with a larger amount of outer tegument proteins covering potential motor-binding sites on the inner tegument. Capsids with lower amounts of outer tegument recruit more kinesin-1 from a cytosolic brain extract than capsids with more outer tegument [11]. Furthermore these outer tegument proteins might lead to an association with cytoplasmic membranes, although our quantitative electron microscopy data indicate that while HSV1-ΔUL20 was impaired in completing secondary envelopment, there was no higher association with wrapping membranes when compared to the respective parental strains. Those capsids associated with wrapping membranes might have been connected to larger membrane systems in neurons, which might have reduced their motility, and thus their chances to find the axonal exit. Axonal transport in general had not been impaired upon infection with either HSV-1 mutant, since the envelope proteins gB and gD as well as the outer tegument proteins VP13/14 and VP22 had been targeted to axons. Our data are consistent with the notion that cytosolic capsids might rely on the inner tegument for kinesin-1 mediated transport in epithelial cells and the somata, but that transport vesicles harboring complete virions are more efficiently targeted to axons than such cytosolic capsids.

Married versus separate assembly models
Our data indicate that in addition to pUL36 and pUL37, other HSV-1 components and processes depending on pUL20 are required for axonal targeting in adult neurons. The simplest interpretation of our data is that HSV-1 relies also on the married model for axonal targeting and trafficking, and that the membrane of the transport vesicles harboring complete virions harbors a viral or a host receptor for smart axonal microtubule motors on its cytosolic surface. Indeed, kinesin-1 can co-traffic with PRV and HSV-1 tegument and envelope proteins [102][103][104]. In the absence of pUL20, secondary envelopment was not completed, and apparently also the tegument adopted a different conformation or composition, as our electron microscopy analysis revealed a different tegument contrast for capsids associated with wrapping membranes than for complete virions. Furthermore without pUL20, the envelope proteins gD and gH/gL, and possibly also their interaction partners VP22 and VP16 [38], are not properly targeted to cytoplasmic virus assembly sites [105].
Of the alphaherpesvirus envelope proteins, gE/gI and pUS9 are also necessary for efficient axonal spread but not for cytoplasmic capsid envelopment [23]. pUS9 of PRV associates with the neuron-specific kinesin-3 subunit KIF1A, and contributes to initial axonal sorting of PRV particles [106][107][108]. Similarly, pUS9 of HSV-1 is required for efficient axonal targeting, and its large cytosolic domain interacts with the KIF5B subunit of conventional kinesin-1 [74,109]. If the intracellular trafficking and targeting of gE/gI or pUS9 had been impaired in the absence of pUL20, this would explain why even capsids associated with wrapping membranes could not be targeted to the axons upon infection with HSV1-ΔUL20. According to the loading hypothesis, capsids require in the somata in addition to the inner tegument proteins pUL36 and pUL37 also the membrane proteins gE/gI and pUS9 to be loaded onto kinesin-1 [13,109,110].
Vesicles harboring gD and possibly VP22 did neither require pUL36, pUL37 nor pUL20 for axonal targeting. Future work elucidating which assembly intermediates of wild-type HSV-1 as well as -ΔUL36, -ΔUL37, -ΔUL20 and other deletion mutants recruit which smart microtubule motors will reveal specific viral and specific host components that need to be assembled onto an alphaherpesvirus cargo for efficient axonal targeting. Our data are consistent with the notion that cytosolic capsids rely on the inner tegument for dynein and kinesin-1 mediated transport prior to secondary envelopment in epithelial cells and in the neuronal somata, but that transport vesicles harboring complete virions are more efficiently targeted to axons than such cytosolic capsids.

Cell lines
Cell lines were cultured in a humidified incubator at 37˚C and 5% CO 2

Neurons
Primary neurons from dorsal root ganglia (DRG) of adult C57Bl/6JHanZtm mice were cultured using established protocols [111,112]. Briefly, mice were sacrificed, and the DRG from the cervical, thoracic and lumbar level were dissected and collected in 1x HBSS-complete buffer (Hank's balanced salt solution, pH 7.4 with 5 mM HEPES and 10 mM D-Glucose). The DRG of three mice were pooled and treated with 20 mg/ml papain (Sigma-Aldrich, Schnelldorf, Germany; in 0.4 mg/ml L-Cysteine, 0.5 mM EDTA, 1.5 mM CaCl 2 , pH 7.4) for 20 min at 37˚C, and with 10 mg/ml collagenase IV (Invitrogen) and 12 mg/ml dispase II (Sigma-Aldrich) in 1x HBSS-complete buffer for 20 min at 37˚C. DRG and cells were sedimented and re-suspended in 1 ml 1xHBSS-complete buffer and triturated using Pasteur pipettes with narrowed ends. The suspensions were spun for 8 min at 381 x g through 20% (v/v) Percoll (Sigma-Aldrich) cushions in CO 2 -independent medium (Life Technologies Gibco, Carlsbad, CA, USA) containing 10 mM D-glucose, 5 mM HEPES, 10% FBS, 100 U/ml penicillin and 0.1 mg/ml streptomycin. The cells were washed with 2 ml CO 2 -independent medium, sedimented 2 min at 1,000 x g, suspended in Ham's F-12 nutrient mix medium with 10% FBS, 50 ng/ml 2.5S nerve growth factor (Promega Corporation, Fitchburg, WI, USA), 100 U/ml penicillin and 0.1 mg/ml streptomycin, and seeded onto cover slips of 20 mm diameter in 24-well plates or glass bottom dishes (Nunc Lab-Tek II Chambered cover glass 4-chamber #155382, #1.5 borosilicate glass, Thermo Scientific). The cover slips and glass bottom dishes had been pre-coated with 0.01% (w/v) poly-L-lysine (150,000-300,000 g/mol, Sigma-Aldrich) and 7 ng/μl murine laminin (Invitrogen). The cells were cultured in a humidified incubator at 37˚C and 5% CO 2 , and the media were replaced twice a week. 1-β-D-arabinofuranosylcytosine (Sigma-Aldrich) was added at 1 to 2 div to a final concentration of 2μM to suppress proliferation of dividing, non-neuronal cells, but removed prior to HSV-1 infection. , HSV1(17 + )Lox-ΔUL20 (see below) and HSV1(17 + )Lox-CheVP26-ΔUL20 (see below). Our pHSV1(17 + )Lox BAC plasmids contain a BAC cassette with a chloramphenicol resistance gene, a Cre recombinase gene with an intron under the control of a eukaryotic promoter, a single flippase recognition target site (FRT) and LoxP sites at both ends inserted between the genes UL22 and UL23, and an almost complete HSV1(17 + ) genome lacking only the Ori L [84,85]. The Cre recombinase excises the BAC cassette upon transfection into eukaryotic cells. HSV-1 stocks were prepared in BHK-21, HS30 (for -ΔUL36), 80C02 (for -ΔUL37) or Flp-In-CV-1-pUL20 (for -ΔUL20), and extracellular virus was harvested by sedimentation from the supernatant of infected cells as described previously [84,89,90]. After DNase treatment, the inocula of HSV1 (17 + )Lox-ΔUL37 and Lox-CheVP26-ΔUL37 had genome to PFU ratios below 2,300 while that of the other strains had genome to PFU ratios of below 108 [120].

Lentiviral production and transduction
To express EB3 in neurons, we used lentiviral transduction. The spleen focus forming virus promoter was replaced by the human cytomegalovirus immediate early promoter in the plasmid pRRL.PPT.SF.GFPpre . The supernatants were harvested and spun in a Beckman SW 28 rotor at 23,000 rpm or a SW32.Ti rotor at 24,000 rpm for 90 min at 4˚C (Beckman Coulter, Krefeld, Germany). The resuspended lentiviral particles were snap frozen in liquid N 2 and stored at -80˚C. For lentiviral transduction, DRG neurons were prepared and seeded as described and at 1 div neuronal growth medium was replaced by one containing 20 mM HEPES and the lentiviral particles.

Plaque assay
HSV-1 inocula titers were determined by plaque assays [89,90,120]. Vero, Flp-In-CV-1 or the derivative Flp-In-CV-1-UL20-expressing cells were cultured in 6-well dishes for 16 to 20 h to almost confluency. The respective inocula were diluted in CO 2 -independent medium (Life Technologies Gibco) containing 0.1% (w/v) cell-culture grade bovine serum albumin (BSA; Capricorn Scientific, Ebersdorfergrund, Germany), added to the cells for 1 h on a rocking platform at room temperature, and then replaced by growth medium containing 20 μg/ml pooled human IgGs (Sigma-Aldrich) to neutralize HSV-1 in the culture medium. At 3 dpi, the cells were fixed with ice-cold, water-free methanol and air-dried prior to staining with 0.1% (w/v) crystal violet in 2% (v/v) ethanol for 1 min. After removing the excess of crystal violet, the cells were air-dried, and plaques were counted while using a binocular loupe (Nikon, Tokyo, Japan) to calculate the virus titer as plaque forming units (pfu) per ml.
We used the forward primer 5'-CCTTGCGGTTTCGGTCTCCCCACCTCCACCGCACACC CCCTGACCCTGTAGTAATAGCGGGATGACCTTCCTCTGGTTAGGGAAACAGGTAA TCGATTT-3', the reverse primer 5'-TCGTCGACCAGATCTCGATCACCAGAGGAAGGT CATCCCGCTATTACTACAGGGTCAGGGGGTGTGCGGTGGCAGGTGGTGCCAGTGT TACAACCAATTAACC-3', the forward sequencing primer 5'-AAAGACCGGCTGGGTAT G-3', and the reverse sequencing primer 5'-GGGCGTAGGCGTAAATTC-3'. The mutated start codons are underlined and the inserted stop codons are shown in bold. The BAC plasmids were digested with 15 U/μg DNA of AscI, BamHI, XhoI or HindIII for 3.5 h, and analyzed on 0.6% (w/v) agarose gels in 0.5x TBE buffer (0.44 M Tris-HCl, 0.44 mM boric acid, 10 mM EDTA, pH 8) run at 66 mA for 17 h (Peqlab system, Erlangen, Germany). To reconstitute viruses, sub-confluent Vero or Flp-In-CV-1-UL20-expressing cells were transfected with 10 μg BAC-DNA (MBS mammalian transfection kit; Stratagene, La Jolla, CA, USA) per 6 cm dish and cultured until cytopathic effects had developed. Cells and medium were pooled, virus was released by three cycles of freeze-thawing and the resulting cell pellet was used to further propagate the virus according to standard protocols. The genomes of the novel HSV1(17 + )Lox strains were sequenced around the pUL20 start site that had been targeted, and the engineered mutations were confirmed. For the growth curves, sub-confluent Vero, Flp-In-CV-1 or the derivative Flp-In-CV-1-UL20-expressing cells were inoculated at 5 pfu/cell, the cells and the supernatants were harvested at the indicated time points, and virus titers were determined by plaque assay.

HSV-1 infection
Vero cells were seeded on cover slips, 3.5 cm cell culture dishes or glass-bottom chambers (Nunc LabTek II Chambered cover glass 4-chamber #155382, #1.5 borosilicate glass, Thermo Scientific) and infected 16 to 20 hours after the seeding as described before [16,20,29,84,89,120,125]. For synchronous infections, the cells were pre-cooled for 20 min on ice, and inoculated with 10 pfu per cell or mock treated as a control in CO 2 -independent medium containing 0.1% (w/v) BSA for 2 h on ice while rocking. The cells were then shifted to regular growth medium at 37˚C and 5% CO 2 for 1 h. Non-internalized virus was inactivated at 4˚C by a 3 min acid wash (40 mM citrate, 135 mM NaCl, 10 mM KCl, pH 3). Neurons cultured on cover slips in 24-well plates were pre-incubated at room temperature with CO 2 -independent medium for 20 min, and inoculated with 1 to 5 × 10 6 PFU in 200 μl per well in CO 2 -independent medium. After 30 min, the virus-suspension was replaced by 500 μl F-12 medium and cells were incubated again at 37˚C and 5% CO 2 .

Automated image processing
To analyze systematically the degree of co-localization of tegument (anti-VP22, red channel) or envelope proteins (anti-gD, red channel) with capsids (anti-VP26, green channel), and the targeting efficiency of the different viral particles to the axons, we developed a novel image processing pipeline. The semi-automated approach consists of three steps: (i) manual annotation of regions of interest (ROIs) to identify the axons (cf. S4 Fig), (ii) automated particle detection of the capsids, and (iii) automated detection of the co-localizing tegument or envelope protein signal using pixel-wise image classification.
(i) ROI selection: To distinguish the axon from other structures, the regions of interest were manually selected. To simplify the process of region annotation, a ROI is defined by positioning a few control points in the image and adjusting the region width at the control points, as shown in S4 Fig. Usually, three to four control points were sufficient for accurate annotation.
(ii) Capsid particle detection: The capsids have a spherical shape with a characteristic diameter. Due to blurring caused by the image acquisition process, the fluorescence signal of a single capsid was modeled by an isotropic Gaussian distribution. Template matching was applied to discriminate between well-separated viral particles and other signals such as background noise or agglomerations of multiple, non-separable capsids. In detail, a detector response map R σ (x, y) was computed for each pixel (x, y) of the image using normalized cross-correlation of the image signal I capsid (x,y) with a Gaussian shaped template model T s u; v ð Þ ¼ exp À u 2 þv 2 2s 2 Â Ã with variance σ 2 and filter size of 1 + 2 Á dσ Á se. Candidate particles were extracted from the detector response map R σ (x, y) by searching for local maxima in R σ (x, y) using an 8-neighborhood. In case of a plateau, a morphological shrinking operator was used to only consider the central position of the plateau as a detection candidate. To discriminate from noise, the candidate points p i = (x i , y i ) were only accepted as capsids if the detector response R σ (x i ,y i ) was greater than a threshold τ corr and the fluorescence signal I capsid (x i ,y i ) was greater than a threshold τ intens . The parameters σ, s, τ corr , and τ intens were trained on manually annotated test images using grid search. The final parameter values used throughout the experiments were σ = 0.7, s = 1.5, τ corr = 0.4, and τ intens = 0.5.
(iii) Tegument or envelope protein signal detection: We used pixel-wise image classification in order to discriminate between signal and background noise. The classifier used various features derived from the gD or VP22 fluorescence signals I gD (x,y): Gaussian blurred intensity signal I gD s ¼ I gD Ã T s , non-linearly distorted intensity E gD s;s ¼ expðs Á I gD s Þ normalized cross correlation (NCC) of I gD to a Gaussian kernel R gD s ¼ NCCðI gD ; T s Þ, and a high-pass filter clamped to values between 0 and 1. Finally, the feature vector is F ¼ ½R gD 0:5 ; R gD 1 ; R gD 2 ; R gD 4 ; I gD ; E gD 10;0 ; I gD 2 ; H gD 2 ; I gD 5 ; H gD 5 ; E gD 10;5 ; I gD 10 ; H gD 10 ; E gD 20;10 ; I gD 20 ; H gD 20 ; E gD 50;20 : We used a logistic regression classifier which was trained on manually annotated test images. The gD or VP22 regions were extracted from the resulting pixel-wise classification images using a connected components analysis with an 8-neighborhood. A capsid was said to co-localize with gD or VP22 if the capsid center position coincided with a gD or VP22 region dilated by 1 pixel. The dilation operation accounted for uncertainties in both the capsid position estimation and the pixel-wise gD or VP22 classification. Several statistical parameter were derived from the automatically detected capsids and gD or VP22 regions. The number of capsids or gD and VP22 regions per 100 μm was derived by projecting the particle locations orthogonally to the axial center line of the ROI as illustrated in S4 Fig.

Live cell imaging and particle tracking
For differential interference contrast imaging and to avoid evaporation at 37˚C, a glass lid was attached to the chambers with vacuum grease (Dow Corning, Midland, Michigan, USA). Movies of infected Vero cells were recorded at 37˚with a high temporal resolution of 5 images per second and a pixel size of 79 nm using a confocal laser scanning microscope equipped with a heating unit (PeCon, Erbach, Germany) as described before [20]. Movies of infected DRG neurons were recorded at 37˚C (Incubator from PeCon, Erbach, Germany) with high temporal resolution 20 images per second on a Nikon Ti microscope equipped with a Yokogawa (Ratingen, Germany) CSU-X1 spinning disk and an Andor iXion Ultra 897 EMCCD camera (Belfast, UK).

Digital movie processing
Automated tracking of cytoplasmic capsid motility was performed using the ImageJ plugin MOSAIC [63,126]. First, the nuclei were identified by the confined nuclear capsids mobility and the nuclear capsid fluorescence was removed. Second, the Gaussian blur filter of ImageJ was applied (Sigma radius 2 pixel). No further image processing was performed. Third, automated tracking was performed using the Particle Tracking plugin with the following settings: Kernel radius 3, Cutoff radius 3, percentile 0.5, displacement 20, and link range 1. Of the tracks identified, we only considered further tracks with a length of 10 frames or longer. Track length and maximum step velocity were calculated from tracks with a MSD exponent of 1.2 or larger, representing non diffusive transport events.

Electron microscopy
Cells were infected as described above and fixed at the indicated time points. For conventional electron microscopy cells were fixed with 2% glutaraldehyde in cacodylate buffer (130 mM (CH 3 ) 2 AsO 2 H, pH 7.4, 2 mM CaCl 2 , 10 mM MgCl 2 ) for 1 h at room temperature. Cells were washed and contrasted with 1% (w/v) OsO 4 in cacodylate buffer (165 mM (CH 3 ) 2 AsO 2 H, pH 7.4, 1.5% (w/v) K 3 [Fe(CH) 6 ] followed by 0.5% (w/v) uranyl acetate in 50% (v/v) ethanol overnight. The cells were embedded in Epon plastic (Serva, Heidelberg, Germany) and 50 nm ultrathin sections were cut parallel to the substrate. Cytoplasmic capsids were counted and the respective cellular areas that had been sampled were measured using an Image J plugin.
For immunoelectron microscopy, infected cells were fixed at the indicated time points with 2% (w/v) PFA and 0.2% [w/v] glutaraldehyde, in PHEM-buffer (60 mM PIPES, 25 mM HEPES, pH 6.9, 10 mM EGTA, 2 mM MgCl 2 ), embedded, frozen and sectioned. Sections were labeled using specific antibodies and protein-A gold of 10 nm (Cell Microscopy Centre, Utrecht School of Medicine, The Netherlands). Sections were contrasted using 0.5% (w/v) uranyl acetate in 2% methylcellulose (Merck) [127]. Images were acquired with an electron microscope at 200 kV equipped with an Eagle 4k camera (Tecnai G2; FEI, Eindhoven, The Netherlands). Immunogold labeling was quantified by counting each gold particle within a radius of 100 nm around the center of a cytoplasmic capsid [20]. Data were analyzed by using Kruskal-Wallis followed by Dunn's post testing, and the p values were adjusted for multiple testing (software Prism, version 6; Graphpad, San Diego, CA, USA).

Ethic statement
The mice (strain C57Bl/6JHanZtm, not genetically modified) were bred and maintained without any perturbation. On the day of the experiment they were picked up from the animal facility and within 3 h sedated with CO 2 -inhalation prior to killing by cervical dislocation without any prior experimental perturbation. DRG were dissected afterwards. According to the German Animal Welfare Law §4, killing of animals does not need approval if the removal of organs serves scientific purposes and the mice had not undergone experimental treatment before. The animal care and sacrifice was performed in strict accordance with the German regulations of the Society for Laboratory Animal Science (GV-SOLAS), the European Health Law of the Federation of Laboratory Animal Science Association (FELASA) and the German Animal Welfare Law. According to the German Animal Welfare Law, this study does not contain animal experiments that require pre-approval, but the total number of killed mice was Purified HSV1 BAC DNA was digested with the restriction enzymes XhoI, AscI, BamHI or Hin-dIII, and the fragments were analyzed on agarose gels. c, bands containing the mCherry-tag; star, bands containing the mutation. (B) Immunoblot analysis of infected cell lysates. Vero cells were infected with HSV1(17 + )Lox or its derivatives -ΔUL20, -CheVP26, or CheVP26-ΔUL20 at an MOI of 10 pfu/cell (3.2 x 10 7 pfu/mL), and lysed in sample buffer at the indicated time points. The proteins were separated with a 12.5% SDS-PAGE, transferred to a nitrocellulose membrane, and probed with antibodies directed against pUL37, pUL20, or actin. (C) and (D) Single step growth kinetics of parental and mutant strains, either black (squares) or tagged with CheVP26 (circles) on either Vero cells (filled and empty symbols) or on Flp-In™-CV-1-pUL20complementing cells (half-filled symbols). Intracellular infectivity (C) and extracellular infectivity (D). (TIF) S3 Fig. HSV-1-pUL36, pUL37 and pUL20