It is commonly observed that viruses concentrate viral and cellular components for certain 37 processes, either final viral assembly or viral replication, at a defined subcellular compartment. 38
In the case of retroviruses, influenza virus, rhabdoviruses, and filoviruses, the compartment of 39 final assembly is the plasma membrane, where all virion components converge and are 40 incorporated into the virion during viral budding (Welsch et al., 2007) . In the case of reovirus, 41 coronavirus, and hepatitis C virus (HCV), the host ER membrane is rearranged to form visually 42 distinct viral structures that serve as either assembly factories (Tenorio et al., 2019) , or 43 replication vesicles (Knoops et al., 2008 , Paul et al., 2013 . It might be expected that such a 44 concentration of virion components is especially important for the assembly of viruses that 45 contain many diverse structural proteins and have a complex virion structure, such as 46 herpesviruses. The virion of all herpesviruses is composed of at least three layers of structural 47 proteins, which include a protein capsid (composed of 8 different proteins), a tegument layer 48
(composed of as many as 23 different proteins), and an envelope containing at least 12 49 transmembrane proteins, which mostly are glycosylated (Loret et al., 2008) . Curiously, despite 50 the structural complexity of the mature herpesvirion, only human cytomegalovirus (HCMV), a 51 betaherpesvirus, has been shown to form a unitary organized cVAC in infected cells (Sanchez 52 et al., 2000) . 53
The HCMV cVAC is a spatially organized system of membranes formed around the microtubule 54 organization center (MTOC) (Sanchez et al., 2000) , and maintenance of the cVAC is 55 microtubule- (Indran et al., 2010) and dynein motor-dependent (Buchkovich et al., 2010) . The 56 HCMV cVAC contains enveloping capsids and concentrates various viral structural proteins and 57
host cellular markers such as the Golgi markers TGN46, GM130, and ManII; recycling 58 endosome marker Rab11; and early endosome marker EEA1 Pellett, 2011, Sanchez 59 et al., 2000) . The lysosome marker LAMP1 also concentrates to the HCMV cVAC but does not 60 co-localize with viral proteins Pellett, 2011, Sanchez et al., 2000) . Formation of the 61 HCMV cVAC is dependent on the function of specific virally encoded proteins including pUL97 62 and pUL71. pUL97 is an early-expressing multi-functional serine/threonine kinase of which 63 homologs exist in all known herpesviruses (Chee et al., 1989 , Smith and Smith, 1989 , Kato et 64 al., 2006 . pUL97, and its homolog pUL13 in HSV, are both involved in the capsid nuclear 65 maturation and cytoplasmic assembly (Krosky et al., 2003 , Azzeh et al., 2006 . On the other 66 hand, pUL71, and its homolog pUL51 in HSV, are palmitoylated membrane-associated 67 tegument proteins that are involved in viral cytoplasmic envelopment (Womack and Shenk, 68 Results 115
cells. During studies of the function of the HSV-1 pUL51 tegument protein we observed cell-117 type-specific localization patterns for pUL51 and, most interestingly, organization into a unitary 118 structure that might correspond to a cVAC in cells of the mouse neuronal CAD cell line. To 119 determine whether this pUL51-containing structure in CAD cells might be a cVAC, we tested the 120 localization of other viral structural proteins. CAD cells were infected with HSV-1(F) at high 121 multiplicity of infection (m.o.i) and co-stained for various tegument and glycoproteins. As shown 122 in Figure 1A , we observed a unitary perinuclear cluster of viral proteins in most cells (Figure 1C ) 123 in which at least six viral structural proteins including pUL11, pUL16, pUL51, gD, gE, and gL 124 were concentrated, suggesting the viral protein cluster might be a cVAC. Note that for these 125 viral proteins, not all of them localize to the putative cVAC at the same efficiency, for example, 126 pUL51 almost exclusively concentrate to the cluster, while most of the gD localizes to the 127 cytoplasmic space or cell membrane. To determine whether a similar structure forms in 128 neuronal cells of human origin, we tested in the same way for formation of the putative cVAC in 129 SH-SY5Y cells, a human cancerous neuronal cell line ( Figure 1B ). As expected, a similar 130 putative cVAC is observed in most cells, albeit at a lower frequency ( Figure 1C ). In addition, 131 unlike in CAD cells, where the putative cVAC is composed of numerous small viral protein 132 puncta ( Figure 1A) , the viral protein cluster in SH-SY5Y cells typically appeared as a collection 133 of several large granules ( Figure 1B) . Together, the localization pattern of viral proteins in both 134 CAD and SH-SY5Y cells is different from it in cells of epithelial origin, such as Vero cells (Figure 135 1D), suggesting that the mode of viral component trafficking is cell context-dependent. 136
To examine whether the formation of the putative cVAC is a specific property of HSV-1, we 137 infected CAD cells with an HSV-2 bacterial artificial chromosome (BAC)-derived virus (Figure 138 1E). As expected, a cluster of viral proteins was observed in infected cells, although pUL11 and 139 gD did not completely colocalize with each other within the cluster ( Figure 1E ). 140
To characterize the temporal dynamics of the HSV-1 putative cVAC, CAD and SH-SY5Y cell 141 were subjected to synchronized infection and fixed at various time points after infection. As 142 shown in Figure 1F , the putative cVAC was formed as early as 4 hour-post-infection (h.p.i) and 143 was maintained as late as 15 h.p.i, and its gross morphology showed no obvious change 144 despite the progression of the cytopathic effect (CPE) caused by the infection. Further time 145 points are not shown due to severe cytoplasmic space shrinkage associated with cell rounding. 146
The stability of the putative cVAC suggests that it is not an intermediate stage of CPE 147
Immortalized cell lines often differ from their primary counterparts in many ways. To determine 149 whether formation of a putative cVAC is a peculiarity of immortalized neuronal cells, primary 150 mouse cortical neurons were tested. Cortical neurons from newborn mouse pups were 151 harvested as described in Material and Methods and allowed to differentiate in vitro for 3 days 152 on a layer of primary mouse astrocyte feeder cells. The mixed culture was then infected with 153 HSV-1(F) and fixed 12 h.p.i before being subjected to immunofluorescent microscopy. 154
Interestingly, a putative cVAC was formed in cells that were stained positive for neuron marker 155 MAP2 (Figure 2 , B and C, white arrows), while in astrocytes, viral proteins form dispersed 156 puncta that resemble the viral protein distribution in epithelial cells ( Figure 2C , cyan arrow). 157
These results suggest that the formation of a putative cVAC is a property of both primary and 158 immortalized neuronal cells, and may be specific for cells of the neuronal lineage. 159
The viral protein cluster is the site of capsid envelopment. It is well-established that HCMV 160 forms a cVAC in infected cell. The HCMV viral protein cluster is formed around the MTOC 161 (Sanchez et al., 2000) and is the site of capsid envelopment (Gilloteaux and Nassiri, 2000) . For 162 most cell types, the MTOC is formed around centrosome, which can be defined by staining for a 163 centrosome-specific microtubule protein γ-tubulin (Sanchez and Feldman, 2017) . To test the 164 hypothesis that the cluster of viral proteins that were observed in HSV-infected neuronal cells is 165 an HCMV cVAC-like viral assembly compartment, the distribution of viral capsids in relationship 166
to the viral protein cluster and centrosome was investigated. First, CAD cells were infected with 167 a recombinant BAC-derived HSV-1 in which the minor capsid protein pUL35 (VP26) was fused 168 with a red fluorescent protein (RFP), and then the localizations of cytoplasmic capsids, viral 169 tegument protein pUL11, and γ-tubulin were determined. As shown in Figure 3A , most 170 cytoplasmic capsids were concentrated within the "clouds" of pUL11 staining, which surrounded 171 the centrosome, suggesting that the viral protein cluster (putative cVAC) is a viral assembly 172
To confirm that capsid envelopment occurs within a unitary structure in CAD cells, transmission 174 electron microscopy (TEM) was performed on infected CAD cells. Whenever more than ten 175 capsids were observed in a microscopy section, most of them were concentrated at one single 176 perinuclear compartment in the cells, in which various stages of enveloping capsids can be 177 identified ( Figure 3B ). Taken together, these results strongly suggest that HSV forms a cVAC at 178 which capsids are enveloped in infected neurons. 179 HSV putative cVAC is not an autophagosome or aggresome. The double-membrane nature 180 of some structures that were observed in TEM ( Figure 3B ), suggested the possibility that the 181 putative cVAC might instead be an autophagic compartment, as autophagy is a major 182 mechanism of anti-viral response in neuronal cells (Orvedahl et al., 2007) . Autophagy leads to 183 the formation of a double-membrane structure called autophagosome, which engulfs cellular 184 components and intracellular pathogens within itself before fusing with lysosomes for 185 degradation (Tanida, 2011, Parzych and Klionsky, 2014) . In mammalian cells, the membranes 186 that form an autophagosome are defined by the incorporation of an autophagy marker LC3 187 GM130 positive membranes were observed to form a tubular structure partially or completely 223
surrounding, but not coinciding with pUL51 staining. 224
In infected SH-SY5Y cell, Golgin97 positive membranes also concentrated at the same 225 subcellular compartment as the HSV cVAC, however, individual Golgin97 puncta did not co-226 localize with pUL51 puncta ( Figure 5F ). This is consistent with our observation of infected Vero Figure 6F ). Interestingly, in CAD cells, whose larger cytoplasmic space allowed clearer 266 identification of individual puncta signals, Rab5 puncta co-localized with gE puncta that were not 267 part of the cVAC ( Figure 6C , white arrowhead). 268
Since Rab11 was actively transported to the centrosome in a microtubule-dependent manner in 269 uninfected cells (Figure 6 suggesting that HSV cVAC formation is microtubule-dependent. 289
Nocodazole causes cell type-specific growth defect. Since nocodazole profoundly disrupts 290 HSV cVAC formation, its net consequence, namely viral production, was investigated by single-291 step growth (SSG) assays. As shown in Figure 8A , viral progeny production was reduced in SH-292 SY5Y cells in a dose-dependent manner that proportionally correlates with the reduction in 293 cVAC formation, but the same effect was not observed in CAD cells. This trend remained 294 unchanged as late as 24 h.p.i ( Figure 8B ).These results suggest that nocodazole treatment is 295 unable to change certain aspects of the HSV assembly compartment in CAD cells even if this 296 compartment is no longer concentrated at one single region in the cell. Thus, we hypothesized 297 that in nocodazole treated CAD cells, HSV assembly compartment is still able to form as 298 individual periphery sites, similar to it in epithelial cells. To test this hypothesis, nocodazole-299 treated HSV-infected CAD and SH-SY5Y cells were stained for viral protein pUL51 and gE. As Interestingly, gD, which is the most widely distributed of the proteins we tested, does not have a 389 retrieval motif (Crump et al., 2004) suggesting that possession of such a motif might be related 390 to the mechanism of trafficking to the cVAC. 391
It is possible that concentrations of viral structural proteins could also reflect formation of 392 aggresomes or autophagosomes, and be unrelated to virus assembly events. If so, 393
concentration of viral proteins would be accompanied by recruitment of markers of 394 autophagosomes or lysosomes. However, the autophagy marker LC3 does not concentrate to 395 the cVAC (Figure 4) , suggesting these membranes are not canonical autophagosomes. Also, 396 the lysosomal marker LAMP1 does not co-localize with viral protein puncta (Figure 4) , 397
suggesting the cVAC is not a degradation compartment that involves lysosome. Combining 398 these two observations with the stability of the cVAC throughout the infection (Figure 1F ), we 399 conclude that the cVAC is not a canonical degradation compartment. We did observe partial Figure 6C ). It is possible that these puncta represent retrieval vesicles that 473 contain viral glycoproteins being trafficked to the cVAC. 474
neuronal cells forms around the MTOC defined by γ-tubulin staining (Figure 3, 7) , suggesting 476 that it is formed and maintained by retrograde transport of membranes and proteins toward the 477 MTOC by microtubule-associated motors. Consistent with this hypothesis, both nocodazole, a 478 microtubule polymerization inhibitor (De Brabander et al., 1976) , and ciliobrevin D, a dynein 479 motor ATPase inhibitor (Firestone et al., 2012) , are able to disrupt cVAC morphology in CAD 480 cells (Figure 6-9) . However, the requirement for an intact microtubule network and, therefore, for 481 a normally formed cVAC for viral assembly in neuronal cell lines is cell-type dependent. involved in HSV assembly, thus we observed its co-localization with periphery gE puncta 525 ( Figure 6C ); while Rab5 is unlikely to support HCMV assembly as it is not present in the virion 526 (Spearman, 2018) , thus no particular co-localization between HCMV viral proteins and Rab5 527 was observed in infected cells (Das et al., 2007) . 528
The requirement for cytoskeleton components is also similar between HCMV and HSV. In cells 529 infected with either virus, nocodazole is able to disrupt cVAC morphology (Indran et al., 530 2010) (Figure 7, 8) , suggesting that utilization of the microtubule network is a conserved 531 mechanism for herpesvirus assembly. Also, sensitivity of the HSV cVAC to ciliobrevin D 532
suggests that dynein motors are important for cVAC maintenance (Figure 9 ), as discussed 533 above, however the potential off-target effect of the dynein ATPase inhibitor cannot be 534 We hypothesize that homologous viral proteins might also function similarly in cells infected with 542 HCMV and HSV. pUL71 of HCMV, which is a homolog of HSV pUL51, was shown to be 543 important for viral cytoplasmic envelopment and proper cVAC morphology (Schauflinger et al., 544 2011) . Reciprocally, in this study, we have shown that pUL51 in HSV is also important for cVAC 545 maintenance, as mutations in HSV pUL51 disrupts cVAC formation ( Figure 10F ). Interestingly, 546 although the hypothetical tyrosine-based trafficking motif in the N-terminus of pUL51 is 547 conserved in several herpesviruses and has been shown to be a cytoplasmic retrieval motif in 548 HCMV homolog pUL71 (Dietz et al., 2018) , it is as yet unclear whether the same holds true for 549 HSV. It is possible that the tyrosine residue at position 19 in pUL51 is indeed a trafficking motif, 550 which upon mutation prevents pUL51 from concentrating to the HSV cVAC and, in turns, causes 551 failure in cVAC formation. HSV biology. One of the outstanding questions in HSV assembly is that whether tegument and 557 glycoproteins are pre-deposited onto a membranous structure that serves as the budding site, 558 or it is the capsid itself recruits vesicles that contain various viral structural proteins. In this 559 study, we have shown that the formation of the viral assembly compartment is independent of 560 capsid and capsid nuclear egress ( Figure 10D, E) , confirming a tegument-and glycoprotein-561 centric model for organization of HSV assembly centers. Further supporting this model, a 562 mutation in tegument protein pUL51 was able to not only disperse the centralized cVAC, but 563 also disrupt co-localization between viral components ( Figure 10F ), suggesting tegument 564 proteins might be essential for the maintenance of the HSV assembly compartment. All in all, 565
we believe that the cultured neuronal cells will be powerful tools to study HSV assembly. 566 When infecting primary cortex neurons, HSV was diluted in the 1:1 mixture of plating medium 627 and growth medium described above. Cultures were incubated in the inoculum for 1 h before 628 replacing with the same media mixture. 629
Transduction of cells using the CellLight Golgi-GFP baculovirus vector (Thermo-Fisher) that 630 expresses GFP-N-acetylgalactosaminyltransferase was performed according to the 631 manufacturer's instructions 24 hours prior to infection of cells with HSV-1. 632
Indirect immunofluorescence and analysis. Transfected or infected cells on 13-mm-diameter 633 coverslips in 24-well tissue culture plates were fixed with 3.7% formaldehyde for 20 minutes 634 before washing with PBS two times. The immunofluorescence (IF) buffer used to dilute 635 antibodies or antiserum is PBS that contains 1% triton X100, 0.5% sodium deoxycholate, 1% 636 egg albumin, and 0.01% sodium azide. Fixed coverslips were first blocked with IF buffer 637 containing 10% human serum before 1 to 2 hs-long primary antibody incubation (with 10% were infected and treated with DMSO or cytochalasin B following the same protocol as in Figure 8 . Cells 1072
were stained for pUL11 (green) and γ-tubulin (red). White arrowheads indicate individual cVACs. (C) 1073
Percentage of cells that form a unitary cVAC were counted as described in Figure 10 . Error bars 1074