key: cord-0281653-igu99c1k
authors: White, Shaowen; Kawano, Hiroyuki; Harata, N. Charles; Roller, Richard J.
title: HSV Forms an HCMV-like Viral Assembly Center in Neuronal Cells
date: 2020-04-23
journal: bioRxiv
DOI: 10.1101/2020.04.22.055145
sha: f412033f08fe73d4affcfb381114f4ad306d7f07
doc_id: 281653
cord_uid: igu99c1k

Herpes simplex virus (HSV) is a neuroinvasive virus that has been used as a model organism for studying common properties of all herpesviruses. HSV induces host organelle rearrangement and forms dispersed assembly compartments in epithelial cells, which complicates the study of HSV assembly. In this study, we show that HSV forms a visually distinct unitary cytoplasmic viral assembly center (cVAC) in both cancerous and primary neuronal cells that concentrates viral structural proteins and is the site of capsid envelopment. The HSV cVAC also concentrates host membranes that are important for viral assembly, such as Golgi- and recycling endosome-derived membranes. Lastly, we show that HSV cVAC formation and/or maintenance depends on an intact microtubule network and a viral tegument protein, pUL51. Our observations suggest that the neuronal cVAC is a uniquely useful model to study common herpesvirus assembly pathways, and cell-specific pathways for membrane reorganization. Summary This study shows that HSV forms a viral assembly center in neuronal cells by reorganization of host membranes. This system is a novel and powerful tool to study herpesvirus assembly pathways and host cell membrane dynamics.

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

Intracellular traffic of herpes simplex virus 676 glycoprotein gE: characterization of the sorting signals required for its trans-Golgi network 677 localization

The human cytomegalovirus assembly compartment: a masterpiece of viral 679 manipulation of cellular processes that facilitates assembly and egress

682 Redistribution of microtubules and Golgi apparatus in herpes simplex virus-infected cells and 683 their role in viral exocytosis

Structural changes in 685 human cytomegalovirus cytoplasmic assembly sites in the absence of UL97 kinase activity

Nucleocapsid assembly and envelopment of herpes simplex virus

Generation of noncentrosomal microtubule arrays

Contribution of Endocytic Motifs in the Cytoplasmic Tail of

Glycoprotein B to Virus Replication and Cell-Cell Fusion

Roles for herpes simplex type 1 UL34 and US3 proteins in disrupting the 696 nuclear lamina during herpes simplex virus type 1 egress

Adaptor proteins involved in polarized sorting

The role of rab proteins in neuronal cells and in the trafficking 699 of neurotrophin receptors

Role of the Endoplasmic Reticulum Chaperone 701

SUN Domain Proteins, and Dynein in Altering Nuclear Morphology during Human 702 Cytomegalovirus Infection

704 Fragmentation and dispersal of golgi proteins and redistribution of glycoproteins and glycolipids 705 processed through the golgi apparatus after infection with herpes simplex virus 1

Herpes simplex virus 708 2 UL13 protein kinase disrupts nuclear lamins

Alpha-, beta-, and gammaherpesviruses encode a 710 putative phosphotransferase

The cellular distribution and Ser262 712 phosphorylation of tau protein are regulated by BDNF in vitro

Alphaherpesvirus glycoprotein M causes the relocalization of plasma membrane proteins

Spatial relationships between markers for secretory and endosomal 717 machinery in human cytomegalovirus-infected cells versus those in uninfected cells

Three-dimensional structure of the human cytomegalovirus 720 cytoplasmic virion assembly complex includes a reoriented secretory apparatus

The 723 effects of methyl (5-(2-thienylcarbonyl)-1H-benzimidazol-2-yl) carbamate, (R 17934; NSC 724 238159), a new synthetic antitumoral drug interfering with microtubules, on mammalian cells 725 cultured in vitro

Live visualization of 728 herpes simplex virus type 1 compartment dynamics

Plant and mammalian sorting signals for protein 730 retention in the endoplasmic reticulum contain a conserved epitope

Human Cytomegalovirus pUL93 Links Nucleocapsid 732 Maturation and Nuclear Egress

Mutations in herpes simplex virus type 1 734 genes encoding VP5 and VP23 abrogate capsid formation and cleavage of replicated DNA

A null mutation in the UL36 gene of herpes simplex virus type 1 results in accumulation 737 of unenveloped DNA-filled capsids in the cytoplasm of infected cells

A Tyrosine-Based Trafficking 739 Motif of the Tegument Protein pUL71 Is Crucial for Human Cytomegalovirus Secondary 740 Envelopment

Regulation of dynactin through 742 the differential expression of p150Glued isoforms

Herpes Simplex Virus 744 gE/gI and US9 Promote both Envelopment and Sorting of Virus Particles in the Cytoplasm

Two Processes That Precede Anterograde Transport in Axons

Herpes simplex virus protein 747 kinases US3 and UL13 modulate VP11/12 phosphorylation, virion packaging, and 748 phosphatidylinositol 3-kinase/Akt signaling activity

Characterization of herpes simplex virus strains 750 differing in their effects on social behavior of infected cells

Signal-752 mediated, AP-1/clathrin-dependent sorting of transmembrane receptors to the somatodendritic 753 domain of hippocampal neurons

Cytoplasmic Residues of Herpes Simplex Virus

Glycoprotein gE Required for Secondary Envelopment and Binding of Tegument Proteins VP22 756 and UL11 to gE and gD

Small-molecule inhibitors of the AAA+ ATPase 759 motor cytoplasmic dynein

The UL13 and US3

Protein Kinases of Herpes Simplex Virus 1 Cooperate to Promote the Assembly and Release of 762 Mature, Infectious Virions

Human bone marrow fibroblasts infected by cytomegalovirus: 764 ultrastructural observations

Human cytomegalovirus UL97 kinase and nonkinase functions mediate 767 viral cytoplasmic secondary envelopment

Control of HSV-1 latency in human trigeminal ganglia--current overview

771 Endocytic tubules regulated by Rab GTPases 5 and 11 are used for envelopment of herpes 772 simplex virus

Cytomegalovirus miRNAs target secretory 775 pathway genes to facilitate formation of the virion assembly compartment and reduce cytokine 776 secretion

Bicaudal D1-Dependent Trafficking of Human 778

Cytomegalovirus Tegument Protein pp150 in Virus-Infected Cells

A role for the small GTPase Rab6 in assembly of human 781 cytomegalovirus

Identification of a novel herpes simplex virus type 1-induced 783 glycoprotein which complexes with gE and binds immunoglobulin

Binding to cells of virosomes containing herpes 785 simplex virus type 1 glycoproteins and evidence for fusion

Aggresomes: a cellular response to misfolded 787 proteins

Herpes simplex virus 1-encoded protein kinase UL13 phosphorylates viral Us3 protein kinase 790 and regulates nuclear localization of viral envelopment factors UL34 and UL31

SARS-Coronavirus Replication Is Supported by a 794 Reticulovesicular Network of Modified Endoplasmic Reticulum

Making the case: married versus separate 796 models of alphaherpes virus anterograde transport in axons

The human cytomegalovirus UL97 protein kinase, an 798 antiviral drug target, is required at the stage of nuclear egress

HCMV-encoded glycoprotein M (UL100) interacts 800 with Rab11 effector protein FIP4

Simplex Virus Infection and Disease

HSV-1 gM and the gK/pUL20 complex are important for the localization 804 of gD and gH/L to viral assembly sites

Emerin is hyperphosphorylated and redistributed in herpes 807 simplex virus type 1-infected cells in a manner dependent on both UL34 and US3

Identification of an essential domain in the herpesvirus 810

VP1/2 tegument protein: the carboxy terminus directs incorporation into capsid assemblons

Microtubule stability and MAP1B upregulation control neuritogenesis 813 in CAD cells

Comprehensive Characterization of Extracellular Herpes Simplex 815 Virus Type 1 Virions

Cytomegaloviruses Exploit Recycling Rab Proteins in the Sequential Establishment of the 819 Assembly Compartment

Remodeling of host membranes during herpesvirus assembly 821 and egress

Mechanism of action of cytochalasin B on actin

Cellular p32 recruits cytomegalovirus kinase pUL97 to 826 redistribute the nuclear lamina

A leucine zipper 828 motif of a tegument protein triggers final envelopment of human cytomegalovirus

Cytomegaloviral proteins that 831 associate with the nuclear lamina: components of a postulated nuclear egress complex

Structure of the Golgi apparatus is not influenced by a GAG deletion mutation in the 835 dystonia-associated gene Tor1a

Plus-end tracking proteins

Akt mimic regulate herpesvirus-induced stable microtubule formation and virus spread

Subcellular Localization of Herpes Simplex Virus Type 1 UL51 Protein and Role of Palmitoylation 841 in Golgi Apparatus Targeting

Herpes Simplex Virus Type 1 UL51 Protein Is Involved in Maturation and Egress of Virus Particles

HSV-1 ICP34.5 confers neurovirulence by targeting the Beclin 1 autophagy 847 protein

An overview of autophagy: morphology, mechanism, and 849 regulation

Differing effects of herpes simplex virus 1 and 851 pseudorabies virus infections on centrosomal function

Morphological and 853 biochemical characterization of the membranous hepatitis C virus replication compartment

Genetic analysis of the cytoplasmic dynein subunit families

Human cytomegalovirus 858 UL97 Kinase is required for the normal intranuclear distribution of pp65 and virion 859 morphogenesis

The HCMV Assembly Compartment Is a Dynamic Golgi-Derived MTOC that 862 Controls Nuclear Rotation and Virus Spread

Multiscale Analysis of Independent Alzheimer's Cohorts 866 Finds Disruption of Molecular, Genetic, and Clinical Networks by Human Herpesvirus

Parkin protects dopaminergic neurons against 869 microtubule-depolymerizing toxins by attenuating microtubule-associated protein kinase 870 activation

UL31 and UL34 872 proteins of herpes simplex virus type 1 form a complex that accumulates at the nuclear rim and 873 is required for envelopment of nucleocapsids

Ultrastructural 875 localization of the herpes simplex virus type 1 UL31, UL34, and US3 proteins suggests specific 876 roles in primary envelopment and egress of nucleocapsids

in herpes simplex virus type 1 particles lacking individual glycoproteins: No evidence 879 for the formation of a complex of these molecules

Analysis of a charge cluster mutation of 881 herpes simplex virus type 1 UL34 and its extragenic suppressor suggests a novel interaction 882 between pUL34 and pUL31 that Is necessary for membrane curvature around capsids

The HSV-1 UL51 protein interacts with the UL7 protein and plays a role 885 in its recruitment into the virion

The Herpes Simplex Virus 1 UL51 Gene 887 Product Has Cell Type-Specific Functions in Cell-to-Cell Spread

Herpes simplex virus type 1 889 UL34 gene product is required for viral envelopment

Sequence-dependent trafficking and activity of 892 GDE2, a GPI-specific phospholipase promoting neuronal differentiation

Microtubule-organizing centers: from the centrosome to non-894 centrosomal sites

Accumulation of virion tegument and envelope 896 proteins in a stable cytoplasmic compartment during human cytomegalovirus replication: 897 characterization of a potential site of virus assembly

Cytosolic herpes simplex virus capsids not only require binding inner tegument protein 900 pUL36 but also pUL37 for active transport prior to secondary envelopment

The tegument protein UL71 of human cytomegalovirus is involved in late 904 envelopment and affects multivesicular bodies

Human cytomegalovirus UL97 phosphorylates the viral nuclear egress complex

Identification of new protein kinase-related genes in three 909 herpesviruses, herpes simplex virus, varicella-zooster virus, and Epstein-Barr virus

Viral interactions with host cell Rab GTPases

Rab GTPases as coordinators of vesicle traffic

914 Simultaneous tracking of capsid, tegument, and envelope protein localization in living cells 915 infected with triply fluorescent herpes simplex virus 1

Construction of an 917 excisable bacterial artificial chromosome containing a full-length infectious clone of herpes 918 simplex virus type 1: viruses reconstituted from the clone exhibit wild-type properties in vitro 919 and in vivo

Function, Architecture, and Biogenesis of Reovirus Replication 923 Neoorganelles. Viruses

Rab11 regulates recycling through 925 the pericentriolar recycling endosome

Extragenic suppression of a mutation in herpes simplex virus type 927 1 (HSV-1) UL34 that affects lamina disruption and nuclear egress

A herpes simplex virus 2 glycoprotein D mutant generated by bacterial artificial 930 chromosome mutagenesis is severely impaired for infecting neuronal cells and infects only Vero 931 cells expressing exogenous HVEM

More than one door -Budding of enveloped viruses 933 through cellular membranes

Neonatal herpes simplex virus infection

Herpes simplex encephalitis: adolescents and adults

Tubulin Post-Translational Modifications and 937 Microtubule Dynamics

Human cytomegalovirus tegument protein pUL71 is required for 939 efficient virion egress

The Human Cytomegalovirus Nonstructural Glycoprotein UL148 Reorganizes 942 the Endoplasmic Reticulum. mBio

) as described in Figure 1 and stained for pUL51 997 (green) or Golgi markers (red), including GM130, a Golgi matrix protein, and Golgin97, a trans-Golgi 998 marker. (G) Vero cells were transduced with a baculovirus expressing a cis-and medial-Golgi marker 999 EGFP-N-acetylgalactosaminyl transferase (rendered in blue) 24 h before HSV infection

White arrows indicate clusters of pUL51 in adjacent to Golgi membranes. All insets to the right show the 1002 zoomed regions outlined by white dash lines in the overlay images

except at 3 h.p.i, the medium was replaced with medium 1011 containing nocodazole. White arrowheads in (C) indicate a periphery punctum that is positive for both 1012 gE and Rab5

Scale bars represent 10 µm. (B-D) CAD and SH-SY5Y cells were stained 1018 for pUL11 (green) and γ-tubulin (red). (B) Mock-infected cells were treated with DMSO. (C) Cells were 1019 infected with WT HSV-1(F) and treated with DMSO as described in Figure 8. White arrowheads indicate 1020 HSV cVACs. (D) Cells were infected with WT HSV-1(F) and treated with nocodazole as described in Figure 1021 8. 1022 1023 1024 1025 lines present percentage of infected cells that form a unitary cVAC. Each cVAC is defined by pUL11 1030 concentration around the γ-tubulin-defined centrosome. Infected cells were fixed at 12 h.p.i. Error bars 1031 represent standard deviation of three independent experiments. Black columns indicate viral production 1032 determined by single step growth (SSG) at 16 h.p.i as descried in method and material

For nocodazole treated CAD cells, white arrowheads indicate periphery 1037 puncta that are positive for both pUL51 and gE. For nocodazole treated SH-SY5Y cells, green arrowheads 1038 indicate pUL51-only puncta and red arrowheads indicate gE-only puncta. (D-E) Quantification of pUL51-1039 gE co-localization in cells in (C)

White arrowheads indicate centrosomes. Scale bars represent 10 µm. (B) CAD cells were 1047 infected and treated with DMSO or ciliobrevin D following the same protocol as in Figure 8. Cells were 1048 stained for pUL11 (green) and γ-tubulin (red). (C) Percentage of cells that form a unitary cVAC were 1049 counted as described in Figure 10. Error bars represent standard deviations of three

A-B) Western 1055 blot of HSV BAC-derived viruses-infected cells. Lysate of infected SH-SY5Y cells (m.o.i.=5) was harvested 1056 16 h.p.i. (C-F) CAD and SH-SY5Y cells were infected with HSV-1 mutant viruses (m.o.i=5) and stained for 1057 indicated viral proteins at 12 h.p.i. (G-H) Quantification of pUL51-gE colocalization

BAC viruses-infected CAD (G) or SH-SY5Y (H) cells by Pearson co-efficient. (I-J) SSG of indicated HSV-1 1059 BAC-derived viruses on CAD (I) or SH-SY5Y (J) cells. For all statistics

ER-derived membranes are not recruited to the HSV cVAC. CAD and SH-SY5Y cells were 1065 transfected with a GFP-KDEL expression plasmid at least 24 hs before mock or WT HSV-1(F) infection. 1066 Cells were infected as described in Figure 1 and stained for gE