Published Ahead of Print 20 February 2013. 2013, 87(9):5106. DOI: 10.1128/JVI.00102-13. J. Virol. 

Sommer, Jürgen Haas, Tihana L. Rovis and Ann M. ArvinXibing Che, Stefan L. Oliver, Mike Reichelt, Marvin H. Varicella-Zoster Virus InfectionEssential Tegument Protein in ORF11 Protein Interacts with the ORF9

http://jvi.asm.org/content/87/9/5106Updated information and services can be found at:

These include:

REFERENCEShttp://jvi.asm.org/content/87/9/5106#ref-list-1at:

This article cites 44 articles, 31 of which can be accessed free

CONTENT ALERTS more»articles cite this article),

Receive: RSS Feeds, eTOCs, free email alerts (when new

http://journals.asm.org/site/misc/reprints.xhtmlInformation about commercial reprint orders: http://journals.asm.org/site/subscriptions/To subscribe to to another ASM Journal go to:

on June 13, 2014 by guesthttp://jvi.asm

.org/D

ownloaded from

on June 13, 2014 by guest

http://jvi.asm.org/

Dow

nloaded from

ORF11 Protein Interacts with the ORF9 Essential Tegument Protein inVaricella-Zoster Virus Infection

Xibing Che,a Stefan L. Oliver,a Mike Reichelt,a Marvin H. Sommer,a Jürgen Haas,b,c Tihana L. Roviš,d Ann M. Arvina

Departments of Pediatrics and Microbiology & Immunology, Stanford University School of Medicine, Stanford, California, USAa; Max-von-Pettenkofer Institute, Ludwig-Maximilians-Universität Munich, Munich, Germanyb; Division of Pathway Medicine, University of Edinburgh, Edinburgh, United Kingdomc; Department of Histology andEmbryology, University of Rijeka, Rijeka, Croatiad

The tegument proteins encoded by ORF11 and ORF9 of varicella-zoster virus (VZV) are conserved among all alphaherpesvirus.We previously demonstrated that the ORF9 gene is essential, whereas ORF11 is dispensable in vitro but its deletion severely im-pairs VZV infection of skin xenografts in the SCID mouse model in vivo. Here we report that ORF11 protein interacts with ORF9protein in infected cells as well as in the absence of other viral proteins, and we have mapped the ORF11 protein domain in-volved in their interaction. Although ORF11 is an RNA binding protein, the interaction between ORF11 and ORF9 proteins wasnot mediated by RNA or DNA bridging. VZV recombinants with mutations preventing ORF11 protein binding to ORF9 proteinhad no effect on 6-day growth kinetics based on plaque numbers, but plaque sizes were reduced in vitro. However, disruption ofthe ORF11 and ORF9 protein interaction was associated with failure to replicate in skin xenografts in vivo. Further, we demon-strate that in the absence of their interaction, the ORF9 protein displays an identical cellular localization, accumulating in thetrans-Golgi region, whereas the ORF11 protein exhibits aberrant localization, dispersing throughout the cytoplasm. Overall, ourobservations suggest that while complete tegument assembly may not be necessary for VZV replication in vitro, the interactionbetween the ORF11 and ORF9 proteins appears to be critical for the proper localization of ORF11 protein to the assembly com-plex and for production of infectious virus during VZV pathogenesis in skin.

Varicella-zoster virus (VZV) is a human alphaherpesvirus thatcauses varicella (chicken pox) and zoster (shingles) (1, 2).

VZV virions, like those of all herpesviruses, contain a capsid thatharbors the linear double-stranded DNA genome. The capsid issurrounded by the tegument, a proteinaceous layer located be-tween the capsid and the host-derived outer lipid membrane en-velope that expresses the viral glycoproteins which are requiredfor cell entry. The tegument of alphaherpesviruses is predicted toconsist of more than 20 virus-encoded proteins (3, 4). These teg-ument proteins have broad functions, including the regulation ofviral and host cell gene expression during replication, transport ofvirions to and from the nucleus by recruitment of cellular molec-ular motors during entry and egress, and assembly of the infec-tious virus particle (5). VZV tegument proteins that have beenidentified experimentally include the immediate-early regulatoryproteins IE4, IE62, and IE63; the open reading frame 47 (ORF47)and ORF66 viral kinases; and proteins encoded by ORF9, -10, and-11 (6–10). VZV ORF9 to ORF12 belong to a gene cluster that isconserved in the alphaherpesviruses, which is designated UL46-UL49 in the other members of the subfamily.

The VZV ORF11 gene is a homologue of UL47 (11). DeletingUL47 from herpes simplex virus 1 (HSV-1), pseudorabies virus(PRV), and Marek’s disease virus (MDV) resulted in delayed rep-lication compared to that of the parent virus (12–15). In contrast,deleting the ORF11 gene from the VZV parent OKA (pOka) ge-nome did not affect growth kinetics (7), indicating that ORF11functions are dispensable in vitro, as was confirmed subsequently(16). However, replication of the ORF11 deletion mutant was dra-matically impaired when human skin xenografts were infected invivo using our SCID mouse model of VZV pathogenesis (7). Thus,ORF11 is a determinant of VZV virulence in differentiated epider-mal cells in their tissue microenvironment in vivo.

The VZV ORF9 gene is a homologue of UL49, which encodes

the HSV-1 protein VP22. However, ORF9 is essential for VZVreplication, whereas VP22 is dispensable, and ORF9 protein local-izes almost exclusively to the cytoplasm, while VP22 traffics be-tween the cytoplasm and nucleus (17–21). ORF9 protein is a ma-jor component of the VZV tegument and interacts with IE62,which is the predominant VZV transactivating protein; ORF9 alsobinds to glycoprotein E (gE), an essential and abundant envelopeglycoprotein, and to the cellular protein tubulin (7, 17). Our ul-trastructural analysis showed that both ORF9 and gE are presentin the trans-Golgi region, where secondary envelopment occurs inVZV-infected cells. Other ORF9 functions have not been defined.

Most recently, we showed that ORF11 protein is an RNA bind-ing protein with the capacity to bind to both viral and cellularmRNAs (22). Nevertheless, an ORF11 truncation mutant inwhich RNA binding was eliminated was as infectious as the parentvirus, pOka, in skin xenografts, indicating that the ORF11 geneproduct is not involved in RNA biogenesis during VZV infectionand that the RNA binding capacity of ORF11 does not explain whythis protein is required for virulence in skin. The purpose of thisstudy was to further investigate the functions of ORF11 and ORF9proteins in order to better understand their roles in the pathogen-esis of VZV skin infection.

Received 10 January 2013 Accepted 13 February 2013

Published ahead of print 20 February 2013

Address correspondence to Xibing Che, xibing@stanford.edu.

Copyright © 2013, American Society for Microbiology. All Rights Reserved.

doi:10.1128/JVI.00102-13

5106 jvi.asm.org Journal of Virology p. 5106–5117 May 2013 Volume 87 Number 9

on June 13, 2014 by guesthttp://jvi.asm

.org/D

ownloaded from

MATERIALS AND METHODSRecombinant viruses. The complete genome of VZV pOka is containedin four overlapping SuperCos1 cosmid vectors (Stratagene, La Jolla, CA)designated pvFsp73 (nucleotides [nt] 1 to 33128), pvSpe14 (nt 21795 to61868), pvPme2 (nt 53755 to 96035), and pvSpe23 (nt 94055 to 125124)(23). ORF11 (nt 13589 to 15970 of pOka VZV genome) is in the pvFsp73cosmid. Three PCR fragments containing the sequence upstream of theSpeI site at nt 12565 to 14298, 15233, or 15886 were synthesized with thesame upper primer (5=-GTTCCGTTCGATTGCCTAGTCCAC-3=) anddifferent lower primers (5=-CGCTAACATAGAGCCTGTTAGGGT-3=,5=-CAACATTCCTCCACCAAGAGTAAC-3=, and 5=-TAGGTCCACGGAGGGAATGGGCTG-3=). The fourth PCR amplified the regions fromeither the ORF11 stop codon or nt 15925 to a PacI site with differentupstream primers (5=-TAAGGGTTGTGATTTTTTTCATTAG-3= or 5=-GATTGGAAAAGCGTAGCCATGC-3=) and the same lower primer (5=-TACGGCTGATCAGACACACTCGATG-3=). The pvFsp73 cosmid wasthe template for synthesizing these PCR products. The resulting PCRproducts were digested with SpeI or PacI and inserted by triple ligationinto a pT7Blue-3 plasmid (New England BioLabs, Beverly, MA) whichcontained a 6.7-kb NheI-NheI fragment corresponding to VZV genoment 12339 to 19129 and was digested by SpeI and PacI. The three NheI-NheI fragments containing mutated ORF11 genes were used to replace theNheI fragment in the pvFsp73 cosmid. Recombinant viruses were isolatedfrom human melanoma cells transfected with the mutated pvFsp73 cos-mid and three intact cosmids, pvSpe14, pvPme2, and pvSpe23. Mutationswere confirmed by sequencing. Viruses were propagated in human em-bryonic lung fibroblasts (HELF) for infection of skin xenografts in SCIDmice, and the replication kinetics and peak titers of the recombinant vi-ruses were assessed by infectious-focus assay (24).

Construction of plasmids and transfection. Full-length ORF9 pro-tein tagged with histidine (ORF9-His) was generated by PCR with theupper primer 5=-CTACGAAGCTTACCATGGCATCTTCCGACGGTG-3= and lower primers 5=-CTACGCTCGAGCTAATGATGATGATGATGATGTTTTCGCGCATCAGTTCTTG-3=. Eight PCR fragments, includ-ing ORF11 253, ORF11 548, ORF11 600, ORF11 667, ORF11 701, ORF11750, ORF11 766, and ORF11 780, were generated with the same plusprimer, 5=-CTACGAAGCTTGTTATGGCACAGTCGGGTCATTATAAC-3=, and different minus primers, 5=-CTACGCTCGAGTTAAGCAGCTAAATCAATTGCAG-3=, 5=-CTACGCTCGAGTTACCAGGACTCATCAAACCCATTATG-3=, 5=-CTACGCTCGAGTTAGTCGACCTCGCCTAACAGTAA-3=, 5=-CTACGCTCGAGTTATTGTAACAATAAAACGGCCCCC-3=, 5=-CTACGCTCGAGTTAGTAAAGAGGACTTGCTGCCAAC-3=, 5=-CTACGCTCGAGTTACAATCCCACAAAACGGAAGTTC-3=, 5=-

CTACGCTCGAGTTATAGGTCCACGGAGGGAATGG-3=, and 5=-CTACGCTCGAGTTACAAACCCAGAATTTCATTCCTG-3=. The HindIII(AAGCTT) and XhoI (CTCGAG) restriction sites are underlined, and theHis sequence is shown in italics. PCR fragments were digested withHindIII and XhoI enzymes and then inserted into HindIII/XhoI-cutpcDNA3.1 vector. The full-length ORF11 protein expressing vectorpcDNA3.1ORF11 was reported previously (22).

IP and analysis. Melanoma cells were infected with ORF11 mutants orpOka, and protein lysates were collected with extraction buffer containing0.1 M Tris base (pH 8.8), 0.1 M NaCl, 5 mM KCl, 1 mM CaCl2, 0.5 mMMgCl2, 1% SDS, 1% NP-40, and an EDTA-free protease inhibitor cocktail(Roche, CA). Protein lysates from pOka�11-infected or uninfected mel-anoma cells were used as negative controls. Protein lysates were treatedwith RNase A (100 �g/ml) and DNase I (40 U/ml) (Invitrogen, Carlsbad,CA) for 30 min at 37°C before immunoprecipitation (IP) to assess possi-ble RNA or DNA bridging in ORF11 protein binding to ORF9 protein. Arabbit antibody against the ORF11 protein N-terminal amino acids 1 to245 (Ab11) was cross-linked to immobilized protein A (Pierce, Rockford,IL), and then antibody-bound beads were washed and incubated withlysates overnight at 4°C. IP samples were separated by SDS-polyacryl-amide electrophoresis and either processed with GelCode blue staining(Thermo Fisher Scientific, Rockford, IL) or transferred to a nitrocellulosemembrane. Transferred proteins were analyzed by immunoblotting usinga rabbit antibody against full-length ORF9 protein (17); the blot was thenstripped and reprobed with Ab11 antibody specific to ORF11 protein. Forin vitro IP assays, HEK 293 cells were transfected with plasmids expressingORF11 or the ORF11 truncations, along with the ORF9-expressing plas-mid. Lysates were collected in radioimmunoprecipitation assay (RIPA)buffer containing the protease inhibitor cocktail (Roche Inc., Indianapo-lis, IN), and IP was performed with Ab11 and a rabbit anti-His antibody(His Ab) (Santa Cruz Biotech, Inc., Santa Cruz, CA). Normal mouse andrabbit sera (Vector Laboratories, Inc., Burlingame, CA) were used asnegative controls. The antibodies were incubated with 30 �l of proteinA�G beads for 2 h at 4°C (Santa Cruz Biotech, Inc.); the beads werewashed in lysis buffer and then incubated with lysates overnight at 4°C.The samples were separated on a 7.5% polyacrylamide gel and trans-ferred to a polyvinylidene difluoride membrane (Immobilon-P; Mil-lipore, Bedford, MA). For Western blotting, anti-ORF11 protein anti-body Ab11 was used at a dilution of 1:20,000 and His Ab or mouseanti-His monoclonal antibody (His MAb) (Abcam Inc., San Francisco,CA) was used at a dilution of 1:1,000. Primary antibodies were de-tected with horseradish peroxidase-conjugated sheep anti-mouse andsheep anti-rabbit secondary antibodies (Amersham) at a dilution of

FIG 1 Identification of the interaction between ORF11 protein and ORF9 protein. (A) GelCode blue-stained gel of proteins immunoprecipitated from infected(I) or uninfected (U) cells using Ab11 and gB antibodies. The bands present only in infected Ab11 IP extracts (dashed-line box) were cut from the gel andsubjected to mass spectroscopy. The arrow indicates a species of around 110 kDa, the predicted size of ORF11 protein. (B) Proteins immunoprecipitated by Ab11and gB antibody from infected and uninfected cells were analyzed by Western blotting (WB) with Ab11 antibody. The ORF11 gene product (arrow) wasimmunoprecipitated by Ab11 from infected cells. (C) Proteins immunoprecipitated by Ab11 and gB antibody from infected and uninfected cells were analyzedby Western blotting with anti-ORF9 protein antibody; ORF9 protein was coprecipitated with ORF11 protein. Numbers on the left side of the blots indicatemolecular mass, in kDa.

Importance of VZV Tegument Proteins ORF9 and ORF11

May 2013 Volume 87 Number 9 jvi.asm.org 5107

on June 13, 2014 by guesthttp://jvi.asm

.org/D

ownloaded from

1:10,000 to 1:20,000 and visualized by ECL Plus (Amersham) and X-ray film (Kodak). The nuclear and cytoplasmic extracts from cells wereprepared by using a nuclear extraction kit (Active Motif, Carlsbad,CA).

Mass spectrometry. Melanoma cells (5 log10 cells/cm2) were inocu-lated with pOka (3 log10 PFU/cm2) and harvested 48 h postinfection (hpi),and the ORF9/ORF11 complex was immunoprecipitated as describedabove. Samples were separated by SDS-PAGE and proteins visualized us-ing GelCode blue. Bands were excised and digested with trypsin, and theproteins were identified using an LTQ-Orbitrap Velos mass spectrometer(Thermo Scientific, West Palm Beach, FL) at the Vincent Coates Founda-tion Mass Spectrometry Laboratory, Stanford University.

Cryo-immuno-EM and confocal microscopy. For cryo-immuno-electron microscopy (cryo-immuno-EM) experiments, HELF wereseeded in a 75-mm2 flask at a density of 7.5 � 106 cells and infected with4.9 log10 PFU of pOka, pOka�11, and pOka11�549-793 (here referred toas pOka11�549) viruses. Samples for cryo-immuno-EM were collected at2 days after infection and prepared as described previously (7). For im-munogold staining, ultrathin sections (60 nm) were incubated in digoxi-genin-blocking solution (Roche Diagnostics) for 30 min and then for 1 hwith Ab11 or rabbit polyclonal anti-ORF9 protein antibodies at a dilutionof 1:200. Samples were washed in phosphate-buffered saline (PBS) andincubated with protein A conjugated to 10-nm colloidal gold (PAG10nm) from CMC (Utrecht, The Netherlands) at a dilution of 1:60. Sampleswere stained with 3.5% aqueous uranyl acetate for 10 min and with 0.2%lead citrate for 3 min and examined on a Joel 1230 transmission electronmicroscope.

For confocal microscopy, HELF were fixed with 4% formaldehyde at48 hpi. After blocking with 1% fish gelatin for 1 h at room temperature(RT), cells were incubated with a rabbit anti-ORF9 protein antibody andan affinity-purified murine monoclonal anti-ORF11 protein antibody,clone 11N_12, which was raised against the N-terminal 400 amino acids ofORF11 protein. Cells were washed and incubated for 1 h at RT with fluo-rescein isothiocyanate-labeled anti-rabbit and Texas red-labeled anti-mouse secondary antibodies (Jackson ImmunoResearch, Inc.). Cell nucleiwere counterstained with Hoechst 22358 (Invitrogen, Carlsbad, CA).Analysis was performed with a Leica TCSSP2 confocal laser scanning mi-croscope (Heidelberg, Germany).

Infection of human skin xenografts in SCIDhu mice. Skin xenograftswere made in homozygous CB-17scid/scid mice, using human fetal tissue sup-plied by Advanced Bioscience Resources (Alameda, CA) according to federaland state regulations (25). Animal use was in accordance with the AnimalWelfare Act and was approved by the Stanford University AdministrativePanel on Laboratory Animal Care. pOka and ORF11 mutant viruses werepassaged three times in primary HELF before inoculation of the xenografts.The infectious virus titer was determined for each inoculum at the time ofinoculation. Skin xenografts were harvested at 10 and 20 days postinoculationand analyzed by infectious-focus assay (24). Virus recovered from the tissueswas tested by PCR and sequencing to confirm the expected deletions.

RESULTSORF11 protein interacts with ORF9 protein in VZV-infectedcells. Immunoprecipitation (IP) experiments to identify potentialORF11 protein binding partners were done using Ab11, a rabbitpolyclonal antibody that recognizes the ORF11 protein N termi-nus (22), and lysates of VZV-infected and uninfected melanomacells. A rabbit polyclonal antibody to VZV glycoprotein B (gB) wasincluded in IP experiments and served as an unrelated antibodycontrol. IP samples were separated by SDS-PAGE and proteinsvisualized using GelCode blue (Fig. 1A). A 110-kDa protein,which is the expected size of the ORF11 protein, was recoveredfrom infected but not from uninfected cell lysates and gB IP ex-tracts. Several polypeptides exhibiting apparent masses between35 and 40 kDa were also detected in Ab11 infected IP extracts only.When these bands were excised from the SDS-PAGE gel and testedby mass spectroscopy, ORF9 polypeptides were detected, suggest-ing that ORF11 protein forms a complex with the ORF9 tegumentprotein in VZV replication. In addition, the VZV protein IE63 andcellular proteins annexin-2 and rRNA 2=-O-methyltransferasefibrillarin were identified in IPs with ORF11 protein.

To verify the mass spectroscopy results, we next performedWestern blot analyses of protein complexes that were coprecipi-tated from infected and uninfected cell lysates using Ab11 and gBantibody. The viral protein IE63 was not detected by Westernblotting (data not shown), and therefore, the interaction betweenORF11 and IE63 proteins was not confirmed. However, ORF11protein was immunoprecipitated from VZV-infected cell lysates(Fig. 1B) and ORF9 protein was pulled down by Ab11, as shownusing an antibody that recognizes the ORF9 gene product(Fig. 1C). In this experiment, ORF9 protein antibody also reactedwith several polypeptides with sizes between 35 and 40 kDa, whichwas consistent with the mass spectroscopy analysis and a previousinvestigation of ORF9 expression in VZV-infected cells (7). Nosignal was detected by Western blotting using Ab11 or anti-ORF9protein antibody in IPs done with uninfected cell lysates (Fig. 1Band C). IP with a rabbit polyclonal antibody to VZV gB did notyield proteins of the molecular masses that were recovered withAb11 and anti-ORF9 protein antibodies (Fig. 1B and C). Thus,ORF9 protein binds to ORF11 protein in VZV-infected cells.

FIG 2 Assessment of the interaction of ORF11 protein with ORF9 protein. (A)Immunoprecipitation was performed with Ab11 and pOka- or pOka�11-in-fected cell lysates, with or without RNase A and DNase I treatment. Westernblotting was done with anti-ORF9 protein antibody. Multiple forms of ORF9protein were detected from pOka-infected cell lysates with or without RNase Aand DNase I treatment and are indicated by arrowheads. (B) Expression ofORF11 protein and His-tagged ORF9 protein. HEK 293 cells were cotrans-fected with an ORF11-expressing plasmid and a plasmid encoding His-taggedORF9 protein, harvested at 48 h posttransfection, and analyzed by Westernblotting with rabbit anti-ORF11 antibody, Ab11 (left panel), or rabbit anti-Hisantibody (His Ab) (right panel). HEK 293 cells transfected with vectorpcDNA3.1 alone served as a negative control. Molecular mass is indicated onthe sides of the blots. (C) The left blot shows proteins immunoprecipitatedwith Ab11 or normal rabbit serum (control) from cotransfected HEK 293 cells,analyzed by Western blotting with mouse anti-His antibody (His MAb). ORF9His-tagged protein was coprecipitated with ORF11 protein. The right blotshows proteins immunoprecipitated with rabbit anti-His Ab or normal rabbitserum (control) from cotransfected HEK 293 cells, analyzed by Western blot-ting with anti-ORF11 antibody Ab11. ORF11 protein was coprecipitated withHis-tagged ORF9 protein.

Che et al.

5108 jvi.asm.org Journal of Virology

on June 13, 2014 by guesthttp://jvi.asm

.org/D

ownloaded from

The interaction between ORF11 protein and ORF9 protein isindependent of RNA, DNA, and other viral proteins. SinceORF11 protein was previously identified as an RNA binding pro-tein, immunoprecipitations using Ab11 were done with pOka-and pOka�11-infected cell lysates prepared in the presence orabsence of RNase A and DNase I. Multiple forms of ORF9 protein(35 to 40 kDa) was detected in pOka lysates with or without RNaseA and DNase I but not in the pOka�11 lysates (Fig. 2A), indicatingthat the interaction between ORF11 and ORF9 proteins is notdependent on the presence of RNA or DNA.

In order to determine whether ORF11 and ORF9 proteins in-teract directly or through an intermediate viral protein, the pro-teins were expressed from plasmids in transient-transfection ex-periments. ORF11 protein and a His-tagged ORF9 protein werecoexpressed in HEK 293 cells, and the expression of both proteinswas confirmed by Western blot analysis of cell lysates. No signalwas observed from vector alone-transfected HEK 293 cells

(Fig. 2B). IP was then performed with Ab11, followed by Westernblot analysis using a mouse anti-His monoclonal antibody (HisMAb). ORF11 protein was coprecipitated with His-tagged ORF9protein (Fig. 2C). The reciprocal IP was also performed with HisAb followed by Western blotting using Ab11. His-tagged ORF9protein was coprecipitated with ORF11 protein (Fig. 2C). NoORF11 protein or His-tagged ORF9 protein was observed usingcontrol rabbit serum for IP (Fig. 2C). Therefore, ORF11 proteinbinds to ORF9 protein in the absence of other viral proteins.

Mapping of the region of ORF11 protein required for inter-action with ORF9 protein. To investigate the functional signifi-cance of the interaction between ORF11 and ORF9, we sought toidentify the amino acids of ORF11 protein that are required fortheir binding. We first constructed two truncation mutants withdeletions beginning from amino acid 254 or 549 of ORF11 protein(Fig. 3A). Expression of the ORF11 protein and its truncationstogether with His-tagged ORF9 protein was confirmed by West-

FIG 3 Mapping of the ORF11 region required for interaction with ORF9 protein. (A) Schematic diagram of full-length ORF11 protein (wild type [wt]) and itsC-terminal truncation mutants used for this study. The amino acids (aa) of the ORF11 protein RNA binding domain and their location are indicated. Plasmidsexpressing ORF11 or the mutants were separately transfected into HEK 293 cells, along with a plasmid expressing His-tagged ORF9 protein. Cell lysates werecollected at 48 h posttransfection and tested by WB and IP. (B) WB with Ab11 antibody (left) for expression of ORF11, ORF11 548, and ORF11 253 incotransfected HEK 293 cells. A WB with His Ab (right) shows the expression of His-tagged ORF9 protein in the three cotransfection assays. (C) Proteinsimmunoprecipitated with His Ab or control rabbit serum (control) from cotransfected HEK 293 cells were analyzed by WB with Ab11. (D) Western blots forexpression of ORF11 and ORF11 mutant proteins (top) and ORF9 protein (bottom) in cotransfection experiments. (E) IP was performed with His Ab (�) andnormal rabbit serum (�), followed by Western blotting using Ab11.

Importance of VZV Tegument Proteins ORF9 and ORF11

May 2013 Volume 87 Number 9 jvi.asm.org 5109

on June 13, 2014 by guesthttp://jvi.asm

.org/D

ownloaded from

ern blots of lysates from cotransfected cells (Fig. 3B). When thesetwo ORF11 truncations were tested for their ability to bind ORF9protein by IP, both abolished the interaction (Fig. 3C), suggestingthat ORF11 protein C-terminal amino acids 549 to 793 are neces-sary for the binding. To more precisely define the ORF11 proteinregion that binds ORF9 protein, we generated a panel of ORF11truncation mutants by stepwise deletions of the C terminus ofORF11 protein (Fig. 3A). These mutants were then cotransfectedtogether with the plasmid expressing His-tagged ORF9 protein.All of the cotransfected ORF11 mutants and ORF9 protein wereexpressed (Fig. 3D). When the capacity of these ORF11 mutants tobind ORF9 protein was analyzed by IP, none showed an interactionwith ORF9 protein except for the ORF11 mutant that included resi-dues from amino acids 1 to 780. ORF9 protein pulldown by thisORF11 small truncation was similar to that of full-length ORF11protein (1 to 793 amino acids) (Fig. 3E). These results suggested thatthe region including amino acid 767 to 780 within the C terminus ofORF11 protein was necessary for the interaction with ORF9 protein.

Effects of the ORF11 mutations on binding to the ORF9 pro-tein during VZV replication. Based on the in vitro mapping re-sult, three VZV recombinants, pOka11�254-793 (hereinafter re-ferred to as pOka11�254), pOka11�549-793 (hereinafter referredto as pOka11�549), and pOka11�767/780, were generated. Theregion of the ORF11 gene encoding the C-terminal amino acids254 to 793 was deleted to make pOka11�254; the ORF11 se-quences encoding amino acids 549 to 793 and amino acids 767 to780 located within the ORF11 protein C terminus were deleted tomake pOka11�549 and pOka11�767/780. The capacity of themutant forms of ORF11 protein to interact with ORF9 proteinwas assessed in VZV-infected cells by IP using Ab11 antibody andWestern blotting using antibody against the ORF9 gene product.pOka11�254, pOka11�549, and pOka11�767/780 proteins wereexpressed as �40-kDa, �75-kDa, and �100-kDa products, re-spectively (Fig. 4A and B). Full-length and mutant ORF11 pro-teins were immunoprecipitated by Ab11, and ORF9 protein wasonly coprecipitated with ORF11 from pOka-infected cells, butbinding of ORF9 protein to ORF11 protein was not detected inlysates of pOka11�254-, pOka11�549-, or pOka�11-infectedcells or an uninfected control (Fig. 4A). This result is consistentwith our in vitro binding assay and indicates that the interactionrequires residues present within amino acids 549 to 793 of theORF11 protein C terminus. When the interaction with ORF9 pro-tein was assessed in pOka11�767/780- and wild-type pOka-in-fected cells, little ORF9 protein was detected from the IP sample ofpOka11�767/780 (Fig. 4B), suggesting that the absence of 767 to780 residues from the ORF11 protein C-terminal region signifi-cantly affected the ability of ORF11 protein to bind to ORF9 pro-tein during VZV infection.

The interaction between the ORF11 and ORF9 proteins wasalso investigated in cells infected with two previously describedORF11 mutants, pOka11-Nd and pOka11-Ns (22). pOka11-Nd ex-presses ORF11 protein lacking amino acids 1 to 30, andpOka11-Ns has a mutation that disrupts the RNA binding do-main that we mapped to amino acids 8 to 15 in the ORF11protein N terminus. The ORF11-ORF9 protein interaction waspreserved and similar to pOka in cells infected withpOka11-Nd and pOka11-Ns but was absent from pOka�11-infected cells (Fig. 4C).

Characterization of VZV ORF11 mutant viruses in vitro. Thegrowth kinetics and plaque morphology of pOka11�254,

pOka11�549, and pOka11�767/780 were investigated in infectedmelanoma cells. Comparisons of the growth kinetics of pOka withthose of pOka11�254, pOka11�549, and pOka�11 (Fig. 5A.I)and of pOka with pOka11�767/780 and pOka�11 in melanomacells showed no statistically significant differences over 6 days (Fig.5A.III). Thus, as expected given our previous observations withpOka�11 and confirmed in these experiments, none of the partialORF11 deletions altered VZV replication as determined by plaquenumbers in vitro. However, when plaque size was used to evaluatecell-cell spread of the pOka11�254, pOka11�549, pOka11�767/780, and pOka�11 mutants compared to pOka in melanoma cells,all exhibited significantly reduced plaque sizes compared to thoseof pOka (Fig. 5A.II, A.IV, and B). Plaque sizes generated bypOka11�254, pOka11�549, and pOka11�767/780 were similarto those generated by pOka�11. Thus, pOka11�767/780 showedthe same phenotype for growth kinetics and plaque size as theVZV recombinants pOka11�254 and pOka11�549, with largerdeletions.

Effects of eliminating the ORF11-ORF9 protein interactionon the localization of ORF11 and ORF9 proteins in infectedcells. The cellular localization of ORF11 and ORF9 proteins wasexamined by confocal microscopy at 48 h postinfection (Fig. 6).ORF11 protein was present in both nuclei and cytoplasm, whereas

FIG 4 Immunoprecipitation of ORF11 and ORF9 proteins from melanomacells infected with VZV and VZV ORF11 mutants. Melanoma cells were in-fected with VZV ORF11 mutants or pOka. The anti-ORF11 antibody Ab11 wasused for immunoprecipitation, while Ab11 and the rabbit anti-ORF9 antibodywere used for Western blotting. (A) The top blot is a Western blot with Ab11showing that ORF11, ORF11�254, and ORF11�549 proteins were immuno-precipitated by Ab11 from cells infected with pOka, pOka11�254, andpOka11�549 but not from uninfected cells or cells infected with pOka�11.The bottom blot is a Western blot with ORF9 antibody showing that ORF9protein was immunoprecipitated only by Ab11 from the pOka-infected cells.(B) The top blot is a Western blot with Ab11 showing ORF11 and ORF11�767/780 protein detection from the input cell lysates used for IP. No signal wasobserved from uninfected or pOka�11-infected cell lysates. The bottom blot isa Western blot with ORF9 antibody showing that ORF9 protein was immuno-precipitated by Ab11 from pOka-infected cells, and a trace of ORF9 proteinwas also observed from pOka11�767/780-infected cells. (C) The top blot is aWestern blot with Ab11 showing that ORF11, ORF11-Nd, and ORF11-Nsproteins were immunoprecipitated by Ab11 from cells infected with pOka,pOka11-Nd, and pOka11-Ns. The bottom blot is a Western blot demonstrat-ing that ORF9 proteins were immunoprecipitated by Ab11 from melanomacells infected with pOka, pOka11-Nd, and pOka11-Ns but not from cells in-fected with pOka�11. Molecular mass, in kilodaltons, is indicated on the rightsides of the blots.

Che et al.

5110 jvi.asm.org Journal of Virology

on June 13, 2014 by guesthttp://jvi.asm

.org/D

ownloaded from

ORF9 protein had a predominantly cytoplasmic localization incells infected with pOka, pOka11�254, pOka11�549, andpOka11�767/780. This observation indicated that the ORF11protein interaction with ORF9 protein occurs in the cytoplasmand that the ORF11 protein mutations had no detectable effect onthe cellular localization of ORF11 or ORF9 as determined by con-focal microscopy. Uninfected controls showed no reactivity withORF11 or ORF9 protein antibodies (Fig. 6).

To further investigate the intracellular distribution of ORF11 andORF9 proteins, cells infected with pOka11�254, pOka11�549,pOka11�767/780, and pOka were fractionated into nuclear and cy-

toplasmic extracts (Fig. 7). As expected from the confocal microscopyanalysis, ORF11 mutant proteins containing residues 1 to 253 and 1to 548 or with deletions of residues 767 to 780 were present in bothnuclear and cytoplasmic fractions, like intact ORF11 protein. In con-trast, little or no ORF9 protein was detected in the nuclear fractionsfrom cells infected with pOka and the VZV ORF11 mutants, includ-ing pOka11�254, pOka11�549, pOka�11, and pOka11�767/780.�-Tubulin and RCC1 (regulator of chromosome condensation 1)were used to confirm the purity of the fractionations; �-tubulin wasdetected exclusively in the cytoplasmic fraction, while the nuclearprotein RCC1 was only observed in the nuclear fraction (Fig. 7).

FIG 5 Growth kinetics of pOka, pOka11�549, pOka11�254, pOka�11, and pOka11�767/780 in melanoma cells. Growth kinetics of pOka, pOka�11,pOka11�549, and pOka11�254 (A.I) and pOka, pOka�11, and pOka11�767/780 over 6 days (A.III) are shown. Bars indicate standard errors of the means. Theplaque sizes of these viruses in melanoma cells at 4 days postinoculation are shown (A.II and A.IV). Bars indicate mean sizes of 50 plaques measured for each virus,with standard errors of the means. The asterisks indicate differences with a P value of �0.001 compared to the pOka plaque size. (B) Representative plaquemorphologies of pOka, pOka�11, pOka11�549, pOka11�254, and pOka11�767/780 in melanoma cells.

Importance of VZV Tegument Proteins ORF9 and ORF11

May 2013 Volume 87 Number 9 jvi.asm.org 5111

on June 13, 2014 by guesthttp://jvi.asm

.org/D

ownloaded from

Subcellular distribution of ORF11 protein relative to ORF9protein in pOka11�549-infected cells. It has been suggested thatORF9 protein accumulates in the trans-Golgi region of VZV-in-fected cells, where it might recruit other proteins for incorpora-tion into the viral tegument (7), leading to the hypothesis thatORF11 protein binding to ORF9 protein may be required for itsinclusion in the virus particle. The effects of blocking the interac-tion on the subcellular localization of these proteins were assessedby cryo-immuno-EM analysis of HELF at 48 h after infection withpOka11�549 or pOka. The distributions of ORF9 protein weresimilar in cells infected with these viruses; ORF9 was localized toGolgi cisternae and Golgi-derived membranes (Fig. 8A and B). Incontrast, the subcellular localization of ORF11 protein was alteredby deleting the ORF11 C terminus, which contains the interactingdomain as shown in HELF infected with pOka11�549. ORF11 wasdetected primarily with the vesicular membranes present in thetrans-Golgi region of cells infected with pOka (Fig. 8C, D, and E),whereas ORF11 was dispersed irregularly throughout the cyto-plasm in cells infected with pOka11�549 (Fig. 8F and G). When

HELF infected with pOka�11 were examined, it was found thatORF9 retained the trans-Golgi region distribution (Fig. 8H), andORF11 was not detected (Fig. 8I). Either no or very faint back-ground labeling was detected in HELF infected with pOka andtested with the preimmune sera for the rabbit antibodies to ORF9(Fig. 8J) and ORF11 (Fig. 8K) proteins. In addition, ORF11 proteinincorporation into virions was significantly impaired inpOka11�549-infected cells, although the mutant ORF11 proteincould be detected in some virions. pOka virions had a mean of 4.3(standard deviation [SD], 1.6) gold particles/virion, compared to 1.8(SD, 1.0) per pOka11�549 virion (P � 0.001), when the number ofgold particles indicating ORF11 protein association with virions wascounted in pOka- and pOka11�549-infected cells (53 virions each).Furthermore, compared with those infected with pOka, cells infectedwith pOka11�549 had fewer enveloped virions in the typical largecytoplasmic vacuoles observed in VZV-infected cells.

Effects of abrogating the interaction of ORF11 protein withORF9 proteins on VZV virulence in human skin xenografts invivo. Skin xenografts were inoculated with fibroblasts infected

FIG 6 Cellular localization of ORF11 and ORF9 proteins. The localization of ORF9 and ORF11 proteins in cells infected with pOka, pOka11�254, pOka11�549,and pOka11�767/780 was examined by confocal microscopy. Infected or uninfected HELF were fixed 48 hpi and stained with an anti-ORF9 protein (green) anda mouse monoclonal antibody to ORF11 protein (red). Cell nuclei were counterstained with Hoechst 22358 (blue). Size bars, 10 �m.

Che et al.

5112 jvi.asm.org Journal of Virology

on June 13, 2014 by guesthttp://jvi.asm

.org/D

ownloaded from

with pOka, pOka11�254, pOka11�549, or pOka�11. pOka wasthe wild-type control and pOka�11, which has shown to be se-verely attenuated in skin in vivo, was a negative control. The inoc-ulum titers of the four viruses were similar (Fig. 9). Infected skinxenografts were harvested at 10 and 20 days after infection, andvirus yields were determined by an infectious center assay. All skinxenografts infected with pOka yielded infectious virus. The meantiter of pOka was 2.8 log10 PFU/ml at day 10 and 3.6 log10 PFU/mlat day 20 postinoculation. In contrast to the in vitro studies, infec-tious virus was not recovered from any skin xenografts inoculatedwith pOka11�254, pOka11�549, or pOka�11 at either day 10 orday 20 postinoculation (Fig. 9).

DISCUSSION

These experiments demonstrate that the ORF11 tegument proteinof VZV interacts directly with ORF9 tegument protein, which isessential and constitutes a major component of the VZ viriontegument (7, 17). Eliminating the capacity of ORF11 protein tobind ORF9 protein was associated with a severe impairment ofinfection in human skin xenografts in the SCID mouse model. Thedeficiency was comparable to the effect of deleting the ORF11gene on skin virulence (7), which was confirmed in this study.Thus, the ORF11-ORF9 protein interaction is likely to be impor-tant for VZV pathogenesis in skin, whereas interfering with theRNA binding capacity of ORF11 does not alter VZV replication inskin, as we showed previously (22).

The gene cluster that includes ORF9 to ORF12 is conserved inall of the alphaherpesviruses; these genes are UL49, UL48, UL47,and UL46 in HSV-1 and related viruses (1, 4). Analyses of HSV-1virions show that the tegument proteins encoded by UL49, UL48,UL47, and UL46 are present at 1,000 to 2,000 copies, while the

other known tegument proteins are present at fewer than 1,000copies (26). These high concentrations suggest that the UL46 toUL49 proteins are likely to play a structural role in HSV-1 parti-cles. Studies of PRV mutants that do not express these gene prod-ucts also suggest their importance as structural components of thetegument (27–29). Quantitative analyses of VZV virions havebeen limited by the difficulty of recovering sufficient numbers ofVZV virions from infected cells. However, the transcripts ofORF11 and ORF9 genes are among the most highly abundant viralmRNAs produced during lytic infection (30). Our previous reportdemonstrated that ORF11 and ORF9 proteins are readily detectedin the VZV virion tegument and appear to be major componentsof the VZV virion tegument, like their HSV-1 and PRV homo-logues (7, 22).

In vitro binding assays indicated that the ORF11 protein C-ter-minal region of amino acids 549 to 793 is required for ORF11protein interaction with ORF9 protein. Further mapping identi-fied ORF11 protein amino acids 767 to 780 as the residues in-volved in ORF9 protein binding. Although plaque numbers werenot reduced, pOka11�767/780, like pOka11�254, pOka11�549,and the full ORF11 deletion mutant, exhibited reduced capacityfor cell-cell spread, based on the small-plaque phenotype. Thiscapacity for cell-cell spread is important for VZV pathogenesis inskin.

We showed that ORF11 protein has both a nuclear and cyto-plasmic distribution in pOka-, pOka11�254-, pOka11�549-, andpOka11�767/780-infected cells, whereas ORF9 is detected pre-dominantly in the cytoplasm, implying that the interaction be-tween these two proteins occurs in the cytoplasm. Like ORF11, theUL47 homologue is present in the nucleus and cytoplasm ofHSV-1 infected cells (18). Precise information about the intracel-lular distribution of ORF11 and ORF9 proteins was provided byultrastructure analysis and showed that both proteins were foundpredominantly with vesicle membranes in the trans-Golgi area ofpOka-infected cells, which is the site of secondary envelopment.In contrast, ORF11 protein lacking the C terminus did not bind toORF9 protein and was predominantly dispersed throughout thecytoplasm. Notably, this difference in the cytoplasmic distribu-tion of ORF11 was not detected by confocal microscopy, whichlacks the precision of cryo-immuno-EM. These observations sug-gest that ORF9 protein may be required to recruit ORF11 proteinto the trans-Golgi region for virion assembly.

The tegument of alphaherpesvirus virions consists of numer-ous viral and several cellular proteins that appear to be recruitedinto the virus particle through a network of protein-protein inter-actions (31). Although the tegument has been described as amor-phous, recent evidence suggests that this network of interactionscauses an ordered addition of proteins that form the tegumentduring assembly. In HSV-1, PRV, and equine herpesvirus 1(EHV-1)-infected cells, UL48 protein (VP16) interacts with cap-sid proteins, tegument proteins (including the other three en-coded by the UL46-UL49 gene cluster), and viral glycoproteins(32–35). UL48 protein is a component of primary enveloped viri-ons and is essential for addition of the outer tegument (36–39).Moreover, UL48 protein is critical for HSV-1 and PRV viral as-sembly (11). The sequence of events by which tegument proteinsare incorporated into the VZV virion tegument is unknown. VZVORF10 protein, the UL48 protein homologue, was associated withthe tegument by analysis of purified virions (9). However, ORF10protein is dispensable for viral replication in cultured cells, al-

FIG 7 Analysis of ORF9 and ORF11 proteins in nuclear and cytoplasmicfractions of VZV-infected cells. Cellular fractions were prepared from mela-noma cells 48 h after infection with pOka, pOka11�254, pOka11�549,pOka�11, or pOka11�767/780, separated on SDS-PAGE gels, and probedwith either anti-ORF9 or anti-ORF11protein antibodies. The membranes werethen stripped and reprobed for cellular protein �-tubulin and nuclear proteinRCC1 to determine the purity of the fractions. Molecular mass is shown on theleft. Nu, nuclear fractionation; Cyto, cytoplasmic fractionation.

Importance of VZV Tegument Proteins ORF9 and ORF11

May 2013 Volume 87 Number 9 jvi.asm.org 5113

on June 13, 2014 by guesthttp://jvi.asm

.org/D

ownloaded from

5114 jvi.asm.org Journal of Virology

on June 13, 2014 by guesthttp://jvi.asm

.org/D

ownloaded from

though it is a virulence factor for skin infection (40). Thus, ORF10protein does not share the same properties as UL48 protein inother alphaherpesvirus orthologues.

ORF9 protein interacts with both tegument proteins and gly-coproteins, suggesting that ORF9 protein is critical for facilitatingtegument assembly and secondary envelopment of VZV virions.ORF9 protein binds to IE62 protein, the major VZV transactiva-tor, indicating that ORF9 protein may recruit IE62 into the tegu-ment and thereby incorporate the other known tegument proteinsthat bind to IE62 protein, including IE4, IE63, and ORF47 proteinkinase (41–43). Moreover, the orthologues of HSV-1 and PRVUL49 proteins have been found to interact with glycoproteins(44–47). However, these studies did not determine whether UL49protein was involved in recruiting viral proteins for assembly.ORF9 protein binds to gE, a major VZV envelope glycoprotein,suggesting that ORF9 protein is present in the outer tegumentlayer. ORF9 protein is an essential protein, which limits the gen-eration of ORF9 mutants (21). ORF9 protein appears to be highlymodified during infection, based on detection of multiple formsof ORF9 protein with anti-ORF9 antibody. Whether all or onlysome of these various-molecular-weight species of ORF9 proteinbind to ORF11 protein was not determined.

Ultrastructural analysis showed that the interaction with ORF9protein is not absolutely required for bringing ORF11 protein tothe site of secondary assembly in cultured cells in vitro, but incor-poration of ORF11 into the virion tegument is markedly dimin-ished and reduced ORF11 protein in virions is likely associatedwith an impaired production of enveloped VZV virions. Nochanges in the morphology of the mature virions could be de-

tected, suggesting that other tegument or cellular proteins com-pensated for limited ORF11 protein in the tegument structure, ashas been reported for HSV-1 and PRV (14, 48). Importantly, thecapacity of ORF11 protein to bind to ORF9 protein appears to becritical for VZV infection of skin xenografts, and any mechanismsthat may have compensated for the disruption of the ORF11-ORF9 protein interaction in vitro are not sufficient for replicationin the human skin tissue microenvironment in vivo. This require-ment for ORF11 protein was not detected in skin organ culturesinfected in vitro (16) but was present for VZV skin pathogenesis invivo under conditions where the viruses must overcome intrinsicantiviral responses. Since it is not yet known why ORF9 protein isan essential VZV protein, it is possible that ORF11 protein mightaffect its recruitment as a structural component, resulting in im-paired skin infection. That the ORF11-ORF9 protein interactionis needed helps to account for the severe attenuation of the ORF11deletion mutant in vivo. As a major tegument component, ORF11protein may be involved in recruitment of other viral proteins forvirion assembly. The deleted ORF11 protein region appears tohave functions that contribute to the phenotype caused by ORF11protein C-terminal truncation.

In summary, we have shown that the ORF11 and ORF9 pro-teins form a complex in VZV infection, which highly depends onthe presence of amino acid residues 767 and 780 in the ORF11C-terminal domain. The loss of the ORF11-ORF9 protein inter-action markedly affected the deposition of ORF11 protein in thetrans-Golgi region, the site for secondary assembly. While ORF11protein may have other functions, its inclusion in the virion teg-ument at normal levels appears to be needed for skin infection in

FIG 8 Ultrastructural analysis of HELF infected with pOka, pOka11�549, and pOka�11. Cells infected with pOka, pOka11�549, and pOka�11 were labeledwith antibodies against ORF9 (anti-ORF9) and ORF11 (Ab11) gene products or preimmune serum corresponding to the anti-ORF9 protein antibody or Ab11,followed by inoculation with gold-tagged secondary antibody, and evaluated by cryo-immuno-EM. (A and B) ORF9 protein cytoplasmic distribution in cellsinfected with pOka (A) and pOka11�549 (B). (C) Overview of ORF11 protein distribution in a pOka-infected cell. A representative extracellular VZV particlelabeled with ORF11 was detected in pOka-infected cells and is shown in upper right corner. (D and E) Higher magnification to show the ORF11 cytoplasmicdistribution in pOka-infected cells. (F and G) ORF11�549 cytoplasmic distribution in pOka11�549-infected cells. A representative extracellular VZV particlelabeled with ORF11 protein was detected in pOka11�549-infected cells and is presented in upper right corner of panel F. (H) ORF9 cytoplasmic distribution inpOka�11-infected cells. (I) Cryo-immuno-EM detection with Ab11 antibody in pOka�11-infected cells. (J) Cryo-immuno-EM detection with preimmuneserum to anti-ORF9 protein antibody in pOka-infected cells. (K) Cryo-immuno-EM detection with preimmune serum to Ab11 in pOka-infected cells. Scale bars,0.2 �m (A, B, C, F, and G) and 0.1 �m (D, E, H, I, J, and K). Nuc, nucleus; Cyt, cytoplasm.

FIG 9 Replication of pOka, pOka�11, pOka11�254, and pOka11�549 in human skin xenografts in vivo. Titers of pOka, pOka�11, pOka11�254, andpOka11�549 in human skin xenografts at 10 and 20 days after inoculation were determined by infectious focus assay; six xenografts were tested for each virus ateach time point. “X” indicates that no virus was recovered from skin xenografts. Each bar represents the mean viral titer (log10), and error bars represent standarddeviations. Inoculum titers of the viruses tested were not significantly different.

Importance of VZV Tegument Proteins ORF9 and ORF11

May 2013 Volume 87 Number 9 jvi.asm.org 5115

on June 13, 2014 by guesthttp://jvi.asm

.org/D

ownloaded from

vivo. The proteins encoded by the conserved alphaherpesvirusgenes in the cluster corresponding to the VZV ORF9 to ORF12genes have some virus-specific differences in their individual con-tributions to tegument assembly. However, these observationsabout VZV ORF11 and ORF9 proteins and the reports of func-tions of related proteins in other alphaherpesviruses suggest thatthey play overlapping roles in tegument assembly. Finally, inter-actions of tegument proteins may not be required in vitro but maybe critical for replication in differentiated host tissues, as shownfor VZV infection of skin in vivo.

ACKNOWLEDGMENTS

We thank Leigh Zerboni, Michelle Lai, and Phillip Sung for their help withthe animal experiments and Kevin Mo for his help with the viral replica-tion assay.

This work was supported by NIH grant AI053846 to the Arvin labo-ratory and a grant from the Bayerisches Staatsministerium für Wissen-schaft, Forschung und Kunst (BayGene) to the Haas laboratory.

REFERENCES1. Cohen I, Straus SE, Arvin AM. 2007. Herpes simplex viruses, p 2773–

2818. In Knipe DM, Howley PM, Griffin DE, Lamb RA, Martin MA, Roiz-man B, Straus SE (ed), Fields virology, 5th ed. Lippincott Williams &Wilkins, Philadelphia, PA.

2. Kinchington PR, Cohen JI. 2000. Viral proteins, p 74 –104. In Arvin AM,Gershon AA (ed), Varicella zoster virus virology and clinical management.Cambridge University Press, Cambridge, United Kingdom.

3. Mettenleiter TC, Klupp BG, Granzow H. 2009. Herpesvirus assembly: anupdate. Virus Res. 143:222–234.

4. Roizman B, Knipe DM, Whitley RJ. 2007. Herpes simplex viruses, p2501–2601. In Knipe DM, Howley PM, Griffin DE, Lamb RA, Martin MA,Roizman B, Straus SE (ed), Fields virology, 5th ed. Lippincott Williams &Wilkins, Philadelphia, PA.

5. Kelly JB, Fraefel C, Cunningham AL, Diefenbach RJ. 2009. Functionalroles of the tegument proteins of herpes simplex virus type 1. Virus Res.145:173–265.

6. Besser J, Sommer MH, Zerboni L, Bagowski CP, Ito H, Ku CC, ArvinAM. 2003. Differentiation of varicella-zoster virus ORF47 protein kinaseand IE62 protein binding domains and their contributions to replicationin human skin xenografts in the SCID-hu mouse. J. Virol. 77:5964 –5974.

7. Che X, Reichelt M, Sommer MH, Rajamani J, Zerboni L, Arvin AM.2008. Functions of the ORF9-to-ORF12 gene cluster in varicella-zostervirus replication and in the pathogenesis of skin infection. J. Virol. 82:5825–5834.

8. Kinchington PR, Bookey D, Turse SE. 1995. The transcriptional regu-latory proteins encoded by varicella-zoster virus open reading frames(ORFs) 4 and 63, but not ORF 61, are associated with purified virus par-ticles. J. Virol. 69:4274 – 4282.

9. Kinchington PR, Hougland JK, Arvin AM, Ruyechan WT, Hay J. 1992.The varicella-zoster virus immediate-early protein IE62 is a major com-ponent of virus particles. J. Virol. 66:359 –366.

10. Spengler M, Niesen N, Grose C, Ruyechan WT, Hay J. 2001. Interac-tions among structural proteins of varicella zoster virus. Arch. Virol.Suppl. 17:71–79.

11. Roizman B, Campadelli-Fiume G. 2007. Alphaherpes viral genes andtheir functions, p 70 –92. In Arvin AM, Campadelli-Fiume C, Mocarski E,Moore PS, Roizman B, Whitley R, Yamanishi K (ed), Human herpesvi-ruses. Cambridge University Press, Cambridge, United Kingdom.

12. Dorange F, Tischer BK, Vautherot JF, Osterrieder N. 2002. Character-ization of Marek’s disease virus serotype 1 (MDV-1) deletion mutants thatlack UL46 to UL49 genes: MDV-1 UL49, encoding VP22, is indispensablefor virus growth. J. Virol. 76:1959 –1970.

13. Kopp M, Klupp BG, Granzow H, Fuchs W, Mettenleiter TC. 2002.Identification and characterization of the pseudorabies virus tegumentproteins UL46 and UL47: role for UL47 in virion morphogenesis in thecytoplasm. J. Virol. 76:8820 – 8833.

14. Zhang Y, McKnight JL. 1993. Herpes simplex virus type 1 UL46 and UL47deletion mutants lack VP11 and VP12 or VP13 and VP14, respectively,and exhibit altered viral thymidine kinase expression. J. Virol. 67:1482–1492.

15. Zhang Y, Sirko DA, McKnight JL. 1991. Role of herpes simplex virus type1 UL46 and UL47 in alpha TIF-mediated transcriptional induction: char-acterization of three viral deletion mutants. J. Virol. 65:829 – 841.

16. Zhang Z, Selariu A, Warden C, Huang G, Huang Y, Zaccheus O, ChengT, Xia N, Zhu H. 2010. Genome-wide mutagenesis reveals that ORF7 is anovel VZV skin tropic factor. PLoS Pathog. 6:e1000971. doi:10.1371/journal.ppat.1000971.

17. Cilloniz C, Jackson W, Grose C, Czechowski D, Hay J, Ruyechan WT.2007. The varicella-zoster virus (VZV) ORF9 protein interacts with theIE62 major VZV transactivator. J. Virol. 81:761–774.

18. Donnelly M, Elliott G. 2001. Nuclear localization and shuttling of herpessimplex virus tegument protein VP13/14. J. Virol. 75:2566 –2574.

19. Elliott G, Hafezi W, Whiteley A, Bernard E. 2005. Deletion of the herpessimplex virus VP22-encoding gene (UL49) alters the expression, localiza-tion, and virion incorporation of ICP0. J. Virol. 79:9735–9745.

20. Pomeranz LE, Blaho JA. 2000. Assembly of infectious herpes simplexvirus type 1virions in the absence of full-length VP22. J. Virol. 74:10041–10054.

21. Tischer B, Kaufer B, Sommer M, Wussow F, Arvin AM, Osterrieder N.2007. A self-excisable infectious bacterial artificial chromosome clone ofvaricella-zoster virus allows analysis of the essential tegument protein en-coded by ORF9. J. Virol. 81:13200 –13208.

22. Che X, Oliver SL, Sommer MH, Rajamani J, Reichelt M, Arvin AM.2011. Identification and functional characterizations of varicella-zostervirus ORF11 gene product. Virology 412:156 –166.

23. Niizuma T, Zerboni L, Sommer MH, Ito H, Hinchliffe S, Arvin AM.2003. Construction of varicella-zoster virus recombinants from parentOka cosmids and demonstration that ORF65 protein is dispensable forinfection of human skin and T cells in the SCID-hu mouse model. J. Virol.77:6062– 6065.

24. Moffat JF, Stein MD, Kaneshima H, Arvin AM. 1995. Tropism ofvaricella-zoster virus for human CD4� and CD8� T lymphocytes andepidermal cells in SCID-hu mice. J. Virol. 69:5236 –5242.

25. Moffat JF, Zerboni L, Kinchington PR, Grose C, Kaneshima H, ArvinAM. 1998. Attenuation of the vaccine Oka strain of varicella-zoster virusand role of glycoprotein C in alphaherpesvirus virulence demonstrated inthe SCID-hu mouse. J. Virol. 72:965–974.

26. Loret S, Guay G, Lippe R. 2008. Comprehensive characterization ofextracellular herpes simplex virus type 1 virions. J. Virol. 82:8605– 8618.

27. Fuchs W, Klupp BG, Granzow H, Hengartner C, Brack A, Mundt A,Enquist LW, Mettenleiter TC. 2002. Physical interaction between enve-lope glycoproteins E and M of pseudorabies virus and the major tegumentprotein UL49. J. Virol. 76:8208 – 8217.

28. Fuchs W, Granzow H, Mettenleiter TC. 2003. A pseudorabies virusrecombinant simultaneously lacking the major tegument proteins en-coded by the UL46, UL47, UL48, and UL49 genes is viable in cultured cells.J. Virol. 77:12891–12900.

29. Fuchs W, Granzow H, Klupp BG, Kopp M, Mettenleiter TC. 2002. TheUL48 tegument protein of pseudorabies virus is critical for intracytoplas-mic assembly of infectious virions. J. Virol. 76:6729 – 6742.

30. Cohrs RJ, Hurley MP, Gilden DH. 2003. Array analysis of viral genetranscription during lytic infection of cells in tissue culture with varicella-zoster virus. J. Virol. 77:11718 –11732.

31. Mettenleiter TC. 2006. Intriguing interplay between viral proteins duringherpesvirus assembly or: the herpesvirus assembly puzzle. Vet. Microbiol.113:163–169.

32. Elliott G, Mouzakitis G, O’Hare P. 1995. VP16 interacts via its activationdomain with VP22, a tegument protein of herpes simplex virus, and isrelocated to a novel macromolecular assembly in coexpressing cells. J.Virol. 69:7932–7941.

33. Hafezi W, Bernard E, Cook R, Elliott G. 2005. Herpes simplex virustegument protein VP22 contains an internal VP16 interaction domain anda C-terminal domain that are both required for VP22 assembly into thevirus particle. J. Virol. 79:13082–13093.

34. Kato K, Daikoku T, Goshima F, Kume H, Yamaki K, Nishiyama Y.2000. Synthesis, subcellular localization and VP16 interaction of the her-pes simplex virus type 2 UL46 gene product. Arch. Virol. 145:2149 –2162.

35. Lee J, Vittone V, Diefenbach E, Cunningham A, Diefenbach R. 2008.Identification of structural protein-protein interactions of herpes simplexvirus type 1. Virology 378:347–354.

36. Gross ST, Harley CA, Wilson DW. 2003. The cytoplasmic tail of herpessimplex virus glycoprotein H binds to the tegument protein VP16 in vitroand in vivo. Virology 317:1–12.

Che et al.

5116 jvi.asm.org Journal of Virology

on June 13, 2014 by guesthttp://jvi.asm

.org/D

ownloaded from

37. Kamen D, Gross S, Girvin M, Wilson D. 2005. Structural basis for thephysiological temperature dependence of the association of VP16 with thecytoplasmic tail of herpes simplex virus glycoprotein H. J. Virol. 79:6134 –6141.

38. Naldinho-Souto R, Browne H, Minson T. 2006. Herpes simplex virustegument protein VP16 is a component of primary enveloped virions. J.Virol. 80:2582–2584.

39. Zhu Q, Courtney RJ. 1994. Chemical cross-linking of virion envelope andtegument proteins of herpes simplex virus type 1. Virology 204:590 –599.

40. Che X, Zerboni L, Sommer MH, Arvin AM. 2006. Varicella-zoster virusopen reading frame 10 is a virulence determinant in skin cells but not in Tcells in vivo. J. Virol. 80:3238 –3248.

41. Baiker A, Bagowski C, Ito H, Sommer M, Zerboni L, Fabel K, Hay J,Ruyechan W, Arvin AM. 2004. The immediate-early 63 protein of vari-cella-zoster virus: analysis of functional domains required for replicationin vitro and for T-cell and skin tropism in the SCIDhu model in vivo. J.Virol. 78:1181–1194.

42. Lynch JM, Kenyon TK, Grose C, Hay J, Ruyechan WT. 2002. Physicaland functional interaction between the varicella zoster virus IE63 and IE62proteins. Virology 302:71– 82.

43. Spengler ML, Ruyechan WT, Hay J. 2000. Physical interaction between

two varicella zoster virus gene regulatory proteins, IE4 and IE62. Virology272:375–381.

44. Chi J, Harley C, Mukhopadhyay A, Wilson D. 2005. The cytoplasmic tailof herpes simplex virus envelope glycoprotein D binds to the tegumentprotein VP22 and to capsids. J. Gen. Virol. 86:253–261.

45. Farnsworth A, Wisner TW, Johnson DC. 2007. Cytoplasmic residues ofherpes simplex virus glycoprotein gE required for secondary envelopmentand binding of tegument proteins VP22 and UL11 to gE and gD. J. Virol.81:319 –331.

46. O’Regan KJ, Bucks MA, Murphy MA, Wills JW, Courtney RJ. 2007. Aconserved region of the herpes simplex virus type 1 tegument proteinVP22 facilitates interaction with the cytoplasmic tail of glycoprotein E(gE). Virology 358:192–200.

47. Stylianou J, Maringer K, Cook R, Bernard E, Elliott G. 2009. Virionincorporation of the herpes simplex virus type 1 tegument protein VP22occurs via glycoprotein E-specific recruitment to the late secretory path-way. J. Virol. 83:5204 –5218.

48. Michael K, Klupp BG, Mettenleiter TC, Karger A. 2006. Composition ofpseudorabies virus particles lacking tegument protein US3, UL47, orUL49 or envelope glycoprotein E. J. Virol. 80:1332–1339.

Importance of VZV Tegument Proteins ORF9 and ORF11

May 2013 Volume 87 Number 9 jvi.asm.org 5117

on June 13, 2014 by guesthttp://jvi.asm

.org/D

ownloaded from