The Human Cytomegalovirus-Specific UL1 Gene Encodes a Late-PhaseGlycoprotein Incorporated in the Virion Envelope

Medya Shikhagaie,a Eva Mercé-Maldonado,b Elena Isern,c Aura Muntasell,d M. Mar Alba,d,e,f Miguel López-Botet,a,d Hartmut Hengel,b

and Ana Anguloc

Immunology Unit, Pompeu Fabra University, Barcelona, Spaina; Institut für Virologie, Heinrich Heine Universität Düsseldorf, Düsseldorf, Germanyb; Institut d’InvestigacionsBiomèdiques August Pi i Sunyer, Barcelona, Spainc; IMIM (Hospital del Mar Research Institute), Barcelona, Spaind; Research Programme in Biomedical Informatics, PompeuFabra University, Barcelona, Spaine; and Catalan Institution for Research and Advanced Studies (ICREA), Barcelona, Spainf

We have investigated the previously uncharacterized human cytomegalovirus (HCMV) UL1 open reading frame (ORF), a mem-ber of the rapidly evolving HCMV RL11 family. UL1 is HCMV specific; the absence of UL1 in chimpanzee cytomegalovirus(CCMV) and sequence analysis studies suggest that UL1 may have originated by the duplication of an ancestor gene from theRL11-TRL cluster (TRL11, TRL12, and TRL13). Sequence similarity searches against human immunoglobulin (Ig)-containingproteins revealed that HCMV pUL1 shows significant similarity to the cellular carcinoembryonic antigen-related (CEA) proteinfamily N-terminal Ig domain, which is responsible for CEA ligand recognition. Northern blot analysis revealed that UL1 is tran-scribed during the late phase of the viral replication cycle in both fibroblast-adapted and endotheliotropic strains of HCMV. Wecharacterized the protein encoded by hemagglutinin (HA)-tagged UL1 in the AD169-derived HB5 background. UL1 is expressedas a 224-amino-acid type I transmembrane glycoprotein which becomes detectable at 48 h postinfection. In infected human fi-broblasts, pUL1 colocalized at the cytoplasmic site of virion assembly and secondary envelopment together with TGN-46, amarker for the trans-Golgi network, and viral structural proteins, including the envelope glycoprotein gB and the tegumentphosphoprotein pp28. Furthermore, analyses of highly purified AD169 UL1-HA epitope-tagged virions revealed that pUL1 is anovel constituent of the HCMV envelope. Importantly, the deletion of UL1 in HCMV TB40/E resulted in reduced growth in a celltype-specific manner, suggesting that pUL1 may be implicated in regulating HCMV cell tropism.

Human cytomegalovirus (CMV) (HCMV) belongs to the Her-pesviridae family and has the largest genome of any charac-

terized human virus, comprised of around 200 predicted openreading frames (ORFs). The 230-kb linear, double-stranded DNAgenome consists of long unique (UL) and short unique (US) se-quences, each flanked by the inverted repeats referred to as TRL/IRL and IRS/TRS, resulting in the overall genomic configurationTRL-UL-IRL-IRS-US-TRS (35). In spite of the high levels of se-quence variability, the gene content is relatively well conservedacross related herpesviruses, and all family members share a set ofcore genes involved in basic metabolic and structural functions(1). Genes that are specific to a given virus or group of viruses aretypically nonessential for replication in cell culture and are fre-quently involved in immune evasion. Certain immunoevasivegenes show significant similarity to genes found in host genomes,possibly dating from events of molecular piracy (27).

Sequence analyses of the HCMV genome showed that as manyas 70 putative glycoprotein-encoding ORFs are found in clinicalisolates (11) and that 57 are found in the AD169 strain (13).HCMV structural glycoproteins can be divided into two broadclasses, those conserved between members of the family Herpes-viridae (including gB, gH, gL, gM, and gN) and subgenus-specificglycoproteins without homology to other herpesviruses, com-prised of, among others, gpTRL10 (51), gpRL13 (53), gpUL132(52), UL74-encoded gO (29), UL4-encoded gp48 (12), US27 (24),and UL33 (34). Many of the HCMV-specific glycoproteins, incontrast to the conserved glycoproteins, are not essential for invitro replication in fibroblasts and presumably participate in celland tissue tropism or pathogenicity (17).

The CMV-specific glycoproteins gpRL13 and gpUL4 are mem-bers of the primate CMV RL11 gene family, located on the extrem-

ity of the CMV genome. This RL11 gene family is comprised of 11members conserved in both HCMV and chimpanzee CMV(CCMV) (RL11, RL12, RL13, UL4, UL5, UL6, UL7, UL8, UL9,UL10, and UL11), and HCMV-specific genes (UL1, RL5A, andRL6) presumably originated by gene duplication in the last 6 to 5million years, which separates both CMV species (13, 14). Themembers of the RL11 gene family were initially assembled due tothe presence of a defined motif in their sequence resembling cel-lular Thy-1 within a region conserved between other immuno-globulin (Ig) superfamily (IgSF) members (13). Subsequently, in amore detailed analysis, this shared core motif was further exam-ined in the context of the neighboring sequences, allowing thecharacterization of an RL11 domain (RL11D) (14). The RL11Dconsists of a region of variable length (65 to 82 amino acids) con-taining three characteristic conserved residues (a tryptophan andtwo cysteines) and several potential N-linked glycosylation sites.Regarding the distribution and functions of the RL11 familymembers, UL4-encoded gp48 and gpRL13 have been described tobe virion envelope glycoproteins (12, 53). UL10 and RL13 areso-called temperance genes, with cell type-specific virus growthinhibition functions (17, 53). TRL11 encodes an IgG-Fc bindingglycoprotein (3, 33). UL11 encodes a hypervariable protein ex-

Received 14 September 2011 Accepted 31 January 2012

Published ahead of print 15 February 2012

Address correspondence to Ana Angulo, aangulo@ub.edu.

Supplemental material for this article may be found at http://jvi.asm.org/.

Copyright © 2012, American Society for Microbiology. All Rights Reserved.

doi:10.1128/JVI.06291-11

0022-538X/12/$12.00 Journal of Virology p. 4091–4101 jvi.asm.org 4091

pressed on the cell surface of infected cells (26). UL7 is a glycopro-tein with structural homology to the signaling lymphocyte activa-tion molecule (SLAM) family receptor CD229, with the capacityto mediate adhesion to leukocytes and interfere with cytokine pro-duction (21). Of note, RL11 genes, dispensable for virus growth incell culture, are among the most variable HCMV genes (16, 36),and RL5A, RL13, and UL9 are members of a restricted set ofHCMV hypervariable genes (16).

Although there is no information on the expression and func-tion of pUL1, sequence similarity was found with members of thehuman carcinoembryonic antigen (CEA) family (27). The CEAfamily, a structural subgroup of the Ig superfamily, is composed of29 members located on the human chromosome 19q13.2 (32). Ingeneral, these molecules possess a leader peptide, one N-terminalIg variable (IgV)-like domain, and a variable number of two dif-ferent types of Ig constant (IgC)-like domains. Members of thisfamily are broadly divided into the CEA-related cell adhesionmolecule (CEACAM) and the pregnancy-specific glycoprotein(PSG) subgroups, involved in immune responses and reproduc-tion. CEACAMs primarily mediate intracellular adhesion throughhomophilic and/or heterophilic interactions. Interestingly, mem-bers of the CEA family have been reported to function as receptorsfor bacterial or viral pathogens (7, 32).

In the present study, we have approached the molecular char-acterization of HCMV-specific UL1. To this end, an HCMV(AD169-derived HB5 background) recombinant with hemagglu-tinin (HA) epitope-tagged UL1 and a mutant with a full UL1 de-letion in the endotheliotropic HCMV TB40/E strain were gener-ated. Our data reveal that the UL1 ORF is transcribed with latekinetics. pUL1 is glycosylated and localizes at the site of virusassembly and secondary envelopment in infected cells, formingpart of the envelope of HCMV virions. An HCMV mutant with atargeted deletion of UL1 exhibits a growth defect phenotype inretinal pigment epithelium cells but not in fibroblasts, indicatingthat this ORF encodes a cell type-specific tropism factor.

MATERIALS AND METHODSCell lines and viruses. The MRC-5 human fibroblast cell line(ATCC CCL-171, passages 5 to 15) and the U373-MG cell line derivedfrom glioblastomas (ECACC 89081403) were grown in Dulbecco’s mod-ified Eagle’s medium (DMEM) (Gibco, Spain) supplemented with 10%(vol/vol) heat-inactivated fetal bovine serum (FBS), penicillin (100U/ml), and streptomycin (10 �g/ml). The telomerase-immortalized hu-man retina pigment epithelial (RPE) cell line (generously provided byMartin Messerle, University of Hannover, Germany) was grown inDMEM-Ham’s F-12 (1:1) medium (Gibco) supplemented with 5% (vol/vol) FBS, penicillin (50 U/ml), streptomycin (50 �g/ml), 0.25% (vol/vol)sodium bicarbonate, and 2 mM glutamine. The following HCMV strainswere used: Towne (ATCC VR-977), Toledo (41), endotheliotropic strainTB40/E (49), TB40/E reconstituted from the respective bacterial artificialchromosome (BAC) clone TB40/E BAC4 (48), and AD169 reconstitutedfrom the respective BAC clone pHB5 (6). Infectious viral stocks werepropagated as follows: MRC-5 cells were infected at a low multiplicity ofinfection (MOI), and the supernatants of infected cells that exhibited100% cytopathic effects were cleared of cellular debris by centrifugation at6,000 � g for 10 min. The cell-free supernatant was centrifuged at20,000 � g for 3 h to pellet the virus particles. The amount of infectiousvirus was determined by counting MRC-5 cells positive for the HCMVimmediate-early (IE) protein IE1 using monoclonal antibody (MAb)B810R (Chemicon) against this protein. Briefly, MRC-5 cells were incu-bated for 2 h with graded volumes of virus-containing supernatants, theinoculums were washed away with a phosphate-buffered saline (PBS)

rinse after the adsorption period, and the cells were incubated withDMEM supplemented with 3% FBS. After 20 h, cells were fixed for 20 minat room temperature (RT) with 4% paraformaldehyde (PFA). Sampleswere blocked with PBS containing 4% bovine serum albumin (BSA), per-meabilized in 0.5% Triton X-100, and incubated with the IE1 MAb andAlexa 488-conjugated rabbit anti-mouse (Fab=)2 fragments. Nuclei werecounterstained with DAPI (4=-6=-diamidine-2-phenylindole; Sigma-Al-drich) for 5 min at room temperature. Slides were examined with a LeicaDM6000B fluorescence microscope. Images were analyzed with Leica flu-orescence workstation software (Leica FW4000).

Construction and generation of TB40/E �UL1 and AD169 UL1-HAviruses. HCMV BAC mutagenesis was carried out by homologous recom-bination between a linear DNA PCR fragment and endotheliotropicHCMV-TB40/E-derived BAC4 (TB40/E BAC4) or the laboratory-adapted AD169 BAC (pHB5) in Escherichia coli. Mutagenesis was per-formed as previously described by Wagner et al. (58). TB40/E BAC4 (48)was used for the generation of UL1 deletion viruses, removing the com-plete UL1 gene, and pHB5 was used to generate recombinant AD169 withthe UL1 HA epitope-tagged virus. To delete the UL1 sequence in TB40/EBAC4 and generate TB40/E BAC4 �UL1, the following oligonucleotideswere used: 5=-GTG GTT TTT ATA GGT TAA CCA CTT ATG GTG TAAAGT AGG ATA TTC ATA GTT ATT GAA AAC CGG CCA GTG AATTCG AGC TCG GTA CCC-3= and 5=-TGA GAG CTA CAC AAA GTAGCA CAA ATT TAT CCT GAA TAA ACT AGC GTC GAT ACT TTGTAA GAC CAT GAT TAC GCC AAG CTC CC-3=. For the construction ofthe UL1 HA-tagged virus in pHB5, a forward primer containing the HAtag epitope sequence followed by a stop codon (5=-CGT TCC GGC AACTCT GAG ACA CAA ACT ACG AAC TAG AAA CAA CGT AAA TCGCAT AGC GGC CTA CCC ATA CGA TGT TCC AGA TTA CGC TTAACC AGT GAA TTC GAG CTC GGT AC-3=) and a reverse primer con-taining the homology region downstream of UL1 (without the stopcodon) (5=-GAG AAC CGC ACG GAG TAA TCA AAT TTT ATC TTGGAT AAA TTA GTG TCG ATA CTT TGT AAG ACC ATG ATT ACGCCA AGC TCC-3=) were used. PCR fragments using plasmid pSLFRTKnas a template containing a kanamycin resistance (Kanr) gene (flanked byminimal FLP recombination target [FRT] sites) were inserted into TB40/EBAC4 or pHB5 by homologous recombination in E. coli (58). The TB40/EBAC4 �UL1 and pHB5 UL1-HA mutants were digested with XbaI, andthe pattern of the restriction enzyme was compared to that of wild-typeTB40/E BAC4 or wild-type pHB5. The presence of the Kanr gene wasfurther confirmed by probing after Southern blotting with a kanamycincassette-specific probe. The Kanr gene was excised from both viruses byFLP-mediated recombination (58), and correct mutagenesis was verifiedby restriction pattern analysis followed by Southern blotting and PCRanalysis. Recombinant HCMV was reconstituted from HCMV BAC mu-tants by the Superfect transfection method as described previously (6).Sequencing to confirm that the appropriate mutagenesis process tookplace and that TB40/E �UL1 and the parental virus had not acquiredadditional mutations in the UL128 gene locus was performed with DNAextracted from infected cells after the reconstitution of the viruses.

Purification of HCMV particles and analysis of virion protease sen-sitivity. HCMV particles were further purified by negative-positive glyc-erol-tartrate gradient centrifugation as previously described (54). The iso-lated tartrate gradient-purified AD169 virions were treated withproteinase K. Protease digestion was performed with 10 �g of proteinaseK (Roche) per ml for 20 min at 37°C. Some virion samples were solubi-lized with sodium dodecyl sulfate (SDS), added to a final concentration of0.5% (wt/vol). Virion preparations were subjected to SDS-PAGE and im-munoblot analysis.

UL1 RNA analysis. For Northern blot analysis, whole-cell RNA wasisolated from mock-infected or infected MRC-5 cells at 8, 24, 48, and 72 hpostinfection (p.i.) using QIAshredder columns and an RNeasy minikit(Qiagen, Hilden, Germany). For gel electrophoresis, 10 �g of each samplewas used, and the preparations were separated on 1% agarose gels con-taining 2% formaldehyde. Northern blot analysis was carried out accord-

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ing to standard procedures. Hybridization and detection were performedby using the digoxigenin (DIG) system (Roche) according to the manu-facturer’s instructions. Briefly, UL1-specific DNA hybridization probeswere labeled with digoxigenin-dUTPs using a DIG DNA labeling kit(Roche). Probes were prepared by PCR with UL1-specific primers (5=-GCG AAC TTT GTG GAT GGA ACG G-3= and 5=-ACG TTA TTT CTAGTT CGT AGT-3=), using pHB5 as a template. Late-phase gene expres-sion was inhibited by the use of phosphonoacetic acid (PAA; Sigma-Al-drich). A total of 250 �g/m1 of PAA was added to the medium at the timeof infection and maintained throughout infection.

Antibodies. Commercial MAbs specific for IE1 (B810R; Chemicon)and pp28 (ab6502; Abcam) were employed. gB-specific MAb 27-78 andMAb 58-15 (generously provided by William J. Britt, University of Ala-bama, Birmingham) were used for immunofluorescence microscopy andimmunoblotting, respectively. Reagents specific for calnexin (catalognumber 610524; BD Bioscience), GM-130 (catalog number 558712; BDBioscience), TGN-46 (catalog number AHP500G; AbDseroTec), and ER-GIC-53 (catalog number E6782; Sigma-Aldrich) were generously pro-vided by Julia V. Blume (Center for Genomic Regulation, Barcelona,Spain). Rabbit anti-HA (catalog number H6908; Sigma-Aldrich) anti-body was used to detect UL1-HA. Secondary antibodies used forimmunofluorescence microscopy were Alexa Fluor 555 goat anti-mouseIgG(H�L) (catalog number A21422; Invitrogen) and Alexa Fluor 488goat anti-rabbit IgG(H�L) (catalog number A11008; Invitrogen). Anti-mouse or anti-rabbit IgG horseradish peroxidase-labeled antibodies(Amersham Bioscience) were used for immunoblotting.

Immunofluorescence microscopy. MRC-5 cells grown to 90% con-fluence in 13-mm-diameter coverslips were mock infected or infectedwith AD169 UL1-HA for 72 h. Coverslips were rinsed with PBS, and cellswere then fixed for 20 min at RT in 4% paraformaldehyde freshly pre-pared in PBS. Cells were blocked with PBS containing 2% BSA for 20 minat RT and permeabilized with 0.5% Triton X-100 in PBS for 5 min. Cov-erslips were incubated for 1 h at RT with a primary antibody diluted inPBS containing 2% BSA, followed by 45 min of incubation with a fluoro-chrome-conjugated secondary antibody in PBS containing 2% BSA. Cov-erslips were mounted in Mowiol solution and analyzed under a confocallaser scanning microscope (Confocal SP2; Leica) at a magnification of�40.

SDS-PAGE and immunoblotting. Virus-infected cell proteins wereobtained from AD169 UL1-HA-infected MRC-5 cells grown in 6-welltissue culture dishes. Cell lysates of mock-infected MRC-5 cells were usedas controls. Cells were lysed under reducing conditions and heated to100°C. Proteins were separated by gradient SDS-polyacrylamide gel elec-trophoresis (10 to 15% SDS-PAGE) and transferred onto nitrocellulosemembranes that were blocked with PBS containing 0.05% Tween 20 and3% BSA (or 5% milk powder). Antibodies were diluted in PBS containing0.05% Tween 20. For the detection of primary antibody binding, horse-radish peroxidase-conjugated goat anti-rabbit or anti-mouse IgG fol-lowed by enhanced chemiluminescence detection (West Pico Supersignalkit; Pierce, Rockford, IL) was used according to the manufacturer’s in-structions. The removal of N-linked oligosaccharides from cell lysates wascarried out by using N-glycosidase F (PNGase F; Roche), according to themanufacturer’s specifications. Briefly, the samples were denatured in gly-coprotein-denaturing buffer at 100°C for 10 min and cooled to 4°C for 5min. The samples were then digested for 4 h at 37°C with PNGase F beforebeing analyzed by immunoblotting.

Viral growth kinetics. MRC-5, RPE, and U373-MG cells seeded into24-well plates were infected with TB40/E or TB40/E �UL1 at differentMOIs (0.025 for MRC-5 cells, 0.1 and 5 for RPE cells, and 0.3 forU373-MG cells). After a 2-h adsorption period, the inoculums were re-moved, and infected monolayers were rinsed with PBS and incubated withDMEM supplemented with 3% FBS. At various times postinfection, thesupernatants of the infected cells were harvested, cleared of cellular debris,and frozen at �70°C. In the case of U373-MG cells, the cell-associatedviral progeny was measured. The quantification of infectious virus was

determined by a fluorescence focus unit (FFU) assay, counting HCMVIE1-positive cells as indicated above. The number of infected cells (fluo-rescence focus) was determined by microscopy.

Sequence analysis and evolutionary studies. Primate CMV genes andproteins were retrieved from the NCBI database (40), and human proteinswere retrieved from the Ensembl Human database (28). NCBI accessionnumbers for the HCMV AD169 strain (13, 15) and chimpanzee cytomeg-alovirus (CCMV) are NC001347 and NC003521.1, respectively. HiddenMarkov models (HMMs) corresponding to members of the Ig domain-containing family, including the V-set (variable), C1-set (constant-1),C2-set (constant-2), and I-set (intermediate) subsets, were downloadedfrom PFAM (23). A total of 1,314 Ig domain-containing human andHCMV proteins were obtained by using the program HMMsearch (19)with an E value cutoff of 10�3. The BLASTP program was used to searchfor putative human homologues of RL11 proteins using an E value cutoffof 0.1 (2). Sequence alignments of homologous Ig domain-containingHCMV proteins and cellular proteins were generated with the help of theCLUSTALW program (56). The number of amino acid substitutions persite (K) was calculated for orthologous pairs of HCMV and CCMV pro-teins by using the PHYLIP program Protdist with default parameters (22).N-glycosylation sites, signal sequences, and transmembrane domainswere predicted by using NetNGlyc 1.0 (25), SignalP 3.0 (37), andTMHMM 2.0 (http://www.cbs.dtu.dk/services/), respectively.

RESULTSStructure of pUL1. The UL1 gene is predicted to encode a 224-amino-acid type I transmembrane glycoprotein, comprising a sig-nal peptide (amino acids 1 to 26), an Ig-like domain (amino acids45 to 152) with 8 potential N-linked glycosylation sites, a trans-membrane region (amino acids 184 to 206), and a short cytoplas-mic tail (amino acids 207 to 224) without apparent signalingmotifs (Fig. 1A). Since sequence similarity of pUL1 to pregnancy-specific glycoprotein 5 and other members of the human CEAfamily (27) was previously noted, we sought to explore this findingin more detail. We performed BLASTP searches of all RL11 genefamily members against all human Ig-containing proteins. A sig-nificant sequence similarity (E value of �10�3) was found be-tween the Ig-like domain in pUL1 and the N-terminal IgV-likedomain in the CEA family members (shown for CEACAM-1, -3,-5, and -6 in Fig. 1B). As an example, the pUL1 Ig-like domainexhibits 61% amino acid similarity and 28% amino acid identitywith the human N-terminal CEACAM-1 IgV domain (E value of0.0004) (Fig. 1B). For the rest of the HCMV RL11 members, sig-nificant similarity to CEA proteins was not detected. The align-ment of the pUL1 Ig-like domain and the three-dimensionalcrystal structure of the soluble ectodomain of the murineCEACAM-1a isoform (55) indicated that the formation of Ig-like�-sheets is conserved and that the conserved residues are mostlyburied in the hydrophobic core, maintaining the Ig fold.CEACAM positions that appear to be critical for the formation ofthe typical �-sheets in IgV-like domains are present in pUL1 (Fig.1C). Furthermore, multiple alignments of the Ig-like domain ofpUL1 and the N-terminal IgV domain of several CEA proteinsshowed that a number of well-conserved residues in members ofthe CEA family are also present in pUL1, whereas they are absentfrom other RL11 proteins (Fig. 1B and data not shown).

Origin and evolution of pUL1. The pULl sequence is foundexclusively in HCMV genomes but lacks a counterpart in otherCMV lineages and betaherpesviruses; therefore, the origin andevolution of the UL1 gene was investigated. The RL11 gene familyis a rapidly evolving and highly variable virus gene family, both interms of gene members and in terms of sequence divergence

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across related viruses or strains (16). To better understand theorigin and evolution of the RL11 family, we determined the num-ber of amino acid substitutions per site (K) between RL11 HCMV-CCMV orthologous proteins and between pULl and RL11 HCMVparalogous proteins (Table 1). The estimated number of aminoacid substitutions per site (K) between RL11 HCMV-CCMV or-thologous proteins is, on average, 1.5, about 15 times higher thanthe average amino acid substitution rate of a prototypical well-conserved herpesvirus gene (i.e., the UL54 gene, encoding DNApolymerase). Among paralogs, the lowest K values are for thealignments of pULl with pTRL11, pTRL12 and pTRL13 (1.9 in thethree cases). This, together with the fact that the UL1 gene is lo-cated just next to the TRL13 gene, suggests that pUL1 may haveoriginated by duplication from an ancestor gene from the TRLcluster.

UL1 is transcribed with late gene kinetics. In the HCMVAD169 genome, UL1 extends from nucleotide 11835 to 12509(675 bp). To analyze potential transcripts arising from the UL1ORF, total RNA was isolated from MRC-5 cells at different timesafter infection with HCMV AD169 and subjected to Northern blotanalysis using a UL1-specific probe. As shown in Fig. 2A, large UL1transcripts, of approximately 5.8 kb and 3.8 kb, could be detectedno earlier than 48 to 72 h p.i. (Fig. 2A). Interestingly, a similar

pattern of large transcripts was also observed previously for otherUL1-related genes, such as TRL10 (51) and TRL/IRL13 (61).

HCMV lytic gene expression is conventionally divided intothree major kinetic classes of viral genes, immediate-early, early,

FIG 1 pUL1 shares key conserved residues with CEA proteins. (A) Structural predictions for HCMV pUL1. A schematic drawing of the 224-amino-acid UL1protein is shown, with the signal peptide (SignalP) (amino acids 1 to 26), the Ig-like domain (amino acids 45 to 152), and the transmembrane region (TM) (aminoacids 184 to 206) indicated. (B) Multiple-sequence alignment showing the sequence similarity between the HCMV UL1 protein Ig-like domain (amino acids 45to 152) and the N-terminal IgV domains of four representative members of the CEA family, human CEACAM-1, -3, -5, and -6. Boldface type indicates highlyconserved residues in the RL11 family, and the potential N-glycosylated residue in the RL11 family is underlined (14). Highly conserved residues in CEACAM-1and other IgSF members that appear to be critical for the formation of the �-sheets in IgV-like domains are boxed (30). The arrows above the sequence alignmentindicate the �-sheet structure of CEACAM-1a as determined by a previous crystal structure analysis (56). An asterisk indicates identical amino acids, a colonindicates conserved amino acids, and a period indicates less conserved amino acids. (C) Alignment of the pUL1 RL11D and the three-dimensional crystalstructure of murine CEACAM-1a. Conserved residues are indicated on the �-sheets, boldface type indicates conserved buried residues, and italic type indicatesconserved surface residues.

TABLE 1 Amino acid substitutions per site in viral protein pairwisecomparisonsa

Orthologue ofHCMV-CCMV

K (no. of aasubstitutions/site)

Paralogue ofUL1-HCMVRL11

K (no. of aasubstitutions/site)

RL5a 3.5RL6 3.3

TRL11 0.7 TRL11 1.9TRL12 1.6 TRL12 1.9TRL13 1.2 TRL13 1.9UL4 2.5 UL4 2.8UL6 1.2 UL6 3.1UL7 1.3 UL7 2.3UL9 2.0 UL9 2.6UL10 2.3 UL10 2.0UL11 1.0 UL11 2.7UL54 0.1a aa, amino acid.

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and late. The transcription of UL1 RNA in AD169-infected cellswas completely blocked by the viral DNA polymerase inhibitorphosphonoacetic acid (PAA), consistent with the classification ofUL1 as a true late-phase gene, as previously described and repro-duced here for UL99 (Fig. 2B) (31). The presence of UL1 tran-scripts in MRC-5 cells infected with a panel of HCMV strains,including laboratory strains AD169 and Toledo and endothelio-tropic strain TB40/E, was studied. The Towne strain, previouslyreported to be UL1 defective due to a deletion in this ORF (47),was used as a negative control. Total RNAs from cells infected withHCMV TB40/E, AD169, Toledo, and Towne were isolated at 72 hp.i. and analyzed by Northern blotting using a UL1 (AD169)-specific probe. As shown in Fig. 2C, the two predominant 5.8-kband 3.8-kb UL1 transcripts were detected in cells infected byHCMV TB40/E, AD169, and Toledo (Fig. 2C). The signal inten-sities of these two RNA species were relatively lower in Toledo-infected cells, and as expected, UL1 transcripts were undetectableupon infection with Towne.

Expression of the UL1 glycoprotein during HCMV replica-tion. The 224-amino-acid-long UL1 polypeptide (molecular massof 25.5 kDa) is predicted to be expressed as a type I glycoproteinwith a signal peptide and a membrane anchor. We investigated theexpression kinetics of the protein product of UL1 during produc-tive HCMV infection. For this purpose, we generated an HCMV(AD169-derived HB5 background) recombinant virus with an

HA tag fused to the UL1 C-terminal end. Reconstituted AD169UL1-HA was used to infect MRC-5 cells, and the kinetics of pUL1expression were monitored by immunoblot analysis using an an-ti-HA antibody in cell extracts harvested at different time pointsranging from 4 to 72 h p.i. HCMV-specific MAbs that are reactivewith the major immediate protein IE1 pp72 (UL123) and the pp28(UL99) late phosphoprotein were used as controls. As shown inFig. 3A, the anti-HA antibody showed two protein species, a fast-er-migrating nonspecific band, which was present in bothHCMV-infected and mock-infected MRC-5 cells, and a band of�55 kDa, which was weakly detectable at 48 h p.i. but was abun-dantly found at 72 h p.i, coincident with the expression kinetics ofpp28 (31). This 55-kDa HA-tagged UL1 band was absent in im-munoblots of wild-type AD169-infected cells (data not shown),further confirming its specific detection. At time points after in-fection when signals for pUL1 were not observed, IE1 pp72 waspresent, as expected, already being expressed after 4 h p.i. andremaining for the duration of the HCMV replication cycle. Con-sistent with the observed kinetic pattern and the results obtainedat the transcriptional level, a metabolic blockade with PAA re-sulted in the disappearance of the 55-kDa band (Fig. 3B), indicat-ing that the UL1 protein is expressed late during HCMV replica-tion.

Many of the HCMV structural glycoproteins form high-mo-lecular-weight disulfide-linked oligomers, such as the gCI com-

FIG 2 Kinetics of UL1 gene expression. (A) Whole-cell RNA was isolated from uninfected (mock) MRC-5 cells or cells infected at an MOI of 2 with HCMVAD169 at the indicated hours postinfection. Equivalent amounts of RNA were subjected to agarose gel electrophoresis and subsequently analyzed by Northernblotting using an HCMV UL1-specific probe. (B) UL1 transcription is inhibited by PAA. Whole-cell RNA was isolated from HCMV AD169-infected cells at 72h p.i., incubated with or without PAA, and processed as described above for panel A. In addition to the UL1-specific probe, RNA was hybridized with a probespecific for the late HCMV gene UL99. (C) UL1 transcripts in MRC-5 cells infected with different HCMV strains (AD169, TB40/E, Toledo, and Towne) for 72h were analyzed as described above for panel A. The bottom panels show ethidium bromide staining of 28S rRNA (4.8-kb) and 18S rRNA (1.8-kb) species.

FIG 3 Expression of the UL1 glycoprotein in HCMV-infected fibroblasts. (A) Uninfected (mock) and AD169-infected MRC-5 cells at an MOI of 5 wereharvested at the indicated times p.i. Equivalent amounts of cell lysates were subjected to SDS-PAGE and analyzed by immunoblotting with antibodies reactivewith HA or the HCMV IE1 and pp28 proteins. �-Actin detection was used as a protein loading control. (B) Experiments were performed as described above forpanel A to assess the effect of PAA treatment (�PAA) on late-phase protein expression. (C) Whole-cell lysates of AD169 UL1-HA-infected MRC-5 cells (72 h p.i.)were left untreated or digested with PNGase F. The preparations were subjected to immunoblot analysis using an anti-HA antibody.

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plex, composed of multidimers of gB (9, 39); therefore, we ana-lyzed UL1 protein migration patterns under both nonreducingand reducing (with �-mercaptoethanol) conditions. The UL1protein was �-mercaptoethanol resistant, implying that it is notpart of a disulfide-linked oligomer in infected MRC-5 cells (datanot shown). The high number of putative N-linked glycan sites(n � 9) present in pUL1 and the difference between the apparentmolecular mass (55 kDa) observed for this protein by immuno-blotting and the calculated molecular mass (25.5 kDa) of the 224-amino-acid polypeptide backbone suggested that pUL1 under-goes an extensive posttranslational glycosylation process. Indeed,digestion with PNGase F, which removes complex and high-man-nose N-linked sugars, enhanced the migration of the specific bandfrom 55 kDa to 25 kDa (Fig. 3C). These results confirmed thatpUL1 is expressed as a glycoprotein.

Subcellular localization of pUL1 in HCMV-infected cells. Toanalyze the localization of pUL1 in HCMV-infected cells, the HAepitope of the AD169 UL1-HA virus was employed to trace itssubcellular distribution by confocal microscopy in comparisonwith a panel of molecular markers of the exocytic and endocyticpathways. In MRC-5 cells following fixation at 72 h p.i., pUL1-HAaccumulated in a large cytoplasmic compartment (Fig. 4). Thiscompartment was not detectable in uninfected and wild-typeAD169-infected MRC-5 cells, thus confirming the specificity ofthe observed staining pattern (see Fig. S1 in the supplementalmaterial). pUL1-containing structures failed to colocalize withseveral intracellular markers, including the endoplasmic reticu-lum (ER) marker calnexin (Fig. 4A) and ERGIC-53 (Fig. 4B), anintegral protein localized in the ER-Golgi-intermediate compart-ment (ERGIC). The staining of GM-130, a cis-Golgi marker, ap-peared in a dotted ring forming structures in the proximity ofpUL1 but did not exhibit a superimposing pattern indicative ofcolocalizing proteins (Fig. 4C). As observed previously by otherinvestigators, alterations in the morphologies of the Golgi com-partment and the ERGIC were seen during HCMV infection (45).During its final envelopment, HCMV acquires membranes con-taining endosomal and trans-Golgi network markers, while lyso-somes and cis- and medial-Golgi markers are not present andsurround the virus envelopment and assembly site (10). Markersfor the endocytic pathway, such as the early endosomal markerEEA-1, and recycling endosomes (transferrin receptor) did notcolocalize with pUL1 (data not shown). Furthermore, pUL1 colo-calization with the lysosomal protein LAMP-1 was also ruled out(data not shown). However, a partial colocalization of pUL1-con-taining structures and the trans-Golgi marker TGN-46 (Fig. 4D)was observed.

During late stages of the infectious cycle, pUL1 did not colo-calize with markers of the cis- or medial-Golgi compartment(ERGIC-53 and GM-130). To further confirm these results, in-fected cells were incubated with brefeldin A (BFA), and the distri-butions of UL1-HA-, GM-130-, and ERGIC-53-positive mem-branes were compared. BFA interferes with exocytic cellulartransport, and the treatment resulted in the vesiculation of thering-structured GM-130- and ERGIC-53-positive membranes,whereas such changes were not observed for the pUL1-HA-con-taining structure (data not shown). The envelopment of the infec-tious HCMV particles was proposed previously to take placewithin a juxtanuclear cytoplasmic site (44, 45), resembling thepUL1 distribution pattern. Altogether, these results suggest thatpUL1 in infected cells, associated with the trans-Golgi apparatus,

accumulates mainly in a juxtanuclear site argued to constitute avirus assembly compartment.

Colocalization of pUL1 with tegument and envelope HCMVproteins. Based on the cytoplasmic compartmentalization ofpUL1, we explored whether other structural HCMV proteinsgathered at the pUL1-containing site. To this end, antibodies re-active with the tegument phosphoprotein pp28 and the majorvirion envelope glycoprotein gB (UL55) were employed. HCMVpp28 was reported previously to colocalize with other tegumentand viral envelope proteins at the virus assembly site during thelate phase of the infectious cycle (45, 46), and it is also acquired bythe virion in the cytoplasm. Interestingly, pUL1 colocalized withpp28 (Fig. 5A) and gB (Fig. 5B), consistent with its presence at thesite of viral assembly and final envelopment, thus suggesting that itmight be integrated into the HCMV virion.

pUL1 is a component of the HCMV virion. To explorewhether pUL1 is a virion structural glycoprotein, we purifiedAD169 UL1-HA particles by negative-viscosity–positive-glycerol-tartrate gradient centrifugation and analyzed them by immuno-blotting with the anti-HA antibody. This purification processfacilitates the isolation of intact virions, separated from non-infectious enveloped particles (NIEPs) and dense bodies (DBs),and the removal of contaminating host cell debris. Purified wild-type AD169 virions were used as a negative control due to theabsence of the HA epitope. In these experiments, the 55-kDa HA-tagged UL1-specific band observed in lysates of AD169 UL1-HA-infected cells was used as a positive control, the detection of whichwas reproduced both in lysates and in AD169 UL1-HA virions, inparallel with the pp28 structural protein but not IE1 pp72, a non-structural protein present only in preparations of infected cells(Fig. 6A, lanes 1 and 3). As expected, the 55-kDa HA-tagged UL1band was not detected in wild-type AD169 virions, while the teg-ument phosphoprotein was (Fig. 6A, lane 4). In order to define thevirion incorporation of pUL1, additional experiments were per-formed. Previous studies have shown that extracellular virion en-velope glycoproteins are sensitive to protease digestion, while cap-sid and tegument proteins are protected (4, 60). Thus, we analyzedthe sensitivity of virion pUL1 to protease digestion. AD169UL1-HA virions were treated with detergent (SDS) to disrupt theenvelope and then digested with proteinase K (Fig. 6B). Whensamples were untreated, the tegument protein pp28 and the enve-lope glycoprotein gB were detectable (Fig. 6B, lane 1). Upon pro-teinase K digestion and in the absence of detergent, pp28 re-mained intact, whereas pUL1 was no longer detected, and thehighly abundant gB (oligomeric form) was largely digested (Fig.6B, lane 2). In samples treated with SDS only, both tegument andenvelope proteins remained intact (Fig. 6B, lane 3). In contrast,when initially solubilized and then digested with proteinase K,pUL1, gB, and pp28 were no longer detected (Fig. 6B, lane 4).Altogether, these findings provided evidence that pUL1, in a man-ner similar to that of gB, is sensitive to protease treatment andlikely exposed on the HCMV envelope.

Deletion of UL1 results in HCMV growth defects in epithelialcells. A number of the HCMV envelope glycoproteins have beenshown to play a role in critical steps of the viral life cycle, i.e., inprocesses such as cell entry, assembly, and envelopment of virionsor cell-to-cell spread. To assess the impact that the pUL1 envelopeglycoprotein may have on the HCMV replication cycle, we con-structed an HCMV mutant lacking the UL1 ORF using the TB40/E-derived BAC (48). Unlike the fibroblast-adapted AD169 strain,

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TB40 virions exhibit an intact tropism for endothelial as well asepithelial cells, the entry of which is mediated by the pentamericgH/gL/UL128/UL130/UL131 envelope complex, which is defi-cient in AD169 (43). The complete UL1 sequence was removedfrom the genome of endotheliotropic strain TB40/E, generatingthe recombinant virus TB40/E �UL1. We then infected humanMRC-5 cells, retinal pigment epithelial (RPE) cells, and U373-MGcells, derived from glioblastomas, at a low MOI with TB40/E�UL1 or parental strain TB40/E. An MOI of 0.025 was used toinfect the fully permissive fibroblasts, while RPE and U373-MG

cells (which support HCMV replication to a lesser extent) wereinfected at MOIs of 0.1 and 0.3, respectively. The production ofextracellular (MRC-5 and RPE cells) or cell-associated (U373-MGcells) virus was analyzed over the course of the replicative cycle. Asshown in Fig. 7B, in RPE cells, the growth levels of the UL1-defi-cient virus were significantly reduced (up to 100-fold) in compar-ison to those obtained with the parental virus (P � 0.05 at thedifferent time points). Consistent with this finding, TB40/E �UL1produced smaller plaques than those obtained after TB40/E infec-tion of this cell type (data not shown). In contrast, under these

FIG 4 Subcellular localization of pUL1-HA in a cytoplasmic compartment. MRC-5 cells grown on glass coverslips were infected with AD169 UL1-HA at an MOIof 5 and fixed at 72 h p.i. Uninfected MRC-5 cells were processed similarly (mock) (left panel of each row). The various cellular compartments in the secretorysystem were stained with the following antibodies: calnexin for the ER (A), ERGIC-53 for the ERGIC (B), GM-130 for the Golgi apparatus (C), and TGN-46 forthe trans-Golgi network (D). The cellular markers were detected with Alexa 488 goat anti-mouse IgG, and pUL1-HA was detected with Alexa 555 goat anti-rabbitIgG. Colocalization is indicated by a yellow signal in the merge channel.

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conditions of multistep growth, infections of MRC-5 andU373-MG cells demonstrated that TB40/E �UL1 and parentalstrain TB40/E grew with comparable kinetics (Fig. 7A and D). Toexamine whether the inefficient growth displayed by TB40/E�UL1 in RPE cells could also be observed when high viral dosesare used, RPE cells were infected with TB40/E �UL1 or parentalstrain TB40/E at an MOI of 5. As shown in Fig. 7C, under thesehigh-MOI conditions, the TB40/E �UL1 growth defect, althoughapparent, was substantially diminished (see the differences of be-tween 2- and 3-fold obtained during TB40/E and TB40/E �UL1infections at high MOIs versus the 1- to 2-log differences displayedat low MOIs). These results indicate that pUL1 is required forefficient HCMV growth in a cell type-specific manner.

DISCUSSION

Growing numbers of viral glycoproteins have been reported to becomponents of the HCMV envelope, including molecules con-served between members of the Herpesviridae family (gB, gH, gL,gM, and gN) and others that are HCMV specific (57) (e.g., gO[29], gpTRL10 [51], gpRL13 [53], UL4/gp48 [12], gpUL128 [59],gpUL130 [38], gpUL132 [52], US27 [24], and UL33 [34]). It isbelieved that genus-specific envelope glycoproteins contribute tothe broad cell/tissue tropism of HCMV and its pathogenicity. Inthis study, we have characterized the HCMV UL1-encoded glyco-protein, transcribed in fibroblast-adapted strains AD169 andToledo as well as in the endotheliotropic strain TB40/E. pUL1 is aPNGase F-sensitive glycoprotein, with a putative C-terminaltransmembrane anchor domain, which is synthesized late duringproductive HCMV infection. In infected human fibroblasts, pUL1is targeted to a cytoplasmic site together with several HCMV teg-ument and envelope proteins, consistent with its final destinationat the previously described distinct compartment of virion assem-bly and final envelopment (reviewed in reference 8). Conse-quently, pUL1 can be incorporated into purified AD169 virions

FIG 6 pUL1 is a structural glycoprotein that forms part of the HCMV enve-lope. (A) Analysis of pUL1 in cell lysates of AD169 UL1-HA-infected MRC-5cells for 72 h (lane 1) and AD169 UL1-HA virions (lane 3). Mock-infected cells(lane 2) and wild-type AD169 virions (lane 4) were used as negative controls.Western blots were developed by using anti-HA, anti-pp28, or anti-IE1 anti-bodies. (B) AD169 UL1-HA virions were treated with SDS (lanes 3 and 4) orleft untreated (lanes 1 and 2). One-half of each virus preparation was subse-quently digested with proteinase K (lanes 2 and 4) or left undigested (lanes 1and 3). The preparations were subjected to immunoblot analysis using anti-HA, anti-pp28, or anti-gB antibodies.

FIG 5 Colocalization with tegument and envelope HCMV proteins. MRC-5 cells grown on glass coverslips were infected with AD169 UL1-HA at an MOI of 5and fixed at 72 h p.i. Following fixation, samples were incubated with MAbs specific for the tegument protein pp28 and HA (A) or the envelope glycoprotein gBand HA (B). pp28 and gB were further detected with Alexa 488 goat anti-mouse IgG, and UL1-HA was further detected with Alexa 555 goat anti-rabbit IgG.Colocalization is indicated by a yellow signal in the merge channel.

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and becomes exposed in the HCMV envelope. Based on theseresults, we propose that pUL1 constitutes a novel HCMV struc-tural envelope glycoprotein.

UL1 belongs to the highly variable and rapidly evolving RL11gene family. The fact that UL1 is an HCMV-specific gene lacking acounterpart in other CMV lineages and betaherpesvirusesprompted us to surmise that UL1 may have arisen from a geneduplication event, which likely occurred after the speciation ofhuman and chimpanzee. Of note, two other HCMV RL11 familymembers, RL6 and RL5A, are not found in chimpanzee CMV or inthe more distantly related rhesus macaque CMV and are thuslikely to have been formed within the RL11 cluster as well in thelast 5 to 6 million years. Based on evolutionary estimates of theRL11 family in primate CMVs, we propose that UL1 may haveoriginated from a gene from the RL11-TRL cluster (TRL11,TRL12, or TRL13) and subsequently diverged at a high rate. This issupported by the K values found between UL1 and its closest ho-mologues (other genes in the TRL cluster), which are higher thanthose typically observed between orthologous HCMV and CCMVgenes. Indeed, a substantial heterogeneity of pUL1 among differ-ent HCMV strains has been appreciated (16, 47). Phylogeneticanalyses of UL1 in a variety of clinical HCMV isolates allowed thedifferentiation of three distinct genotypes, with the existence of aconsiderable number (4/31; 12.9%) of isolates containing im-paired versions of the UL1 sequence due to internal stop codon

mutations (47). In addition, the fibroblast-adapted Towne strain,which has lost virulence and experienced substantial genome al-terations during extensive in vitro passaging (41), bears a deletionin UL1 (47). The hypothesis that the UL1 polymorphisms existingin clinical isolates may have an effect on HCMV tissue tropismand pathogenesis remains to be substantiated in the future.

Both the transcriptional analysis of UL1 and the time course ofpUL1 synthesis in HCMV-infected MRC-5 cells proved exclusiveexpression in the late phase of the replication cycle. The absence ofUL1 transcription and translation after treatment with the potentviral DNA polymerase inhibitor PAA corroborated that UL1 be-longs to the late class of CMV genes. In infected MRC-5 cells,pUL1 does not seem to be organized in higher-order disulfide-linked complexes. We show that gpUL1 acquires a molecular massof 55 kDa. The glycoprotein is sensitive to PNGase F, producing a25-kDa polypeptide. Based on these results, one could concludethat most if not all of the 9 potential N-glycosylation sites thatpUL1 bears are modified by N-linked carbohydrates, with eight ofthem residing in the Ig-like domain and the remaining one resid-ing in the 32-amino-acid stretch that connects the Ig-like domainto the transmembrane sequence. It must also be noted that in thispUL1 region expanding from amino acids 153 to 183, several po-tential O-glycosylation sites are present. After entering the secre-tory pathway, the UL1 glycoprotein exits the ER to accumulate inclose vicinity to other components of the HCMV particle, such as

FIG 7 Growth kinetics of TB40/E �UL1 in different cell types. MRC-5 (A), RPE (B and C), and U373-MG (D) cells were infected with TB40/E or TB40/E �UL1at MOIs of 0.025 (A), 0.1 (B), 5 (C), and 0.3 (D). On the designated days p.i., cultures were collected, and yields of extracellular (MRC-5 and RPE cells) orcell-associated (U373-MG cells) infectious virus present were determined by FFU assays. Each data point represents the average and standard deviation of datafrom three separate cultures. The dashed line indicates the limit of detection. The data are representative of three independent experiments.

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the tegument protein pp28 and the envelope glycoprotein gB, atthe juxtanuclear cytoplasmic site of virion assembly. The HCMVenvelope is still incompletely defined (8). Based on previous massspectrometric studies (57), at least 19 structural glycoproteins ofAD169 virions could be identified. It is interesting to note thatpUL1, among several other bona fide virion glycoproteins (51,53), was not resolved by this approach. We find that after tartrategradient viral particle purification, pUL1 can be detected as a vi-rion constituent. Moreover, biochemical analyses of purified viri-ons revealed the same features of pUL1 as those demonstrated forgB, allowing the assignment of pUL1 to the virion envelope.

pUL1 shares a striking similarity to the N-terminal IgV domainof cellular CEA family molecules that are involved in cellular ad-hesion by homotypic and heterotypic interactions with eachother. The CEACAM subfamily includes inhibitory and activatingreceptors widely expressed in different cell types and tissues, andmembers of this family have been reported to be critical modula-tors of several physiological processes (32). Multiple alignmentsshowed that a number of well-conserved residues found in severalmembers of the CEA family are also present in pUL1. CEACAMpositions that appear to be critical for the formation of the typical�-sheets in Ig-like domains are conserved as well. This sequencesimilarity raises the possibility that pUL1 acts as a decoy receptormimicking host CEA proteins. Molecular mimicry is a strategyfrequently employed by viruses to exploit or subvert host proteinfunctions (20). As other members of the RL11 family, despite con-taining RL11-characteristic features and conserved residues of theIgSF in their Ig-like domains, do not exhibit a significant degree ofsimilarity to CEA proteins (E values below cutoff levels of 0.1 inour BLASTP searches), we hypothesize that pUL1 may haveevolved to mimic members of the CEA family by sequence con-vergence, gaining CEA-like features along the million years of vi-rus-host coevolution. The molecular targets and the biologicalsignificance of the sequence similarity between pUL1 and mem-bers of the CEA family remain to be explored. Notably, a numberof pathogens take advantage of the CEA extracellular domains tobind and infect their target cells; e.g., mouse CEACAM-1a is tar-geted by mouse hepatitis virus (18). Moreover, neisserial patho-gens have been shown to use CEACAM-1 in bronchial epithelialcells to suppress Toll-like receptor 2 (TLR-2) signaling (50).TLR-2 has been described to sense HCMV entry through a phys-ical interaction with gB and gH, which results in the activation ofNF-�B and cellular inflammatory cytokine secretion (5). Thus,pUL1 might contribute to HCMV access to host cells by interact-ing with members of the CEA family.

UL1 is dispensable for HCMV growth in cell culture (42). Ourresults indicate, however, that pUL1 might be of significant rele-vance for HCMV to establish efficient infections in certain celltypes. As a virion envelope glycoprotein, pUL1 has the potential tomodulate HCMV host cell tropism. We demonstrate that anHCMV TB40/E mutant lacking UL1 displayed attenuated growthin RPE cells, which was not observed for MRC-5 or U373-MGcells. This differential growth phenotype is MOI dependent, beingmeasurable although less apparent at high viral doses. Notably, aninvolvement in cell tropism was reported previously for othermembers of the RL11 glycoprotein family: a UL10 deletion mu-tant grew better than the parental virus in an epithelial cell line(17), and the RL13 envelope glycoprotein was shown to repressHCMV replication in certain cell types (53). The nature of theinefficient growth associated with the UL1 deficiency is at present

unknown. A possibility is that the UL1 glycoprotein may have arole in entry or egress when replicating in epithelial cells, as rep-resented by RPE cells. As indicated above, and in line with thehomology of the UL1 glycoprotein with members of theCEACAM family, an attractive hypothesis to test is whether thisviral protein could be interacting with a member of this family atthe host cell surface. In addition, the possibility that pUL1 mayrepresent a relevant target of humoral immune responses duringHCMV infection should be considered. Future studies will beaimed at clarifying the function(s) of pUL1 during HCMV repli-cation.

ACKNOWLEDGMENTS

This work was supported by the Spanish Ministry of Science and Innova-tion (SAF2010-22153 to M.L.-B. and SAF2008-00382 and SAF2011-25155 to A.A.), the Marie Curie Training Network, European Union(MRTN-CT-2005-019284), and the Carlos III Health Institute/EuropeanRegional Development Fund (ERDF) (ISCIII/FEDER; Red HERACLES).M.S. and E.M.-M. were supported by MRTN-CT-2005-019284.

We thank Albert Zimmermann and Vu Thuy Khanh Le for scientificadvice, William J. Britt and Julia V. Blume for providing antibodies, C.Cothia for revising the alignment between pUL1 and murine CEACAM-1a, and Xavier Sanjuan and Gemma Heredia for technical support.

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