JOURNAL OF VIROLOGY, Dec. 1996, p. 8402–8410
Copyright ? 1996, American Society for Microbiology
Vol. 70, No. 12
Accessory Human Cytomegalovirus Glycoprotein US9 in the Unique
Short Component of the Viral Genome Promotes Cell-to-Cell
Transmission of Virus in Polarized Epithelial Cells
EKATERINA MAIDJI,1SHAROF TUGIZOV,1THOMAS JONES,2ZHENWEI ZHENG,1AND LENORE PEREIRA1*
Department of Stomatology, School of Dentistry, University of California San Francisco, San Francisco, California 94143-0512,1
and Department of Molecular Biology, Wyeth-Ayerst Research, Pearl River, New York 109652
Received 17 June 1996/Accepted 12 August 1996
Human cytomegalovirus (CMV) encodes accessory glycoproteins that are dispensable for virus growth in
nonpolarized cells in culture. We report that CMV deletion mutants lacking the gene for accessory glycoprotein
US9 in the unique short component of the viral genome are impaired in plaque formation in polarized human
retinal pigment epithelial (ARPE-19) cells. Comparison of CMV deletion mutants in US9 with herpes simplex
virus type 1 deletion mutants lacking glycoproteins gE and gI showed that both of these mutants are impaired
in altering junctional complexes and increasing paracellular permeability in polarized ARPE-19 cells cultured
on permeable filter supports. Results of functional studies indicate that CMV US9 and homologs of gE have
analogous roles in promoting virus spread across lateral membranes of polarized epithelial cells.
Human cytomegalovirus (CMV) is a major cause of serious
congenital and perinatal infections in newborns, of disease in
immunosuppressed organ transplant recipients, and of gener-
alized infections in patients with AIDS, affecting the central
nervous system, lung, bowel, and other organs (1, 18). Human
CMV, varicella-zoster virus, and herpes simplex virus type 1
(HSV-1) are the major viral causes of retinitis in patients with
AIDS (27). Infection disseminates through all layers of the
neuronal retina and, if untreated, spreads to the retinal pig-
mented epithelium (RPE), composed of polarized cells de-
rived from the neuroectoderm (70). Most studies of herpesvi-
rus replication are done with nonpolarized cells, and little is
known about virus spread in polarized epithelial cells. CMV
productively infects primary human RPE cells, but replication
proceeds more slowly than in human foreskin fibroblasts
(HFF) (14, 47, 65). We have shown that CMV infects a polar-
ized RPE cell line, ARPE-19, grown on filter supports; the
virus enters cells predominantly from the apical membrane,
alters the paracellular permeability of the polarized mono-
layer, and spreads across lateral membranes (65). In contrast
to our previous findings in studies of nonpolarized HFF (51),
a panel of potent neutralizing monoclonal antibodies (MAbs)
to CMV gB, a key glycoprotein that functions in virion pene-
tration of cell membranes, fails to impede virus transmission
across lateral membranes of polarized ARPE-19 cells (65). A
distinguishing structural feature of polarized epithelial cells is
the tight junctions and undercoat of submembrane proteins
that are found in junctional complexes of lateral membranes
and are linked to the cortical actin cytoskeleton (26, 43). The
asymmetry in the protein composition of polarized cell mem-
branes (58) suggests that the initial process of virion attach-
ment and penetration across apical membranes differs from
transmission of progeny virions across lateral membranes.
CMV and HSV-1 are enveloped DNA viruses with genomes
consisting of two components, a short (US) and a long (UL)
unique sequence flanked by inverted repeats (38, 59). The US
components contain genes encoding glycoproteins that are dis-
pensable for replication in nondifferentiated cell cultures. How
these glycoproteins function is largely unknown, but it is
thought that they enhance virus transmission, increase patho-
genesis, and allow evasion of the immune response in vivo.
US6 through US11 are dispensable for CMV infection of HFF
but function in immune evasion (32–34, 36). US11 down-reg-
ulates the major histocompatibility complex class I molecules
from the cell surface, increases their turnover, and dislocates
class I molecules from the endoplasmic reticulum to the cyto-
plasm (31, 68, 69). HSV-1 glycoproteins in US include gI (US7)
and gE (US8) (41, 42). Homologs of these two glycoproteins
are found in the US components of varicella-zoster virus, pseu-
dorabies virus (PrV), and other neurotropic herpesviruses of
animals (28, 40, 67, 71). These glycoproteins form het-
erodimers, and one of their functions is to bind the immuno-
globulin G Fc fragment. Recent studies with HSV-1 deletion
mutants lacking gE and gI (gE?gI?mutants) showed that
these glycoproteins also play a key role in viral pathogenesis in
the central nervous system and the epithelium (4, 15, 16). PrV
gE?gI?mutants are avirulent in the rat visual system because
of an impairment in transneuronal transport from the retina to
certain visual centers in the brain (6, 7, 67).
Here we present evidence that CMV US9 and HSV-1 gE
have functional similarity and play a key role in the transmis-
sion of infection across lateral membranes of polarized epithe-
lial cells. Mutants with deletions of CMV US9 and HSV-1 gE
were severely impaired in cell-to-cell spread, failing to alter the
paracellular permeability of polarized cell monolayers and the
pattern of the actin cytoskeleton in ARPE-19 cells.
MATERIALS AND METHODS
Viruses and epithelial cells. Construction and properties of mutants with
deletions in the US components of CMV strain AD169 and HSV-1 strain F have
been published (33, 41, 42) and are summarized in Table 1. Derivation of human
RPE (ARPE-19) cells and propagation on permeable filter supports are reported
elsewhere (19, 65). Briefly, ARPE-19 cells were cultured on 12-mm-diameter
Transwell filters (Costar no. 3401, 0.45-?m pore size) coated with mouse laminin
(10 ?g/cm2) (Sigma) until they were fully polarized, approximately 6 to 8 weeks.
MDCK cells were grown on filter supports in Dulbecco’s modified Eagle’s me-
dium with 5% fetal bovine serum and were used 4 to 8 days after plating.
Transepithelial resistance of ARPE-19 and MDCK cells on permeable filter
* Corresponding author. Mailing address: Department of Stomatol-
ogy, School of Dentistry, University of California San Francisco, 513
Parnassus Ave., San Francisco, CA 94143-0512. Phone: (415) 476-
8248. Fax: (415) 476-4204. Electronic mail address: firstname.lastname@example.org
supports was monitored with an Epithelial Voltohmmeter (Millipore). The para-
cellular permeability of infected epithelial cells on filter supports was measured
with3H-inulin (0.25 ?Ci/ml; ICN) as described previously (65).
Immune reagents. The MAbs used to detect expression of CMV proteins in
foci of infected ARPE-19 cells were CH160-5 (IE72, UL122/123), CH16-1
(ICP36, UL44), CH12-1 (matrix, UL83), and CH19-1 (pp28, UL99) (17, 35, 48,
53). Immune reagents to cellular proteins were added to the apical or the basal
compartments of the filter. For ARPE-19 cells, these included rabbit antisera to
human ZO-1 as marker for tight junctions (ZO-1 amino acids 463 to 1109;
Zymed) and to chicken cadherin as a marker for adherens junctions (Sigma). For
MDCK cells, rat MAbs to mouse ZO-1 (Chemicon) and mouse E-cadherin
(Sigma) were used. Rabbit antiserum to ?-catenin was gift from Inka Nathke,
Stanford, Calif.; goat anti-mouse, -rat, and -rabbit antisera conjugated with
fluorescein isothiocyanate (FITC) or Texas red were from Jackson ImmunoRe-
search. Cellular actin was stained for 30 min with FITC-phalloidin. Cells were
incubated with primary antibody (1:100 to 1:200) for 1 h at 37?C, washed three
times with phosphate-buffered saline (PBS; pH 7.2), and then incubated with
appropriate secondary antibodies for 30 min. For double staining, cells were
incubated with two primary antibodies simultaneously followed by secondary
antibodies labeled with FITC and Texas red.
Plaque formation. Polarized ARPE-19 cells were infected with CMV (0.1 PFU
per cell) or deletion mutants (0.5 PFU per cell) or with HSV-1 (0.01 PFU per
cell) from the apical membrane. Polarized MDCK cells were infected with
HSV-1 (50 PFU per cell) from the basolateral membrane. Dulbecco’s modified
Eagle’s medium-F12 supplemented with 0.1% human gamma globulin (Sigma),
which contained neutralizing antibodies to human CMV and HSV-1, was applied
to the apical and basal compartments to prevent formation of secondary plaques.
Plaques were defined as five or more cells that showed a cytopathic effect
(HSV-1) or expressed late viral proteins (CMV).
Confocal microscopy. Cells on filters were fixed stepwise on ice with 3%
paraformaldehyde in 2% sucrose and 0.1% Triton X-100 (5 min), in 3% para-
formaldehyde in 2% sucrose (15 min), and then in 50 mM NH4Cl in PBS (10
min). Antibodies were added to the apical or the basal compartments of the
filter, and cells were incubated with FITC-labeled anti-mouse and Texas red-
labeled anti-rabbit conjugate at 37?C for 30 min. Filters were analyzed by using
a krypton-argon laser coupled with a Bio-Rad MRC600 confocal head.
Impaired cell-to-cell spread of CMV deletion mutants in
US9. We previously reported and confirmed that plaques
formed by CMV mutants with deletions in US6 through US11
were indistinguishable from plaques of CMV strain AD169 in
nonpolarized HFF (33, 54). To study the growth properties of
CMV deletion mutants in polarized epithelial cells, we prop-
agated strain AD169 and mutants lacking genes in US (Table
1) in ARPE-19 cells. We compared plaque formation by count-
ing the number of infected cells that reacted with MAbs to
CMV proteins, as demonstrated by immunofluorescence and
confocal microscopy. Simultaneous staining of the polarized
cells for ZO-1 protein allowed changes in the pattern of ZO-1
staining to be used as a cellular marker for infection. Plaques
formed in strain AD169-infected ARPE-19 cells contained an
average of 20 to 25 infected cells after 3 weeks (Fig. 1A). In
contrast, CMV deletion mutants RV61, RV35, and RV80 in-
fected single cells, or at most two or three cells, but did not
form plaques on polarized ARPE-19 cells after 3 weeks (Fig.
1C, E, and G). ZO-1 was disaggregated from tight junctions
within cells infected with CMV and the deletion mutants (Fig.
1B, D, F, and H). CMV mutants with deletions of other genes,
RV67 and RV69, formed plaques as large as those of strain
AD169 on ARPE-19 cells (data not shown). Results of these
experiments demonstrated that deletion mutants RV35, lack-
ing US6 through US11, RV80, lacking US8 and US9, and
RV61, lacking US9 alone, were impaired in spread across
lateral membranes of polarized epithelial cells (Table 1).
Impaired cell-cell spread of HSV-1 gE?mutants. The sim-
ilar positions of CMV US9, HSV-1 gE, and gE homologs of the
alphaherpesviruses in the US components of the respective
viral genomes (9, 13, 20, 39, 45, 55) suggested that US9 may
have a function analogous to that of gE in virus spread in
epithelial cells (4, 15) and neurons (21, 67). CMV US9 is
predicted to be a type I membrane-anchored glycoprotein, 247
amino acids (aa) in length, with a hydrophobic signal sequence
(24 aa), a transmembrane sequence (30 aa), two potential
N-glycosylation sites in the ectodomain (168 aa), and a casein
kinase site in the intracellular carboxyl terminus (25 aa) (9).
Comparison of the amino acid sequence of CMV US9 with
those of the alphaherpesvirus gE homologs showed 44% sim-
ilarity and 13% identity over 160 aa of the entire US9 se-
quence. However, the low degree of sequence similarity cou-
pled with the absence of three-dimensional structure for the
gE homologs did not permit further evaluation of the related-
ness of US9 to these glycoproteins.
Given the similar positions of CMV US9 and the gE ho-
mologs in the US components, we tested whether plaque for-
mation was impaired in two polarized cell types, ARPE-19
(Fig. 2A to F) and MDCK (Fig. 2G to L), infected with HSV-1
(F) mutants R7032, R7048, and others (Table 1). Changes in
the pattern of ZO-1 staining were used as a cellular marker for
infection. Plaques formed by strain F in ARPE-19 cells were
large, ranging from 80 to 150 infected cells at 24 h (Fig. 2A). In
contrast, plaques of mutant R7032 contained an average of 15
cells, and plaques of R7048 contained fewer than 5 cells, in
polarized ARPE-19 cells (Fig. 2C and E). Similarly, in polar-
ized MDCK cells, strain F produced plaques ranging from 25
to 30 cells (Fig. 2G), whereas mutants R7032 (Fig. 2I) and
R7048 (Fig. 2K) produced plaques that contained fewer than
15 and 5 cells, respectively. ZO-1 was disaggregated from tight
junctions within ARPE-19 (Fig. 2B, D, and F) and MDCK cells
(Fig. 2H, J, and L) infected with HSV-1 and the deletion
mutants. Unrelated HSV-1 deletion mutants R7037 and
R7016, which lack genes encoding gG and gM, respectively
(Table 1), formed plaques similar in size to those formed by
HSV-1(F) in both types of polarized cells (data not shown).
The results (summarized in Table 1) showed that HSV-1 mu-
tants R7032, lacking gE, and R7048, lacking gE and gI, were
impaired in spread across lateral membranes of both polarized
human ARPE-19 cells, which are a nonimmortalized, limited-
passage cell line, and immortalized polarized MDCK cells.
Mutants lacking US9 and gE are impaired in altering junc-
tional complexes. We reported that plaque formation by CMV
strain AD169 and HSV-1(F) in polarized ARPE-19 cells per-
meabilizes cellular tight junctions, reducing the transepithelial
resistance and increasing the paracellular permeability of the
cell monolayer (65). The failure of CMV and HSV-1 deletion
mutants to spread across lateral membranes of polarized epi-
TABLE 1. Impaired cell-cell spread in polarized epithelial cells of
mutants with deletions of genes mapping in the US components of
CMV and HSV-1 genomes
US8 and US9
US10 and US11
gE, gI (US7, US8)
aFrom references 3, 33, and 42.
bPolarized epithelial cells include ARPE-19 (CMV and HSV-1) and MDCK
(HSV-1) grown on permeabilized filter supports.
VOL. 70, 1996ROLE OF CMV US9 IN VIRUS TRANSMISSION 8403
thelial cells suggested that the mutants might be impaired in
permeabilizing cell-cell junctions. We therefore evaluated the
paracellular permeability of polarized epithelial cells infected
with CMV(AD169), HSV-1(F), or their respective deletion
mutants by measuring the apical-to-basolateral transfer of3H-
inulin. CMV(AD169)-infected ARPE-19 cells showed greater
paracellular permeability than cells infected with deletion mu-
tants RV35, RV80, and RV61 at 10 and 15 days postinfection
(Fig. 3A). In fact, the values obtained with mutant-infected
cells were similar to those of uninfected polarized ARPE-19
cells. Results with HSV-1(F)-infected ARPE-19 cells from 24
to 72 h were similar (Fig. 3B). The movement of3H-inulin
from apical to basolateral membranes was significantly in-
creased in strain F-infected cells by 24 h but not in cells in-
fected with deletion mutants R7032 and R7048, which showed
values slightly higher than those for uninfected cells. The
HSV-1 deletion mutants showed similar defects in infected
MDCK cells at 72 h (Fig. 3C). Together, these results showed
that deletion mutants in CMV US9 and HSV-1 gE are im-
paired in altering tight junctions and increasing the paracellu-
lar permeability of polarized epithelial cells grown on perme-
able supports, which confirms the finding that the mutants are
impaired in cell-to-cell spread.
Tight junctions and adherence junctions of epithelial cells
contain proteins that are key in the maintenance of cell polar-
ity (44, 49). The increased paracellular permeability of
ARPE-19 cells following infection with CMV and HSV-1 sug-
gested that proteins in the junctional complexes, ZO-1, B-
cadherin, and ?-catenin, as well as the actin cytoskeleton of
infected cells had been altered. Analysis of CMV(AD169)-
infected cells by immunofluorescence confocal microscopy
showed noticeable changes in the pattern of ZO-1, indicated
by a loss of staining along cell junctions (Fig. 4A and B).
?-Catenin (Fig. 4I and J) and B-cadherin (Fig. 4M and N)
accumulated within infected cells, as did actin (Fig. 4E and F),
giving strong, irregular staining patterns. Cells infected with
CMV mutant RV61 (US9?) showed similar changes in the
pattern of ?-catenin (Fig. 4K and L) and B-cadherin staining
(Fig. 4O and P), whereas the patterns of ZO-1 (Fig. 4C and D)
and actin (Fig. 4G and H) showed moderate changes. Com-
parison of the staining patterns of proteins in HSV-1(F)-in-
fected cells gave similar results (Fig. 5). Proteins in junctional
complexes, ZO-1 (Fig. 5A and B), ?-catenin (Fig. 5I and J),
and B-cadherin (Fig. 5M and N), and the actin cytoskeleton
(Fig. 5E and F) were altered in ARPE-19 cells. Likewise, cells
infected with HSV-1 mutant R7048 (gE?gI?) showed altered
patterns of ?-catenin (Fig. 5K and L) and B-cadherin (Fig. 5O
and P), whereas moderate changes were detected in the dis-
tribution of ZO-1 (Fig. 5C and D) and actin (Fig. 5G and H).
Results of these experiments indicate that proteins forming
cell-cell junctions and maintaining the polarity of epithelial
cells are altered following CMV and HSV-1 infection. In con-
trast, mutants with deletions of US9 and gE, which form small
plaques in ARPE-19 cells, are somewhat impaired in altering
ZO-1 and actin. Whether this defect is related to deletions of
US9 and gE glycoproteins or is a consequence of the small-
plaque phenotype of the mutants remains to be determined.
Transmission of CMV and HSV-1 across lateral membranes
of polarized epithelial cells confers a survival advantage. In
this study, we show that CMV US9 and HSV-1 gE, glycopro-
teins that are dispensable for virus growth in nonpolarized
cells, function to promote the cell-cell spread of progeny viri-
ons in polarized epithelial cells. Comparison of polarized cells
infected with wild-type viruses and cells infected with mutants
with deletions of US9 and gE indicates that both the paracel-
FIG. 1. Comparison of plaque sizes and ZO-1 changes by immunofluoresence staining of CMV-infected polarized ARPE-19 cells on permeable filters, using
confocal microscopy. Cells were infected with CMV(AD169) (0.1 PFU per cell) and deletion mutants RV35, RV61, and RV80 (0.5 PFU per cell) as indicated. At 3
weeks, infected cells were stained with both a pool of MAbs to CMV proteins (A, C, E, and G) and antisera to ZO-1 (B, D, F, and H).
8404MAIDJI ET AL.J. VIROL.
lular permeability of the polarized epithelial cell monolayer
and virus transmission across lateral membranes are signifi-
cantly increased in the presence of these glycoproteins. The
findings that both US9 and gE promote virus spread across
lateral membranes of polarized cells and have comparable
locations in the US components of the respective viral ge-
nomes indicate that US9 and gE have similar functions.
Little is known about the role of the essential and the ac-
cessory viral glycoproteins in herpesvirus infection of polarized
epithelial cells. In a recent study, we showed that in polarized
ARPE-19 cells, CMV infection proceeds predominantly from
the apical membrane, whereas HSV-1 infects both apical and
basolateral membranes with approximately equal efficiency
(65). The conserved gB homolog, which is required for infec-
tion of other cell types, also functions in fusion of the virion
envelope with the surface of polarized epithelial cells (for a
review, see reference 52). HSV-1 gC (UL44), which is expend-
able for growth in nonpolarized cells, functions in virion at-
tachment to asymmetrical receptors on polarized MDCK cells
(61). It was reported that in animals, HSV-1 gE is required for
spread from the site of inoculation, i.e., in the epithelium of the
ear pinna or cornea (4, 15). When the spread of HSV-1 gE?
gI?mutants was compared with that of wild-type virus in
cultures of nonpolarized BHK-21 cells and human epithelial
cells, a marked failure of the mutants to spread from cell to cell
was observed, as evidenced by reduced plaque size and virus
yield per infected cell (4). It was suggested that the gE-gI
complex functions in interactions with the plasma membrane,
as cells infected with a syncytial mutant in gB showed reduced
cell fusion but not reduced virion adsorption or penetration.
In this study, detailed examination of CMV and HSV-1
transmission in ARPE-19 and MDCK cells on permeable filter
supports indicates that US9 and gE are required for cell-cell
transfer of virus in polarized epithelial cells. Spread of progeny
virions across lateral membranes in the presence of virus-spe-
cific antibodies could confer a growth advantage in the infected
host. Whereas neutralizing MAbs to CMV and HSV-1 gB
block cell-cell spread of virus in nonpolarized HFF and Vero
cells (50, 51), MAbs to CMV gB block virus penetration of the
apical membrane of polarized ARPE-19 cells on filters but
have little or no effect on virus spread across lateral mem-
branes (65). With respect to HSV-1, although plaque size is
somewhat reduced in the presence of neutralizing MAbs in
polarized ARPE-19 cells, transfer of progeny virions from cell
to cell nevertheless occurs, which may result from the efficient
replication of HSV-1 in this human cell type (43a).
Studies are ongoing to elucidate the function of CMV US9
in the absence of other viral proteins (64a). Western blot
(immunoblot) analysis of MDCK cells stably expressing
epitope-tagged US9 showed that the protein was of the ex-
FIG. 2. Comparison of plaque sizes and ZO-1 changes by immunofluorescence staining of HSV-1-infected polarized ARPE-19 and MDCK cells on permeable
filters, using confocal microscopy. ARPE-19 cells were infected (0.01 PFU per cell) from the apical membrane with HSV-1(F) and deletion mutants R7032 (gE?) and
R7048 (gE?gI?) as indicated. Polarized MDCK cells were infected with HSV-1 (50 PFU per cell) from the basolateral membrane. The cell types used were ARPE-19
cells at 24 h postinfection (A to F) and MDCK cells at 48 h postinfection (G to L). Infected cells were stained with MAbs to HSV-1 ?4 protein (A, C, E, G, I, and
K) and with antisera to ZO-1 (B, D, F, H, J, and L).
VOL. 70, 1996ROLE OF CMV US9 IN VIRUS TRANSMISSION8405
pected molecular mass (30 to 36 kDa) and that its mobility
increased following endoglycosidase treatment, indicating that
it is glycosylated as predicted by the amino acid sequence (9).
Immunofluorescence analysis of MDCK cells expressing US9
in the absence of other viral glycoproteins showed that junc-
tional complexes were permeabilized and the pattern of the
actin cytoskeleton was altered. Together, the findings of these
studies suggest that retention of US9 in the CMV genome
confers a significant survival advantage in vivo insofar as viri-
ons that cross lateral membranes are sequestered from anti-
bodies and escape neutralization, whereas virions that are re-
leased from the lumenal and serosal surfaces come into contact
with antiviral antibodies and would be neutralized.
Transmission of human CMV and the alphaherpesviruses
alters cell-cell junctions. When our results are compared with
reports on the avirulence in animals of PrV mutants lacking gE
and gI homologs, striking parallels emerge for the role of US9
in invasion of polarized epithelial cells in vivo—a role that is
directly related to pathogenesis. PrV gE and gI are required
for virus spread from retinorecipient neurons to axon terminals
that synapse on these neurons in the central nervous system of
rats (6, 7, 21, 67). In support of these observations, PrV gE?
gI?mutants, which are used as live vaccines, are avirulent in
pigs (46, 57). The finding that deletion of either gE or gI
restricts PrV neurotropism demonstrates that the gE-gI het-
erodimer plays a central role in pathogenesis of the alphaher-
pesviruses in neurons (67). Observations with PrV relevant to
our studies are that virus spread in the retina occurs primarily
in columns of individual ganglion cells that are synaptically
linked, not as focal cell-cell spread (7), and that gE and gI are
required for a step in anterograde transneuronal infection,
which may involve transport or release of virions from infected
primary neurons in the retina (21). It is notable that neuronal
cells are highly polarized and that vectorial secretion to the
apical membrane of polarized epithelial cells and that to the
axon terminals of neurons are similar (58). One question
raised by our results is how defects in virion spread across
lateral membranes of polarized epithelial cells relate to failure
of the mutant alphaherpesviruses to spread into the central
nervous system. Recent reports indicate that, in addition to
microtubule-based motors, actin microfilaments play an impor-
tant role in transport of Golgi-derived vesicles in polarized
epithelial cells and neurons because microtubules rarely ex-
tend into the terminal web of actin filaments at the apical
membrane or presynaptic terminals (44). Sequential transport
on both microtubules and actin filaments delivers secretory
vesicles from Golgi to the apical membrane (24) or to axon
terminals (37). Interestingly, CMV infection in HFF causes a
rapid progressive disruption of the actin cytoskeleton, which
correlates with actin depolymerization and facilitates viral in-
fectivity (30). Our results showed that the cortical actin cy-
toskeleton was altered in infected cells and suggest that this
modifies proteins in junctional complexes as well. It is tempting
to speculate that the types of changes that US9 and the gE
homologs effect in the actin cytoskeleton of polarized epithe-
lial cells may have a counterpart in neurons that facilitates
transport of virion-containing secretory vesicles to membranes
and spread of virus from cell to cell.
Virulence factors increase pathogenesis in vivo. Our studies
provide evidence that CMV US9 plays a role similar to that of
gE in promoting cell-cell spread in epithelial cells and suggest
that these glycoproteins function as virulence factors. The vir-
ulence cassettes of certain bacteria encode proteins that in-
crease the pathogenesis of strains that colonize the intestinal
epithelium (60). Cholera, a severe diarrheal disease caused by
Vibrio cholerae, results from a potent enterotoxin, cholera
toxin, which stimulates adenylate cyclase activity in intestinal
epithelial cells. In addition, two accessory V. cholerae entero-
toxins, the zonula occludens toxin (Zot) and the accessory
cholera enterotoxin (Ace), were recently identified and map
adjacent to each other upstream of cholera toxin in the viru-
lence cassette (5, 22, 64). Zot increases the paracellular per-
meability of epithelial cells, and Ace increases fluid secretion
in the intestinal mucosa. Bacterial invasion of the intestinal
epithelium is increased by Zot, which modulates tight junctions
in vitro through a protein kinase C-dependent actin reorgani-
zation (22, 23). Conservation of these accessory proteins em-
FIG. 3. Change in paracellular permeability of polarized ARPE-19 and
MDCK cells on filters following infection with CMV(AD169), HSV-1(F), and
mutants with deletions of CMV US8 and US9 and of HSV-1 gE and gI (Table
1). Passage of3H-inulin from the apical to the basolateral medium was measured
in ARPE-19 cells infected from the apical membrane with CMV (0.1 PFU per
cell) and deletion mutants (0.5 PFU per cell) (A), or with HSV-1 and deletion
mutants (0.01 PFU per cell) (B), and in MDCK cells infected from the basolat-
eral membrane with HSV-1 and deletion mutants (50 PFU per cell) (C). The
paracellular permeability of infected epithelial cells on filter supports was mea-
sured with3H-inulin (0.25 ?Ci/ml; ICN) as described previously (65). At 24-h
intervals,3H-inulin was added to medium in the apical compartment, the baso-
lateral medium compartment was sampled after 9 min, and radioactivity was
counted. Values are means of three determinations.
8406MAIDJI ET AL.J. VIROL.
FIG. 4. Analysis of the patterns of cellular proteins and viral plaque size by immunofluorescence staining of CMV-infected ARPE-19 cells on permeable filter
supports, using confocal microscopy. Polarized ARPE-19 cells were infected from the apical membrane with CMV(AD169) (left) and mutant RV61 (US9?) (right).
At 3 weeks postinfection, cells were fixed and costained with antibodies to cellular and viral proteins. (A and C) ZO-1; (E and G) actin; (I and K) ?-catenin; and (M
and O) B-cadherin. (B, F, J, and N) AD169 plaques; (D, H, L, and P) RV61 plaques.
FIG. 5. Analysis of the patterns of cellular proteins and viral plaque size by immunofluorescence staining of HSV-1-infected ARPE-19 cells on permeable filter
supports, using confocal microscopy. Polarized ARPE-19 cells were infected from the apical membrane with HSV-1(F) (left) and mutant R7048 (gE?gI?) (right). At
24 h postinfection, cells were fixed and costained with antibodies to cellular and viral proteins. (A and C) ZO-1; (E and G) actin; (I and K) ?-catenin; (M and O)
B-cadherin. (B, F, J, and N) HSV-1(F) plaques; (D, H, L, and P) R7048 plaques.
phasizes their importance in pathogenesis (29), as evidenced
by attenuation of vaccine strains of V. cholerae lacking the toxin
genes in vivo (11, 66). Several groups recently reported that
intracellular bacterial and viral pathogens, including Listeria
monocytogenes, Shigella flexneri, and vaccinia virus, have devel-
oped mechanisms to exploit the actin cytoskeleton of host cells
by forming actin tails that confer motility and facilitate the
direct cell-cell spread of infection (8, 10, 12, 56, 63).
It has been proposed that accessory glycoproteins may have
been acquired as a gene cluster in a DNA fragment and di-
verged to fulfill functions relevant to survival of the herpesvi-
ruses in the environmental niches of their hosts, rather than
being necessary for virus replication (41, 42, 59). This hypoth-
esis is supported by the finding that deletion of large gene
blocks from the US components of HSV-1 and human CMV
fails to alter virus replication in cell culture (33, 41, 42). Re-
gions in the genomes of human CMV and the alphaherpesvi-
ruses that are flanked by repeats could be remnants of recom-
bination events. It was recently reported that the origin of lytic
(oriLyt) replication in UL components of HSV-1, varicella-
zoster virus, human CMV, and human herpesvirus 6 contains
an imperfect direct repeat, which suggests that oriLyt was ac-
quired by transposition (62). It is notable that genomes of the
lymphotropic viruses Epstein-Barr virus (2) and human her-
pesvirus 6 (25) and of a betaherpesvirus, murine CMV, all of
which lack US components, do not contain genes encoding
proteins similar to gE and gI, which supports the unique role of
these glycoproteins in dissemination of the herpesviruses in
polarized epithelial cells and neurons. Following transfer of
genes that confer a survival advantage, these genes would then
diverge with the respective herpesviruses. In addition to US11
(31), the US component of the CMV genome encodes other
glycoproteins, which permit immune evasion by downregula-
tion of class I molecules from the surface of infected cells
(30a). It is anticipated that detailed investigation of other gene
products mapping in the US component will identify novel
functions that enhance CMV invasion and pathogenesis in
We thank Bernard Roizman for HSV-1 deletion mutants and Patri-
cia Babbitt for assistance with amino acid sequence comparison of the
This study was supported by Public Health Service grants EY10138
and EY11223, and by a grant from the Universitywide AIDS Research
Program (R94-SF-051) to the University of California San Francisco.
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