JOURNAL OF VIROLOGY, July 2011, p. 7203–7215
Copyright © 2011, American Society for Microbiology. All Rights Reserved.
Vol. 85, No. 14
Herpes Simplex Virus 1 pUL34 Plays a Critical Role in Cell-to-Cell
Spread of Virus in Addition to Its Role in Virus Replication?
Alison C. Haugo,1Moriah L. Szpara,2Lance Parsons,3Lynn W. Enquist,2and Richard J. Roller1*
Department of Microbiology, University of Iowa, Iowa City, Iowa 522421; Department of Molecular Biology and
the Princeton Neuroscience Institute, Princeton University, Princeton, New Jersey 085442; and
Lewis-Sigler Institute for Integrative Genomics, Princeton University, Princeton, New Jersey 085443
Received 4 February 2011/Accepted 2 May 2011
Herpes simplex virus (HSV) pUL34 plays a critical role in virus replication by mediating egress of nucleo-
capsids from the infected cell nucleus. We have identified a mutation in pUL34 (Y68A) that produces a major
defect in virus replication and impaired nuclear egress but also profoundly inhibits cell-to-cell spread and
trafficking of gE. Virion release to the extracellular medium is not affected by the Y68A mutation, indicating
that the mutation specifically inhibits cell-to-cell spread. We isolated extragenic suppressors of the Y68A
plaque formation defect and mapped them by a combination of high-throughput Illumina sequencing and
PCR-based screening. We found that suppression is highly correlated with a nonsense mutation in the US9
gene, which plays a critical role in cell-to-cell spread of HSV-1 in neurons. The US9 mutation alone is not
sufficient to suppress the Y68A spread phenotype, indicating a likely role for multiple viral factors.
Dissemination of herpes simplex virus (HSV) during recur-
rent disease in the host is dependent upon efficient viral rep-
lication and on the ability of the virus to spread from cell to cell
in the face of the host innate and adaptive immune defenses.
Cytoplasmic envelopment of HSV-1 virions is followed by
vesicular transport of virions to the cell surface and secretion
by fusion of the vesicle membrane with the plasma membrane
(8, 23, 44). Transport of virions to cell membranes in contact
with the extracellular medium results in release of free virions.
Transport to surfaces apposed to other cells results in cell-to-
cell spread of virus infection. The mechanism by which virions
are sorted to junctional or basolateral surfaces in epithelial and
fibroblast cells is poorly characterized. About half of the virus-
encoded proteins play critical roles in virus replication, but
relatively few have been found to have specific functions in
cell-to-cell spread of virus. The essential components of the
virion entry apparatus, gB, gD, and gH/gL, are required for
cell-to-cell spread (7, 17, 30, 52). It is likely that this is because
cell-to-cell spread requires interaction of the virus entry pro-
teins with cellular receptors and subsequent fusion of the vi-
rion envelope with the plasma membrane of the naïve host cell.
A few additional viral proteins have been shown to be required
for efficient cell-to-cell spread at least in some cell types.
HSV-1 gE and gI form a heterodimeric complex that is re-
quired for efficient cell-to-cell spread in the nervous system in
vivo (10, 11, 21, 22, 36). The gE/gI complex is also required for
spread in cultured neuronal cells and in epithelial and fibro-
blast cells that form well-defined cell junctions (1, 10, 12, 36).
The gE spread phenotype in epithelial cells requires sequences
in the cytoplasmic tail of gE and also requires sorting of gE to
basolateral cell surfaces and adherens junctions, where it co-
localizes with ?-catenin (12, 16, 37, 64). Deletion of US9 in
pseudorabies virus (PRV) is associated with failure of viral
spread in neuronal cultures and in vivo (5, 33). In HSV-1, the
effect of US9 deletion on neuronal spread is less clear, and
the degree of inhibition of neuronal spread may depend on the
experimental system (36, 60). The function of US9 appears to
be tied in neurons to sorting of virus components from the
neuronal cell body into axons (5, 33, 60, 63).
The HSV-1 UL34 gene, along with its homologs in other
herpesviruses, is required for efficient viral replication in all
cultured cells tested, presumably because it is required for
efficient egress of capsids from the infected cell nucleus (15, 25,
41, 43, 51). The UL34 protein (pUL34) is targeted specifically
to the inner nuclear membrane (INM) by a mechanism that
requires its interaction with HSV pUL31 (48, 49), and this
dependence is a conserved feature of herpesvirus envelopment
(18, 26, 48, 49, 55, 56, 65). In addition to their localization at
the nuclear envelope in infected cells, pUL31 and pUL34 of
HSV and PRV have been shown to be structural components
of the perinuclear virion (18, 25, 49). The proteins are lost
from the egressing virion upon deenvelopment at the outer
nuclear membrane (ONM), and pUL34 and pUL31 and their
homologs are not detected in mature virions (15, 18, 19, 25,
49). Localization of these two proteins at the INM results in
the recruitment of other proteins, including protein kinase C
delta and pUS3, to the nuclear membrane to form a nuclear
envelopment complex (NEC) (41, 45, 54). Deletion of the HSV
UL34 gene causes failure to disrupt the nuclear lamina and
essentially complete failure of nuclear egress, with accumula-
tion of nucleocapsids in the infected cell nucleus (3, 27, 38, 39,
45, 47, 51, 57, 58). The concentration of pUL34 and pUL31 at
the nuclear membrane during infection suggests that the nu-
clear envelope (NE) is likely to be their most important func-
Complete deletion of any gene whose product is required at
multiple steps in infection will result in arrest of infection at
the first of those steps, making identification and analysis of
* Corresponding author. Mailing address: Department of Microbi-
ology, The University of Iowa, 3-432 Bowen Science Building, Iowa
City, IA 52242. Phone: (319) 335-9958. Fax: (319) 335-9006. E-mail:
?Published ahead of print on 11 May 2011.
later events impossible. Point mutations in that gene will some-
times result in proteins with full or partial function at early
steps and failure of function at later steps, thereby allowing
characterization of those later steps. This strategy has been
useful in analysis of UL34 gene function, since careful analysis
of point mutations has allowed identification of UL34 gene
functions in nuclear egress that follow nuclear lamina disrup-
tion, including mediation and regulation of membrane curva-
ture around capsids (50). Analysis of point mutations has the
additional advantage that extragenic suppressors of the mutant
phenotypes can be selected and mapped, allowing identifica-
tion of functionally important interactions. This genetic ap-
proach has had limited use in analysis of herpesvirus morpho-
genesis, principally because of the difficulty of mapping the
extragenic suppressor mutations by marker transfer. Applica-
tion of the method so far has yielded useful results only when
the position of the suppressor could be predicted based on
already known or suspected interactions (9, 20). Two recent
technical advances allow for more extensive and powerful use
of extragenic suppressor analysis. The first of these is the use of
high-throughput sequencing methods for rapid and convenient
sequencing of whole herpesvirus genomes for identification of
single nucleotide polymorphisms (SNPs) between the parental
and suppressor mutant viruses (61). The second is the use of
molecular clones of herpesvirus genomes in the form of bac-
terial artificial chromosomes (BACs) as the parent genomes
for marker transfer identification of the relevant SNP.
Here, we show that a mutation in UL34 that results in
substitution of alanine for a highly conserved tyrosine at
pUL34 position 68 results in a surprising phenotype. This mu-
tation is associated with a major virus replication defect and
inhibition of nuclear egress but also results in a profound
defect in virus cell-to-cell spread and in trafficking of gE. We
isolated extragenic suppressors of the cell-to-cell spread phe-
notype and showed by whole-genome sequencing and PCR-
based screening that phenotypic suppression is correlated with
a nonsense mutation in the US9 gene. This mutation alone is
not sufficient to suppress the UL34 Y68A phenotype.
MATERIALS AND METHODS
Cells and viruses. Vero and HEp-2 cells were maintained as previously de-
scribed (51). The properties of HSV-1(F), vRR1072(TK?), and UL34(?) BACs
have been previously described (14, 51).
Plasmids and cell lines. pRR1072 and pRR1072Rep were previously de-
scribed (2, 51). To construct an infection-inducible UL34Y68A-expressing cell
line, we built the plasmid pRR1374. To achieve this, the 1,250-bp XbaI/Klenow-
BspEI fragment of pRR1293 that contains the Y68A UL34 gene on the
pRR1072Rep background was ligated into AseI/Klenow-NgoMIV-cut pTuner-
IRES2 (Clontech). The resulting plasmid expresses bicistronic pUL34-IRES2-
enhanced green fluorescent protein (EGFP) mRNA from the UL34 promoter/
regulatory sequences. Clonal cell line Y68A-DD was constructed by transfection
of pRR1374 into Vero cells followed by selection with G418 and isolation of
clones by limiting dilution. Expressing cell clones were initially screened by assay
for EGFP expression 20 h after infection with HSV-1(F). Cell clones that ex-
pressed EGFP were further screened for pUL34 expression by immunofluores-
cence assay of cells 20 h after infection with the UL34-null virus vRR1072(TK?).
Although several Y68A pUL34-expressing cell clones were isolated, only one,
Y68A-DD, expressed pUL34 after UL34-null virus infection at a level compa-
rable to that seen in wild-type (WT) virus infection. The Y68A-DD cells are
referred to in the remainder of this report simply as Y68A UL34-expressing cells.
The wild-type pUL34-expressing cell line, called RepAC, was previously de-
scribed (50), and these cells are referred to as WT UL34-expressing cells.
Plaque assays. Six-well tissue culture wells were seeded with 1.8 ? 106Vero
cells, WT UL34-expressing cells, or Y68A UL34-expressing cells the day before
infection. Infection was initiated by removal of growth medium and the addition
of 1 ml of virus diluted in V medium (Dulbecco’s modified Eagle medium
[DMEM] containing 1% heat-inactivated calf serum). The virus inoculum was
removed after 90 min and replaced with 2.5 ml V medium containing a 1:250
dilution of pooled human immunoglobulin as a source of HSV-neutralizing
antibody (GamaSTAN S/D; Talecris Biotherapeutics). At the indicated times
after infection, monolayers were washed twice with phosphate-buffered saline
(PBS) and then fixed by incubation for 15 min in 3.7% formaldehyde in PBS.
After fixation, monolayers were washed three times with 2 ml PBS. Plaques were
stained by indirect immunofluorescence using a 1:5,000 dilution of mouse mono-
clonal anti-gD DL6 (gift of Gary Cohen and Rosalyn Eisenberg) as the primary
antibody and a 1:1,000 dilution of Alexa Fluor 488 goat anti-mouse IgG (Invi-
trogen) as the secondary antibody.
Single-step growth measurement. Measurement of replication and release of
HSV-1(F), vRR1072(TK?), and Y68ARev viruses on Vero cells, WT UL34-
expressing cells, and Y68A UL34-expressing cells after infection at high multi-
plicity was performed as previously described (29).
Indirect immunofluorescence. Immunofluorescence was performed as previ-
ously described using either a 1:2,000 dilution of mouse monoclonal anti-gE
ascites fluid (gift of Lenore Pereira), a 1:500 dilution of mouse monoclonal
anti-emerin clone H-12 (Santa Cruz), or a 1:5,000 dilution of mouse monoclonal
anti-gD DL6 (2, 48).
Immunoblotting. Nitrocellulose sheets bearing proteins of interest were
blocked in 5% nonfat milk plus 0.2% Tween 20 for at least 2 h. The membranes
were probed either with a previously described chicken polyclonal antibody
directed against pUL34 (1:1,000) (48), followed by reaction with alkaline phos-
phatase-conjugated anti-chicken secondary antibody (Aves Laboratories), or
with mouse monoclonal antibody directed against the HSV-1 scaffolding protein
(1:2,000) (Serotec), followed by reaction with alkaline phosphatase-conjugated
anti-mouse secondary antibody (Sigma).
Transmission electron microscopy (TEM) of infected cells. Confluent
monolayers of Vero or Y68A UL34-expressing cells were infected with
vRR1072(TK?) at a multiplicity of infection (MOI) of 10 for 20 h and then fixed
by incubation in 2.5% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.4) for 2 h.
Cells were postfixed in 1% osmium tetroxide, washed in cacodylate buffer,
embedded in Spurr’s resin, and cut into 95-nm sections. Sections were mounted
on grids, stained with uranyl acetate and lead citrate, and examined with a JEOL
1250 transmission electron microscope.
Sequencing of the Y68ARev genome. Y68ARev genomic DNA was isolated
from C capsids from infected HEp-2 cell nuclei purified on sucrose gradients as
previously described (51). Viral DNA was sequenced on an Illumina Genome
Analyzer II (GAII), as described previously (61). Briefly, viral nucleocapsid
DNA was prepared for sequencing using Illumina’s genomic DNA sample prep
kit, loaded onto one lane of a flow cell, and sequenced for 75 cycles using
standard data acquisition by the Illumina Pipeline software version 1.3. Sequence
reads were filtered to remove mononucleotides, and the human aligning se-
quence derived from the HEp-2 cells was used to prepare viral DNA. Then
sequence reads were aligned to the HSV-1(F) viral genome (GenBank identifier
GU734771) using the Mapping and Alignment with Qualities (MAQ) software
package (28). Default MAQ settings were used to call SNPs in the Y68ARev
sequence compared to the HSV-1(F) genome. Default MAQ filtering excludes
SNP calls in any region where sequence reads align to more than one location.
This situation occurs in the large inverted repeat regions of HSV-1, where
sequence reads can align to either the terminal or internal copy of the repeat. For
this reason, MAQ filtering was lifted for the repeat regions, and all potential SNP
calls were considered in the repeat regions, which includes the RS1, RL1, and
BAC construction. An HSV-1 BAC genome, carrying a UL34 gene deletion
and a mutation in the US9 gene creating an R58Stop substitution, was engi-
neered using Red recombineering on the background of a UL34-null BAC as
previously described (50, 62). The UL34-null BAC was mutagenized at the US9
locus by insertion and scarless excision of a gentamicin (Gm) resistance (Gmr)
The Gmrcassette with the mutant US9 flanking sequence was constructed in
several steps. First, a Gm resistance cassette containing the Gmrpromoter,
protein coding sequence, and terminator flanked at the 5? end with an SceI
homing nuclease site was amplified as previously described (50). Second, PCR
products containing the 5? and 3? halves of the Gm resistance gene were ampli-
fied from the Gmrcassette template using the primers US9R58StGm For/Gm
mid Rev and Gm mid For/US9R58StGm Rev, respectively. The two resulting
PCR products overlap in the Gmrcoding sequence. The complete Gm resistance
cassette with the US9 flanking sequence was then assembled in a PCR using the
overlapping partial genes and the primers US9R58St unique For and US9R58St
7204HAUGO ET AL.J. VIROL.
unique Rev (Table 1). The resulting PCR product was recombined into the
UL34-null BAC, Gm-resistant recombinants were picked, and genomes were
tested for insertion of the Gm cassette by diagnostic PCR by using the flanking
primers US9 test Fwd and US9 test Rev (Table 1). Correct insertion of the Gm
cassette was confirmed by direct sequencing of the BAC DNA. Scarless excision
of the Gm cassette, leaving an intact US9 gene carrying the R568Stop mutation,
was carried out as described previously (50), and Gm-sensitive, kanamycin
(Kan)-resistant clones were tested for correct structure at both the US9 and
UL34 loci by diagnostic PCR using the UL34 test For, UL34 test Rev, US9 test
Fwd, and US9 test Rev primers. Correct structure was confirmed by direct
sequencing of the BAC DNA at both loci.
Viruses were rescued from the WT BAC, UL34-null BAC, and UL34-null/
US9R58Stop BAC by transfection into UL34-expressing complementing cells.
The sequence of the rescued UL34-null/US9R58Stop virus at the US9 locus was
verified by sequencing of a PCR product containing the US9 gene.
Y68A mutant pUL34 is deficient in its ability to support
virus production and cell-to-cell spread. We have previously
described a strategy for evaluation of mutant UL34 function
based on UL34-null virus infection of cell lines that express
mutant pUL34 (50). The Y68A mutant pUL34 was evaluated
as part of a larger effort to examine the function of UL34
mutants in which individual conserved amino acids were re-
placed with alanine. Tyrosine 68 is in the most conserved
region of pUL34 and is absolutely conserved among alphaher-
pesviruses (Fig. 1A). In order to evaluate the function of Y68,
we constructed a cell line that expresses Y68A pUL34 under
the control of its own promoter regulatory sequences. We
compared pUL34 expression relative to a previously con-
structed WT pUL34-expressing cell line, and to wild-type virus
infection, by infecting cells with 10 PFU/cell of either HSV-
1(F) or the UL34-null virus vRR1072(TK?) for 18 h. Total
protein was determined in each extract, and equivalent
amounts were separated on an SDS-PAGE gel, transferred to
nitrocellulose, and probed for pUL34 (Fig. 1B). HSV-1 scaf-
folding protein was used as a loading control. The UL34-null
virus-infected Y68A UL34-expressing cells expressed as much
pUL34 as WT virus-infected Vero cells (Fig. 1B, compare
lanes 1 and 4) and considerably more than UL34-null virus-
infected WT UL34-expressing cells (lane 3). We could, there-
fore, directly compare the activity of Y68A pUL34 to that of
WT pUL34 both in WT virus-infected cells and in WT UL34-
expressing cells. Y68A mutant phenotypes could not be as-
cribed to insufficient levels of the mutant protein.
Virus replication and spread were measured in single-step
growth and plaque formation assays (Fig. 2 and 3) with several
interesting results. Figure 2 shows single-step growth kinetics
and the sizes of representative plaques. Figure 3 shows the
results of measurement of plaque areas (note that the y axis of
Fig. 3 has a logarithmic scale). As expected, wild-type virus
grew efficiently and formed robust plaques on all cell lines
tested (Fig. 2A to D and 3A). This indicates that the Y68A
UL34-expressing cells show no UL34-independent defect in
virus growth and no dominant negative effect of the UL34
mutant on virus growth. UL34-null virus replication on Y68A
pUL34-expressing cells was less efficient than WT virus repli-
cation on any of the cell types, producing about 60-fold-less
virus at peak times (compare curves in Fig. 2A with the triangle
marker curve in Fig. 2E). Since roughly equivalent amounts of
pUL34 were expressed in both cases, this suggests that Y68A
pUL34 is deficient in its ability to support single-step growth of
HSV-1. This inhibition of single-step growth was, however,
much less than that seen in the absence of pUL34 (compare
triangle and circle marker curves in Fig. 2E).
Interestingly, the single-step growth of UL34-null virus on
Y68A UL34-expressing cells (triangle marker curve in Fig. 2E)
was very similar to that obtained on WT UL34-expressing cells
(square marker curve in Fig. 2E), probably because of the low
level of WT pUL34 expression (Fig. 1B). The latter observa-
tion was in striking contrast to the result of plaque formation
assays. While UL34-null virus formed robust plaques on WT
pUL34-expressing cells, it formed only tiny foci of one to a few
infected cells on Y68A pUL34-expressing cells (compare Fig.
2G and H and 3B). Y68A pUL34-expressing cells supported
plaque formation no better than cells that express no pUL34 at
all (compare Fig. 2F and H and 3B). Since infected cultures for
plaque formation assays were incubated in the presence of
HSV-neutralizing antibody to make plaque formation exclu-
sively dependent on cell-to-cell spread, this result suggests that
the Y68A mutation in UL34 results in a severe defect in cell-
to-cell spread. Spread of WT virus in Y68A UL34-expressing
cells was just as efficient as that in Vero cells (Fig. 2D and 3A),
indicating that Y68A UL34-expressing cells have no UL34-
independent defect in virus spread and showing that the Y68A
UL34 has no dominant negative effect on virus spread.
Failure in plaque formation could be due to specific inhibi-
tion of virion trafficking to cell junctions, or it could reflect
inhibition of virion trafficking to any cell surface. In order to
determine whether Y68A pUL34 is associated with a general
virion release defect, amounts of total infectious virus and
infectious particles released to the medium at 18 h after infec-
TABLE 1. Primers used for construction and analysis of mutant US9 BAC
Primer name Primer sequencea
Gm mid For ....................................5?-GGTCGTGAGTTCGGAGACGTAGC-3?
Gm mid Rev....................................5?-CACTACGCGGCTGCTCAAACC-3?
R58St unique For...........................5?-TCCTCGTACGCATGGGCCG-3?
R58St unique Rev ..........................5?–GACAGGCGATCACCATGCCGAC-3?
US9 test Fwd...................................5?-AGTCACTGCGACCGCAACTTCC-3?
US9 test Rev...................................5?-AAGGCTGGGTGCAAATTGCGG-3?
aUnderlined sequences have homology to the US9 gene. Lowercase indicates nucleotides altered to create the R58Stop mutation and an NheI restriction enzyme
cleavage site. Sequences not underlined have homology to the Sce-Gmrcassette.
VOL. 85, 2011HSV-1 pUL34 CRITICAL FOR CELL-TO-CELL SPREAD OF VIRUS7205
tion were measured in cells that express WT or Y68A pUL34
following infection with either UL34-null virus or Y68ARev
(Fig. 4). Again, virus replication was slightly depressed in
Y68A-expressing cells compared to that in cells that express
WT pUL34 (Fig. 4, gray bars). As is typical for Vero cells, only
a small fraction of total virus was released to the medium
(white bars). The amounts of virus released from UL34-null
virus-infected cells were the same regardless of whether WT or
Y68A pUL34 was expressed, indicating that the Y68A muta-
tion does not interfere with release of mature virus from the
cell. Furthermore, the efficiency of release (i.e., the fraction of
total virus produced that is released to the medium) from
UL34-null virus-infected Y68A UL34-expressing cells (0.33%)
is similar to that seen in wild-type virus infection of either Vero
(0.53%) or Y68A UL34-expressing (0.58%) cells. This indi-
cates that there is no UL34-independent inhibition of virus
release occurring in the UL34-expressing cell lines. Given the
severe inhibition of plaque formation, these data suggest that
the Y68A mutation is associated with a major defect in cell-
Y68A mutant pUL34 induces exaggerated disruption of the
nuclear envelope and a nuclear egress defect. The Y68A mu-
tation is outside the region of pUL34 that is necessary and
sufficient for nuclear envelope targeting (29). To test whether
FIG. 1. Position of the Y68A mutation and expression of pUL34 by cell lines. (A) Schematic diagrams of pUL34 showing the locations of
relevant sequence features. Protein sequence of pUL34 is indicated as a bar with the N terminus at the left. Sequences in pUL34 that mediate
nuclear envelope targeting of the NEC are indicated as a stippled region. Position of Y68 is indicated below the bar. Positions of conserved regions
are indicated immediately below each of the bars. The conservation plot shows conservation of biochemical properties of amino acids using all
available herpesvirus sequences aligned by using the program MUSCLE (13). (B) Expression of wild-type and Y68A mutant pUL34 by stable cell
lines. Digital images of Western blots are shown. Vero cells (lanes 1 and 2) or cells stably expressing WT pUL34 (lane 3) or Y68A pUL34 (lane
4) were infected with WT HSV-1(F) (lane 1) or UL34-null vRR1072(TK?) virus (lanes 2 to 4). Blotted infected cell proteins were probed for either
scaffolding protein (top) or pUL34 (bottom).
7206 HAUGO ET AL.J. VIROL.
the Y68A mutation interferes with proper localization of
pUL34, WT UL34-expressing cells and Y68A UL34-expressing
cells that were mock infected or infected with 10 PFU/cell of
HSV-1(F) or UL34-null viruses for 16 h were fixed and assayed
for localization of pUL34 or the host cell nuclear envelope
protein emerin by immunofluorescence (Fig. 5). As we have
seen with other cell lines that express pUL34 from its own
promoter/regulatory sequences, pUL34 expression in both
wild-type UL34- and Y68A UL34-expressing cells is strictly
dependent upon infection and therefore cannot be detected in
uninfected cells (Fig. 5A and D). As previously reported, wild-
type pUL34 was tightly localized to the nuclear rim in cells
FIG. 2. Single-step growth and plaque formation on Vero, WT
pUL34-expressing, or Y68A mutant pUL34-expressing cell lines. For
single-step growth replicate, cultures of Vero cells, WT UL34-express-
ing cells, or Y68A UL34-expressing cells were infected at an MOI of 5
with HSV-1(F) (A), the UL34-null virus vRR1072(TK?) (E), or
Y68ARev (I). Residual virus was removed or inactivated with a
low-pH wash, and at the indicated times total culture virus was titrated
on WT UL34-expressing cells. Virus yields are expressed as PFU per
milliliter. Each data point represents the mean of results from three
independent experiments. Error bars indicate the range of values. For
plaque formation assays, digital micrographs of infected cell monolay-
ers stained for glycoprotein D are shown. The cell line infected is
indicated to the left of each panel. In panels B to D, the infecting virus
was HSV-1(F). In panels F to H, the infecting virus was the UL34-null
virus vRR1072(TK?). In panels J to L, the infecting virus was
Y68ARev. All plaques were fixed and stained at 2 days after infection.
All plaque images are shown at the same magnification.
FIG. 3. Quantitation of plaque formation on wild-type and mutant
UL34-expressing cell lines. Histograms of mean plaque sizes on Vero,
WT pUL34-expressing, and Y68A pUL34-expressing cells are shown.
Plaques stained 2 days after infection, as described in the legend to Fig.
2, were photographed, and plaque areas in image pixels were de-
termined using ImageJ. For each bar, 20 randomly selected plaques
from two independent experiments (40 plaques total) were mea-
sured. Brackets indicate pairwise statistical comparisons performed
using a Student t test. All of the indicated comparisons showed a
highly significant difference (P ? 0.001). Note that the y axis has a
VOL. 85, 2011HSV-1 pUL34 CRITICAL FOR CELL-TO-CELL SPREAD OF VIRUS7207
infected with either UL34-null virus or HSV-1(F) (Fig. 5B and
C). Y68A pUL34 was also tightly localized to the nuclear rim,
but the appearance of the rim was changed such that there
were numerous, sometimes large blebs on the outer nuclear
envelope, indicating an exaggerated disruption of the architec-
ture of the NE (Fig. 5E). Interestingly, the same effect was
observed when Y68A pUL34-expressing cells were infected
with wild-type virus (Fig. 5F), indicating that the mutant phe-
notype dominates even when WT pUL34 is present. The blebs
formed in Y68A cells include the host NE protein emerin (Fig.
5H and I), and their induction depends upon infection since
the appearance of the nuclear envelope is normal in uninfected
cells (Fig. 5G). The exaggerated NE disruption due to the
Y68A mutation is not likely to contribute significantly to the
virus production or cell-to-cell spread defects, since WT HSV-
1(F) replicated normally on Y68A UL34-expressing cells (Fig.
2A) and formed plaques normally (Fig. 2D and 3), even though
the same degree of NE disruption was induced.
Examination of Y68A pUL34-expressing cells that were in-
fected with UL34-null virus by TEM revealed two interesting
phenotypes. First, as suggested by the immunofluorescence
localization experiments above, there was blebbing of the nu-
clear membrane into the cytoplasm (Fig. 6A, white arrow-
heads). These blebs were formed by distension of both inner
and outer nuclear membranes into the cytoplasm. In some
cases, we also observed multilamellar structures containing
multiple thicknesses of nuclear envelope separated by an elec-
tron-dense layer (Fig. 6B, white arrowheads). In all these cy-
toplasmic structures, the inner and outer nuclear envelopes
appeared to be properly spaced, suggesting distortion of the
nuclear envelope as a whole rather than just the inner or outer
nuclear membranes. This phenotype is in distinct contrast to
the behavior of UL34-null mutants in cells that express no
pUL34, where the nuclear envelope is generally of uniform
shape (27, 51). Second, in most Y68A UL34-expressing cells
infected with UL34-null virus, we observed no cell surface
virions or cytoplasmic egress intermediates despite the pres-
ence of numerous A, B, and C capsids in the infected cell
nucleus (C capsids are indicated in Fig. 6A, black arrowheads).
In this regard, Y68A-expressing cells were similar in appearance
to UL34-null mutant-infected cells that express no UL34 at all,
suggesting that the Y68A mutation causes a major defect in nu-
clear egress. The lack of surface virions and cytoplasmic egress
intermediates was unsurprising given the magnitude of the single-
FIG. 4. Virus release to the medium mediated by wild-type and mutant pUL34. Replicate cultures of Vero, wild-type pUL34-expressing, or
Y68A mutant pUL34-expressing cells were infected at an MOI of 5 with wild-type virus HSV-1(F), UL34-null virus vRR1072(TK?), or Y68ARev.
Residual virus was removed or inactivated with a low-pH wash, and at 18 h after infection total culture virus (gray bars) or culture supernatant
(white bars) was titrated on WT UL34-expressing cells. Virus yields are expressed as PFU/ml. Each data point represents the mean of results from
three independent experiments. Error bars indicate the range of values. The values in parentheses above each pair of bars indicate the percentages
of total virus released to the medium calculated as the mean amount of released virus divided by the mean amount of total virus times 100. Low
pH wash completely removed residual extracellular virus, as the titer of culture supernatant at 2 h postinfection was undetectable.
7208HAUGO ET AL. J. VIROL.
step growth defect associated with the Y68A mutation (Fig. 2).
Even in EM examination of wild-type virus-infected Vero cells,
we would ordinarily observe less than 50 such structures per cell.
Our failure to observe extranuclear virus here was consistent with
the 60-fold single-step growth defect conferred by the Y68A mu-
tation and suggests that the single-step growth defect is due
largely or entirely to a defect in nuclear egress.
pUL34 function is required for proper localization of glyco-
protein E. Cell-to-cell spread of HSV-1 is correlated with traf-
ficking of glycoprotein E, and probably virions, to cell junc-
tions. While gE is not required for cell-to-cell spread in Vero
cells, we reasoned that alterations in its localization might
reflect defects in proper trafficking of viral or cellular compo-
nents required for cell-to-cell spread. To determine whether
pUL34 function is necessary for proper gE sorting, we infected
Vero and WT or mutant pUL34-expressing cells and assayed
for gE and gD localization at 16 h after infection by immuno-
fluorescence (Fig. 7). In Vero cells infected with wild-type virus
(Fig. 7A), gE was localized on intracellular membranes, includ-
ing the nuclear membrane in some cells. It was most promi-
nent, however, on the plasma membrane, especially at junc-
tional surfaces (Fig. 7A, arrowheads). In Vero cells infected
with UL34-null virus, plasma membrane staining was much less
prominent. Instead, gE accumulated in large membrane aggre-
gates in the cytoplasm (Fig. 7B, arrowheads), and in most cells,
junction staining was not evident. Plasma membrane and junc-
tion staining was restored when Vero cells that express WT
pUL34 were infected with UL34-null virus (Fig. 7C), indicating
that abnormal gE trafficking was due to lack of pUL34 expres-
sion. Infection of cells that express Y68A pUL34 with UL34-
null virus resulted in an intermediate phenotype (Fig. 7D). gE
accumulated more on cytoplasmic membranes than in a wild-
type infection, but there was more cell surface and junctional
staining than what was seen in infections with no pUL34.
In addition, Y68A pUL34 expression resulted in more pro-
nounced nuclear rim staining (Fig. 7D, arrowhead) than that
seen in infections with wild-type or with no pUL34. These
effects on gE trafficking did not reflect general disruption of
cell surface trafficking of viral glycoproteins, since gD localized
normally to the plasma membrane regardless of pUL34 expres-
FIG. 5. Localization of WT pUL34, Y68A mutant pUL34, and
emerin in infected cells. Digital confocal images of WT UL34-express-
ing cells (A to C) or Y68A UL34-expressing cells (D to I) are shown.
Cells were mock infected (A, D, and G) or infected for 16 h with
UL34-null virus (B, E, and H) or with HSV-1(F) (C, F, and I) and then
fixed and immunofluorescently stained for pUL34 (A to F) or emerin
(G to I). All images are shown at the same magnification.
FIG. 6. TEM analysis of cells that express Y68A UL34. Digital micrographs show Y68A UL34-expressing cells infected with the UL34-null
virus vRR1072(TK?) for 20 h. White arrowheads in panel A point to examples of blebbing of the nuclear membrane into the cytoplasm. Black
arrowheads point to examples of C capsids in the nucleus. The boxed area in panel A is enlarged (?4) in panel B. The white arrowheads in panel
B point to an instance of a bleb with two thicknesses of nuclear envelope separated by an electron-dense layer.
VOL. 85, 2011HSV-1 pUL34 CRITICAL FOR CELL-TO-CELL SPREAD OF VIRUS7209
sion (Fig. 7E and F). gB and gH also did not differ in local-
ization in WT and UL34-null virus-infected cells (not shown).
Isolation and characterization of an extragenic suppressor
of the Y68A mutation. Plaque formation defects allow selection
of extragenic suppressors of the growth defect. In order to
isolate suppressors, 10 replicate cultures of Y68A UL34-ex-
pressing cells were infected with 1 ? 107PFU of the UL34-null
recombinant vRR1072(TK?) for 24 h. At the end of the in-
fection, virus stocks were prepared and serial dilutions were
plated onto Y68A UL34-expressing cells. Suppressors were
rare; after 3 days, only one of the selection stocks gave rise to
minute plaques on the mutant pUL34-expressing cells. The
suppressor virus (designated Y68ARev) was plaque purified
twice on Y68A UL34-expressing cells and then amplified to a
high titer stock on WT pUL34-expressing cells. The behavior
of the Y68ARev virus in single-step growth and plaque forma-
tion are shown in Fig. 2I and J and in Fig. 3C. In single-step
growth, Y68ARev replicated only slightly less well in Y68A
UL34-expressing cells than in the WT UL34-expressing cells
and much less well in cells that express no pUL34 at all (Fig.
2I). In this respect, Y68ARev behaves exactly the same as its
UL34-null virus parent, indicating that the suppressor muta-
tion(s) do not improve virus replication.
Y68ARev could form plaques on Y68A UL34-expressing
cells, but those plaques were minute (Fig. 2L and 3C). The
plaques formed by the Y68ARev virus in Y68A UL34-express-
ing cells were about 15-fold larger than those formed by UL34-
null virus in the same cells but about 25 times smaller than
those formed by WT virus. These results indicate that the
Y68A plaque formation phenotype is suppressed, albeit inef-
ficiently, by mutations in the Y68ARev virus. The Y68ARev
virus formed robust plaques in WT pUL34-expressing cells
(Fig. 2K and 3), indicating that the genetic changes leading to
suppression do not interfere with the function of WT pUL34.
We further compared virus production and extracellular re-
lease by Y68ARev and found levels comparable to those of its
UL34-null parent strain (Fig. 4). Taken together, these data
suggest that genetic changes in the Y68ARev strain might
provide insight specifically into the cell-to-cell spread (plaque
formation) defect of the Y68A UL34 mutation.
Extragenic suppression of the Y68A mutation correlates
with a mutation in US9. Suppression of the plaque formation
defect of Y68A UL34 was not strong enough to allow mapping
of the relevant mutation(s) by marker transfer. Accordingly,
we sequenced the whole genome of the Y68ARev virus and
compared it to the recently published HSV-1(F) genomic se-
quence (61). Surprisingly, we found 139 SNPs compared to the
sequence of HSV-1(F), 34 of which produce amino acid
substitutions in known viral proteins (Table 2). We further
reasoned that many of these SNPs would have accumulated
during the multistep construction of the UL34-null virus,
vRR1072(TK?), which was the immediate parent of Y68ARev
(51). Since these were far too many SNPs to test individually by
construction of recombinant viruses, we chose to focus on 11
gene products with known roles in virus assembly, egress, or
cell-to-cell spread (indicated by boldface in Table 2). We
PCR amplified the relevant regions from the genome of
vRR1072(TK?) to determine which of the SNPs were found
only in the suppressor virus genome and found that eight of the
SNPs were suppressor specific (Table 2). Finally, we repeated
the suppressor selection procedure in five separate experi-
ments to isolate an additional five Y68A suppressor viruses.
We then PCR amplified and sequenced the regions containing
suppressor-specific SNPs to see which, if any, were present in
most or all suppressor genomes (Table 2). Only one SNP,
found in the US9 protein coding sequence, was present in
more than one of the suppressor genomes. This SNP, which
changes the arginine codon at position 58 to a stop codon, was
found in five of the six independently selected suppressors,
suggesting that this substitution might be responsible for phe-
To test this hypothesis, we introduced a stop codon at posi-
tion 58 in the US9 sequence into our previously characterized
UL34-null BAC (50) and rescued mutant virus on WT pUL34-
expressing complementing cells. The replacement of the UL34
gene with the kanamycin resistance gene cassette in the UL34-
null and UL34-null/US9R58Stop viruses was confirmed by
PCR amplification of the UL34 locus, and PCR products of the
expected sizes were produced (Fig. 8). To confirm the presence
of the expected mutation at the US9 locus and to assess the
purity of the rescued virus, the US9 gene was PCR amplified
FIG. 7. Wild-type pUL34 expression is required for normal local-
ization of gE but not gD. Vero cells (A, B, E, and F), WT UL34-
expressing cells (C), and Y68A UL34-expressing cells (D) were in-
fected with HSV-1(F) (A and E) or UL34-null virus (B to D and F) for
16 h and then fixed and immunofluorescently stained for gE (A to D)
or gD (E and F). Arrowheads in panel A point to junctional surfaces,
where gE concentrates in wild-type infection. Arrowheads in panel B
point to cytoplasmic membranes, where gE concentrates during UL34-
null infection. Arrowhead in panel D points to the nuclear envelope.
7210 HAUGO ET AL. J. VIROL.
from the rescued virus genome and digested with HincII re-
striction enzyme. Introduction of the US9R58Stop mutation
resulted in elimination of an HincII restriction enzyme site.
PCR amplification of the US9 locus from UL34-null and
UL34-null/US9R58Stop mutant viruses resulted in a product
of 395 bp, as expected (Fig. 8, lanes 2 and 4). The wild-type
US9 product from the UL34-null virus was cut by HincII into
two fragments of 216 and 179 bp, as expected, but no digestion
products were detected from the UL34-null/US9R58Stop (Fig.
8, compare lanes 3 and 5). The UL34-null, UL34-null/
US9R58Stop, and Y68ARev viruses were tested for suppres-
sion in a plaque assay on Y68A pUL34-expressing cells in
which plaques were allowed to develop for 1 week (Fig. 9).
UL34-null virus formed only minute plaques after 1 week,
whereas the Y68ARev virus forms quite large plaques with this
extended growth time (compare Fig. 9A and B). However,
addition of the US9R58Stop mutation to the UL34-null virus
does not improve its ability to form plaques on Y68A UL34-
expressing cells (Fig. 9C). This suggests that the phenotypic
improvement in plaque size in the Y68ARev strain is polygenic
and that the UL34-null/US9R68Stop virus will be an important
tool in mapping additional contributors to this phenotype.
Mutation of conserved tyrosine 68 of pUL34 reveals new
functions for pUL34. It has been clear for some time that the
nuclear envelopment complex (NEC) consisting of pUL34,
pUL31, and other viral and cellular proteins is multifunctional.
HSV-1 pUL34 and pUL31 have each been shown to have the
ability to disrupt the nuclear lamina and to function at multiple
steps in capsid envelopment, including capsid docking, curva-
ture of the membrane around capsids, and deenvelopment at
the outer nuclear membrane (3, 24, 27, 40, 45, 47, 50, 51, 58,
59). All of these functions are consistent with the concentra-
tion of pUL34 and pUL31 at the nuclear envelope in infected
cells and consistent with a role as master regulators of the
nuclear egress process. Tyrosine 68 is one of the most con-
served residues in pUL34, and therefore it is unsurprising that
it may be important for multiple pUL34 activities.
TABLE 2. Amino acid sequence-altering SNPs in Y68ARev compared to the HSV-1(F) sequence
Amino acid changed
Frequency in 5 additional
C to T (?)
G to A (?)
G to T (?)
T to G (?)
C to T (?)
C to A (?)
G to A (?)
G to A (?)
G to A (?)
A to G (?)
C to T (?)
G to A (?)
G to A (?)
T to C (?)
G to A (?)
G to A (?)
G to A (?)
T to C (?)
C to T (?)
G to A (?)
G to A (?)
T to C (?)
C to A (?)
G to A (?)
C to T (?)
C to T (?)
C to T (?)
C to T (?)
A to C (?)
A to C (?)
G to A (?)
G to A (?)
G to C (?)
A to G (?)
GCG to GTG
AGC to AAC
GCC to GTC
ATC to ACC
GCC to GTC
GCC to GTC
GAA to AAA
GAT to AAT
GTG to ATG
CTG to CCG
GCA to ACA
CGC to TGC
ACG to ATG
ATG to GTG
TCG to TTG
CGT to TGT
CGG to TGG
TGC to CGC
GCT to GTT
AGC to AAC
AGC to AAC
ACG to GCG
CCC to ACC
ACG to ATG
GCT to GTT
GCG to GTT
CGA to TGA
CTC to TTC
CAG to CCG
GAG to GCG
CGC to CAC
ACC to GCG
GCG to CCG
GAC to GGC
aGenes indicated in boldface have reported functions related to virus assembly, egress, or cell-to-cell spread and were tested to determine whether they were
bAll SNPs are listed as nucleotide changes on the top strand of the prototype arrangement of the HSV-1(F) genome. In some cases, this will be the sense strand
(?), in other cases, this will be the antisense strand (?), and in the case of RS1, both.
cAll codons are rendered on the sense strand of the gene.
dAmino acids are numbered from the first amino acid encoded by the open reading frame. Amino acid differences indicated in boldface also occur in the published
sequence of HSV-1 (17) (GenBank accession NC_001806).
eY, yes; N, no.
VOL. 85, 2011HSV-1 pUL34 CRITICAL FOR CELL-TO-CELL SPREAD OF VIRUS 7211
While most of the functions of NEC components have been
identified using analysis of deletion mutants, functional anal-
yses can be greatly enhanced by analysis of more subtle muta-
tions. Analysis of small insertion mutations in the murine cy-
tomegalovirus homologs of UL34 and UL31 (M50 and M53,
respectively) has allowed identification of functional domains
of these proteins required for their interaction with each other,
nuclear envelope localization, capsid envelopment, and capsid
maturation (6, 32, 46, 53). These studies showed that the res-
idue homologous to HSV-1 Y68 (M50 residue Y57) is critical
for function of the protein (6). Surprisingly, however, the effect
of mutation at this site is to disrupt interaction with M53 and
consequently prevent proper localization of M50 and M53 at
the nuclear envelope (6). In HSV pUL34, the sequences re-
quired for interaction with pUL31 and proper localization to
the nuclear envelope have been mapped to a different part of
the protein (29), and we observed no defect in recruitment of
pUL34 to the nuclear envelope (Fig. 5). This suggests that
despite its conservation, tyrosine 68 has different functions in
different herpesvirus families. Those functions may, however,
overlap. Because M50 function is disrupted very early by mu-
tation of Y57, downstream functions, including possible roles
in capsid envelopment and virus spread, may be masked.
Our data show three phenotypes for the Y68A substitution
mutation in pUL34. (i) There is an exaggerated disruption of
nuclear envelope structure, resulting in the formation of blebs
that protrude from the nucleus into the cytoplasm. These ap-
pear from EM analysis to be extensions of both INM and
ONM, still properly spaced with respect to one another, into
the cytoplasm. Blebs like this are seen during normal infections
with the wild-type virus and are likely the result of disconnec-
tion of nuclear envelope from the underlying lamina (44).
Their much more extensive formation in infection with Y68A
pUL34 suggests that this mutation may result in exaggerated
disruption of the structure of the lamina. The lamina disrup-
tion activity of the NEC is tightly controlled and limited, pre-
sumably to maintain nuclear functions necessary for virus rep-
lication (3, 39). Lamina disruption is limited in part by the
pUS3 protein kinase activity (3), but it is unsurprising that
intrinsic sequence features of pUL34 might also play a role. (ii)
There is a major defect in virus replication, and EM analysis
suggests that this defect is caused by inhibition of nuclear
egress, since capsids were only rarely observed outside the
nucleus. Y68A mutant pUL34 accumulates normally at the
nuclear envelope, suggesting that it makes a normal targeting
interaction with pUL31. Since nuclear envelope disruption was
observed, but no nuclear egress intermediates (e.g., docked
capsids, perinuclear virions, etc.) accumulated, the Y68A nu-
clear egress defect likely follows lamina disruption but pre-
cedes docking of DNA-containing capsids at the inner nuclear
membrane. (iii) There is a major defect in cell-to-cell spread of
virus infection. This cannot be accounted for by the Y68A
replication defect, since a similar growth defect that is associ-
ated with low-level wild-type pUL34 expression is not accom-
panied by inhibition of plaque formation. The spread defect
FIG. 8. Characterization of mutation at the UL34 and US9 loci.
Digital images show electrophoretically separated PCR products.
(A) Amplification products from the UL34 locus using genomes from
rescued viruses as the template. The BAC used for rescue is indicated
above each lane. The sizes of the PCR products are indicated to the
right of the gel. Lambda BstEII digest size standards are shown in lane
1, and the sizes of standard bands are indicated to the left of the gel.
(B) Amplification products from the US9 locus that are either undi-
gested (lanes 2 and 4) or digested with restriction enzymes HincII
(lanes 3 and 5). A 100-bp ladder is shown in lane 1.
FIG. 9. Growth of BAC-derived UL34-null and US9R58Stop virus
on WT and mutant pUL34-expressing cells. Digital micrographs are
shown of representative plaques formed for 1 week on Y68A pUL34-
expressing cells. Plaques were immunofluorescently stained for gD.
The infecting virus is indicated in each panel.
7212HAUGO ET AL. J. VIROL.
also cannot be accounted for by a general inhibition of virus
release from the cell, since the efficiency of release in cells that
express Y68A pUL34 is just the same as that seen in cells that
express WT pUL34 and that form robust plaques. The defect
in cell-to-cell spread also cannot be accounted for by some
UL34-independent peculiarity of the Y68A UL34-expressing
cell line. These cells support completely normal replication
and plaque formation by WT HSV-1, indicating that they have
no general defect in the ability to support growth and cell-to-
cell spread. The spread phenotype is evident only upon infec-
tion with UL34-null virus, clearly demonstrating that the
spread phenotype is tied to the function of the mutant pUL34.
pUL34 in cell-to-cell spread. pUL34 is the first viral protein
other than those required for viral entry that is required for
efficient cell-to-cell spread in Vero cells, and its effect on
spread is far more dramatic than that seen for gE/gI in epithe-
lial or fibroblast cells. The pUL34 mutant cell-to-cell spread
defect seems not to be mediated by a pathway that requires
either gE or US9, since deletion of either of these genes has no
apparent effect on growth or spread in Vero cells (10, 31). A
role for pUL34 in cell-to-cell spread is unexpected, since its
localization at the nuclear envelope makes it unlikely to par-
ticipate directly in trafficking of virions or viral components to
the cell surface. It is more likely that the phenotype is indirect
and reflects defective synthesis or trafficking of other essential
viral or cellular components in the mutant-infected cell.
pUL34 expression is also required for proper localization of
gE (Fig. 7). gE is required for efficient cell-to-cell spread in
some cells (not including Vero cells), and this activity requires
gE sorting to junctional surfaces, where it colocalizes with
?-catenin (12, 16, 37, 64). The mechanism of sorting is not
characterized but requires sequences in the cytoplasmic tail of
gE (64). The requirement for gE in cell-to-cell spread is not
absolute; gE-null mutants have only moderately diminished
plaque sizes in all cells tested. Since gE is not essential for
efficient spread in Vero cells, the pUL34 effect on gE local-
ization is not likely the root of the spread defect. However,
it suggests that localization of proteins at the cell periphery
can be influenced by pUL34. The target(s) of pUL34 func-
tion that affects cell-to-cell spread in Vero cells remains to
Mapping of extragenic suppressors. To our knowledge, this
is the first demonstration of the use of high-throughput se-
quencing (HTS) to facilitate mapping of extragenic suppressor
mutations in HSV-1. As anticipated, this method revealed po-
tential interactors with the UL34 pathway that would not have
been considered in a candidate-based approach. The availabil-
ity of sequences from several different HSV-1 strains allowed
us to filter down the list of potential candidate mutations
further, and this approach will be further aided as additional
HSV-1 genomes are added to the databases. As sequencing
technology and analysis improve, it may become practical to
apply this technique further, for example, to an HTS screen in
parallel with the parental strain and the additional five Y68A
suppressors described here. Although it is beyond the scope of
this paper, this approach might reveal a polygenic basis to the
observed phenotype, if for example the additional suppressors
shared not only the US9 R58Stop mutation tested for by PCR
but also an untested SNP from Table 2 or even a noncoding
mutation. The UL34-null/US9R58Stop strain generated
here provides an important substrate for further testing of
such mutations, since conserved candidates from further
HTS screening can be added sequentially to the BAC and
tested phenotypically. Since polygenic mutations are typically
lost from mutant and suppressor screens due to failure to
complement, future application of HTS may greatly improve
this aspect of mutational mapping.
The appearance of a US9 nonsense mutation in five out of
six pUL34 Y68A suppressor mutants is consistent with the
observed cell-to-cell spread phenotype for the Y68A mutant. It
was unexpected, however, because pUS9 is not thought to
influence cell-to-cell spread in cells other than neurons, and
there is no other evidence for a physical or functional interac-
tion between pUS9 and pUL34. pUS9 is a type II membrane
protein found on cytoplasmic membranes, including Golgi
membranes, whereas pUL34 is highly concentrated at the nu-
clear membrane (4, 34, 48).
Phenotypic suppression of missense mutations can occur by
several mechanisms. True reversion (restoration of the original
UL34 sequence) and intragenic suppression (restoration of
function by other mutations in UL34) were eliminated by our
selection strategy. Extragenic suppressors are mutations in a
different gene, and the US9 mutation is a candidate for this
type of suppressor. In some cases of extragenic suppression, an
initial mutation in one protein disrupts a critical functional
interaction with a second protein, and the suppressor mutation
changes the sequence of a second protein, restoring the inter-
action or its functional consequence. In this scenario, however,
deletion of either gene would be expected to result in the same
defective phenotype. Deletion of US9 has no cell-to-cell
spread phenotype in Vero cells, making it unlikely that pUS9
and pUL34 interact in some way. A more likely possibility is
that the US9 nonsense mutation is a bypass suppressor. This
suppressor class opens an alternative pathway for achievement
of the same biological result (35).
How US9 mutation might open an alternative spread path-
way depends on the effect of the R58Stop mutation. The exact
R58Stop mutation described here as correlated with the
Y68ARev phenotype has been previously found by Negatsch
and colleagues in the supposedly wild-type strain HSV-1 KOS
(42). They showed that the truncated protein is not detectable
in HSV-1 KOS-infected cells and suggested that the mutation
creates a null allele. If the US9R58Stop mutant is indeed a null
allele of US9, it may be that US9 expression normally inhibits
the function of an alternative cell-to-cell spread pathway that is
exposed only in a US9-null infection. However, there are many
single nucleotide substitutions that would result in even more
severely truncated proteins and abrogate US9 expression.
Truncation of US9 in the same position in these disparate
strains suggests that this specific mutation is advantageous for
viral growth in some circumstances, that the viral DNA has
features facilitating mutation at this position, or both. This
truncation preserves US9’s endocytosis motif and key phos-
phorylation sites but removes its transmembrane domain and
the preceding cluster of basic, positively charged amino acids.
It is possible that the truncated protein, even if expressed at
very low levels, might interact in interesting ways with virion
VOL. 85, 2011HSV-1 pUL34 CRITICAL FOR CELL-TO-CELL SPREAD OF VIRUS7213
We thank the staff of the Central Microscopy Research Facility of
the University of Iowa and especially Jean Ross for expertise and help
with TEM analysis.
These studies were supported by the University of Iowa and Public
Health Service award AI 41478. We acknowledge funding from a
Center Grant (NIH/NIGMS P50 GM071508), the New Jersey Com-
mission on Spinal Cord Research (M.L.S.), NIH P40 RR018604
(L.W.E. and M.L.S.), and a supplement to NIH R01 AI 033063
1. Balan, P., et al. 1994. An analysis of the in vitro and in vivo phenotypes of
mutants of herpes simplex virus type 1 lacking glycoproteins gG, gE, gI or the
putative gJ. J. Gen. Virol. 75:1245–1258.
2. Bjerke, S. L., et al. 2003. Effects of charged cluster mutations on the function
of herpes simplex virus type 1 UL34 protein. J. Virol. 77:7601–7610.
3. Bjerke, S. L., and R. Roller. 2006. Roles for herpes simplex type 1 UL34 and
US3 proteins in disrupting the nuclear lamina during herpes simplex virus
type 1 egress. Virology 347(2):261–276.
4. Brideau, A. D., B. W. Banfield, and L. W. Enquist. 1998. The Us9 gene
product of pseudorabies virus, an alphaherpesvirus, is a phosphorylated,
tail-anchored type II membrane protein. J. Virol. 72:4560–4570.
5. Brideau, A. D., J. P. Card, and L. W. Enquist. 2000. Role of pseudorabies
virus Us9, a type II membrane protein, in infection of tissue culture cells and
the rat nervous system. J. Virol. 74:834–845.
6. Bubeck, A., et al. 2004. Comprehensive mutational analysis of a herpesvirus
gene in the viral genome context reveals a region essential for virus replica-
tion. J. Virol. 78:8026–8035.
7. Cai, J. S., et al. 1999. Identification and structure of the Marek’s disease virus
serotype 2 glycoprotein M gene: comparison with glycoprotein M genes of
Herpesviridae family. J. Vet. Med. Sci. 61:503–511.
8. Darlington, R. W., and L. H. Moss. 1968. Herpesvirus envelopment. J. Virol.
9. Desai, P., and S. Person. 1999. Second site mutations in the N terminus of
the major capsid protein (VP5) overcome a block at the maturation cleavage
site of the capsid scaffold proteins of herpes simplex virus type 1. Virology
10. Dingwell, K. S., et al. 1994. Herpes simplex virus glycoproteins E and I
facilitate cell-to-cell spread in vivo and across junctions of cultured cells.
J. Virol. 68:834–845.
11. Dingwell, K. S., L. C. Doering, and D. C. Johnson. 1995. Glycoproteins E and
I facilitate neuron-to-neuron spread of herpes simplex virus. J. Virol. 69:
12. Dingwell, K. S., and D. C. Johnson. 1998. The herpes simplex virus gE-gI
complex facilitates cell-to-cell spread and binds to components of cell junc-
tions. J. Virol. 72:8933–8942.
13. Edgar, R. C. 2004. MUSCLE: multiple sequence alignment with high accu-
racy and high throughput. Nucleic Acids Res. 32:1792–1797.
14. Ejercito, P. M., E. D. Kieff, and B. Roizman. 1968. Characteristics of herpes
simplex virus strains differing in their effect on social behavior of infected
cells. J. Gen. Virol. 2:357–364.
15. Farina, A., et al. 2005. BFRF1 of Epstein-Barr virus is essential for efficient
primary viral envelopment and egress. J. Virol. 79:3703–3712.
16. Farnsworth, A., and D. C. Johnson. 2006. Herpes simplex virus gE/gI must
accumulate in the trans-Golgi network at early times and then redistribute to
cell junctions to promote cell-cell spread. J. Virol. 80:3167–3179.
17. Forrester, A., et al. 1992. Construction and properties of a mutant herpes
simplex virus type 1 with glycoprotein H coding sequences deleted. J. Virol.
18. Fuchs, W., B. G. Klupp, H. Granzow, N. Osterrieder, and T. C. Mettenleiter.
2002. The interacting UL31 and UL34 gene products of pseudorabies virus
are involved in egress from the host-cell nucleus and represent components
of primary enveloped but not mature virions. J. Virol. 76:364–378.
19. Gonnella, R., et al. 2005. Characterization and intracellular localization of
the Epstein-Barr virus protein BFLF2: interactions with BFRF1 and with the
nuclear lamina. J. Virol. 79:3713–3727.
20. Jacobson, J. G., K. Yang, J. D. Baines, and F. L. Homa. 2006. Linker
insertion mutations in the herpes simplex virus type 1 UL28 gene: effects on
UL28 interaction with UL15 and UL33 and identification of a second-site
mutation in the UL15 gene that suppresses a lethal UL28 mutation. J. Virol.
21. Johnson, D. C., and V. Feenstra. 1987. Identification of a novel herpes
simplex virus type 1-induced glycoprotein which complexes with gE and
binds immunoglobulin. J. Virol. 61:2208–2216.
22. Johnson, D. C., M. C. Frame, M. W. Ligas, A. M. Cross, and N. D. Stow.
1988. Herpes simplex virus immunoglobulin G Fc receptor activity depends
on a complex of two viral glycoproteins, gE and gI. J. Virol. 62:1347–1354.
23. Johnson, D. C., and P. G. Spear. 1982. Monensin inhibits the processing of
herpes simplex virus glycoproteins, their transport to the cell surface, and the
egress of virions from infected cells. J. Virol. 43:1102–1112.
24. Klupp, B. G., et al. 2007. Vesicle formation from the nuclear membrane is
induced by coexpression of two conserved herpesvirus proteins. Proc. Natl.
Acad. Sci. U. S. A. 104:7241–7246.
25. Klupp, B. G., H. Granzow, and T. C. Mettenleiter. 2000. Primary envelop-
ment of pseudorabies virus at the nuclear membrane requires the UL34 gene
product. J. Virol. 74:10063–10073.
26. Lake, C. M., and L. M. Hutt-Fletcher. 2004. The Epstein-Barr virus BFRF1
and BFLF2 proteins interact and coexpression alters their cellular localiza-
tion. Virology 320:99–106.
27. Leach, N., et al. 2007. Emerin is hyperphosphorylated and redistributed in
herpes simplex virus type 1-infected cells in a manner dependent on both
UL34 and US3. J. Virol. 81:10792–10803.
28. Li, H., J. Ruan, and R. Durbin. 2008. Mapping short DNA sequencing reads
and calling variants using mapping quality scores. Genome Res. 18:1851–
29. Liang, L., and J. D. Baines. 2005. Identification of an essential domain in the
herpes simplex virus 1 UL34 protein that is necessary and sufficient to
interact with UL31 protein. J. Virol. 79:3797–3806.
30. Ligas, M. W., and D. C. Johnson. 1988. A herpes simplex virus mutant in
which glycoprotein D sequences are replaced by ? galactosidase sequences
binds to but is unable to penetrate into cells. J. Virol. 62:1486–1494.
31. Longnecker, R., and B. Roizman. 1987. Clustering of genes dispensable for
growth in culture in the S component of the HSV-1 genome. Science 236:
32. Lo ¨tzerich, M., Z. Ruzsics, and U. H. Koszinowski. 2006. Functional
domains of murine cytomegalovirus nuclear egress protein M53/p38.
J. Virol. 80:73–84.
33. Lyman, M. G., B. Feierbach, D. Curanovic, M. Bisher, and L. W. Enquist.
2007. Pseudorabies virus Us9 directs axonal sorting of viral capsids. J. Virol.
34. Lyman, M. G., C. D. Kemp, M. P. Taylor, and L. W. Enquist. 2009. Com-
parison of the pseudorabies virus Us9 protein with homologs from other
veterinary and human alphaherpesviruses. J. Virol. 83:6978–6986.
35. Manson, M. 2000. Allele-specific suppression as a tool to study protein-
protein interactions in bacteria. Methods 20:18–34.
36. McGraw, H. M., S. Awasthi, J. A. Wojcechowskyj, and H. M. Friedman.
2009. Anterograde spread of herpes simplex virus type 1 requires glycopro-
tein E and glycoprotein I but not Us9. J. Virol. 83:8315–8326.
37. McMillan, T. N., and D. C. Johnson. 2001. Cytoplasmic domain of herpes
simplex virus gE causes accumulation in the trans-Golgi network, a site of
virus envelopment and sorting of virions to cell junctions. J. Virol. 75:1928–
38. Morris, J. B., H. Hofemeister, and P. O’Hare. 2007. Herpes simplex virus
infection induces phosphorylation and delocalization of emerin, a key inner
nuclear membrane protein. J. Virol. 81:4429–4437.
39. Mou, F., T. Forest, and J. D. Baines. 2007. Us3 of herpes simplex type 1
encodes a promiscuous protein kinase that phosphorylates and alters local-
ization of lamin A/C in infected cells. J. Virol. 81:6459–6470.
40. Mou, F., E. Wills, and J. D. Baines. 2009. Phosphorylation of the U(L)31
protein of herpes simplex virus 1 by the U(S)3-encoded kinase regulates
localization of the nuclear envelopment complex and egress of nucleocap-
sids. J. Virol. 83:5181–5191.
41. Muranyi, W., J. Haas, M. Wagner, G. Krohne, and U. H. Koszinowski. 2002.
Cytomegalovirus recruitment of cellular kinases to dissolve the nuclear lam-
ina. Science 297:854–857.
42. Negatsch, A., T. C. Mettenleiter, and W. Fuchs. 2011. Herpes simplex virus
type 1 strain KOS carries a defective US9 and a mutated US8A gene. J. Gen.
43. Neubauer, A., J. Rudolph, C. Brandmuller, F. T. Just, and N. Osterrieder.
2002. The equine herpesvirus 1 UL34 gene product is involved in an early
step in virus egress and can be efficiently replaced by a UL34-GFP fusion
protein. Virology 300:189–204.
44. Nii, S., C. Morgan, and H. M. Rose. 1968. Electron microscopy of herpes
simplex virus. II. Sequence of development. J. Virol. 2:517–536.
45. Park, R., and J. Baines. 2006. Herpes simplex virus type 1 infection induces
activation and recruitment of protein kinase C to the nuclear membrane and
increased phosphorylation of lamin B. J. Virol. 80:494–504.
46. Popa, M., et al. 2010. Dominant negative mutants of the murine cytomega-
lovirus M53 gene block nuclear egress and inhibit capsid maturation. J. Vi-
47. Reynolds, A. E., L. Liang, and J. D. Baines. 2004. Conformational changes in
the nuclear lamina induced by herpes simplex virus type 1 require genes
UL31 and UL34. J. Virol. 78:5564–5575.
48. Reynolds, A. E., et al. 2001. UL31 and UL34 proteins of herpes simplex virus
type 1 form a complex that accumulates at the nuclear rim and is required for
envelopment of nucleocapsids. J. Virol. 75:8803–8817.
49. Reynolds, A. E., E. G. Wills, R. J. Roller, B. J. Ryckman, and J. D. Baines.
2002. Ultrastructural localization of the herpes simplex virus type 1 UL31,
UL34, and US3 proteins suggests specific roles in primary envelopment and
egress of nucleocapsids. J. Virol. 76:8939–8952.
7214HAUGO ET AL.J. VIROL.
50. Roller, R. J., S. L. Bjerke, A. C. Haugo, and S. Hanson. 2010. Analysis of a
charge cluster mutation of herpes simplex virus type 1 UL34 and its extra-
genic suppressor suggests a novel interaction between pUL34 and pUL31
that is necessary for membrane curvature around capsids. J. Virol. 84:3921–
51. Roller, R. J., Y. Zhou, R. Schnetzer, J. Ferguson, and D. DeSalvo. 2000.
Herpes simplex virus type 1 UL34 gene product is required for viral envel-
opment. J. Virol. 74:117–129.
52. Roop, C., L. Hutchinson, and D. C. Johnson. 1993. A mutant herpes simplex
virus type 1 unable to express glycoprotein L cannot enter cells and its
particles lack glycoprotein H. J. Virol. 67:2285–2297.
53. Rupp, B., et al. 2007. Random screening for dominant-negative mutants of
the cytomegalovirus nuclear egress protein M50. J. Virol. 81:5508–5517.
54. Ryckman, B. J., and R. J. Roller. 2004. Herpes simplex virus type 1 primary
envelopment: UL34 protein modification and the US3-UL34 catalytic rela-
tionship. J. Virol. 78:399–412.
55. Santarelli, R., et al. 2008. Identification and characterization of the product
encoded by ORF69 of Kaposi’s sarcoma-associated herpesvirus. J. Virol.
56. Schnee, M., Z. Ruzsics, A. Bubeck, and U. H. Koszinowski. 2006. Common
and specific properties of herpesvirus UL34/UL31 protein family members
revealed by protein complementation assay. J. Virol. 80:11658–11666.
57. Scott, E. S., and P. O’Hare. 2001. Fate of the inner nuclear membrane
protein lamin B receptor and nuclear lamins in herpes simplex virus type 1
infection. J. Virol. 75:1818–1830.
58. Simpson-Holley, M., R. C. Colgrove, G. Nalepa, J. W. Harper, and D. M.
Knipe. 2005. Identification and functional evaluation of cellular and viral
factors involved in the alteration of nuclear architecture during herpes sim-
plex virus 1 infection. J. Virol. 79:12840–12851.
59. Simpson-Holley, M., J. Baines, R. Roller, and D. Knipe. 2004. Herpes sim-
plex virus 1 UL31 and UL34 promote the late maturation of viral replication
compartments to the nuclear periphery. J. Virol. 78:5591–5600.
60. Snyder, A., K. Polcicova, and D. C. Johnson. 2008. Herpes simplex virus
gE/gI and US9 proteins promote transport of both capsids and virion glyco-
proteins in neuronal axons. J. Virol. 82:10613–10624.
61. Szpara, M. L., L. Parsons, and L. W. Enquist. 2010. Sequence variability in
clinical and laboratory isolates of herpes simplex virus 1 reveals new muta-
tions. J. Virol. 84:5303–5313.
62. Tischer, B. K., J. von Einem, B. Kaufer, and N. Osterrieder. 2006. Two-step
Red-mediated recombination for versatile high-efficiency markerless DNA
manipulation in Escherichia coli. Biotechniques 40:191–197.
63. Tomishima, M. J., and L. W. Enquist. 2001. A conserved alpha-herpesvirus
protein necessary for axonal localization of viral membrane proteins. J. Cell
64. Wisner, T., C. Brunetti, K. Dingwell, and D. C. Johnson. 2000. The extra-
cellular domain of herpes simplex virus gE is sufficient for accumulation at
cell junctions but not for cell-to-cell spread. J. Virol. 74:2278–2287.
65. Yamauchi, Y., et al. 2001. Herpes simplex virus type 2 UL34 protein requires
UL31 protein for its relocation to the internal nuclear membrane in trans-
fected cells. J. Gen. Virol. 82:1423–1428.
VOL. 85, 2011 HSV-1 pUL34 CRITICAL FOR CELL-TO-CELL SPREAD OF VIRUS 7215