JOURNAL OF VIROLOGY, Apr. 2010, p. 3921–3934
Copyright © 2010, American Society for Microbiology. All Rights Reserved.
Vol. 84, No. 8
Analysis of a Charge Cluster Mutation of Herpes Simplex Virus Type 1
UL34 and Its Extragenic Suppressor Suggests a Novel Interaction
between pUL34 and pUL31 That Is Necessary for Membrane
Curvature around Capsids?
Richard J. Roller,1* Susan L. Bjerke,2Alison C. Haugo,1and Sara Hanson3
Department of Microbiology, University of Iowa, Iowa City, Iowa 522421; Department of Biology, Washburn University, Topeka,
Kansas 666212; and Department of Biology, University of Iowa, Iowa City, Iowa 522423
Received 5 August 2009/Accepted 20 January 2010
Interaction between pUL34 and pUL31 is essential for targeting both proteins to the inner nuclear mem-
brane (INM). Sequences mediating the targeting interaction have been mapped by others with both proteins.
We have previously reported identification of charge cluster mutants of herpes simplex virus type 1 UL34 that
localize properly to the inner nuclear membrane, indicating interaction with UL31, but fail to complement a
UL34 deletion. We have characterized one mutation (CL04) that alters a charge cluster near the N terminus
of pUL34 and observed the following. (i) The CL04 mutant has a dominant-negative effect on pUL34 function,
indicating disruption of some critical interaction. (ii) In infections with CL04 pUL34, capsids accumulate in
close association with the INM, but no perinuclear enveloped viruses, cytoplasmic capsids, or virions or cell
surface virions were observed, suggesting that CL04 UL34 does not support INM curvature around the capsid.
(iii) Passage of UL34-null virus on a stable cell line that expresses CL04 resulted in selection of extragenic
suppressor mutants that grew efficiently using the mutant pUL34. (iv) All extragenic suppressors contained an
R2293L mutation in pUL31 that was sufficient to suppress the CL04 phenotype. (v) Immunolocalization and
coimmunoprecipitation experiments with truncated forms of pUL34 and pUL31 confirm that N-terminal
sequences of pUL34 and a C-terminal domain of pUL31 mediate interaction but not nuclear membrane
targeting. pUL34 and pUL31 may make two essential interactions—one for the targeting of the complex to the
nuclear envelope and another for nuclear membrane curvature around capsids.
Egress of herpesvirus capsids from the nucleus occurs by
envelopment of capsids at the inner nuclear membrane (INM)
and is followed by de-envelopment at the outer nuclear mem-
brane (ONM). This process can be broken down into a path-
way of discrete steps that begin with recruitment of the viral
envelopment apparatus to the INM. Herpes simplex virus type
1 (HSV-1) UL34 and UL31 and their homologs in other her-
pesviruses are required for efficient envelopment at the INM
(7, 13, 22, 23, 29). HSV-1 pUL31 and pUL34 are targeted
specifically to the INM by a mechanism that requires their
interaction with each other (27, 28), and this mutual depen-
dence is a conserved feature of herpesvirus envelopment (9,
14, 27, 28, 32, 33, 39). 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
(22, 24, 30). The sequences in HSV-1 pUL34 that mediate
interaction with UL31 and that lead to nuclear envelope tar-
geting were mapped to amino acids (aa) 137 to 181 (16). The
sequences in the murine cytomegalovirus (MCMV) homolog
of UL31, M53, that mediate the nuclear envelope targeting
interaction with the UL34 homolog, M50, were mapped to the
N-terminal third of the protein in the first of four conserved
regions (17), and Schnee et al. subsequently showed that this
same region of pUL31 homologs from other families of her-
pesviruses mediates interaction with the corresponding pUL34
After the targeting of the pUL34/pUL31 complex to the
INM, subsequent steps in nuclear egress include, it is thought,
(i) local disruption of the nuclear lamina to allow capsid access
to the INM, (ii) recognition and docking of capsids by the
envelopment apparatus at the INM, (iii) curvature of the inner
and outer nuclear membranes around the capsid, (iv) scission
of the INM to create an enveloped virion in the space between
the INM and ONM, (v) fusion of the virion envelope with the
outer nuclear membrane, and (vi) capsid release into the cy-
At least some of the viral and cellular factors critical for
nuclear lamina disruption and for de-envelopment fusion have
been identified. pUL34, pUL31, and pUS3 of HSV-1 have all
been implicated in changes in localization, interaction, and
phosphorylation of nuclear lamina components, including
lamins A/C and B and the lamina-associated protein, emerin
(3, 15, 19, 20, 24, 26, 34, 35). pUS3, pUL31, and glycoproteins
B and H have been implicated in de-envelopment of primary
virions at the ONM (8, 21, 28, 30, 38).
pUL34 and pUL31 are thought to be involved in steps be-
tween lamina disruption and de-envelopment, but genetic ev-
idence in infected cells has so far been lacking. Klupp et al.
have shown that overexpression of alphaherpesvirus pUL31
and pUL34 in the absence of other viral proteins can induce
formation of small vesicles derived from the INM, suggesting a
* 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 27 January 2010.
role for these two proteins in membrane curvature around the
capsid (12). Tight membrane curvature is an energetically un-
favorable event and is thought to be accomplished by coupling
curvature to energetically favorable interactions between
membrane-bound proteins or protein complexes (reviewed in
reference 40). The data of Klupp et al. suggest the possibility
that upon recognition of a capsid, pUL31 and pUL34 may
interact in a way that induces tight curvature of the INM. Here
we present data in support of this hypothesis, showing that a
specific point mutation in UL34 induces accumulation of
docked capsids at the INM, extragenic suppression of the mu-
tant phenotype is associated with a mutation in UL31, and
pUL31 and pUL34 can interact via sequences that are not
involved in their INM targeting interaction.
We previously published a characterization of a library of 19
charge cluster mutants of pUL34. In each of these mutants,
one charge cluster (defined as a group of five consecutive
amino acids in which two or more of the residues have charged
side chains) was mutated such that the charged residues were
replaced by alanine. Six of the 19 charge cluster mutants tested
failed to complement replication of UL34-null virus, indicating
that they disrupt essential functions of pUL34. Interestingly,
five of the six noncomplementing mutants were synthesized at
levels comparable to that of wild-type UL34 and localized
normally to the nuclear envelope, suggesting that they were
unimpaired in their ability to make a nuclear envelope target-
ing interaction with UL31. In order to identify essential func-
tions of pUL34 downstream of nuclear envelope targeting, we
have undertaken a detailed study of the behavior and interac-
tions of these mutants.
MATERIALS AND METHODS
Cells and viruses. HEp-2 and Vero cells were maintained as previously de-
scribed (29). The properties of HSV-1(F) and vRR1072 (TK?), referred to here
as UL34-null virus, have been described previously (6, 29).
Plasmids and cell lines. pRR1072, pRR1072Rep, and pRR1162, which con-
tains the CL04 charge cluster mutation in UL34 on the pRR1072Rep back-
ground, were previously described (2, 29).
pRR1340, used for construction of an infection-inducible CL04-expressing cell
line, was constructed by ligation of the 1,820-bp XbaI-PmlI fragment of
pRR1162 that contains the CL04 UL34 gene between the XbaI and NruI sites of
the pcDNA3 vector. An otherwise identical plasmid for expression of wild-type
(wt) pUL34 was constructed using pRR1072Rep as the source of the insert.
Clonal cell lines RepAC (expressing wt pUL34) and CL04AI (expressing CL04)
were constructed by transfection of the corresponding plasmid into Vero cells,
selection with G418, and isolation of clones by limiting dilution. Expressing cell
clones were identified by an immunofluorescence (IF) assay for pUL34 expres-
sion 20 h after infection with the UL34-null virus vRR1072(TK?).
pRR1238 for constitutive expression of wt pUL34 was constructed by digestion
of pRR1072Rep with NcoI and BspEI, treatment with Klenow enzyme in the
presence of deoxynucleoside triphosphates (dNTPs) to create blunt ends, and
ligation of the UL34-containing fragment into EcoRV-digested pcDNA3.
pRR1328 for constitutive expression of CL04 pUL34 was constructed by InFu-
sion (Clontech) cloning of a PCR fragment containing the CL04 pUL34 coding
sequence amplified from pRR1162 between the HindIII and XhoI sites of
pcDNA3. PCR primers used for amplification of the CL04 pUL34 coding se-
quence were 5?-AGGGAGACCCAAGCTCCATGGCGGGACTGGGCAA
G-3? and 5?-TAGATGCATGCTCGATTATAGGCGCGCGCCAGCAC-3?.
pRR1330, in which the coding sequences for amino acids 91 to 228 are deleted
from the UL34 gene (Fig. 1, line 3), was constructed by digestion of pRR1238
with SgrAI and HpaI, treatment with Klenow enzyme in the presence of dNTPs
to create blunt ends, and religation of the large fragment of the plasmid.
pRR1331, in which the same coding sequences are deleted from the CL04 gene
(Fig. 1, line 4), was created in the same way as pRR1330, except that the parent
plasmid was pRR1328.
pRR1344, in which amino acids 91 to 275 of wt pUL34 expressed from
pcDNA3 are deleted and replaced by the emerin transmembrane domain (Fig. 1,
line 5), was constructed by PCR amplification of sequences coding for amino
acids 219 to 254 of emerin and ligation of the BspEI- and XbaI-digested PCR
product between the SgrAI and XbaI sites of pRR1238. The primers used for
amplification of the emerin TM sequence were 5?-GATCTCCGGACAGGATC
GCCAGGTCCCGC-3? and 5?-TAGCTCTAGACTAGAAGGGGTTGCCTTC
TTCAGCCTG-3?. pRR1345, which is the equivalent construct for CL04 UL34
(Fig. 1, line 6), was made in the same way, except that the parent plasmid was
pRR1346, in which amino acids 64 to 275 of wt pUL34 expressed from
pcDNA3 are deleted and replaced by the emerin transmembrane domain
(Fig. 1, line 7) was made by insertion of an overlap PCR product into
pcDNA3. For the first step in insert construction, sequences encoding amino
acids 1 to 63 of UL34 fused to 19 nucleotides (nt) of the emerin sequence
were amplified using pRR1238 as the template and the primers HSV1-
63LEFT (5?-AGCTCTCGAGATGGCGGGACTGGGCAAGCCC-3?) and
HSV1-63RIGHT (5? GCGGGACCTGGCGATCCTGAAACGACTCGTCG
GACCCGTCATG-3?). Next, sequences encoding amino acids 219 to 254 of
emerin were amplified using the primers EmTMLEFT (5?-CAGGATCGCC
AGGTCCCGC-3?) and EmTMRIGHT (5?-GGCCTCTAGACTAGAAGGG
GTTGCCTTCTTCAGCC-3?). The resulting PCR products were gel purified
and then combined as the template for a PCR using HSV1-63LEFT and
EmTMRIGHT as primers. The resulting PCR product was digested with
XbaI and XhoI and cloned between the XbaI and XhoI sites of pcDNA3.
pRR1347, which is equivalent to pRR1346 except for the presence of the
CL04 mutation in UL34 sequences (Fig. 1, line 8), was constructed in the
same way, except that the template for amplification of UL34 sequences was
Plasmids for marker transfer of tagged UL31 wild-type or mutant sequences to
the viral genome were constructed in several steps. First, a parent plasmid
(pRR1336) containing UL31 wild-type sequences and flanking sequences from
the UL30 and UL32 genes was constructed by reaction of pRR1060 (29) with
BstEII and HindIII and with Klenow enzyme and dNTPs and by religation of the
8.64-kb fragment containing the vector, UL30, UL31, and UL32 sequences. Next,
pRR1337, in which the 3? end of the UL31 coding sequence was modified to
include tandem FLAG epitope 8?His tags was made by insertion of an overlap
PCR product into pRR1336. For the first step in insert construction, sequences
between the AvrII site in UL31 and the 3? end of the UL31 coding sequence and
containing the 5? part of the tandem tag were amplified using pRR1336 as the
template and the primers TagUL31#1 (5?-CCGGGATGGGCTACTACCTAG
GC-3?) and TagUL31#2 (5? GTGATGGTGATGGTGCGCTGCTTTGTCGTC
GTCCTTATAGTCCGGCGGAGGAAACTCGTCGAATGTTG-3?). For the
second step in insert construction, sequences encoding the 3? part of the tandem
tag and sequences between the 3? end of UL31 and the PstI site in UL30 were
amplified using the primers TagUL31#3 (5?-GACGACGACAAAGCAGCGC
GCTCTATGCAACATTCG-3?) and TagUL31#4 (5?-CCTGCCCGAGGGACT
GCAG-3?). The resulting overlapping PCR products were gel purified and then
combined as the template for a PCR using the TagUL31#1 and TagUL31#4
primers. The resulting PCR product was digested with AvrII and PstI and cloned
between the AvrII and PstI sites of pRR1336. pRR1338, which encodes the
UL31 R229L mutation in the context of pRR1337, was made by insertion of an
overlap PCR product into pRR1337. For the first step in insert construction,
sequences between the AvrII site in UL31 and the site of the R229L mutation
were amplified using pRR1337 as the template and the primers TagUL31#1 and
CG-3?) (lowercase indicates nucleotides altered to create the R229L mutation
and an AseI restriction enzyme cleavage site). For the second step in insert
construction, sequences from the site of the R229L mutation in UL31 to the PstI
site in UL30 were amplified using the primers UL31R229L#2 (5?-CACCTGC
ACTACttattaATAGACCGGATGCTCACCGCGTG-3?) and TagUL31#4. The
design of the primers created an overlap between the two PCR products that
included the mutated sequences. The engineered mutated sequences included an
AseI restriction site introduced at the site of the R229L mutation. The resulting
overlapping PCR products were gel purified and then combined as the template
for a PCR using the TagUL31#1 and TagUL31#4 primers. The resulting over-
lap PCR product was digested with AvrII and PstI and cloned between the AvrII
and PstI sites of pRR1337.
pRR1334 and pRR1335, which contain the coding sequences for wt and
R229L mutant tagged UL31, respectively, in pcDNA3 (Fig. 1, lines 9 and 10),
were constructed by PCR amplification of the tagged UL31 coding sequences
from pRR1337 and pRR1338 and cloning between the XhoI and XbaI sites of
3922 ROLLER ET AL.J. VIROL.
pcDNA3. pRR1327 and pRR1328, which code for tagged UL31 wt and R229L
mutant amino acids 127 to 306, respectively (Fig. 1, lines 11 and 12), were
constructed by PCR amplification of the corresponding sequences from
pRR1337 and pRR1338 using the primers 5?-GATCCTCGAGATGGCGGGA
GACGGGCG-3? and 5?-GTACTCTAGACTAATGGTGATGGTGATGGTG
ATGGTGC-3?. PCR products were digested with XhoI and XbaI and cloned
into the XhoI and XbaI sites of pcDNA3.
For complementation assays using combinations of wt and CL04 mutant UL34
and wt and R229L mutant UL31, plasmids were constructed that carry both the
UL34 and UL31 genes, each driven by its own promoter/regulatory sequences.
pRR1348, which carries both wt UL34 and wt, tagged UL31, was constructed by
ligation of the 2.76-kb BstBI-PvuII fragment of pRR1337 into pRR1072Rep that
had been cut with BstBI and PmlI. pRR1349, which carries wt UL34 and mutant,
tagged UL34 was constructed in the same way, except that the UL31-containing
insert was derived from pRR1338. pRR1350 and pRR1351, which carry CL04
UL34 and wt or mutant tagged UL31, respectively, were constructed in the same
way as pRR1348 and pRR1349, except that pRR1162 was used as the parent
Single-step growth measurement. Measurement of replication of HSV-1(F),
vRR1072(TK?), and CL04Rev viruses on Vero, RepAC, and CL04AI cells after
infection at high multiplicity was performed as previously described (29).
Complementation assays. Twelve-well cultures of Vero cells containing
400,000 cells were transfected with a total of 650 ng of plasmid mixtures using 5
?l of Lipofectamine according to the manufacturer’s instructions. Complemen-
tation assays were performed on transfected cultures as previously described (2).
Marker transfer assays. Genomes of UL34-null virus vRR1072(TK?) for
marker transfer transfection were prepared in the form of a detergent-extracted,
sonicated nuclear lysate from infected cells. A T25 culture of Vero cells was
infected for 24 h with 5 PFU/cell of vRR1072(TK?), washed once with phos-
phate-buffered saline (PBS), and scraped into 1 ml of PBS. Cells were pelleted
at 6,000 rpm in a microcentrifuge, resuspended in 1 ml PBS containing 0.2%
NP-40, and placed on ice for 1 min. Nuclei were pelleted at 6,000 rpm for 2 min
and then washed twice by resuspension in PBS without detergent and centrifu-
gation at 6,000 rpm in the microcentrifuge. The pellet of washed nuclei was
resuspended in 100 ?l of PBS, lysed by one freeze-thaw cycle, and sonicated.
Debris was pelleted by centrifugation at 14,000 rpm in the microcentrifuge for 2
min, and 2 ?l of the supernatant was used for each transfection experiment.
For mapping of the extragenic suppressor, gel-purified restriction fragments of
the CL04Rev genome (200 ng) were cotransfected with sonicated nuclear lysate
from cells infected with vRR1072(TK?) into 12-well cultures, each containing
400,000 RepAC cells using 5 ?l of Lipofectamine according to the manufactur-
er’s protocol. Four days after transfection, when cytopathic effect was observed
with most cells, virus stock was prepared and the titer was determined by plaque
assay on RepAC cells. One thousand PFU (as measured on RepAC) cells were
then plated onto confluent monolayers of CL04AI cells in 100-mm dishes. Four
days after infection, cell monolayers were fixed with methanol for 10 min and
FIG. 1. Wild-type and mutant UL31 and UL34 pcDNA3 expression constructs. Schematic drawings of expression constructs are shown. All
constructs were expressed from the human cytomegalovirus immediate-early promoter in pcDNA3 and were constructed as described in Materials
and Methods. The plasmids encoding each gene are the following: line 1, ppRRR1238; line 2, pRR1328; line 3, pRR1330; line 4, pRR1331; line
5, pRR1344; line 6, pRR1345; line 7, pRR1346; line 8, pRR1347; line 9, pRR1334; line 10; pRR1335; line 11, pRR1327; line 12, pRR1328.
VOL. 84, 2010 NOVEL pUL31/pUL34 INTERACTION IN MEMBRANE CURVATURE3923
then air dried. Fixed monolayers were stained with a solution of 0.04% amido
black in 40% methanol and 10% acetic acid in water and then destained with
water. Wild-type and recombinant R229L mutant UL31 sequences were tested in
the same way, except that 200 ng of either PstI-linearized pRR1337 or PstI-
linearized pRR1338 was used instead of genome restriction fragments.
Indirect IF. IF was performed as previously described, with some variations (3,
27). Cells were fixed with 4% formaldehyde for 20 min and then washed with
PBS. Cells were permeabilized and blocked in the same step by incubating in
10% Blokhen (Aves Labs) in IF buffer. Primary antibodies were diluted as
follows in IF buffer: chicken anti-UL34 (1:1,000) and mouse monoclonal IgG
anti-FLAG (1:1,000) (Sigma). Secondary antibodies were also diluted in IF
buffer as follows: Alexa Fluor 594 goat anti-chicken IgG (1:1,000) was used to
detect pUL34, and Alexa Fluor 488 goat anti-mouse IgG (1:400) was used to
detect FLAG-UL31 fusions. Slo-fade II (Molecular Probes) was used to mount
coverslips on glass slides. All confocal microscopy work was done with a Zeiss
510 confocal microscope. All images shown are representative of experiments
performed a minimum of three times.
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 with a previously described chicken polyclonal antibody directed
against pUL34 (1:1,000) (27) and subjected to reaction with alkaline phos-
phatase-conjugated anti-chicken secondary antibody (Aves Laboratories), with
mouse monoclonal antibody directed against the HSV-1 scaffolding protein (1:
2,000) (Serotec) and reacted with alkaline phosphatase-conjugated anti-mouse
secondary antibody (Sigma), or with anti-FLAG M2 mouse monoclonal antibody
(Sigma) and reacted with alkaline-phosphatase-conjugated anti-mouse.
Transmission electron microscopy (EM) (TEM) of infected cells. Confluent
monolayers of Vero or CL04AI cells were infected with vRRR1072(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.
BAC construction. An HSV-1 bacterial artificial chromosome (BAC) genome
carrying a UL34 deletion and a mutation in UL31 creating an R229L substitution
was engineered in two steps using Red recombineering as described in reference
37. In the first step, the UL34 gene was disrupted by insertion of a kanamycin
resistance (Kanr) cassette. This construction deletes exactly the same amino
acids deleted in vRR1072(TK?). In the second step, the resulting UL34-null
virus was mutagenized at the UL31 locus by insertion and scarless excision of a
gentamicin resistance (Gmr) cassette.
The Kanrconstruct for insertion into the UL34 locus was generated by PCR
from a pEPKan-S2 template using the primers KanUL34 Fwd and KanUL34 Rev
(Table 1). The resulting PCR product was recombined into the HSV-1 BAC
pYE-BAC102 (kind gift of Y. Kawaguchi ) as described previously (37).
Kan-resistant recombinants were picked, and genomes were tested for insertion
of the Kanrcassette by diagnostic PCR using the flanking primers UL34 test Fwd
and UL34 test Rev (Table 1). Correct insertion of the Kanrcassette was con-
firmed by direct sequencing of the BAC DNA.
The Gm resistance cassette with the mutant UL31 flanking sequence was
constructed in several steps. First, a Gm resistance cassette containing the Gmr
promoter, protein coding sequence, and terminator flanked at the 5? end with an
SceI homing nuclease site was amplified from a pFastBac-1 template (Invitro-
gen) using primers Sce-GmR For and Sce-GmR Rev (Table 1). Second, PCR
products containing the 5? and 3? halves of the Gm resistance gene were ampli-
fied from the Gmrcassette template using the primers R229L-Gm For and Gm
mid Rev and the primers Gm mid For and R229L-Gm Rev, respectively. The two
resulting PCR products overlap in the Gm coding sequence. The complete Gm
resistance cassette with the UL31 flanking sequence was then assembled in a
PCR using the overlapping partial genes and the primers UL31 unique For and
UL31 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 using the flanking
primers UL31 test Fwd and UL31 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 UL31 gene carrying the R229L
mutation, was carried out as described, and Gm-sensitive, Kan-resistant clones
were tested for correct structure at both the UL31 and UL34 loci by diagnostic
PCR using the UL34 test For, UL34 test Rev, UL31test For, and UL31test 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 UL31 R229L
mutant BAC by transfection into UL34-expressing complementing cells.
Coimmunoprecipitation. 293T cells in 6-well culture were transfected with
plasmids encoding portions of UL34 and UL31 using Lipofectamine 2000 ac-
cording to the manufacturer’s instructions. Two days after transfection, trans-
fected monolayers were washed once with PBS, and then cells were lysed by
addition of 0.5 ml immunoprecipitation (IP) buffer (50 mM Tris, 150 mM NaCl,
1 mM EDTA, 1% Triton X-100) per well and sonicated for 10 s with a Fisher
Sonic dismembrator at a power level of 2. Insoluble material was pelleted at top
speed in a microcentrifuge for 2 min, and the supernatant was transferred to a
fresh tube. Ten percent of each sample was removed and stored frozen as the
input sample, and the remainder was immunoprecipitated using anti-FLAG M2
resin (Sigma) according to the manufacturer’s instructions, using an overnight
binding step and elution with 3?FLAG peptide. Immunoprecipitated samples
were separated on 15% SDS-PAGE gels, blotted to nitrocellulose, and probed
with antibodies directed against either FLAG or UL34.
CL04 mutant UL34 has a dominant-negative effect on wild-
type protein function. We previously described construction
and characterization of a library of charge cluster mutants of
UL34 (2). Initial attempts to study mutant UL34 function
focused on construction of recombinant viruses expressing a
mutant pUL34 in place of wt pUL34. Such a recombinant virus
could be isolated for only one of the nonfunctional charge
cluster mutants (CL13). For most of the UL34 charge cluster
mutants, attempts were unsuccessful due to the inability to
amplify the recombinant viruses on UL34-complementing
cells. This failure during amplification suggested that the mutant
pUL34 expressed from the viral genome might interfere with the
function of the wt pUL34 expressed from the complementing cell
genome. To test this hypothesis for the CL04 mutant, replicate
cultures of Vero cells were transfected with a constant amount of
wt pUL34-expressing plasmid pRR1072Rep and with increasing
TABLE 1. Primer used for construction and analysis
of mutant BACs
Primer name Primer sequence
UL34 test Fwd.................5?-CAGCGAACTTTACGGGACACAATCC-3?
UL34 test Rev..................5?-ATAGGCTGCGGGGTAAACGTGT-3?
R229L Gm Fwdc..............5?- GTACGTCATCTTTCCCGGCACGTCCGCCCA
R229L Gm Revc..............5?-CGAACCGGTACCCCGGGCACGCGGTGAGC
Gm mid For.....................5?-GGTCGTGAGTTCGGAGACGTAGC-3?
Gm mid Rev.....................5?-CACTACGCGGCTGCTCAAACC-3?
UL31 unique For.............5?-GTACGTCATCTTTCCCGGCACG-3?
UL31 unique Rev............5?-CGAACCGGTACCCCGGGCAC-3?
UL31 test Fwd.................5?-TGCCCCTGGTGAAGACCAC-3?
UL31 test Rev..................5?-GCTACGGCGGAGGAAACTCG-3?
aUnderlined sequences have homology to the Kanrcassette. Sequences not
underlined have homology to the UL34 gene.
bUnderlined sequences have homology to the Gmrcassette.
cUnderlined sequences have homology to the UL31 gene. Lowercase indi-
cates nucleotides altered to create the R229L mutation and an AseI restriction
enzyme cleavage site. Sequences not underlined have homology to the Sce-Gmr
3924 ROLLER ET AL.J. VIROL.
amounts of test plasmids that expressed a fully functional charge
cluster mutant (CL19), the charge cluster mutant for which re-
combinant virus isolation was possible (CL13), or the putative
dominant-negative mutant (CL04). Cultures were also trans-
fected with pRR1072, which carries the enhanced green fluores-
cent protein (EGFP) gene in place of UL34 in the 1072Rep
background, so that the total amount of UL34 promoter-
containing plasmid was constant in each culture. Transfected
cultures were infected with the UL34-null virus, and produc-
tion of infectivity 20 h after infection was measured by a plaque
assay using complementing cells. As shown in Fig. 2, expres-
sion of increasing amounts of functional pUL34 (CL19) along
with wt pUL34 results in increasingly efficient complementa-
tion of the UL34-null virus (Fig. 2A). Expression of increasing
amounts of nonfunctional but apparently noninterfering mu-
tant CL13 does not interfere with the ability of wt pUL34 to
complement the UL34-null virus (Fig. 2B). Increasing expres-
sion of the CL04 mutant, however, results in a dose-dependent
decrease in the ability of wt pUL34 to complement, suggesting
that the CL04 mutation exhibits a dominant-negative pheno-
type (Fig. 2C).
UL34 expression by stable cell lines. To better study CL04
function during infection, clonal cell lines expressing either wt
pUL34 or CL04 pUL34 under the control of its own promoter-
regulatory sequences were constructed by stable transfection
of Vero cells. Two stable lines called RepAC and CL04AI,
expressing wt and CL04 UL34, respectively, were isolated. To
compare their pUL34 expression relative to each other and to
wild-type virus infection, Vero cells were infected with 10 PFU/
cell HSV-1(F), and Vero, wtUL34-expressing, and CL04
UL34-expressing cells were infected with 10 PFU/cell UL34-
null virus for 18 h. Total protein was determined for each
extract, and equivalent amounts were separated on an SDS-
PAGE gel, transferred to nitrocellulose, and probed for
pUL34. HSV-1 scaffolding protein was used as a loading con-
trol (Fig. 3). Neither of the clonal cell lines infected with
UL34-null virus expressed as much pUL34 as wt virus-infected
cells, but CL04 pUL34 expression was higher than that of wt
pUL34 expression. This ensures that any putative CL04 phe-
notype observed in comparison of these two cell lines could not
be ascribed to lower levels of CL04 protein expression.
Characterization of the CL04 mutation effect on virus
growth. To test the ability of CL04 pUL34-expressing cells to
support plaque formation, serial dilutions of wt HSV-1(F) and
UL34-null virus were plated on Vero cells, wt pUL34-express-
ing cells, and CL04 pUL34-expressing cells. Plates were exam-
ined 48 h after infection, and digital images of representative
plaques are shown in Fig. 4. HSV-1(F) forms robust plaques
with similar efficiencies on all three of the cell lines tested (Fig.
4A to C). In contrast, UL34-null virus formed only tiny foci of
one or a few infected cells on noncomplementing Vero cells
(Fig. 4D, black arrowhead) but formed large plaques on cells
expressing wt pUL34 (Fig. 4E). UL34-null virus formed slightly
smaller plaques on wild-type UL34-expressing cells than on
wild type virus (compare Fig. 4B and E), probably due to lower
expression of pUL34 from the cell line genome than from the
FIG. 2. CL04 is a dominant-negative mutant of UL34. Graphs of complementation indices for combinations of cotransfected plasmids are
shown. The complementation index for each condition was calculated by dividing the infectivity produced in each culture by the infectivity
produced when the wt UL34-expressing plasmid is transfected in the absence of mutant UL34. The numbers below each bar indicate the number
of ng ? 100 of the corresponding plasmid transfected into the culture. In addition to the transfected plasmids indicated below each graph, all
cultures were transfected with pCMV? and assayed for ?-galactosidase activity to control for differences in transfection efficiency. Transfection
efficiencies were similar (within 30%) in all samples. One representative of three independent experiments is shown.
VOL. 84, 2010 NOVEL pUL31/pUL34 INTERACTION IN MEMBRANE CURVATURE3925
viral genome. UL34-null virus formed two populations of
plaques on CL04-expressing cells. At dilutions for which
plaques were easily visible on wt UL34-expressing cells, only
small aggregations of a few infected cells could be seen on
CL04-expressing cells (examples are indicated with black ar-
rowheads in Fig. 4F). However, at high virus inputs (10,000
PFU/well), a few robust plaques developed on CL04-express-
ing cells (an example is indicated with the white arrowhead in
Fig. 4F), suggesting the existence of a small subpopulation of
the UL34-null virus that could replicate using CL04 mutant
pUL34. After infection of CL04-expressing cells with UL34-
null virus, robust plaques were purified by three rounds of
plaque picking and amplifying. One of the amplified viruses,
called CL04Rev, was selected for further analysis. Like its
parent, vRR1072(TK?), CL04Rev could form only tiny foci of
infection on noncomplementing Vero cells (Fig. 4G, black
arrowhead) but efficiently formed large plaques on both wild
type- and CL04-expressing cells (Fig. 4H and I), indicating that
the UL34 deficiency in this virus could be complemented by
either wt or CL04 mutant UL34.
The single-step growth kinetics of HSV-1(F), vRR1072(TK?),
and CL04Rev were measured with Vero, wild-type UL34-ex-
pressing RepAC, and mutant UL34-expressing CL04AI cells
(Fig. 5). As shown previously, the UL34-null virus vRR1072
FIG. 3. Expression of wild-type and CL04 mutant UL34 by stable
cell lines. Digital images of Western blots are shown. Vero cells (lanes
1 and 2) or cells stably expressing wt UL34 (lane 3) or CL04 UL34
(lane 4) were infected with wt HSV-1(F) (lane 1) or UL34-null
vRRR1072(TK?) virus (lanes 2 to 4). Blotted infected cell proteins
were probed for either scaffolding protein (top) or UL34 (bottom).
FIG. 4. Formation of plaques on wt UL34 or CL04 mutant UL34-expressing cell lines. Digital micrographs of infected cell monolayers stained
with amido black are shown. The cell line infected is indicated above each column of panels, and the infecting virus is indicated to the left of each
row. Black arrowheads indicate minute plaques comprised of a few cells. The white arrowhead (F) indicates a robust plaque formed by an
extragenic suppressor virus.
3926 ROLLER ET AL.J. VIROL.
(TK?) replicates poorly on Vero cells (29). CL04Rev replicates
no better than vRR1072(TK?), indicating that it is not different
from its parent in ability to replicate in the absence of pUL34. All
three viruses replicate efficiently on RepAC cells, indicating that
both UL34 null and CL04Rev viruses can be complemented by
wild-type pUL34. Both UL34-null viruses produce about 10-fold
less virus than wt HSV-1(F), however, probably because of the
low level of pUL34 expressed by the complementing cells.
CL04Rev replicates efficiently on CL04-expressing cells, achiev-
ing a peak titer similar to that attained by wt virus, whereas
vRR1072(TK?) achieves a peak titer about 100-fold less than
that of wt virus. This result indicates that CL04Rev can be effi-
ciently complemented by CL04 UL34, while its parent cannot.
An extragenic suppressor of the CL04 mutation maps to
UL31. Marker transfer experiments were performed by co-
transfection of vRR1072(TK?) nuclear lysate and gel-purified
restriction fragments from the CL04Rev genome into RepAC
cells. Viral progeny of cotransfection were plated on CL04-
expressing cells and scored for formation of robust plaques.
Only the 40.9-kb HindIII fragment, containing all of the UL20
through UL36 genes and a portion of the UL19 gene, repro-
ducibly transferred the robust plaque phenotype. Genes having
reported connections to DNA packaging or nuclear egress
(UL19, UL20, UL25, UL26, UL28, UL31, UL33, UL35, and
UL36) were then sequenced from the parental vRR1072
(TK?) strain and from CL04Rev. Only UL31 showed a se-
quence difference compared to the parental virus—a single
nucleotide substitution, changing the arginine at position 229
to leucine. Revertant viruses were isolated in six independent
selections and purifications. All six carried the same amino
acid substitution in UL31, suggesting that the R229L mutation
was necessary for the revertant phenotype. Two approaches
were used to determine whether the R229L mutation in UL31
might be sufficient for the revertant phenotype. For the first
approach, marker transfer was performed by transfecting
cloned plasmids containing either the wt UL31 sequence or
containing an engineered R229L mutation and genomes of the
UL34-null virus vRR1072(TK?) onto cells that express wt
UL34. The viral progeny recovered were then tested by plating
at low multiplicity on CL04-expressing cells and looking for the
ability to form robust plaques. Only transfection with the mu-
tant UL31 resulted in formation of robust plaques on CL04-
expressing cells (compare Fig. 6C with A and B), suggesting
that the UL31 R229L mutation was sufficient to suppress the
CL04 phenotype. To confirm this result, a second approach
using transient complementation of UL34-null virus was used.
Replicate cultures of Vero cells were transfected with one of
six plasmids that express either wt pUL34 alone, CL04 pUL34
alone, both wt pUL34 and wt pUL31, both wt pUL34 and
pUL31R229L, both CL04 pUL34 and wt pUL31, or both CL04
pUL34 and pUL31R229L. One day after transfection, cultures
were infected with UL34-null virus for 18 h, and viral infectiv-
ity was measured by plaque assay on wt pUL34-expressing
RepAC cells (Fig. 6D). As previously shown, wt pUL34 com-
plements replication of a UL34-null virus much better than
CL04 pUL34. We expected that if the UL31 R229L mutant
were sufficient to suppress the CL04 UL34 mutant phenotype,
then complementation by the plasmid expressing both CL04
pUL34 and the pUL31 R229L mutant would be significantly
better than that of the plasmid that expressed CL04 pUL34
and wt pUL31. Surprisingly, CL04 pUL34 complemented
poorly, regardless of whether it was expressed with wt pUL31
or the pUL31 R229L mutant.
The conflicting results of the marker transfer and comple-
mentation experiments led us to test for sufficiency of the
R229L mutation by characterization of a recombinant virus
rescued from a BAC that had been mutagenized so that it was
UL34 null and carried the UL31 R229L mutation. We con-
structed a UL34-null BAC with the same UL34 deletion as that
in vRR1072(TK?) and then introduced the R229L mutation
FIG. 5. Single-step growth of wild-type and mutant viruses on complementing and noncomplementing cells. Replicate cultures of Vero (A),
RepAC (B), or CL04AI cells (C) were infected at an MOI of 5 with HSV-1(F) (open circles), vRR1072(TK?) (closed circles), or CL04Rev (open
squares). Residual virus was removed or inactivated with a low-pH wash, and at the indicated times, total culture virus was titrated on RepAC cells.
Virus yields are expressed as the number of PFU per milliliter. Each data point represents the mean of three independent experiments. Error bars
indicate the range of values.
VOL. 84, 2010 NOVEL pUL31/pUL34 INTERACTION IN MEMBRANE CURVATURE3927
into the UL31 gene. Wild-type and UL34-null and UL34-null/
UL31 R229L mutant viruses were rescued by transfection of
the BAC clones into UL34-expressing complementing cells.
Introduction of the R229L mutation into the UL31 locus was
accompanied by introduction of an AseI restriction enzyme
site. To confirm the presence of the mutation, the UL31 locus
was PCR amplified from the wild-type and UL34-null/UL31
R229L mutant viruses, and the PCR products were digested
with AseI (Fig. 7A). Amplification from both wt and mutant
BAC DNAs resulted in a product of 672 bp, as expected (Fig.
7, lanes 2 and 4). The wild-type PCR product was not digested
(Fig. 7, lane 3), whereas the UL34-null/UL31 R229L mutant
PCR product was digested to release the expected fragments of
435 and 237 bp (Fig. 7, lane 5). To confirm the presence of the
Kan replacement of UL34 sequences, the UL34 locus was PCR
amplified from wild-type, UL34-null, and UL34-null/UL31
R229L mutant BAC genomes, and the PCR products were
digested with HindIII (Fig. 7B). The wild-type UL34 locus
does not contain a HindIII site, but the introduced Kanrcas-
sette contains a single HindIII site. As expected, the wild-type
sequence was not digested with HindIII (Fig. 7, lanes 2 and 3),
whereas the UL34-null (not shown) and UL34-null/UL31
R229L mutant PCR products were digested into the expected
fragments of 1,117 and 781 bp (Fig. 7, lane 5).
To determine whether the presence of the R229L mutation
confers the ability to grow using CL04 UL34, Vero, wild-type
UL34-expressing, and CL04-expressing cells were infected at
low multiplicity with BAC-derived wild-type, UL34-null, and
UL34-null/R229L mutant viruses. After two days, plaques were
detected by indirect immunofluorescence using primary anti-
body directed against glycoprotein D. Representative plaques
are shown in Fig. 7C to K. BAC-derived wild-type virus was
able to form plaques on all three cell lines (Fig. 7C, F, and I),
although the plaques observed with CL04-expressing cells (Fig.
7I) were slightly smaller than those seen with Vero or wild-type
UL34-expressing cells. Like vRR1072(TK?), the UL34-null
BAC-derived virus formed large plaques on wild-type UL34-
expressing cells (Fig. 7G) but showed only single infected cells
or minute plaques of only a few cells on either Vero or CL04-
expressing cells (Fig. 7D and J, respectively). The UL34-null/
UL31 R229L mutant BAC-derived virus was unable to form
plaques on Vero cells (Fig. 7E) but efficiently formed large
plaques on cells expressing either wild-type or CL04 UL34
(Fig. 7H and K, respectively), suggesting that in the context of
a recombinant virus, the R229L mutation is sufficient to allow
efficient viral replication using the CL04 mutant UL34.
Localization of the CL04 mutant UL34. We previously re-
ported that the CL04 mutant pUL34 localized normally to the
nuclear envelope in transient transfection/infection assays. The
mapping of an extragenic suppressor mutation to UL31 after
selection of virus on a cell line stably expressing CL04 led us to
examine the localization of the CL04 mutant protein in in-
fected cells. CL04-expressing cells or wt UL34-expressing cells
were infected with either vRR1072(TK?) (UL34-null mutant
and UL31 wt) or CL04Rev (UL34-null mutant and UL31
R229L mutant) at an MOI of 10 for 16 h, fixed, and processed
for immunofluorescence using antibody directed against
pUL34 (Fig. 8). As reported previously, wt pUL34 in a wild-
type virus-infected cell localizes almost exclusively to the nu-
clear envelope and is distributed evenly (Fig. 8A). Consistent
FIG. 6. Marker transfer and complementation assays for UL31
R229L mutant function. Digital micrographs of CL04AI cell monolay-
ers infected with the progeny of cotransfection of UL34-null
vRR1072(TK?) viral genomes with either no plasmid (A), plasmid
carrying wt UL31 (B), or plasmid carrying UL31R229L (C). (D) Graph
of complementation indices for transfected plasmids is shown.
Complementation index for each plasmid was calculated by dividing
the infectivity produced in each culture by the infectivity produced
when plasmid that expressed wt UL34 alone was transfected. Plasmids
for each condition are the following: no UL34/no UL31, pRR1072; wt
UL34/no UL31, pRR1072Rep; CL04/no UL31, pRR1162; wt UL34/wt
UL31, pRR1348; wt UL34/UL31 R229L, pRR1349; CL04/wt UL31,
pRR1350; CL04/UL31 R299L, pRR1351. Each bar represents the
mean of five independent experiments. Error bars indicate the stan-
3928 ROLLER ET AL.J. VIROL.
FIG. 7. Growth of BAC-derived UL34-null, UL31 R229L mutant virus on wt and mutant UL34-expressing cells. (A and B) Digital images show
electrophoretically separated PCR products that are either digested with restriction enzyme (lanes 3 and 5) or undigested (lanes 2 and 4). The sizes
of the undigested and digested products are indicated on the right of the gel. Lambda BstEII digest size standards are shown in lane 1, and the
sizes of standard molecular weight bands are indicated on the left of the gel. (A) PCR products from the UL31 locus in virus rescued from wild-type
HSV-1 BAC (lanes 2 and 3) or UL34-null/UL31 R229L mutant (A, lanes 4 and 5) are shown. (B) PCR products from the UL34 locus in the same
viruses are shown. Digital micrographs of immunofluorescently stained plaques formed on Vero (C to E), wt UL34-expressing RepAC cells (F to
H), or CL04-expressing CL04AI cells (I to K) by viruses rescued from wt HSV-1(F) BAC (C, F, and I), UL34-null/UL31 wt BAC (D, G, and J),
or UL34-null/UL31 R229L mutant BAC (E, H, and K).
VOL. 84, 2010 NOVEL pUL31/pUL34 INTERACTION IN MEMBRANE CURVATURE3929
with earlier transient transfection/infection assays, CL04
pUL34 expressed in the CL04AI cell line shows the same
localization as wt pUL34 when expressed in the context of a
wild-type infection (Fig. 7B). The localization of both wt and
CL04 pUL34 changes when they are expressed in cells infected
by the CL04Rev virus carrying the UL31 R229L mutation (Fig.
8C and D). In both cases, pUL34 is still found almost exclu-
sively at the nuclear envelope, but it is distributed in discrete
CL04 mutant UL34 envelopment. In order to characterize
the defect in CL04 UL34, Vero cells or CL04-expressing cells
were infected with the UL34-null mutant virus at an MOI of 10
for 20 h and then prepared for analysis by transmission elec-
tron microscopy (Fig. 9). As shown previously, Vero cells in-
fected with UL34-null virus do not produce cell surface or
cytoplasmic virions or capsids, even though nuclear empty and
DNA-containing capsids are present (Fig. 9A). These nuclear
capsids are found distributed throughout the nucleoplasm, and
though a few may be closely associated with the nuclear mem-
brane, most capsids are not found in close association with the
INM (Fig. 9A; Table 2). CL04-expressing cells infected with
the UL34-null virus also produce neither cell surface nor cy-
toplasmic virions or capsids but differ from normal Vero cells,
in that most accumulated nuclear capsids are found in close
association with the INM (Fig. 9B; Table 2). In many cases, the
association of capsids with INM was accompanied by slight
curvature of the nuclear membrane (Fig. 9B, inset), a feature
not seen with UL34-null-infected Vero cells. Interestingly,
close association of capsids with the nuclear envelope in these
cells shows no dependence upon packaged DNA. Three types
of capsids may be distinguished in these preparations: (i)
DNA-containing capsids (C capsids) appearing as capsids with
an electron-dense center (black arrowhead in Fig. 9B), (ii)
empty capsids having an electron-transparent interior (A cap-
FIG. 8. Localization of wt UL34 and CL04 mutant UL34 in trans-
fected, infected cells. Digital confocal images of cells transfected with
pRR1072Rep expressing wt (A and C) or pRR1162 expressing CL04
mutant UL34 (B and D), subsequently infected with either
vRR1072(TK?) (A and B) or CL04RevB (C and D), and immunofluo-
rescently stained for UL34 are shown.
FIG. 9. TEM analysis of nuclear egress from cells that express
CL04 UL34. Digital micrographs show Vero (A) or CL04AI (B) cells
infected with the UL34-null virus vRR1072(TK?) for 20 h. Black
arrowheads (A) point to examples of intranuclear capsids. Black ar-
rowheads (B) point to examples of capsids docked at the INM. The
white arrowhead (B) points to an instance of a capsid docked with
slight curvature of the membrane. Scale bars are shown at the lower
left of each panel.
TABLE 2. Percentage of INM-associated capsids in UL34-infected
Vero and CL04AI cells
aINM-associated capsids are defined as capsids having one side no more than
one-fourth of a capsid diameter from the inner nuclear membrane.
3930 ROLLER ET AL.J. VIROL.
sids) (white arrowhead in Fig. 9B), and (iii) capsids with mod-
erate electron density in the interior (B capsids) (gray arrow-
head in Fig. 9B). For all capsid types, the frequency of such
capsids in close association with the INM is equivalent to their
frequency among total intranuclear capsids (Table 3).
Interactions between deletion mutants of UL34 and UL31.
Reversion of a point mutation in the coding sequence of one
protein by an extragenic suppressor in the coding sequence
of another strongly suggests that the proteins encoded by
the two mutant genes interact and that the interaction, or its
functional consequence, is mediated at least in part by the
altered amino acids (11). pUL34 and pUL31 are known to
interact, but the previously described interacting domains
have been mapped to the central part of pUL34 and the
N-terminal half of pUL31 (summarized in Fig. 1). Both the
CL04 mutation in UL34 and the R229L mutation in UL31
are outside these previously characterized interaction do-
mains (Fig. 1, lines 1, 2, 9, and 10). In order to look for an
additional interaction between pUL34 and pUL31, medi-
ated by N-terminal sequences of UL34 and C-terminal se-
quences of UL31, deletion mutants were constructed that
lack the previously characterized interaction domains. In-
ternal deletions of wt and CL04 UL34 that lack amino acids
91 to 228 were constructed (Fig. 1, lines 3 and 4). In order to
exclude a possible contribution of the UL34 transmembrane do-
main to this interaction, deletion mutants of UL34 and CL04 that
the UL34 transmembrane domain is replaced by that of the cel-
lular nuclear lamina-associated protein emerin were also con-
structed (Fig. 1, lines 5 to 8). wt and R229L mutant UL31 N-
terminal truncations that delete amino acids 2 to 126 and that add
a FLAG-His tag to the C terminus of the protein were con-
structed (Fig. 1, lines 11 and 12).
Plasmid transfection. Transfection of the plasmids described
above, alone and in combination, revealed the following.
(i) wt pUL34. wt pUL34 lacking amino acids 91 to 229 or
lacking amino acids 91 to 275 and carrying the emerin transmem-
brane domain when expressed alone localizes like full-length wt
pUL34 (compare Fig. 10A through C). As shown previously,
UL34 expressed in the absence of any UL31 construct is found
mostly on cytoplasmic membranes, although some in a patchy
distribution on the nuclear membrane can be found as well. All of
the UL34 constructs tested here show very similar localizations,
although pUL34 lacking amino acids 64 to 275 shows a greatly
reduced tendency to localize in patches (Fig. 10D).
(ii) Full-length wt pUL31 and N-terminal truncation mu-
tant. Whereas full-length wt pUL31 localizes exclusively to the
nucleoplasm when expressed alone (Fig. 10E), the N-terminal
truncation mutant shows some concentration in the nucleus
but also shows diffuse cytoplasmic localization (Fig. 10F), sug-
gesting that sequences required for specific pUL31 nuclear
targeting are located in the N-terminal 125 amino acids.
TABLE 3. Percentage of docked A, B, and C capsids
% of total
% of docked
FIG. 10. Interaction between wild-type and mutant UL34 deletion constructs and the C-terminal domain of UL31. Digital confocal micrographs
of transfected Vero cells are shown. Cells were transfected either with a single plasmid (A to F) or with combinations of two plasmids (G to O)
The plasmids used are those depicted in Fig. 1 and are indicated within each panel. All UL31 constructs were FLAG tagged and were detected
with anti-FLAG primary antibody and either green (E-F, G to I, and K to N) or red (J and O) secondary antibody. All UL34 constructs were
detected with anti-UL34 primary antibody and red secondary antibody. EGFP emerin was detected by GFP fluorescence in green (J and O). For
combination transfections, the color of the text in the panel corresponds to the color of the antibody used to detect that construct. Only merged
images are shown.
VOL. 84, 2010 NOVEL pUL31/pUL34 INTERACTION IN MEMBRANE CURVATURE3931
(iii) Colocalization of pUL34 constructs with N-terminally
truncated pUL31. pUL34 constructs lacking the previously
identified UL31 interaction domain can colocalize with N-
terminally truncated pUL31. Expression of the N-terminal de-
letion of wt pUL31 with full-length pUL34 (Fig. 10K), inter-
nally deleted pUL34 (Fig. 10L), or C-terminally truncated
pUL34 lacking amino acids 91 to 275 (Fig. 10M) results in
recruitment of the truncated pUL31 to sites of pUL34 local-
ization and complete colocalization of the two proteins. While
simple colocalization of two proteins may occur by coinci-
dence, the complete recruitment of the UL31 construct from
its normal site of localization to the site of UL34 localization is
strongly suggestive of a stable, physical interaction between aa
1 to 90 of pUL34 and aa 126 to 305 of pUL31. Deletion of
amino acids 65 to 275 from pUL34, however, resulted in loss
of ability to recruit truncated pUL31 from the nucleus and loss
of colocalization (Fig. 10N), suggesting that sequences of
pUL34 between aa 65 and 90 are necessary for this interaction.
(iv) Failure of pUL34 deletion constructs to colocalize with
full-length pUL31. All pUL34 constructs lacking the previously
identified UL31 interaction domain do not colocalize with full-
length pUL31. As reported by Liang and Baines, internally
deleted wt pUL34 fails to colocalize with full-length pUL31
and neither protein is properly targeted to the nuclear mem-
brane (Fig. 10G) (16). Failure to colocalize with full-length
pUL31 was also seen for the pUL34 truncation mutants that
lack amino acids 91 to 275 (Fig. 10H) and 64 to 275 (Fig. 10I).
(v) Similar patterns of colocalization and recruitment. The
same patterns of colocalization and recruitment were seen
regardless of whether wt pUL34, CL04 pUL34, wt pUL31, or
the pUL31 R229L mutant was expressed (Table 4), suggesting
that the CL04 mutation does not result in loss of interaction.
As a specificity control, EGFP-emerin was cotransfected
with full-length and truncated pUL31 to see whether these
proteins could be relocalized by overexpression of any type II
membrane protein or if the emerin transmembrane domain
could mediate relocalization of UL31 constructs in the absence
of UL34 sequences (Fig. 10J and O). Coexpression of emerin
at levels high enough to induce formation of large cytoplasmic
aggregates did not perturb the localization of either pUL31
Recruitment of UL31?2-126 by UL34?91-275EmTM strongly
suggests a stable physical interaction between these two proteins.
two constructs alone and in combination and then immunopre-
cipitated with anti-FLAG antibody. Unimmunoprecipitated input
samples and immunoprecipitated proteins were separated on
SDS-PAGE gels, blotted to nitrocellulose, and then probed with
antibodies directed against the FLAG epitope (for detection of
the UL31 construct) or anti-UL34 (Fig. 11). Expression of both
constructs was detected in transfected cells (Fig. 11, lanes 1 to 3),
and UL31?2-126 expressed alone or in combination with
UL34?91-275EmTM was efficiently immunoprecipitated using
anti-FLAG (Fig. 11, lanes 4 to 6, top). UL34?91-275EmTM was
not immunoprecipitated by anti-FLAG when expressed alone
(Fig. 11, lane 5, bottom) but was coimmunoprecipitated when
coexpressed with UL31?2-126.
A dominant-negative phenotype for a mutation usually re-
flects the ability of the mutation to disrupt a subset of the
essential interactions made by the wild-type form of the pro-
tein. The existence of a dominant-negative UL34 mutation
suggests that wt UL34 makes at least two essential interactions,
one of which is altered by the mutation. To this point, the only
essential interaction known for the UL34 protein was the in-
teraction with UL31 that mediates the targeting of both pro-
teins to the nuclear membrane. This interaction is not dis-
rupted by the CL04 mutation, inasmuch as targeting of CL04
pUL34 to the nuclear membrane occurs normally in the pres-
ence of wt pUL31 (Fig. 7). pUL34 must make another essential
Evidence presented here suggests that in addition to the
pUL34/pUL31 interaction that mediates nuclear envelope tar-
geting, pUL34 and pUL31 make another interaction that is
required for a step in nuclear egress subsequent to targeting to
the nuclear envelope. There are two lines of evidence in sup-
TABLE 4. Interactions of UL34 and UL31 wild-type and mutant constructs
UL31 ?2-126 mutant
UL31 R229L mutant
UL31 R229L ?2-126 mutant
FIG. 11. Digital images of Western blots are shown. Unfraction-
ated lysates (lanes 1 to 3) or proteins immunoprecipitated with anti-
FLAG antibody (lanes 4 to 6) were separated by SDS-PAGE and
probed with antibody directed against FLAG epitope to detect UL31
(top row) or with antibody directed against pUL34 (bottom row).
3932ROLLER ET AL.J. VIROL.
port of this hypothesis. First, the CL04 mutation can be extra-
genically suppressed, and all suppressor viruses carry a muta-
tion in UL31 that converts the arginine at position 229 to a
leucine, thereby converting a positive-charge side chain to a
hydrophobic side chain of similar bulk. The CL04 mutation
converts two negatively charged residues, aspartic acid 35 and
glutamic acid 37, to alanine. One of these two, aspartic acid 35,
is mutated to alanine in the fully functional charge cluster
mutant CL03, suggesting that the critical mutated residue in
CL04 is glutamic acid 37 (2). It is tempting to speculate that a
charge interaction between glutamic acid 37 of UL34 and ar-
ginine 229 of UL31 is critical for the function of the pUL34/
pUL31 complex. Second, the regions of pUL31 and pUL34
sequences that contain the mutations physically interact when
expressed in constructs that lack the interaction domains that
mediate nuclear envelope targeting.
Three experimental approaches designed to determine
whether the R229L mutation in pUL31 is sufficient to suppress
the CL04 mutation in pUL34 gave contradictory results (Fig. 5
and 6). The assays used differ, in that infected cells in the
complementation assay may express both mutant suppressor
pUL31 (from the complementing plasmid) and wt pUL31
(from the infecting virus), whereas in the marker transfer assay
and assays with BAC recombinant viruses, only one type of
pUL31 is expressed in each infected cell. It is possible that
nonproductive interactions between mutant pUL34 and wild-
type pUL31 will “poison” the system, even when otherwise
productive mutant pUL34-suppressor pUL31 interactions can
occur. Consistent with this possibility, the interaction assays
shown in Fig. 9 suggest that the CL04 mutant pUL34 can
interact with wt pUL31. This may suggest that the CL04 mu-
tation does not disrupt the physical interaction between the N
terminus of pUL34 and the C terminus of pUL31 but does
prevent the functional consequence of that interaction in nu-
The interaction between the N-terminal domain of pUL34
and the C-terminal domain of UL31 occurs when amino acids
1 to 90 of UL34 are present, but not when amino acids 1 to 63
are present, suggesting that the C-terminal boundary of the
interaction domain is located between amino acids 64 and 90.
Interestingly, the pUL34 N terminus/pUL31 C terminus
(UL34 N/UL31 C) interaction does not occur in the context of
full-length pUL31 when pUL34 and pUL31 are the only viral
proteins expressed (Fig. 9). For this interaction to function in
nuclear egress, interaction with some other infected cell struc-
ture, perhaps the docking capsid, must change the conforma-
tion of the full-length pUL34 or pUL31 to allow the UL34
N/UL31 C interaction to take place.
The CL04 mutation in UL34 reveals a function for pUL34
after capsid docking. In Vero cells infected with UL34-null
virus, virus capsids are unable to efficiently egress from the
nucleus, and they accumulate in the nucleoplasm but not in
close association with the INM. trans-complementation of the
UL34-null virus with CL04 UL34 also results in the failure of
nuclear egress but leads to the accumulation of a specific
egress intermediate—capsids docked at the INM. This pheno-
type suggests two specific functions for UL34. (i) The obser-
vation that capsids cannot dock in the absence of pUL34 but
can when a mutant pUL34 is present suggests that the presence
of pUL34 is required for some step in envelopment leading to
capsid docking. This step might be the docking step itself, it
might be that pUL34-mediated nuclear lamina disruption is
a necessary prerequisite to capsid docking, or it might be
both. (ii) Accumulation of docked capsids in cells that ex-
press CL04 pUL34 suggests that the CL04 mutation disrupts
a pUL34 function required for the step immediately follow-
ing capsid docking at the INM, perhaps curvature of the
nuclear membrane around the capsid. A function for a
pUL34/pUL31 interaction in nuclear membrane curvature
would be consistent with the results of Klupp et al., who
observed that expression of pUL34 and pUL31 of HSV or
pseudorabies virus (PRV) in the absence of other viral pro-
teins can result in vesicularization of the INM into the
perinuclear space in a membrane curvature event topolog-
ically similar to nuclear envelopment (12).
One model that accounts for observations reported here and
those of Klupp et al. would include the following steps leading
up to membrane curvature around the capsid. (i) Interaction
between the previously mapped interaction/targeting domains
of pUL34 and pUL31 mediates targeting to the nuclear mem-
brane. At this stage, interaction between the pUL34 N domain
and the pUL31 C domain is blocked by sequences in the
N-terminal domain of pUL31. (ii) Following nuclear lamina
disruption, capsids dock at the inner nuclear membrane in a
process dependent upon pUL34 and perhaps on other viral or
cellular factors. (iii) Capsid docking triggers a change in the
structure of the pUL34/pUL31 complex allowing interaction
between the pUL34 N domain and the pUL31 C domain. This
interaction is not sensitive to the CL04 mutation in pUL34. (iv)
The pUL34N/pUL31C interaction permits multimerization of
pUL31/pUL34 complexes (perhaps by induction of a confor-
mational change in one or both proteins), and geometry of
these interactions drives curvature of the nuclear membrane
around the capsid. This oligomerization-enabling step is dis-
rupted by the CL04 mutation in pUL34 and can be suppressed
by the R229L mutation on pUL31. This model accounts for
several features of our observations. First, it is consistent with
the dominant-negative character of the CL04 mutation. The
presence of a noninteracting pUL34 could presumably poison
the formation of multimers, even in the presence of wt pUL34.
This model is also consistent with the failure of the pUL31
R229L mutant to suppress the CL04 phenotype when wild-type
pUL31 is present, for the same reason.
One of the intriguing properties of the nuclear egress system
is its selectivity. Primary envelopment apparently requires
completion of the DNA packaging process. In wt virus-infected
cells, empty capsids are enveloped more rarely than full cap-
sids, and a variety of viral mutants that synthesize capsids but
fail to complete the DNA packaging process do not efficiently
envelop the capsids that are produced (1, 4, 5, 10, 18, 25, 31).
The phenotype of the CL04 mutation was surprising, in that
there was apparently no selectivity in the type of capsid that
could dock at the INM. This suggests either that selection
occurs subsequent to docking or that the CL04 mutation im-
pairs docking selectivity. The first of these explanations seems
unlikely since, if true, docked, empty capsids should accumu-
late at the INM in wild-type infection, and this has not been
VOL. 84, 2010NOVEL pUL31/pUL34 INTERACTION IN MEMBRANE CURVATURE 3933
ACKNOWLEDGMENTS Download full-text
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. We are grateful to Yasushi Kawaguchi for provid-
ing the HSV-1(F) BAC and to Klaus Osterrieder for other plasmids
required for BAC mutagenesis.
These studies were supported by the University of Iowa and Public
Health Service award AI 41478. S.L.B. was supported by the Washburn
University Mary B. Sweet Sabbatical Program. S.H. was supported by
NSF REU site grant DBI-0097361.
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3934ROLLER ET AL.J. VIROL.