JOURNAL OF VIROLOGY, Sept. 2009, p. 9567–9576
Copyright © 2009, American Society for Microbiology. All Rights Reserved.
Vol. 83, No. 18
The Flexible Loop of the Human Cytomegalovirus DNA Polymerase
Processivity Factor ppUL44 Is Required for Efficient DNA
Binding and Replication in Cells?‡
Gualtiero Alvisi,1,2* Daniela Martino Roth,2Daria Camozzi,1Gregory S. Pari,3Arianna Loregian,4
Alessandro Ripalti,5† and David A. Jans2,6†
Dipartimento di Ematologia e Scienze Oncologiche L.A. Seragnoli, Universita ` Degli Studi di Bologna, Bologna, Italy1; Department of
Biochemistry and Molecular Biology, Monash University, Clayton, Victoria, Australia2; Department of Microbiology and
Immunology and the Cell and Molecular Biology Graduate Program, University of Nevada, Reno, Reno, Nevada3;
Dipartimento di Istologia, Microbiologia e Biotecnologie Mediche, Universita ` di Padova, Padua, Italy4;
Azienda Ospedaliera Universitaria di Bologna Policlinico S. Orsola–Malpighi, Dipartimento di
Ematologia, Oncologia e Medicina di Laboratorio–Unita ` Operativa di Microbiologia, Bologna,
Italia5; and ARC Centre of Excellence in Biotechnology and Development6¶
Received 1 April 2009/Accepted 19 June 2009
Phosphoprotein ppUL44 of the human cytomegalovirus (HCMV) DNA polymerase plays an essential role in
viral replication, conferring processivity to the DNA polymerase catalytic subunit pUL54 by tethering it to the
DNA. Here, for the first time, we examine in living cells the function of the highly flexible loop of ppUL44
(UL44-FL; residues 162 to 174 [PHTRVKRNVKKAP174]), which has been proposed to be directly involved in
ppUL44’s interaction with DNA. In particular, we use a variety of approaches in transfected cells to charac-
terize in detail the behavior of ppUL44?loop, a mutant derivative in which three of the five basic residues
within UL44-FL are replaced by nonbasic amino acids. Our results indicate that ppUL44?loop is functional
in dimerization and binding to pUL54 but strongly impaired in binding nuclear structures within the nucleus,
as shown by its inability to form nuclear speckles, reduced nuclear accumulation, and increased intranuclear
mobility compared to wild-type ppUL44. Moreover, analysis of cellular fractions after detergent and DNase
treatment indicates that ppUL44?loop is strongly reduced in DNA-binding ability, in similar fashion to
ppUL44-L86A/L87A, a point mutant derivative impaired in dimerization. Finally, ppUL44?loop fails to
transcomplement HCMV oriLyt-dependent DNA replication in cells and also inhibits replication in the
presence of wild-type ppUL44, possibly via formation of heterodimers defective for double-stranded DNA
binding. UL44-FL thus emerges for the first time as an important determinant for HCMV replication in cells,
with potential implications for the development of novel antiviral approaches by targeting HCMV replication.
The Betaherpesviridae subfamily member human cytomegalo-
virus (HCMV) is a major human pathogen, causing serious
disease in newborns following congenital infection and in immu-
nocompromised individuals (28, 42). Replication of its double-
stranded DNA (dsDNA) genome occurs in the nuclei of infected
cells via a rolling-circle process mediated by 11 virally encoded
proteins (32, 33), including a viral DNA polymerase holoenzyme,
comprising a catalytic subunit, pUL54, and a proposed processiv-
ity factor, ppUL44 (14). ppUL44 is readily detectable in virus-
strong dsDNA-binding ability (30, 45). Defined as a “polymerase
accessory protein” (PAP) whose function is highly conserved
among herpesviruses, ppUL44 is an essential factor for viral rep-
lication in cultured cells and hence represents a potential thera-
peutic target to combat HCMV infection (39). It is a multifunc-
tional protein capable of self-associating (5, 10), as well as
interacting with a plethora of viral and host cell proteins, in-
cluding the viral kinase pUL97 (29), the viral transactivating
protein pUL84 (15), the viral uracil DNA glycosylase ppUL114
(37), and the host cell importin ?/? (IMP?/?) heterodimer, which
is responsible for its transport into the nucleus (4). The activ-
ities of ppUL44 as a processivity factor, including the ability to
dimerize, as well as bind to, pUL54 and DNA, reside in the
N-terminal portion (26, 45), whereas the C terminus is essen-
tial for phosphorylation-regulated, IMP?/?-dependent nuclear
targeting of ppUL44 monomers and dimers (4–6). Once within
the nucleus, ppUL44 is thought to tether the DNA polymerase
holoenzyme to the DNA, thus increasing its processivity (14).
Recent studies have identified specific residues responsible
for ppUL44 interaction with pUL54, as well as for the inter-
action with IMP?/? and homodimerization (4, 10, 27, 41). The
crystal structure of ppUL44’s N-terminal domain (Fig. 1A)
reveals striking similarity to that of other processivity factors,
such as proliferating cell nuclear antigen (PCNA) and its her-
pes simplex virus type 1 (HSV-1) homologue UL42 (10, 46).
Unlike the PCNA trimeric ring, however, both ppUL44 and
UL42, which bind to dsDNA as dimers and monomers, respec-
tively, have an open structure, which is believed to be the basis
for their ability to bind to dsDNA in the absence of clamp
* Corresponding author. Mailing address: Department of Molecular
Virology, University of Heidelberg, Im Neuenheimer Feld 345, Hei-
delberg 69120, Germany. Phone: 49 622156-6306. Fax: 49 622156-4570.
† A.R. and D.A.J. contributed equally to this study.
‡ Supplemental material for this article may be found at http://jvi
?Published ahead of print on 1 July 2009.
loaders and ATP (9, 10, 46). Both ppUL44 and UL42 share a
very basic “back” face, which appears to be directly involved in
DNA binding via electrostatic interactions (19, 22, 23, 38, 46).
One striking difference between ppUL44 and UL42 is the
presence on the former of an extremely basic flexible loop
(UL44-FL, PHTRVKRNVKKAP174) protruding from the ba-
sic back face of the protein (Fig. 1A). Comparison of ppUL44
homologues from different betaherpesviruses, including human
herpesvirus 6 (HHV-6) and 7 (HHV-7), showed that all possess
similar sequences in the same position (44) (Fig. 1B), implying
A recent study revealed that substitution of UL44-FL basic
residues with alanine residues strongly impairs the ability of a
bacterially expressed N-terminal fragment of UL44 to bind 30-bp
dsDNA oligonucleotides in vitro, suggesting that UL44-FL could
be involved in dsDNA-binding during viral replication (22). How-
ever, the role of UL44-FL in mediating the binding of full-length
UL44 to dsDNA in cells and its role in DNA replication have not
been investigated. We use here a variety of approaches to delin-
eate the role of UL44-FL in living cells, our data revealing that
UL44-FL is not required for ppUL44 dimerization or binding to
the catalytic subunit pUL54 but is crucial for HCMV oriLyt-
dependent DNA replication, being required for the formation of
nuclear aggregates, nuclear accumulation/retention, and DNA
binding of ppUL44. Importantly, ppUL44?loop exhibits a trans-
dominant-negative phenotype, inhibiting HCMV oriLyt-depen-
dent DNA replication in the presence of wild-type ppUL44, pos-
sibly via formation of heterodimers defective for dsDNA binding.
This underlines ppUL44-FL as an important determinant for
HCMV replication in a cellular context for the first time, with
potential implications for the development of novel antiviral ap-
MATERIALS AND METHODS
Generation of molecular graphics images. A molecular graphic image of
ppUL44 was generated by using the UCSF Chimera package (35) from the
Resource for Biocomputing, Visualization, and Informatics at the University of
California, San Francisco (supported by NIH P41 RR-01081).
FIG. 1. The highly conserved flexible loop (residues 162 to 174) within ppUL44 protrudes from ppUL44 basic face and is important for efficient
nuclear accumulation and localization in nuclear speckles. (A) Schematic representation of ppUL44 N-terminal domain (residues 9 to 270, protein data
bank accession no. 1T6L) generated using the Chimera software based on the published crystal structure (10, 35). Color: yellow, ?-sheets; red, ?-helices.
Residues involved in ppUL44 dimerization (P85, L86, L87, L93, F121, and M123), as well as basic residues potentially involved in DNA binding (K21,
R28, K32, K35, K128, K158, K224, and K237), are represented as spacefill in orange and green, respectively. Residues P162 and C175, in black, are
indicated by arrowheads, while residues 163 to 174 are not visible in the electron density maps and could potentially extend in the cavity formed by
ppUL44’s basic face to directly contact DNA. Residues forming ppUL44 connector loop (128–142) are in blue. (B) Sequence alignment between
HCMVUL44-FL and the corresponding region of several betaherpesvirus ppUL44 homologues. The single-letter amino acid code is used, with basic
residues in boldface. (C) COS-7 cells were transfected to express the indicated GFP fusion proteins and imaged live 16 h after transfection using CLSM
and a 40? water immersion objective lens. (D) Quantitative results for the Fn/c and speckle formation for GFP-UL44 fusion proteins. The data for the
Fn/c ratios represent the mean Fn/c relative to each protein indicated as a percentage of the mean Fn/c relative to GFP-UL44wt ? the standard error
of the mean, with the number of analyzed cells in parentheses. (E) HEK 293 cells expressing the indicated GFP-UL44 fusion proteins were lysed,
separated by PAGE, and analyzed by Western blotting as described in Materials and Methods, using either the anti-GFP or the anti-?-tubulin MAbs.
9568ALVISI ET AL.J. VIROL.
Construction of expression plasmids. Plasmid pDNR207-UL44?loop encod-
ing a point mutant derivative of ppUL44 in which three of the five basic residues
within UL44-FL have been mutated (PHTRVKRNVKKAP1743 PHTgVngNV
KKAP174), was generated by using a QuikChange mutagenesis kit (Stratagene)
according to the manufacturer’s recommendations, with appropriate oligonucle-
otide pairs and plasmid pDNR207-UL44 as a template (5). Plasmid pDNR207-
UL44?loop was used to perform LR Gateway system (Invitrogen) recombination
reactions with the Gateway system compatible expression plasmids pDEST-
FLAG, pEPI-DEST-GFP (36), and pBkCMV-DsRed2 (18), according to the
manufacturer’s recommendations, in order to generate mammalian expression
vectors encoding N-terminally tagged fusion proteins. Plasmids pDEST-FLAG-
UL44, pEGFPC1-H1E, pEPI-GFP-UL44?NLS, pEPI-GFP-UL54(1213-1242),
GFP-UL44L86A/L87A, DsRed2-UL44wt, DsRed2-UL44I135A, and DsRed2-
UL44L86A/L87A have been described (4, 5, 7, 16, 41).
Cell culture and transfection. Daoy and COS-7 cells were maintained in
Dulbecco modified Eagle medium supplemented with 5% (vol/vol) fetal bovine
serum (FBS), 50 U of penicillin/ml, 50 U of streptomycin/ml, and 2 mM L-
glutamine. Human embryonic kidney 293 (HEK 293) and human foreskin fibro-
blast (HFF) cells were cultured in Dulbecco modified Eagle medium supple-
mented with 10% (vol/vol) FBS, 50 U of penicillin/ml, 50 U of streptomycin/ml,
and 2 mM L-glutamine.
For all experiments, cells were treated with trypsin, and 8 ? 104cells were used
to seed respective plates 1 day before transfection, which was performed using
Lipofectamine 2000 (Invitrogen) according to the manufacturer’s specifications.
For live cell imaging by confocal laser scanning microscopy (CLSM), COS-7 cells
were seeded onto coverslips in six-well plates. For Western blotting and nuclear
matrix extraction experiments, HEK 293 cells were seeded in six-well plates, and
for imaging of nuclear matrix-associated proteins, Daoy cells were seeded onto
2-mm glass-bottom Willcodishes (Willcowells). For HCMV oriLyt-dependent
DNA replication assays, HFF cells were plated onto 6-cm-diameter dishes at a
cell density of 105per plate 24 h before transfection that was performed by using
the calcium phosphate method (41).
CLSM/image analysis. To determine the nuclear to cytoplasmic fluorescence
ratio (Fn/c) values relative to GFP-UL44 fusion proteins when expressed in
COS-7 cells, CLSM was used as previously described (4, 5, 7). Cells were ana-
lyzed by using an Olympus Fluoview 1000 equipped with a heated Planapo 60?
water immersion lens (Nikon) in combination with a heated stage. The Fn/c
ratios were calculated by using ImageJ 1.38 public domain software (National
Institutes of Health) from single cell measurements for the nuclear (Fn) and
cytoplasmic (Fc) fluorescence levels, subsequent to the subtraction of fluores-
cence due to autofluorescence and/or background.
FRAP analysis. Fluorescence recovery after photobleaching (FRAP) analysis
was performed in COS-7 cells transiently expressing GFP-UL44 wild-type and
mutant derivatives by using an Olympus Fluoview 1000 (Olympus) microscope
equipped with an Argon ion laser (40 mW) and a 100? oil immersion lens
(Nikon) in combination with a heated stage. Three images were collected by
using 3% total laser power with excitation at 488 nm (2? zoom, scanned at a rate
of 8 ?s/pixel) before photobleaching. The bleaching was performed by zooming
100-fold in a small area covering ca. 5% of the nucleus and by using 100% of the
laser power (10 scans, at a rate of 8 ?s/pixel). After bleaching, cells were
immediately scanned, and fluorescence recovery was monitored by acquiring
subsequent images at 20-s intervals for 6 min using detector and laser settings
identical to those prior to photobleaching. Image analysis was performed as
described above. The results were expressed as the fractional recovery of Fb/Fnb
ratios (i.e., the fluorescence of the bleached area divided by the fluorescence
of the nonbleached area) at several time points, divided by the prebleach
Fb/Fnb value. FRAP data were fitted exponentially according to the formula
y ? a(1 ? e?bx) as previously described (11, 24, 34) to determine the
fractional recovery and t1/2values.
Subcellular fractionation and Western blotting. HEK 293 cells transfected to
express the green fluorescent protein (GFP) fusion proteins of interest were
harvested by gentle pipetting. After three washes in phosphate-buffered saline,
cells were either resuspended in 100 ?l of radioimmunoprecipitation assay buffer
(50 mM Tris-HCl [pH 7.4], 150 mM NaCl, 1% NP-40, 0.25% sodium deoxy-
cholate, 1 mM phenylmethylsulfonyl fluoride [PMSF], 10 ?g of leupeptin/ml, 10
?g of aprotinin/ml), sonicated five times for 2 min on ice, and incubated for 1 h
on ice to obtain whole-cell lysates or permeabilized for 15 min at room temper-
ature using 100 ?l of buffer A (10 mM Tris-HCl [pH 7.4], 150 mM NaCl, 5 mM
MgCl2, 1% NP-40, 1 mM PMSF, 10 ?g of leupeptin/ml, 10 ?g of aprotinin/ml).
Soluble proteins (S1) were isolated by centrifugation at 3,000 rpm for 10 min.
Cell pellets were washed with 100 ?l of buffer B (10 mM Tris-HCl [pH 7.4], 150
mM NaCl, 5 mM MgCl2, 1 mM PMSF, 10 ?g of leupeptin/ml, 10 ?g of aprotinin/
ml), and pellets were separated from soluble proteins (S2) by centrifugation.
DNA was digested by incubating pellets with buffer C (10 mM Tris-HCl [pH 7.4],
150 mM NaCl, 5 mM MgCl2, 100 U of DNase I [Fermentas]/ml, 1 mM PMSF,
10 ?g of leupeptin/ml, 10 ?g of aprotinin/ml) 1 h at room temperature. Digested
DNA and DNA-bound proteins (S3) were separated from cellular pellets by
centrifugation at 3,000 rpm for 10 min. Pellets were then washed with 100 ?l of
buffer B (S4). The nuclear matrix was separated from tightly DNA-bound pro-
teins (S5) by treatment with buffer D (10 mM Tris-HCl [pH 7.4], 2 M NaCl, 5
mM MgCl2, 1 mM PMSF, 10 ?g of leupeptin/ml, 10 ?g of aprotinin/ml) and
centrifugation at 3,000 rpm for 10 min. Matrix-associated proteins (S6) were
solubilized with 150 ?l of buffer E (90 mM Tris-HCl [pH 6.8], 100 mM dithio-
threitol, 2% sodium dodecyl sulfate [SDS], 20% glycerol, 1 mM PMSF, 10 ?g of
leupeptin/ml, 10 ?g of aprotinin/ml) and heating for 5 min at 95°C. Laemmli
loading buffer was added to each fraction, the samples were boiled for 5 min at
95°C, and the mixtures were loaded onto 10% bis-Tris acrylamide gel prior to
separation by polyacrylamide gel electrophoresis (PAGE). Electrophoretically
separated proteins were then transferred to Hybond-P membranes (Amersham)
as previously described (39). Membranes were blocked in blocking buffer F (5%
[wt/vol] skin dry milk and 1? Tris-buffered saline) for 1 h at room temperature
and washed three times with buffer G (0.05% Tween and 1? Tris-buffered
saline). GFP fusion proteins were detected by incubating the membranes suc-
cessively with either the anti-GFP (clone sc-9996; Santa Cruz Biotechnology;
1:400) or anti-? tubulin (clone B-5-1-2; Sigma; 1:1,000) monoclonal antibodies
(MAbs) and horseradish peroxidase-coupled secondary antibody (Sigma; 1:500).
Immunoblots were developed with the horseradish peroxidase substrate 4-Cl-1-
naphthol (Bio-Rad) in the presence of H2O2. The intensity of bands relative to
each fusion protein was quantified by using the ImageJ (National Institutes of
Nuclear matrix preparation from intact cells. Daoy cells grown on Willcod-
ishes (Willcowells) were incubated for 10 min at 37°C in 5% CO2with Hoechst
33342 (Invitrogen) at 1 ?g/ml dissolved in Dulbecco modified Eagle medium
containing 5% FBS. The subcellular localization of GFP fusion proteins was
analyzed by using a Nikon Eclipse E600 inverted microscope (Nikon), equipped
with a Nikon DXN1200 digital camera and a Nikon Plan Fluor ?40 objective
lens (Nikon). Cells were then permeabilized with buffer A for 15 min at room
temperature and washed twice with buffer B to remove the soluble proteins, and
the subcellular localization of GFP fusion proteins was investigated by fluores-
cence microscopic analysis as described above. Cells were subsequently treated
with buffer F (10 mM Tris-HCl [pH 7.4], 150 mM NaCl, 5 mM MgCl2, 40 U of
DNase I [Fermentas]/ml, 1 mM PMSF, 10 ?g of leupeptin/ml, 10 ?g of aprotinin/
ml) for 1 h at room temperature. DNA-bound proteins were then removed by
incubating cells 10 min at room temperature with buffer D and washing them
with buffer B, and the subcellular localization of the nuclear matrix-associated
GFP fusion proteins was visualized as described above.
Immunoprecipitation of FLAG-tagged proteins. At 48 h posttransfection,
HEK 293 cells were harvested and washed with PBS. Cells were lysed in lysis
buffer (50 mM HEPES [pH 7.4], 100 mM NaCl, 1% Nonidet-P 40, Complete
Mini EDTA-free protease inhibitor cocktail tablets [Roche Applied Science]) for
10 min on ice and sonicated at low intensity for 10 s. After clarification, super-
natants were incubated with 4 ?g of the anti-FLAG at 4°C, with gentle rocking.
The following day, 30 ?l of protein A/G beads (Santa Cruz Biotechnology) was
added, followed by incubation for 4 h. After washes with lysis buffer, the beads
were resuspended in 2? Laemmli buffer, boiled at 95°C, and centrifuged at
16,000 ? g for 5 min before analyzing the supernatant by SDS–10% PAGE and
Western blotting using the anti-GFP (clone sc-9996; Santa Cruz Biotechnology;
1:10,000) and anti-FLAG (Sigma; 1:5,000) MAbs, followed by incubation with a
peroxidase-coupled secondary antibody (Sigma; 1:10,000). The immunoblots
were developed with ECL plus (Amersham).
oriLyt-dependent DNA replication transcomplementation assays. To investi-
gate the role of ppUL44 basic loop in HCMV DNA replication, HFF cells were
transfected with the pSP50 plasmid, which contains the HCMV oriLyt DNA
replication origin (8), a set of plasmids expressing the HCMV proteins essential
for oriLyt-dependent DNA replication except for ppUL44 (32, 33) and either
pEPI-GFP-UL44wt or pEPI-GFP-UL44?loop plasmid. To test for the ability of
ppUL44 mutants to interfere with the functionality of wild-type ppUL44, the
GFP-UL44?loop and the dimerization defective GFP-UL44L86A/L87A mu-
tants were expressed in the presence of a set of plasmids encoding all of the
HCMV proteins required for oriLyt-dependent DNA replication, including
ppUL44, and of the pSP50 plasmid. Replication of pSP50 was detected by
treatment of transfected cell DNA with DpnI, which cleaves only unreplicated,
dam-methylated input DNA (32, 33), followed by Southern blotting as described
VOL. 83, 2009IN VIVO FUNCTION OF HCMV ppUL44 FLEXIBLE LOOP9569
The highly conserved ppUL44 flexible loop is crucial for
ppUL44 localization in nuclear speckles and nuclear reten-
tion. Based on the presence of a proline residue upstream of a
stretch of basic amino acids, the N-terminal portion of UL44-FL
(PHTRVKR168) had initially been proposed as a putative nu-
clear localization signal (NLS), which proved to be nonfunc-
tional in terms of nuclear import (4, 25). Indeed, mutation of
basic residues within UL44-FL does not prevent either binding
to the IMP?/? heterodimer nor ppUL44 nuclear import. How-
ever, there is a difference in intranuclear distribution of
ppUL44?loop, a mutant derivative in which three of five
UL44-FL basic residues were mutated, compared to wild-type
ppUL44 (4). When fused to GFP, UL44?loop showed diffuse
nucleoplasmic localization rather than a speckled nuclear ap-
pearance, which has been described for PAPs from several
herpesviruses and is believed to be the consequence of binding
to dsDNA (1, 3–5, 7, 25, 44). Intriguingly, alanine substitution
of residues L86 and L87, which are essential for dimerization
of ppUL44, results in a similar loss of speckled pattern upon
overexpression of the GFP-UL44L86A/L87A fusion protein in
mammalian cells (41). Since ppUL44 dimerization defective
mutants are also impaired for dsDNA in vitro (10), we hypoth-
esized that the similar intranuclear localization of GFP-
UL44?loop and GFP-UL44L86A/L87A could be the result of
impaired DNA binding. We therefore transiently expressed the
aforementioned ppUL44 derivatives as GFP fusion proteins in
COS-7 cells and analyzed their subcellular localization by
CLSM (Fig. 1C). We also expressed GFP-UL44I135A, a point
mutant derivative fully capable of dimerization and DNA
binding but impaired in pUL54 binding as a control (27, 41).
As expected, all proteins localized mainly in the nucleus
of transfected cells. However, while GFP-UL44wt and GFP-
UL44I135A often localized in distinctive intranuclear speckles
(in ?50% of cells [see Fig. 1C and D and reference 41]), both
GFP-UL44L86A/L87A and GFP-UL44?loop mutants did not
(speckles were observed in ca. 10% of cells [see Fig. 1C and
D]), a finding consistent with the idea that this results from
impaired intranuclear binding. To verify this, we quantified the
levels of nuclear accumulation for each protein. Both GFP-
UL44L86A/L87A and GFP-UL44?loop accumulated to signif-
icantly lower levels (P ? 0.05) compared to GFP-UL44wt
(Fn/c ca. 65 and 75%, respectively, of that of GFP-UL44wt [see
Fig. 1D]), whereas the I135A substitution had no effect on
ppUL44 nuclear accumulation (Fn/c ca. 90% of that of GFP-
UL44wt). Thus, UL44-FL appears to play a key role in local-
izing ppUL44 in nuclear speckles and thereby increasing nu-
clear accumulation. Importantly, when we compared the level
of expression of wild-type and mutant ppUL44 proteins by
Western blot analysis, all proteins were expressed to compa-
rable levels (Fig. 1E), indicating that the impaired ability to
form nuclear speckles and accumulate into the nucleus ob-
served for GFP-UL44L86A/L87A and GFP-UL44?loop was
not due to reduced expression levels.
UL44-FL is involved in ppUL44 intranuclear binding. We
reasoned that the reduced nuclear accumulation and speckle
formation ability of GFP-UL44?loop and GFP-UL44L86A/
L87A could be due to reduced intranuclear binding (see above).
To test this hypothesis, we analyzed their intranuclear mobility
when transiently expressed in COS-7 cells using the FRAP tech-
nique. A small region within the nucleus was bleached and the
recovery of the fluorescent signal monitored over time to cal-
culate the fractional recovery (Fig. 2; see Movies S1 to S3 in
the supplemental material). Using the laser power at 100% (10
scans, 8 ?s/pixel), it proved possible to bleach a small area
within the nucleus of cells expressing GFP-UL44 fusions (Fig.
2A). This was not possible in cells expressing GFP alone,
where the treatment resulted in bleaching of fluorescence in
the whole-cell (data not shown), which is consistent with the
idea that ppUL44 binds to nuclear components such as dsDNA
that markedly reduce its intranuclear mobility. Recovery of
fluorescence on the bleached area of cells expressing GFP-
UL44wt was three times slower than that of GFP-UL44L86A/
L87A (t1/2of ca. 40 s and 13 s, respectively; Fig. 2B and C, see
also Movies S1 to S3 in the supplemental material), in keeping
with ppUL44wt’s ability to bind tightly to dsDNA. Similarly,
GFP-UL44?loop was also more mobile than GFP-UL44wt,
with a t1/2of ca. 20 s, implying that mutation of the UL44-FL
reduces intranuclear binding and hence increases the intranu-
clear mobility of the protein. Consistent with the idea that
ppUL44 binds to nuclear components, the fractional recovery
of fluorescence in cells expressing GFP-UL44wt was ca. 0.6,
indicating that ca. 40% of ppUL44 is immobile within the cell
nucleus. In contrast, cells expressing GFP-UL44L86A/L87A ex-
hibited significantly higher fractional recovery (ca. 0.8), support-
ppUL44 dimerization increases its intranuclear mobility. Cells
expressing GFP-UL44?loop also exhibited significantly higher
(ca. 0.8) fractional recovery, a finding consistent with impaired
binding of the protein to nuclear components/DNA and further
supporting the critical role of UL44-FL in intranuclear binding.
UL44-FL is required for intranuclear retention of ppUL44.
Our FRAP results strongly suggested that ppUL44 binds to
intranuclear structures in living cells. We therefore decided to
verify this hypothesis by imaging detergent-permeabilized cells.
Since treatment of cells with detergents results in the release of
nucleoplasmic proteins, we expressed GFP-UL44, GFP-UL44?
loop, and GFP-UL44L86/87A in Daoy cells and analyzed their
subcellular localization before and after cell permeabilization
with Nonidet P-40 (NP-40). As a negative control, we also ana-
lyzed the subcellular localization of GFP, which does not bind to
nuclear components. As a further control, we expressed a fusion
protein between GFP and the linker histone H1 variant H1E,
which binds tightly to cellular chromatin, and it is therefore re-
tained in the nucleus upon cell permeabilization (31).
As expected, before cell permeabilization GFP localized dif-
fusely throughout the cytoplasm and the nucleus, whereas
GFP-H1E strongly localized within the cell nucleus, occasion-
ally colocalizing with cellular heterochromatin (Fig. 3). On the
other hand, all GFP-UL44 mutant derivatives strongly local-
ized within the cell nucleus, with GFP-UL44wt often colocal-
izing with cellular chromatin, and GFP-UL44?loop and GFP-
UL44L86A/L87A mainly localizing with a diffuse pattern (Fig.
3). Upon cell permeabilization, GFP alone was no longer de-
tectable, indicating that GFP does not bind to nuclear compo-
nents within the cell. As expected, GFP-H1E was mainly re-
tained within the cell nucleus, as a consequence of its ability to
strongly bind to cellular chromatin via its C-terminal tail (2).
Importantly, a considerable fraction of GFP-UL44wt was still
9570ALVISI ET AL.J. VIROL.
FIG. 2. UL44-FL is required for ppUL44 intranuclear binding as determined by FRAP analysis. The intranuclear mobility of the indicated GFP
fusions was analyzed by FRAP 16 h after transfection of COS-7 cells. (A) Visualization using CLSM of the return of intranuclear fluorescence after
photobleaching in a specific area of the nucleus (black boxes) (see Materials and Methods). (B) Quantification of the recovery over time of specific
nuclear fluorescence after photobleaching expressed in terms of the fractional recovery of the Fb/Fnb ratio (Fb [fluorescence of the bleached area
above background] divided by Fnb [fluorescence of the unbleached area above background] at the indicated time points divided by the prebleach
Fb/Fnb value). (C) Pooled data (average ? the standard error of the mean, n ? 9) for the fractional recovery and half-time of the return of
fluorescence (t1/2) for the wild type and the mutants, with significant differences denoted by the P values.
VOL. 83, 2009 IN VIVO FUNCTION OF HCMV ppUL44 FLEXIBLE LOOP9571
retained within the nucleus in a typical speckled pattern after
cell permeabilization, whereas most of GFP-UL44L86A/L87A
was lost, possibly as a consequence of its inability to efficiently
bind to dsDNA (10). Similarly, cell permeabilization resulted in a
strong decrease of the fluorescence relative to GFP-UL44?loop
(Fig. 3), a finding consistent with the involvement of UL44-FL
in binding to dsDNA in vitro (22). These results clearly indi-
cate that GFP-UL44, but not GFP-UL44L86A/L87A and
GFPUL44?loop, binds strongly to nuclear structures. Impor-
tantly, upon permeabilization, the colocalization of GFP-UL44
with cellular chromatin became clearer (see Fig. S1 in the
supplemental material), in keeping with the idea that UL44
interacts with nuclear components. To verify that GFP-UL44 is
able to bind to cellular dsDNA when transiently expressed in
the absence of other viral proteins as suggested by previous
studies (25), cells where further treated with DNase I before
being washed with 2 M NaCl. A significant decrease in the
signal relative to both GFP-H1E and GFP-UL44 was observed
upon such treatment, suggesting that a portion of ppUL44
could bind to cellular dsDNA when expressed in the absence of
other viral proteins.
ppUL44-FL binds to dsDNA in cells. To verify the involvement
of UL44-FL in DNA binding in cells, we transiently expressed
several GFP-UL44 fusion proteins in HEK 293 cells and, at
48 h posttransfection, quantitatively analyzed the amount of
proteins released after cell permeabilization and DNase I
treatment by Western blotting. GFP and GFP-H1 were also
expressed as controls. As expected, almost all of GFP (?90%
[Fig. 4A and B]) was detectable in the S1 and S2 fractions,
corresponding to completely soluble proteins. On the other
hand, only ca. 20% of GFP-H1E was detectable in the soluble
fractions (S1 and S2), and a considerable amount was detect-
able in the DNA-containing (S3 to S5, more than 30% of total)
and matrix-associated (S6, ca. 50%) fractions. Importantly only
ca. 50% of total GFP-UL44 could be detected in the soluble
fractions, with a significant amount of protein (ca. 25% each)
being found in the DNA- and nuclear matrix-containing frac-
tions, confirming that ppUL44 can interact with cellular chro-
matin in the absence of viral DNA. As expected, the dimer-
ization defective GFP-UL44L86A/L87A mutant derivative was
also impaired in DNA binding, being detectable almost exclu-
sively (ca. 90%) in the soluble fractions. Similar results were
obtained for the GFP-UL44?loop mutant derivative, which
was mainly (ca. 85%) detectable in the soluble fractions and
was almost completely absent (ca. 3% of total fusion protein)
from the DNA-containing fractions. On the other hand, sig-
nificant amounts of the control molecule GFP-UL44P85G,
which is not impaired for dimerization and DNA binding, were
detectable in both the DNA- and the matrix-associated frac-
tions (ca. 15 and 25% of the total fusion protein, respectively).
These results show that, when expressed in the absence of
other viral proteins, ppUL44 can bind to nuclear components
such as DNA and the nuclear matrix through a process that
requires dimerization of the protein and its highly flexible loop.
FIG. 3. GFP-UL44 associates with dsDNA and the nuclear matrix, in contrast to GFP-UL44L86A/L87A and GFP-UL44?loop. The intracel-
lular localization of the indicated GFP fusion proteins was investigated 24 h after transfection of Daoy cells, prior to treatment (NT), or after
treatment with 1% NP-40 for 15 min at room temperature (NP-40) or after incubation with 40 U of DNase I/ml for 1 h at room temperature,
followed by incubation with NaCl 2 M for 5 min (DNase I, NaCl). The bright-field (BF) and the green channel (GFP) are shown, with cellular
dsDNA being shown in blue (Hoechst).
9572 ALVISI ET AL.J. VIROL.
UL44-FL is not required for ppUL44 dimerization and bind-
ing to pUL54. We next decided to analyze the ability of the
ppUL44?loop mutant derivative to bind to the catalytic sub-
unit pUL54 (14) and to homodimerize (10), using our recently
described live cell CLSM-based assays (5, 7, 41). We analyzed
the subcellular localization of GFP-UL54(1213-1242) when ex-
pressed alone (Fig. 5A, upper panels) or in the presence of
DsRed2-UL44wt and mutant derivatives thereof (Fig. 5A). As
expected, GFP-UL54(1213-1242), a fusion protein containing
the minimal binding site for ppUL44 but lacking the pUL54
NLS (7), localized both in the nucleus and in the cytoplasm of
transfected cells with a diffuse pattern (Fig. 5A, upper panels).
However, when GFP-UL54(1213-1242) was coexpressed with
DsRed2-UL44wt and DsRed2-UL44L86A/L87A, the two pro-
teins strongly colocalized within the cell nucleus (Fig. 5A),
indicating functional interaction. A similar result was ob-
tained upon coexpression of GFP-UL54(1213-1242) with
DsRed2-UL44?loop, indicating that the mutant derivative
could still interact with pUL54. In contrast, no colocaliza-
tion was observed when GFP-UL54(1213-1242) was coex-
pressed with DsRed2-UL44I135A, a fusion protein carrying
a point mutation within the binding site for pUL54 that is
sufficient to prevent the ppUL44-pUL54 interaction (41).
The ability of DsRed2-UL44?loop to dimerize was tested by
investigating its ability to influence the subcellular localization
of GFP-UL44?NLS, a mutant derivative unable to interact
with IMP?/? and whose localization is therefore entirely re-
stricted to the cytoplasm (4). As expected, GFP-UL44?NLS
localized to the cytoplasm when expressed alone (Fig. 5B, upper
panels), but coexpression with DsRed2-UL44wt and DsRed2-
UL44I135A resulted in its marked relocalization to the cell
nucleus as a consequence of the ability of ppUL44 to dimerize
before being translocated into the nucleus (5). Coexpression of
GFP-UL44?NLS with DsRed2-UL44?loop, but not with
DsRed2-UL44L86A/L87A, resulted in a similar relocalization
(Fig. 5B). These results suggest that the basic residues within
UL44-FL are essential neither for binding to pUL54 nor for
ppUL44 homodimerization. They also suggest that the ppUL44?
loop mutant protein is not affected pleiotropically in multiple
functions, and hence it is highly unlikely that its impaired DNA
binding is the result of general misfolding. To verify this further,
we analyzed the ability of FLAG-UL44?loop to coimmunopre-
cipitate with GFP-UL54(1213-1242) compared to that of FLAG-
UL44wt. Immunoprecipitation of both FLAG-UL44 and FLAG-
UL44?loop (see Fig. S2 in the supplemental material), using an
anti-FLAG MAb, resulted in coimmunoprecipitation of GFP-
UL54(1213-1242), further demonstrating that the UL44?loop
mutant derivative is not misfolded, with the effects of the muta-
tion on intranuclear mobility/retention likely to be attributable to
its impaired ability to bind to dsDNA.
UL44-FL is required for HCMV oriLyt-dependent DNA rep-
lication in cells. ppUL44 is believed to confer processivity to
the DNA polymerase holoenzyme by tethering the catalytic
subunit pUL54 on the dsDNA, meaning that mutations af-
fecting its ability to bind to dsDNA could affect its activity as
a processivity factor. We decided to investigate the ability of
GFP-UL44?loop to support HCMV oriLyt-dependent DNA
replication using a cotransfection replication assay in HFF
cells (41). Briefly, the pSP50 plasmid, bearing the HCMV
oriLyt DNA replication origin (8), was cotransfected with a
plasmid encoding wild-type or mutant GFP-UL44 (pEPI-GFP-
UL44wt or pEPI-GFP-UL44?loop), together with a set of
plasmids encoding the remaining HCMV proteins essential for
oriLyt-dependent DNA replication (32, 33). Replication of
pSP50 was detected by treatment of transfected cells DNA
with DpnI, which cleaves only unreplicated, dam-methylated
input DNA (8). As expected, a DpnI-resistant replication
product was detected in the presence of wild-type GFP-UL44
(Fig. 6A). On the other hand, oriLyt-mediated DNA replica-
tion was not detected in the presence of GFP-UL44?loop (Fig.
6A), clearly indicating that the basic residues within UL44-FL
are required for efficient HCMV oriLyt DNA replication in
cells. Based on the fact that the GFP-UL44?loop is capable of
binding pUL54 (Fig. 5A and see Fig. S2 in the supplemental
material), as well as homodimerizing (Fig. 5B), an interesting
ppUL44?loop could result in the formation of ppUL44wt-
FIG. 4. ppUL44 dimerization and UL44-FL are required for in
vivo DNA binding. (A) HEK 293 cells expressing the indicated fusion
proteins were harvested 48 h after transfection, and cellular fraction
collection and SDS-PAGE/Western blot analysis were performed as
described in Materials and Methods. An anti-GFP MAb was used to
detect the GFP fusion proteins extracted after incubation of the cells
with the following buffers: S1, NP-40; S2, wash; S3, DNase I; S4, wash;
S5, NaCl 2 M; S6, Laemmli buffer; and L, whole-cell lysates. (B) Im-
ages such as those in A were analyzed as described in Materials and
Methods to calculate the relative amounts of the indicated GFP fusion
protein present in the specified cellular fraction. The data represent
the means of three independent experiments, where the amount of
soluble (S1 and S2), DNA-bound (S3 to S5), and matrix-associated
(S6) proteins are expressed as a percentage of the total. The data
represent the means of three independent experiments.
VOL. 83, 2009 IN VIVO FUNCTION OF HCMV ppUL44 FLEXIBLE LOOP9573
ppUL44?loop heterodimers, which potentially may be impaired
in dsDNA binding. We decided to test this possibility by testing
the effect of overexpressing GFP-UL44?loop to interfere with
HCMV oriLyt-dependent DNA replication in HFF cells in the
presence of wild type in our cotransfection replication assay. The
dimerization-defective GFP-UL44L86A/L87A fusion protein was
used as a control. The pSP50 plasmid and a set of plasmids
encoding all of the HCMV proteins essential for oriLyt-depen-
dent DNA replication, including ppUL44, were transfected in
HFF cells in the absence or presence of GFP-UL44?loop or
GFP-UL44L86A/L87A. As expected, replicated pSP50 was de-
tectable when cells were transfected to express to HCMV repli-
cating proteins in the absence of the aforementioned ppUL44
mutants (Fig. 6B, left lane). Expression of GFP-UL44L86A/
L87A, which is not capable of mediating oriLyt-dependent DNA
replication but is defective for dimerization (41), did not affect
pSP50 replication in the presence of ppUL44wt (Fig. 6B, middle
lane). Importantly, the expression of GFP-UL44?loop prevented
the replication of pSP50 even in the presence of ppUL44wt (Fig.
6B, right lane), presumably due to the formation of DNA-bind-
ppUL44 is a multifunctional protein that is capable of
interacting with dsDNA, through a process that is believed
to involve electrostatic interactions between the dsDNA
backbone and basic residues located both on ppUL44’s basic
face and within its highly flexible basic loop (UL44-FL) (4,
15, 29, 37, 45). Our results support this hypothesis, indicat-
ing a direct involvement of UL44-FL in DNA binding in
living transfected cells and in a cell-based HCMV replica-
tion system (Fig. 6). Mutation of three of the five basic
residues (PHTgVngNVKKAP174) does not affect ppUL44’s
ability to dimerize or to bind to the catalytic subunit pUL54
(Fig. 5 and see Fig. S2 in the supplemental material), but it is
sufficient to impair nuclear accumulation of ppUL44, as well as
to decrease its ability to form nuclear speckles (Fig. 1C and D).
Since the level of nuclear accumulation of a protein is a prod-
FIG. 5. UL44-FL is not required for ppUL44 binding to pUL54 and homodimerization. COS-7 cells were transfected to express GFP-
UL54(1213-1242) (A) or GFP-UL44?NLS (B), in the absence or the presence of the indicated DsRed2-UL44 fusions. Cells were imaged 24 to
36 h after transfection by CLSM. Merged images of the green (GFP) and red (DsRed2) channels are shown on the right, with yellow coloration
indicative of colocalization.
FIG. 6. UL44-FL is required for HCMV oriLyt-dependent DNA
replication. (A) HFF cells were transiently transfected with the pSP50
plasmid, which contains the HCMV oriLyt DNA replication origin, a
plasmid expressing GFP-UL44 (left lane) or GFP-UL44?loop (right
lane), as well as a set of plasmids expressing all other essential HCMV
replication proteins. After DNA extraction and digestion with DpnI,
undigested, replicated DNA (indicated by an arrow) was visualized by
Southern blotting. (B) HFF cells were transiently transfected with the
pSP50 plasmid, which contains the HCMV oriLyt DNA replication
origin, a set of plasmids expressing all of the essential HCMV repli-
cation proteins, in the absence (left lane) or in the presence of the
indicated GFP-UL44 mutant derivatives (middle and right lanes). Af-
ter DNA extraction and digestion with DpnI, undigested, replicated
DNA (indicated by an arrow) was visualized by Southern blotting.
9574 ALVISI ET AL.J. VIROL.
uct of its nuclear import and export rates, as well as of its
ability to be retained within the nucleus upon nuclear entry
(12, 40), and mutation of UL44-FL does not affect ppUL44
binding to the IMP?/? heterodimer, the reduced nuclear ac-
cumulation of GFP-UL44?loop clearly implicates a role for
UL44-FL in intranuclear binding.
Mutation of UL44-FL resulted in an increase in ppUL44
intranuclear mobility (Fig. 2) due to reduced binding to in-
tranuclear components as supported by detergent extraction
and/or fractionation experiments, where a significant amount
of GFP-UL44 is retained in the nucleus associated with cellular
chromatin after permeabilization of transfected Daoy cells (Fig.
3). In contrast, both GFP-UL44?loop and GFP-UL44L86A/
L87A are almost completely released upon cell permeabilization,
as is GFP alone. This finding is also in keeping with the observa-
tion that HSV-1 UL42 nuclear localization is also resistant to
detergent treatment in HSV-1-infected cells and that mutations
affecting its binding to dsDNA in vitro also affect its resistance to
detergent treatment (13, 21). Our quantitative analysis indicates
that ca. 50% of GFP-UL44 is retained in the nucleus after deter-
gent permeabilization of HEK 293 cells (Fig. 4). This is consistent
with a fractional recovery of ca. 0.6 calculated for GFP-UL44 in
COS-7 cells in FRAP experiments (see Fig. 2C), indicating that
L87A are released by cell permeabilization (see Fig. 4), a finding
consistent with their significantly higher fractional recoveries (ca.
0.8) determined in FRAP experiments and indicating that only a
small fraction of the proteins is bound to intranuclear structures.
A significant fraction of GFP-UL44wt, but not of GFP-UL44?
loop and GFP-UL44L86A/L87A, is released from permeabilized
cells after DNase I treatment (see Fig. 4). Although we cannot
exclude the possibility that UL44-FL’s DNA-binding activity in
live cells is indirect, i.e., through interaction with other nuclear
proteins immobilized on DNA, our data strongly support the idea
that UL44-FL is directly involved in the ppUL44-DNA interac-
tion, as implicated by in vitro experiments (22). Our finding that
ppUL44 can bind to cellular dsDNA when expressed even in the
absence of viral DNA is not surprising, since ppUL44 appears to
be able to bind to dsDNA without any sequence specificity (22).
Underlining the physiological significance of the results, GFP-
UL44?loop failed to support HCMV oriLyt-dependent DNA
replication in cells (Fig. 6). Similarly, mutations preventing
ppUL44 homodimerization in a cellular context also prevented
ppUL44 from transcomplementing viral DNA replication in a
transient-transfection assay (41), most likely due to the inabil-
ity of these mutants to bind to dsDNA (see Fig. 3 and 4).
Although the experiments here were not performed using the
preferred experimental system of recombinant viruses, the re-
sults obtained are clearly consistent with a recent report show-
ing that mutations impairing HSV-1 UL42 ability to bind to
dsDNA also impair replication of a recombinant virus (20) and
thus strong evidence for an important role for ppUL44-FL in
HCMV replication. Importantly, the defect of ppUL44?loop
in mediating oriLyt-dependent DNA replication is not due to
misfolding of the protein, as indicated by its ability to both bind
to pUL54 and to heterodimerize with ppUL44wt (see Fig. 5AB
and see Fig. S2 in the supplemental material). Hence, our results
suggest that the inability of ppUL44?loop to transcomplement
oriLyt-dependent DNA replication depends directly on its re-
duced affinity for dsDNA (41; the present study). We cannot
formally exclude the possibility that ppUL44 is impaired in bind-
ing to other viral or cellular proteins implicated in viral DNA
replication despite still being able to bind to pUL54 (43), but
its transdominant-negative phenotype in the replication assay
is consistent with the defect of ppUL44?loop in mediating
HCMV DNA replication being at least partially due to its
decreased ability to bind to dsDNA; the transdominant-nega-
tive effect can be explained by the formation of ppUL44?loop/
ppUL44wt heterodimers upon coexpression of the two pro-
teins (see Fig. 5B and 6B). Formation of inactive protein
complexes has been reported to be the molecular basis for the
transdominant-negative phenotype of several viral multimeric
proteins, including the human immunodeficiency virus type 1
protein Rev (17).
Whether UL44-FL is solely implicated in the binding of
ppUL44 to dsDNA or also to cellular and viral proteins, inter-
action between herpesvirus PAPs and viral DNA represents a
potential therapeutic target to hinder viral replication. In par-
ticular, UL44-FL appears as a potentially important determi-
nant of ppUL44 biological activity in vivo and thus a target of
great interest for the future.
This study was supported by the University of Bologna and the
Italian Ministry of Education (60 and 40%), the AIDS Project of the
Italian Ministry of Public Health, and the Australian National Health
and Medical Research Council (fellowship 384109 and project grant
We thank Valerio Leoni (Bologna, Italy) for help with the Chimera
software and Simone Avanzi (Bologna, Italy) for tissue culture.
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