JOURNAL OF VIROLOGY, Oct. 2009, p. 10187–10197
Copyright © 2009, American Society for Microbiology. All Rights Reserved.
Vol. 83, No. 19
Human Cytomegalovirus UL28 and UL29 Open Reading Frames
Encode a Spliced mRNA and Stimulate Accumulation of
Dora P. Mitchell,1John P. Savaryn,2Nathaniel J. Moorman,1Thomas Shenk,1and Scott S. Terhune1,2*
Department of Molecular Biology, Princeton University, Princeton, New Jersey 08544,1and Microbiology and Molecular Genetics and
Biotechnology and Bioengineering Center, Medical College of Wisconsin, Milwaukee, Wisconsin 532262
Received 23 February 2009/Accepted 14 July 2009
We have identified a spliced transcript that contains sequences from the HCMV UL29 and UL28 open
reading frames. It contains amino-terminal UL29 sequences followed by UL28 sequences, and it includes a
poly(A) signal derived from the 3?-untranslated region following the UL26 open reading frame. UL29/28 RNA
is expressed with early kinetics, and a virus containing a FLAG epitope inserted at the amino terminus of UL29
expressed a tagged ?79-kDa protein, pUL29/28, that was detected at 6 h postinfection. The virus also expressed
a less-abundant tagged 41-kDa protein, which corresponds in size to a protein that could be produced by
translation of an unspliced UL29/28 transcript. Consistent with this prediction, both unspliced and spliced
UL29/28 transcript was present in RNA isolated from polysomes. FLAG-tagged protein from the UL29/28 locus
accumulated within nuclear viral replication centers during the early phase of infection. Late after infection it
was present in the cytoplasm as well, and the protein was present and resistant to proteinase treatment in
partially purified preparations of viral particles. Disruption of the UL29/28 locus by mutation resulted in a
10-fold decrease in the levels of DNA replication along with a similar reduction in virus yield. Quantitative
reverse transcription-PCR analysis revealed an ?2-fold decrease in immediate-early gene expression at 4 to
10 h postinfection compared to the wild-type virus, and transient expression of pUL29/28 activated the major
immediate-early promoter. Our results argue that the UL29/28 locus contributes to activation of immediate-
early gene expression.
Human cytomegalovirus (HCMV) is a ubiquitous human
pathogen and the prototypical member of the Betaherpesvirus
family (26). HCMV infection is generally asymptomatic in
healthy adults, but the virus causes disease in immunocompro-
mised adults often leading to pneumonitis, retinitis, or hepa-
titis. It also is responsible for congenital infections that result
in a range of neurological abnormalities. Like all herpesvi-
ruses, HCMV infection leads to life-long latency.
Lytic HCMV replication follows a coordinated series of events.
The first viral gene products to function within an infected cell
are virion tegument proteins (5, 20, 24, 43), some of which
facilitate transcription of the immediate-early class of viral
genes upon delivery of the DNA genome to the nucleus. As
these immediate-early products accumulate, they help to es-
tablish a permissive environment for replication and activate
expression of the early and late classes of viral genes (26).
Proteins encoded by early genes are responsible for viral DNA
replication, as well as regulating cellular responses to infection
(46), and late proteins include virion constituents (26).
The 230-kbp HCMV genome potentially encodes about 200
open reading frames (ORFs) (30). These ORFs include several
families of genes, including the US22 family (7). This gene
family is conserved among betaherpesviruses, and in HCMV it
is comprised of 13 members: UL23, UL24, UL26, UL28,
UL29, UL36, UL43, IRS1, TRS1, US22, US23, US24, and
US26 (7, 13, 15, 39). US22 family proteins contain four con-
served sequence motifs of uncertain function, which consist of
hydrophobic residues interspersed with charged amino acids.
Some US22 family members (UL23, UL24, UL36, UL43, IRS1,
and US22) are dispensable for HCMV replication in fibro-
blasts, while others (UL26, UL28, UL29, TRS1, US23, US24,
and US26) are not essential but are required for optimal virus
yields in these cells (16, 28, 37, 50). Several US22 family mem-
bers in mouse CMV are required for efficient replication in
mouse macrophages (25), suggesting that some of the HCMV
family members that are dispensable for replication in fibro-
blasts might be required for replication in other cell types.
The HCMV US22 family members UL36 and TRS1 have
been extensively studied, and their proteins influence cellular
pathways similarly to their murine CMV orthologues (11, 47).
pUL36 inhibits Fas-mediated apoptosis by blocking caspase-8
activation (44), and pTRS1 binds double-stranded RNA and
inhibits activation of the protein kinase R-mediated antiviral
response (9, 17). The US22 family members UL26, TRS1,
IRS1, and US24 have been shown to influence immediate-early
gene expression (16, 42, 45) and, consistent with this role,
several US22 family members have been found in preparations
of HCMV virions (2, 16, 37, 41, 48). TRS1 and UL26 have also
been shown to facilitate virion assembly and influence the
stability of viral particles (1, 23, 28).
We recently discovered by mass spectrometry an ?79-kDa
protein containing amino acid sequences derived from two
US22 family members: UL28 and UL29 (27). We have now
* Corresponding author. Mailing address: Microbiology and Molec-
ular Genetics and Biotechnology and Bioengineering Center, Medical
College of Wisconsin, 8701 Watertown Plank Rd., P.O. Box 26509,
Milwaukee, WI 53226. Phone: (414) 955-2511. Fax: (414) 955-6568.
?Published ahead of print on 22 July 2009.
identified a spliced mRNA spanning the UL28 and UL29
ORFs. Disruption of this locus results in reduced immediate-
early gene expression, and pUL29/28 can activate expression of
the major immediate-early promoter (MIEP) within trans-
fected cells. In addition, we demonstrate that pUL29/28 is
packaged into HCMV virions. The transcriptional activity of
pUL29/28 and its inclusion in virions gives it the potential to
influence gene expression at the very start of infection.
MATERIALS AND METHODS
Cell culture, viruses and plasmids. Primary human foreskin fibroblasts (HFF)
and human MRC-5 embryonic lung fibroblasts cells were propagated in Dul-
becco modified Eagle medium supplemented with 10% fetal calf serum. Wild-
type virus, BADwt, was derived from an infectious bacterial artificial chromo-
some (BAC) clone of the AD169 strain of HCMV, termed pAD/Cre (51). The
UL28- and UL29-deficient mutants, BADsubUL28 (originally termed DH54)
and BADsubUL29 (originally termed DH56) were produced from the BAC
clone and have been described previously (50). Viruses were propagated on
fibroblasts and concentrated 10-fold by centrifugation through a sorbitol cushion
(20% D-sorbitol, 50 mM Tris-HCl [pH 7.2], 1 mM MgCl2), and titers (infectious
units) were determined by using an immunofluorescence assay to quantify
pUL123 (IE1)-expressing cells (46). Briefly, serial dilutions of virus samples were
plated on MRC-5 fibroblasts, and cells were fixed and permeabilized in methanol
at ?20°C for 15 min at 36 h postinfection (hpi). pUL123-positive cells were
labeled by using a primary mouse antibody to IE1 (1B12) (16) and a secondary
goat anti-mouse antibody conjugated with Alexa Fluor 546 or 488 (Molecular
Probes), and positive cells were counted by using a fluorescence microscope.
Cell-free virus was collected from supernatants of infected cells, and cell-asso-
ciated virus was prepared by three rounds of freezing and thawing.
The BADinUL29F virus, which expresses pUL29 with an N-terminal FLAG tag,
was constructed in the BAC clone of AD169 (51) by using galK selection and
counter selection “recombineering” protocols (49) as modified for application to
HCMV BAC clones (29). Briefly, PCR products were generated with primers con-
taining both galK sequences and HCMV flanking sequence, which targeted recom-
bination of the amplified galK expression cassette to the desired location on the viral
genome. The targeting HCMV-specific primers were 5?-ACTGCTGCTTCTGCTT
a-3? and 5?-GACGAGGAGGAAGACGCCGTGGCCGCCGAGCAGCCCTTGC
GACGGCCGGAtcagcactgtcctgctcctt-3? (capitalized nucleotides correspond to the
HCMV sequence, and lowercase nucleotides are galK specific). Competent AD169-
BAC-containing SW102 cells were transformed with the HCMV-galK PCR product
and selected for growth on M63 minimal salt agar plates supplemented with chlor-
amphenicol and 0.2% galactose. AD169-BAC clones that underwent homologous
recombination were screened for growth on MacConkey medium supplemented
with galactose, and positive colonies were picked and further screened by PCR
to confirm the proper location of the galK insert. Clones that passed the screens
were next transformed with linear DNA comprised of three repeats of the FLAG
epitope tag coding sequence flanked by the HCMV sequences bracketing the
galK integrated sequence. This DNA was generated by annealing the following
GGCCGGActtatcatcatcgtctttatagtcggctttgtcatcgtcatccttataat-3? (with lowercase
nucleotides representing the FLAG sequences) and filling in the ends using a
high-fidelity polymerase. Recombinants were selected by growth on M63 mini-
mal salt agar plates containing 0.2% 2-deoxygalactose. DNA was isolated and
transferred by electroporation into HFF cells (51) to produce BADinUL29F
virus. The BADinUL28F virus expresses UL28 with a C-terminal FLAG epitope
tag. It was constructed using the galk primers 5?-TGTTTTCGGGCCGTAGCG
a-3? and 5?-GCGGTAAAGCCAGACACCGGCTATATAGCTAGTCATCAC
AGTCTCCTCCTtcagcactgtcctgctcctt-3? and the FLAG primers 5?-TTTTCGG
aggatgacgacgataaggcagattataaggatgacgatgacaaag-3? and 5?-AAAGCCAGACAC
gtcggctttgtcatcgtcatccttataatct-3? as described for BADinUL29F.
The MIEP-luciferase plasmid contains 1.1 kb of the MIEP sequence between
nucleotide (nt) 74978 and nt 173724 from HCMV strain AD169 (accession no.
X17403) using the primers 5?-CTGGAATACGACAAGATAACCCG-3? and
5?-GGCAGAGATCTGACGGTTCACTAAACGAGCTCTG-3?. The PCR am-
plicon was digested with MluI and BglII and introduced into the pGL3-Basic
(Promega). The expression vector for pUL29/28 was constructed by PCR using the
primers for UL29 (5?-ATATCTAGATCCGGCCGTCGCAAGG-3? and 5?-cacgac
gagcCTACGCTTTTTGAACG-3?) and UL28 (5?-gcgtagGCTCGTCGTGGGCTA
C-3? and 5?-ATAGGATCCTCACGACGCCCCCGTGCCGC-3?) (lowercase se-
quences represent the overlap between genes). UL29 and UL28 amplicons were
combined and the UL29/28 sequence was amplified by using outer primers. The
product was digested using XbaI and BamHI and introduced into pCGN in-frame
with the amino-terminal hemagglutinin (HA) epitope tag. UL29/28 sequence was
verified by sequencing analysis. The UL29 gene was amplified by using 5?-ATATC
TAGATCCGGCCGTCGCAAGG-3? and 5?-ATAGGATCCTAAACGCTTTTTG
AACGGCAGTC-3? and introduced into pCGN.
Analysis of viral RNA and DNA. For 3?RACE (3? rapid amplification of cDNA
ends) analysis, ?5 ? 106confluent HFF cells were infected at a multiplicity of 3
infectious units/cell, and 6 or 24 h later the total RNA was prepared with TRIzol
reagent (Invitrogen) and treated with DNA-free reagent (Ambion). The purified
RNA was subjected to 3?RACE analysis (Clontech) in which the two gene-
specific primers were located inside the UL28 ORF: 5?-AATCTGCTGCGCGT
ATGTCAG-3? and 5?-CACGCCGGTAGCAAGATCCG-3?. The RACE frag-
ments generated by PCR were cloned into pGEM-T Easy (Promega) and
screened for UL28 sequences by using PCR, and the positive clones were then
RNA levels in infected cells were determined by quantitative real-time
reverse transcription-PCR (qRT-PCR) (46). Total RNA was isolated by using
TRIzol reagent (Invitrogen), and contaminating DNA was removed by using
DNA-free reagent (Ambion). cDNAs were synthesized with TaqMan reverse
transcription reagents and random hexamers according to the manufacturer’s
instructions (Applied Biosystems). Real-time PCR was completed with SYBR
green PCR master mix (Applied Biosystems) and primers specific to UL28
(5?-GGATGGATGGAACCCGTGAACA-3? and 5?-ACGAAACCAGAAGG
AGCCCTGAC-3?), UL29 (5?-CCGATGCTCTCGTAGCGAAAGTC-3? and
5?-GCTGGTGGGGCAGGATAAGTTG-3?), UL83 (5?-GGGACACAACAC
CGTAAAGCCG-3? and 5?-CGTGGAAGAGGACCTGACGATGAC-3?), UL123
(5?-GCCTTCCCTAAGACCACCAAT-3? and 5?-ATTTTCTGGGCATAAGCCA
TAATC-3?), or glyceraldehyde-3-phosphate dehydrogenase (GAPDH; 5?-ACCCA
CTCCTCCACCTTTGAC-3? and 5?-CTGTTGCTGTAGCCAAATTCGT-3?). For
amplification across the splice junction between UL29 and UL28, RT-PCR was
carried out using RNA isolated from infected cells as described above with the
primers UL29 (5?-TTTCGCTACGAGAGCATCGGC-3?) and UL28 (5?-TTGTG
GGTCCAGGCATCACG-3?). Additional primers used for qRT-PCR included
UL26 (5?-TCATCGGCACCATCGGACTC-3? and 5?-GCGCACTACAAAGTTC
TCACGGC-3?), UL30 (5?-AGCGGAGGGGGAGAATAAACAGTT-3? and 5?-G
GGCAACACAAGATAGGGAAATACAA-3?), UL32 (5?-GGTTTCTGGCTCG
TGGATGTCG-3? and 5?-CACACAACACCGTCGTCCGATTAC-3?), UL37x1
(5?-GACGAAGTCCGATGAGGAGGATG-3? and 5?-TGGGACACTGGGCGT
TGTTG-3?), UL44 (5?-TACAACAGCGTGTCGTGCTCCG-3? and 5?-GGCG
TGAAAAACATGCGTATCAAC-3?, UL54 (5?-CCCTCGGCTTCTCACAA
CAAT-3? and 5?-CGAGTTAGTCTTGGCCATGCAT-3?), UL83 (5?-GGGA
CACAACACCGTAAAGCCG-3? and 5?-CGTGGAAGAGGACCTGACGA
TGAC-3?, UL99 (5?-GTGTCCCATTCCCGACTCG-3? and 5?-TTCACAAC
GTCCACCCACC-3?), US3 (5?-TTCCACTCGAAATAGGCTCCGC-3? and
5?-CGAGAAACACTTTGTGAACGTGGG-3?), and UL27 (5?-ATCAGGCT
GTTTAAAGGCGAGGCT-3? and 5?-AAAGTCGCAGAAGGTCTCCACG
Polysomes were isolated from HCMV-infected cells at 24 and 72 hpi as
previously described (3, 21). Briefly, 5 ? 106HFF cells were infected with
wild-type virus at a multiplicity of 3.0 infectious units/ml and, 10 min prior to
sample isolation at the indicated time points, the cultures were treated with 100
?g of cycloheximide/ml in media. Cells were washed once with phosphate-
buffered saline (PBS) containing cycloheximide, collected by scraping and cen-
trifugation at 2,000 rpm for 5 min in PBS containing cycloheximide, and resus-
pended in 2 ml of lysis buffer (15 mM Tris [pH 7.4], 150 mM NaCl, 1.5 mM
MgCl2, 10 mM dithiothreitol, 1% Triton X-100, protease inhibitors, and 50 U of
RNasin [Promega]/ml) containing cycloheximide. A control sample was prepared
in the absence of cycloheximide and using 50 mM EDTA to dissociate poly-
somes. Samples were incubated at 4°C for 10 min, and cells were disrupted by
using a Dounce homogenizer. Nuclei were removed by centrifugation at 3,000 ?
g for 5 min at 4°C. Supernatants were subjected to centrifugation at 14,000 rpm
for 10 min at 4°C to remove particulate material. Samples were loaded onto a 10
to 50% sucrose gradient and separated by centrifugation at 35,000 ? g for 3 h at
4°C. Fractions of 500 ?l were collected, and RNA was isolated from 250 ?l by
using TRIzol reagent (Invitrogen) as described above. Polysomal fractions were
determined by separating RNA fractions by agarose gel electrophoresis and
10188MITCHELL ET AL.J. VIROL.
identifying fractions containing 18S and 28S rRNA bands in the cycloheximide-
treated sample relative to the equivalent fractions from the EDTA-treated con-
Virus DNA was quantified by using qPCR. DNA was prepared from virus
particles starting with 100 ?l of cell-free virus stock or virus stock that was
partially purified by centrifugation through a 20% sorbitol cushion and resus-
pended in PBS. To remove free DNA, samples were pretreated with DNA-free
reagents (Ambion) and then lysed in lysis buffer (400 mM NaCl, 10 mM Tris [pH
8.0], 10 mM EDTA, 0.1 mg of protease K/ml, and 0.2% sodium dodecyl sulfate
[SDS]) at 37°C overnight. To isolate viral DNA from infected cells, cells were
harvested, pelleted, resuspended in lysis buffer, and incubated at 37°C overnight.
DNA from either source was then extracted with phenol-chloroform, treated
with RNase A, extracted again with phenol-chloroform, precipitated with etha-
nol, resuspended in water, and quantified by qPCR using SYBR green (Applied
Biosystems) and primers specific for the UL123 gene or cellular ?-actin (46).
Analysis of viral proteins. For immunofluorescence, cells grown on glass
coverslips were washed once with PBS and fixed in 2% paraformaldehyde for 15
min. Fixed cells were washed twice with PBS and permeabilized with 0.1% Triton
X-100 for 15 min. Cells were washed twice with PBS, incubated in blocking
solution (2% bovine serum albumin in PBS) for 30 min, and then labeled with the
primary antibody in blocking solution for 1 h. Cells were washed again and then
incubated with the secondary antibody in blocking solution for 1 h. Cells were
then washed three times with PBS, rinsed once with H2O, and mounted on slides
with Slow-Fade solution containing DAPI (4?,6?-diamidino-2-phenylindole) to
counterstain the DNA (Molecular Probes). Images were captured by using a
Zeiss LSM510 confocal microscope and Nikon Eclipse Ti. The antibodies used
for immunofluorescence were an anti-FLAG M2 (Sigma) and anti-HA (Sigma)
and a secondary goat anti-mouse antibody conjugated with Alexa Fluor 546 or
488 (Molecular Probes).
To analyze proteins by Western blotting, samples were mixed with sample
buffer (0.05 mM Tris-HCl [pH 6.8], 2% SDS, 10% glycerol, 0.1% bromophenol
blue, 1.25% [vol/vol] 2-mercaptoethanol) and separated by electrophoresis in an
SDS-containing 10% polyacrylamide gel. Resolved proteins were transferred to
membranes and blocked with 5% nonfat milk in Tris-buffered saline containing
0.05% Tween 20. Membranes were incubated with a secondary goat anti-mouse
antibody (Amersham) and visualized by ECL-Plus detection (Amersham) and a
Molecular Dynamics Storm phosphorimager. Proteins were detected with the
primary antibodies to pUL69 (1E12 ), ppUL83 (8F5 ), pUL44 (Virusys),
pUL123 (1B12), FLAG M2 (Sigma), pUL99 (10B429), HA (Sigma), and tubulin
To evaluate the protein constituents of virus particles, virus was centrifuged
through a sorbitol cushion and then dissolved in 500 ?l of TN buffer (50 mM Tris
[pH 7.4], 100 mM NaCl). Undiluted virus and serial dilutions of the pellet were
frozen at ?80°C and subsequently assayed by Western blotting. Some virion
samples were treated with protease prior to analysis of their protein constituents.
For protease treatment, aliquots (5 ?l) of virus were treated with trypsin (25, 40
or 65 ?g/ml), in the presence or absence of Triton X-100 (1%) and incubated at
37°C for 1 h before analysis.
Luciferase assays. 293 cells were seeded onto 12-well plates using 5 ? 104cells
per well and transfected using Fugene 6 (Roche) according to the manufacturer’s
instructions. Cells were transfected with 50 ng of pGL3-MIEP reporter plasmid
and 10, 100, or 500 ng of pCGN empty vector, pCGN-pUL29/28, or pCGN-
pUL29 effector plasmid. Luciferase activity was assayed 48 h posttransfection
using a luciferase reporter assay system (Promega) and a Victor3 luminometer
(Perkin-Elmer) according to the manufacturers’ instructions. Luciferase activity
was measured by using equal protein amounts within each lysate and normalized
to the luciferase activity from empty vector. Expression of pUL29/28 and pUL29
was assayed by Western blot analysis using the same lysates.
UL28 and UL29 ORFs are expressed from a spliced mRNA.
The AD169 UL28 and UL29 ORFs are transcribed from the
same DNA strand between nt 35893 and nt 34754 and between
nt 37005 and nt 35924, respectively, with UL29 residing up-
stream of UL28 (Fig. 1A, top) (7). The UL29 ORF begins with
an AUG and potentially encodes a 360-amino-acid protein,
while the UL28 ORF potentially encodes a 379-amino-acid
protein but lacks a starting methionine. Since we knew that
amino acid sequence from UL29 and UL28 are contained in
one polypeptide (27), we searched for potential RNA splicing
motifs and identified a consensus splice donor site at nt 35927
within the UL29 gene and a consensus acceptor site within the
UL28 gene at nt 35780 (Table 1).
To test for a spliced mRNA, PCR primers were used to
FIG. 1. HCMV-coded transcripts containing the UL28 and UL29
genes. (A) Location of the UL28 and 29 ORFs on the viral genome and
organization of their transcripts. The top of the panel diagrams the coding
region showing the location of the UL26-UL30 ORFs within the viral
chromosome. The coding sequences are indicated by open arrows with
the C terminus of each ORF indicated by an arrowhead. Also indicated
are the locations of three polyadenylation signals (filled circles) and a
putative TATA box element (arrow). The position of deletions in substi-
tution mutations within the UL28 and UL29 genes of the recombinant
viruses, BADsubUL28 and BADsubUL29, are indicated by filled boxes.
The bottom of the panel shows UL28 and UL29-related mRNAs with the
location of splice donor and acceptor sites noted. Splice sites were iden-
tified by 3? RACE analysis using RNA isolated at 6 and 24 hpi from
fibroblasts infected with wild-type HCMV. The nucleotide numbers are
from GenBank accession no. X17403. (B) Expression of unspliced and
spliced transcripts during infection. Total RNA was isolated at indicated
times from fibroblasts infected at a multiplicity of 0.5 infectious unit/cell
with wild-type virus. RT-PCR was performed using primers that span a
predicted splice donor at nt 35927 and an acceptor at nt 35780 and
produced products corresponding to both spliced (188 nt) and unspliced
(334 nt) cDNAs. Primers to GAPDH were used for a loading control.
PCR products for both spliced and unspliced sequences were also ob-
served using cDNA prepared from purified polysomes but not in the
non-RT (N) control. (C) Accumulation of RNAs containing UL28 and
UL29 sequences after infection with wild-type virus. Fibroblasts were
infected under the conditions described above. Total RNA was isolated at
the indicated times after infection, and RNA was quantified by real-time
RT-PCR using primers specific to UL28 and UL29 and normalized to
GAPDH RNA. RNAs containing the UL26, UL30, UL123, and UL83
ORFs were analyzed as controls for the rate of accumulation of viral
RNAs from different kinetic classes.
VOL. 83, 2009 HCMV UL28 AND UL29 FACILITATE GENE EXPRESSION10189
amplify sequences from cDNA preparations that were gener-
ated at various times after infection of fibroblasts. The primers
were designed so that cDNAs derived from unspliced and
spliced RNAs would produce 334- and 188-nt products, respec-
tively. Both products were observed by using standard PCR
throughout the time course (Fig. 1B, left panel), indicating that
both unspliced and spliced RNAs were present. In addition, we
observed both the spliced and the unspliced products using
cDNA prepared from RNA that was isolated from purified
polysomes (Fig. 1B, right panel). This result argues that both
RNAs are translated and rules out the possibility that the
unspliced product is simply the result of amplification from
pre-mRNA sequences. DNA sequence analysis of the spliced
PCR-generated product confirmed the existence of a splice
junction at the predicted site (Table 1 and Fig. 1A, bottom).
This spliced mRNA would produce a 701-amino-acid protein
coded by the UL29 and UL28 ORFs (Fig. 2A), and the un-
spliced mRNA would produce a 360-amino-acid UL29 protein.
In order to identify the 3? ends of RNAs containing the
UL29/UL28 ORF, we performed 3?RACE with primers spe-
cific to UL28 to amplify cDNA isolated at 6 and 24 hpi. Se-
quence analysis of multiple clones located the 3? end of the
transcript at nt 31874. The sequences also identified a second
splicing event near the 3? end of the mRNAs: two splice donor
sites (one just downstream of the UL28 stop codon at nt 34650
and another just within the 5? end of the UL27 ORF at nt
34645) and a common splice acceptor site within the UL26
3?-untranslated region (3?UTR) at nt 31933 (Table 1). The
UL26 gene is downstream of UL28 and expressed in the same
orientation (Fig. 1A, top). Although earlier work identified a
UL26-specific AUUAAA polyadenylation signal at nt 32103
(45), it falls within the intron specified by the splice acceptor
identified within the UL26 3?UTR. A putative CAUAAA poly-
adenylation signal is present at nt 31892, 18 nt from the site of
poly(A) addition identified in the sequence of the 3?RACE
products (Table 1) and within the range expected for the motif
to direct processing (8). In sum, our analysis has identified two
mRNAs that contain the UL29 and UL28 ORFs (Fig. 1A,
bottom). One is spliced to generate a combined UL29/28 cod-
ing region, and the other does not contain a splice at this
position. Both are spliced near their 3? end so that they utilize
a poly(A) addition signal downstream of UL26.
Previous studies have identified multiple RNAs expressed
within the locus spanning from UL29 to UL26 during the early
and late phases of infection (6, 45). We used qRT-PCR with
primers specific for either UL29 or UL28 to assay DNase-
treated total RNA isolated at various times after infection at a
multiplicity of 0.1 infectious unit/cell (Fig. 1C). RNA contain-
ing UL29 or UL28 was detected at 2 hpi, accumulated to
maximal levels by 6 hpi, and remained at that level until 72 hpi,
the last time assayed. Probes to the two ORFs detected the
accumulation of RNA with identical kinetics, which is consis-
tent with our discovery that the two ORFs are carried on the
same RNA. Similar kinetics were evident for accumulation of
immediate-early UL123 RNA, and accumulation of early-late
UL83 RNA occurred with a delay as expected (Fig. 1C). The
RNAs detected at 2 hpi might have been delivered to cells by
infecting virions, which contain numerous virus-coded RNAs
pUL29/28 is a 79-kDa protein localized to the nucleus and
cytoplasm of infected cells. The identification of the in-frame
splicing event (Table 1) between the UL29 and UL28 ORFs
predicts that infected cells contain a 701-amino-acid protein,
pUL29/28. Previous in silico structural and functional analysis
failed to identify substantial homologies within the UL28 or
UL29 protein sequences to mammalian proteins (34). However,
we searched for nuclear localization sequences (NLS) in the pri-
mary sequence by using two different algorithms, PSORT II (36)
and Predict NLS (15) and identified a putative NLS signal,
RRPRRKR, at amino acids 32 to 38 (Fig. 2A). This sequence
has been previously identified and demonstrated to function as
an NLS in the mammalian Sox2 transcription factor (22) and
suggests that pUL29/28 accumulates within the nuclei of in-
fected cells. In addition to the putative NLS, the UL29 and
UL28 protein sequences contain two US22 domains, and these
motifs are also noted in Fig. 2A.
In order to detect expression of the putative pUL29/28 pro-
tein, we introduced a FLAG epitope tag at the amino terminus
of UL29 within the AD169 strain of HCMV to produce
BADinUL29F. The growth of the tagged virus was compared
to BADwt after infection of fibroblasts at a multiplicity of 0.1
infectious unit/cell, and the variant grew with the same kinetics
as its parent (Fig. 2B). This demonstrates that the introduction
of the FLAG epitope at the amino terminus of the UL29 ORF
maintains the function of the protein during viral infection,
since previous studies have shown that disruption of the ORF
results in reduced virus growth (50). We also introduced the
FLAG epitope at the carboxyl terminus of the UL28 ORF, and
this variant grew with the same kinetics as the parental virus
TABLE 1. Identified splice donor and acceptor sites in the UL26-to-UL29 regiona
Donor siteAcceptor site
Junction (coding potential) Poly(A) addition (nt)c
CGT AGˆG TGA GTC 35780GAC AGˆG CTC GTC CGT AG-G CTC GTC
(Arg Arg Leu Val)
CCC GTG GˆAT CAG 31933GGGTGGTˆGGGGATC CCC GTG G-GGGGATC
31874 (CAUAAA nt 31892)
ATG AAC CCˆC GTG 31933GGGTGGTˆGGGGATC ATG AAC CC-GGGGATC
31874 (CAUAAA nt 31892)
aThe indicated nucleotide is underlined in each sequence. The nucleotide numbers are from GenBank accession no. X17403: UL26 (nt 32775 to 32209), UL27
(nt 34657 to 32831), UL28 (nt 35893 to 34754), and UL29 (nt 37005 to 35923). The exon is indicated in boldface. NA, not applicable.
b*, Identified by PCR and sequence analysis; †, identified by 3?RACE analysis.
cThe poly(A) signal sequence is indicated in parentheses.
10190 MITCHELL ET AL.J. VIROL.
Using Western blot analysis, we detected an ?79-kDa pro-
tein in cells infected with BADinUL29F (Fig. 2C), and this is
the predicted size of FLAG-tagged pUL29/28 that would be
expressed from the RNA with spliced UL29/28 ORFs (Fig. 1A,
bottom). Expression of pUL29/28 was observed as early as 6
hpi and continued throughout infection (Fig. 2C), and this is
consistent with the kinetics of RNA expression (Fig. 1C). In
addition, we observed a smaller (41-kDa), less-abundant, FLAG-
tagged protein specific to infection, which was not detected at
6 h but evident at 24 hpi. The polypeptide migrates at the
predicted size for pUL29 expressed from the RNA containing
the unspliced UL29 and 28 ORFs (Fig. 1A, bottom), which we
found to be associated with polysomes (Fig. 1B, right panel). A
nonspecific band was monitored to demonstrate that equal
protein was loaded for each sample (Fig. 1C, asterisk). In
infections using BADinUL28F, we also detected a 79-kDa
protein beginning at 6 hpi (Fig. 2C). However, the smaller
41-kDa band was not observed, as would be expected if the
smaller protein terminates at the end of the UL29 ORF. Im-
munofluorescence using an antibody specific for the FLAG
epitope identified protein expression as early as 6 hpi and
continuing through the course of infection using BADinUL29F
virus (Fig. 3). No differences were observed in protein local-
ization between BADinUL29F and BADinUL28F (Fig. 3).
The fluorescent signal must be generated by pUL29/28 in
BADinUL28F-infected cells, because pUL29 protein would not
be tagged. However, we cannot rule out a contribution from
pUL29 to the fluorescent images observed in BADinUL29F-
infected cells, even though it was not detected at 6 hpi and is
expressed to a significantly lower level than pUL29/28 at later
times (Fig. 2C). Expression of the tagged protein was exclu-
sively within the nucleus between 6 and 48 hpi (Fig. 3), a
finding consistent with the existence of an NLS within the
protein sequence. At 72 hpi, tagged protein was concentrated
within replication centers in the nucleus, and it also was
present in the cytoplasm (Fig. 3). Taken together, our results
FIG. 2. Early expression and predominantly nuclear localization of HCMV pUL29/28. (A) Amino acid sequence of HCMV strain AD169
pUL29/28 encoded by the spliced transcript encoding the UL29 and UL28 genes. The location of the junction between UL29 and UL28 is indicated
by an arrow. A putative nuclear localization signal is in boldface letters, and the US22 family domains found in both UL29 and UL28 are
underlined. (B) Virus expressing epitope-tagged pUL29/28 grows with normal kinetics. Replicate cultures of fibroblasts were infected at a
multiplicity of 0.5 infectious unit/cell with wild-type HCMV (wt) or a recombinant virus containing a FLAG epitope at the N terminus of the UL29
ORF (inUL29F) and at the C terminus of the UL28 ORF (inUL28F). Culture supernatants were harvested at the indicated times, and the
infectious virus progeny was quantified. (C) Accumulation of tagged proteins in cells infected with inUL29F and inUL28F viruses. Fibroblasts were
infected at a multiplicity of 3.0 PFU/cell, harvested at the indicated times, and processed for Western blot assay using a FLAG-specific antibody.
A nonspecific band (asterisks) was monitored to confirm equal protein loading.
VOL. 83, 2009 HCMV UL28 AND UL29 FACILITATE GENE EXPRESSION10191
demonstrate expression of pUL29/28 as a 79-kDa protein dur-
ing infection beginning very early after infection and a less
pUL29/28-deficient viruses produce less viral DNA and prog-
eny. Previously, our laboratory generated two recombinant vi-
ruses, BADsubUL28 and BADsubUL29, which contained
transposon insertions between nt 35568 and nt 35780 and be-
tween nt 36866 and nt 36896, respectively (50) (Fig. 1A, top).
Disruption of either ORF resulted in a small plaque variant
that produced a reduced yield in fibroblasts (50). Since the
mutations are relatively small and contained entirely within the
UL29 and UL28 ORFs, it is unlikely that surrounding ORFs
are affected. Nevertheless, as a test for the possibility that the
mutations influenced expression of additional ORFs from the
complex UL29/28 coding region (Fig. 1A, top), we monitored
expression of the downstream UL26 ORF in BADsubUL28-
infected cells. Western blot assay demonstrated that pUL26
accumulated at 24 and 72 hpi (Fig. 4A). We also observed by
qRT-PCR in BADsubUL28-infected cells expression of RNAs
containing both the UL27 and UL30 ORFs (Fig. 4A).
To investigate the effect of mutations in UL29 and UL28 dur-
ing virus growth, we first determined whether BADsubUL29 and
BADsubUL28 virions exhibited wild-type infectivity by quan-
tifying their genome to PFU ratios relative to their parent,
BADwt. Virions were concentrated and partially purified by
centrifugation through a sorbitol cushion. Three samples for
each virus prepared in the identical way were assayed for
infectivity by plaque assay and genome copy number by qPCR.
The infectivity of mutant virions was very similar (within a
factor of 2) to that determined for wild-type particles (Fig. 4B).
We next determined the kinetics of viral DNA accumulation
after infection with aliquots of mutant and wild-type virus
stocks containing the same number of genomes and equivalent
to a multiplicity of ?0.1 infectious unit/cell. Viral genome
levels during infection were determined through qPCR using
primers to HCMV DNA and normalized to cellular actin DNA
in each sample (46). Both mutants exhibited a 10-fold de-
crease in DNA accumulation compared to the wild-type
virus (Fig. 4C).
Having demonstrated that BADsubUL28 and BADsubUL29
virions are equally infectious and that both mutants exhibit
reduced viral DNA accumulation, we analyzed the growth ki-
netics of BADsubUL28. During a single round of virus growth
after infection with virus aliquots containing the same number
of genomes and equivalent to a multiplicity of ?0.1 infectious
unit/cell, BADsubUL28 produced 10-fold less cell-associated
and cell-free virus compared to BADwt (Fig. 4D). The reduced
yield is likely a direct reflection of reduced viral DNA accu-
mulation since both defects are of the same magnitude.
pUL29/28 is required for efficient immediate-early RNA ac-
cumulation. To more precisely identify the step during the viral
life cycle compromised by the mutation in the UL29/28 coding
region, we determined the expression levels of viral transcripts
during the course of infection. HCMV expresses its genes in a
regulated cascade of immediate-early, early, and late genes, and
qRT-PCR was performed using DNase-treated total RNA iso-
lated from fibroblasts infected with BADwt, BADsubUL28, or
BADsubUL29 virus stocks containing the same number of ge-
nomes and equivalent to a multiplicity of 0.1 infectious unit/cell.
Immediate-early RNAs (UL123 and UL37x1), early RNAs
(UL44 and UL54), and the late RNAs (UL32 and UL99) were all
reduced by a factor of ?5 by 96 hpi in mutant compared to
wild-type virus-infected cells (Fig. 5A). The levels of the UL123
and US3 immediate-early RNAs produced by BADsubUL28 and
BADwt were examined in a second, independent experiment, and
by a factor of ?2 at each time assayed between 4 and 10 hpi
Expression of pUL29/28 activates the MIEP. Because we
observed a decrease in immediate-early gene expression
upon disruption of UL28 during viral replication, we were
next interested in determining whether pUL29/28 and pos-
sibly pUL29 expression would influence the activity of the
FIG. 3. Localization of pUL29/28F in fibroblasts during infection. Cells were infected at a multiplicity of 0.5 infectious unit/cell using either
inUL29F or inUL28F, fixed at the indicated times, and processed for immunofluorescence using a FLAG-specific antibody (inUL29F, green;
inUL28F, red) and DAPI (blue).
10192 MITCHELL ET AL.J. VIROL.
viral MIEP outside the context of an HCMV infection. To
express these proteins in the absence of infection, we intro-
duced the defined coding region into a mammalian expression
vector containing the HA epitope tag (Fig. 6A). Western blot
analysis of lysates from transfected 293T cells demonstrated
expression of a ?79-kDa protein upon transfection using the
UL29/28 coding sequence and a ?41-kDa using the UL29
sequence (Fig. 6A), a finding consistent with our observations
from cells infected with epitope-tagged viruses (Fig. 2C). Im-
munofluorescence using the HA antibody of transfected cells
showed expression of both proteins within the nucleus (Fig.
6B), which was also observed during viral infection (Fig. 3) and
confirms the existence of an NLS within the UL29 coding
To determine whether expression of either protein could
influence MIEP activity, we performed luciferase assays using
a reporter containing the MIEP upstream of the luciferase
gene within the pGL3-Basic vector. Increasing amounts of
empty vector and pUL29 or pUL29/28 expression vector were
transfected into 293T cells. The luciferase activity was deter-
mined at 48 h posttransfection using equal protein levels, and
these data are presented in Fig. 6C. Increasing amounts of
pUL29/28 expression resulted in a fourfold increase in lucifer-
ase activity relative to empty vector. Using the same lysate used
to measure luciferase activity, we observed by Western blot
analysis an increase in the levels of pUL29/28 (Fig. 6C, bot-
tom) relative to cellular tubulin expression. Upon transfection
of pUL29, a twofold increase in luciferase activity (Fig. 6C)
was observed relative to increasing amounts of empty vector,
and Western blot analysis demonstrated pUL29 protein levels
were similar to that of pUL29/28 (Fig. 6C). These observations
demonstrate that pUL29 and pUL29/28 can both influence
MIEP activity, albeit to various degrees.
pUL29/28 is a component of HCMV virions. Previous studies
have demonstrated that several US22 family members are
present in HCMV virion preparations (2, 16, 37, 41, 48). The
defect we observed in immediate-early gene expression in the
absence of pUL29/28 would be consistent with the loss of a
protein delivered to infected cells in virions, so we tested
whether pUL29/28 is present in virions. BADinUL29F virions
were partially purified by centrifugation through a sorbitol
cushion, and proteins associated with the virion preparation
were characterized by Western blot analysis. Multiple dilutions
of virions were assayed to account for differences in protein
abundance and antibody avidity. In Fig. 7A, we demonstrate
that several proteins known to be packaged into HCMV viri-
ons were present in the BADinUL29F preparation: pUL44,
pUL69, pUL83, and pUL99. An anti-FLAG antibody was used
to assay for the pUL29/28 protein, and it detected pUL29/28
within the undiluted virion preparation, as well as the 1:10
dilution sample. Infected cell lysates harvested late after infec-
tion were used as positive controls. The UL123-coded IE1
protein previously has not been observed in virion preparations
(48), and we also failed to demonstrate its presence, even
though it was clearly present in the lysate control. The associ-
ation of pUL29/28 with virions was further analyzed by treat-
ment with trypsin (Fig. 7B). Virions were maintained intact
during the treatment or they were incubated with detergent to
solubilize their envelopes and expose internal proteins. The
known tegument protein, pUL99, was not affected by trypsin
treatment of intact virions, but it was partially degraded upon
disruption of virions with Triton X-100 (Fig. 7B). Under the
same conditions, pUL29/28 was resistant to digestion when
virions remained intact, and, like pUL99, it was degraded when
virions were disrupted with detergent prior to protease treat-
ment (Fig. 7B). The smaller FLAG-tagged protein (presum-
ably pUL29F) was detected in virion preparations, and it also
FIG. 4. Inefficient BADsubUL28 and BADsubUL29 replication.
(A) Expression of UL26, UL27, and UL30 in fibroblasts infected with
subUL28 compared to wild-type virus. Fibroblasts were infected using
equivalent amounts of virus, harvested at the indicated times, and
processed for Western blot analysis with a pUL26-specific antibody.
Expression of RNAs containing UL27 and UL30 ORFs were quanti-
fied by qRT-PCR using total RNA harvested from cells infected by
wild-type and subUL28 viruses. (B) Infectivity of wild-type, subUL28,
and subUL29 viruses. Genome content was determined by using qPCR
and primers against HCMV DNA using partially purified virus, and
infectious units were determined by quantifying IE1-positive cells us-
ing the same virus stock. The results are from duplicate experiments
and are presented relative to wild-type virus. (C) Accumulation of viral
DNA. Fibroblasts were infected with wild type, subUL28, or subUL29
at an input genome number equivalent to 0.1 infectious unit of wild-
type virus/cell. Total cell-associated DNA was isolated, and viral ge-
nomes were quantified by using real-time PCR and normalized to
?-actin DNA. (D) Single-step growth analysis of subUL28. Cell-asso-
ciated (left panel) and cell-free (right panel) virus was assayed. Fibro-
blasts were infected with wild-type or subUL28 virus at an input ge-
nome number equivalent to 0.1 infectious unit of wild-type virus/cell.
Infected cell culture medium was collected as cell-free virus samples,
and cell-associated virus was isolated by freezing and thawing cell
pellets. The amount of virus present in each sample was determined by
counting the number of IE1-positive cells and experiment was com-
pleted in duplicate. wt, wild type.
VOL. 83, 2009HCMV UL28 AND UL29 FACILITATE GENE EXPRESSION10193
became susceptible to trypsin after treatment with detergent
Our results demonstrate that pUL29/28 is packaged within
HCMV encodes a diverse set of proteins that influence the
immediate-early environment of the cell (26). These proteins
are either newly expressed upon initiation of viral gene expres-
sion or are introduced as components of infectious virions
upon virus entry. We have identified here a new HCMV gene
product, pUL29/28, that is expressed from the UL29 and UL28
ORFs through the use of multiple splice donor and acceptor
sites (Fig. 1A). Previous reports suggested that the mRNAs
within the UL29-UL26 region share a common polyadenyla-
tion signal, AUUAAA, within the UL26 3?UTR at nt 32103
(10, 45). This motif is not present in the UL29/28 mRNAs that
we have mapped, and we have identified a second putative
polyadenylation signal at nt 31892, CAUAAA, just upstream
of the site of poly(A) addition, as seen in the 3?RACE products
By using a virus with an epitope tag at the N terminus of the
UL29 sequence, BADinUL29F, we demonstrated expression
of pUL29/28 protein throughout the course of infection at the
predicted size of 79 kDa (Fig. 2C). In addition, a less abundant,
41-kDa protein specific to infected cells was observed (Fig.
2C). We also observed these proteins upon transient transfec-
tion of expression vectors containing the mapped coding se-
quences (Fig. 6A). The 41-kDa protein was not observed dur-
ing infection using a virus with an epitope tag at the C terminus
of the UL28 sequence, BADinUL28F (Fig. 2C). This is the
predicted size of UL29 protein expressed from an unspliced
transcript. We observed both spliced and unspliced RNAs as-
FIG. 5. pUL29/28-deficient viruses express reduced levels of viral transcripts. (A) Accumulation of viral RNAs during a single-step growth
analysis. Fibroblasts were infected with wild-type (wt), subUL28, or subUL29 at an input genome number equivalent to 0.1 infectious unit of
wild-type virus/cell. Total RNA was isolated at the indicated times after infection, and viral RNA was quantified by real-time RT-PCR using
primers specific to the indicated genes and normalized to GAPDH RNA. (B) Accumulation of HCMV RNAs during the immediate-early phase
of infection. Total RNA was isolated at the indicated times postinfection, and RNA levels were quantified as described above using primers specific
to the immediate-early genes UL123 and US3.
10194 MITCHELL ET AL.J. VIROL.
sociated with polysomes isolated from HCMV-infected cells
(Fig. 1B), which is consistent with the finding that a smaller
protein is expressed from this locus. Our results suggest that a
79-kDa protein, pUL29/28, and a 41-kDa protein, pUL29 (Fig.
2C), are expressed through alternative splicing and share a
common amino-terminal domain.
Two mutant viruses, BADsubUL29 and BADsubUL28, unable
to produce pUL29/28, produced less immediate-early RNA com-
pared to wild-type virus (Fig. 5A). The UL29 and UL28 genes
are adjacent to and expressed in the same orientation as the
UL30, UL27, and UL26 genes (Fig. 1A), and earlier work has
demonstrated that disruption of UL26 results in a delay in
immediate-early gene expression and reduced virus yield (28).
Given the similarity in phenotypes, we tested whether the disrup-
tion of the UL29/28 genes influenced pUL26 expression. Western
blot analysis with an antibody to pUL26 demonstrated that the
mutations in BADsubUL29 and BADsubUL28 still allow for ex-
pression of pUL26 during infection (Fig. 4A). We have moni-
tored the expression of UL27 and UL30 by qRT-PCR and
observed RNAs containing both ORFs in BADsubUL28-in-
fected cells (Fig. 4A). Similar levels of expression occurred
between viruses for RNAs containing the UL30 ORF. How-
ever, we observed slightly reduced levels of UL27 RNA ex-
pression. Because pUL29/28-deficient viruses result in reduced
gene expression, we cannot distinguish between reduced levels
of UL27 RNAs mediated from sequence disruptions in
UL29/28 versus an overall reduction in pUL29/28-mediated
gene expression. However, other reports have demonstrated
that disruption of the UL27 ORF results in only a slight growth
attenuation in fibroblasts, and no growth phenotype was ob-
served during virus replication in human tissue implanted in
SCID mice (10, 38). In addition, we have recently identified the
start site of an RNA containing the UL27 ORF that sits down-
stream of the UL28 ORF (unpublished observations). Thus,
the impaired immediate-early gene expression and growth
defect we observed for BADsubUL29 and BADsubUL28
(Fig. 4) almost certainly result from disruption of pUL29/28
and pUL29 function and not from ancillary effects on neigh-
FIG. 6. pUL29/28 activates the MIEP. (A) Transient expression of
pUL29/28 and pUL29. The UL29/28 and UL29 sequences were intro-
duced into mammalian expression vector pCGN in-frame with the HA
tag. 293T cells were transfected using empty vector or the pUL29/
28HA or pUL29HA expression vectors, and whole-cell lysates were
analyzed by Western blotting using an antibody to HA or to cellular
tubulin. (B) Localization of pUL29/28HA and pUL29HA was visual-
ized by indirect immunofluorescence on transfected cells using anti-
HA (green) and DAPI (blue). (C) 293T cells were transfected using 50
ng of pGL3-MIEP reporter plasmid and 10, 100, or 500 ng of pCGN
empty vector, pCGN-pUL29/28, or pCGN-pUL29 effector plasmid.
Luciferase activity was assayed 48 h posttransfection using equal pro-
tein amounts within each lysate and normalized to luciferase activity
from empty vector. The levels of pUL29/28HA and pUL29HA expres-
sion were assayed by Western blot analysis using the same lysates and
antibody to HA or cellular tubulin.
FIG. 7. pUL29 and pUL29/28 are present in HCMV virions.
(A) Partially purified BADinUL29F virion preparations contain
pUL29/28F. Virions were solubilized, and serial dilutions, as well as an
infected cell lysate control, were analyzed by Western blotting with
antibodies to the FLAG epitope and the known virion proteins pUL69,
pUL44, pUL83, and pUL99. pUL123 was assayed as a negative con-
trol. (B) pUL29F and pUL29/28F are located within the virion enve-
lope. inUL29F virions were treated with increasing amounts of trypsin
(T), with trypsin and Triton X-100 (TT), or untreated (U). Virions
were then solubilized, and proteins were analyzed by Western blotting
with a monoclonal antibody specific to the FLAG epitope and, as a
control, a monoclonal antibody to pUL99.
VOL. 83, 2009 HCMV UL28 AND UL29 FACILITATE GENE EXPRESSION10195
boring ORFs. Further evidence for this conclusion comes from
the fact that both of these proteins can induce activity of the
MIEP within transfected cells (Fig. 6C).
RNAs from the UL26-29 region have been reported to be
expressed with early or late kinetics (6). Given the delay in its
expression, how might pUL29/28 and pUL29 be influencing
immediate-early gene expression? Although previous charac-
terization of HCMV virion and dense body components by
using mass spectrometry did not detect pUL29/28 or pUL29
within particles (48), we found an association by using a virus
with an epitope tag at the N terminus of the UL29 sequence.
pUL29/28 and pUL29 both copurify with virions and are pro-
tected from protease digestion by the virion envelope (Fig. 7A
and B). Consistent with their presence in virions, a portion of
the tagged proteins is present in the assembly zone late after
infection and, as expected for a role in activation of immediate-
early gene expression, pUL29/28 is predominantly nuclear at
6 h after infection (Fig. 3). Newly synthesized pUL29/28 might
also influence immediate-early gene expression. We have dem-
onstrated the presence of pUL29/28 RNA (Fig. 1C) and pro-
tein (Fig. 2C and 3) within infected cells very early after infec-
tion and, importantly, the amount of RNA containing UL28
and UL29 sequences increases from 2 to 6 hpi (Fig. 1C). Thus,
even though drug sensitivity experiments have categorized this
RNA as early-late, it is clearly accumulating during the imme-
diate-early phase of infection. In sum, our data indicate that
pUL29/28 and pUL29 are delivered to cells in virions and also
expressed very early in infection, and they are predominantly
nuclear. Thus, the proteins are present at the start of infection
and correctly positioned to assist in the activation of immedi-
There is ample precedent for tegument proteins such as
pUL29/28 and pUL29 to influence immediate-early gene ex-
pression. The pUL26 tegument protein was demonstrated to
contain a strong transcriptional activation domain (45), and a
UL26 deletion virus exhibited a delay in the onset of immediate-
early gene expression (28). In this case, it is unclear whether
pUL26 is directly activating immediate-early expression, since
pUL26 can also influence the stability of viral particles (28, 43).
We observed similar levels of infectivity between wild-type virus
and the BADsubUL28 and BADsubUL29 mutants (Fig. 4A), so
it is not likely that pUL29/28 or pUL29 influences particle stabil-
ity. The pp71 protein has also been demonstrated to activate
immediate-early gene expression (5), and this transactivation ac-
tivity is enhanced by the tegument protein pUL35 (43). pp71
activation depends upon proteasomal degradation of the cellular
Daxx protein, a negative regulator of immediate-early gene ex-
HCMV promoters are, of course, regulated by histone mod-
ifications (12, 19, 31–33, 36, 40), and we recently discovered
that the pUL38 protein interacts with pUL29/28, as well as
with the host cell nucleosome remodeling and histone deacety-
lase (NuRD) complex (27). The NuRD complex contains his-
tone deacetylases and chromatin-remodeling ATPases, and it
can repress transcription (4, 14). Although we have not yet
shown that pUL29/28 and NuRD reside in the same complex,
it is conceivable that pUL29/28 acts to protect the viral chro-
mosome from repressive effects of the NuRD complex.
We thank Eain Murphy for help with the galK system, Mariko
Aoyagi for providing polysomes fractions, Katherine Faust for the
qPCR experiments, and Trish Robinson for help with HCMV anti-
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VOL. 83, 2009 HCMV UL28 AND UL29 FACILITATE GENE EXPRESSION10197