The ULb′ Region of the Human Cytomegalovirus Genome Confers an Increased Requirement for the Viral Protein Kinase UL97

Depeng Wang; Gang Li; Martin Schauflinger; Christopher C Nguyen; Ellie D Hall; Andrew D Yurochko; Jens von Einem; Jeremy P Kamil

doi:10.1128/JVI.03477-12

. 2013 Jun;87(11):6359–6376. doi: 10.1128/JVI.03477-12

The ULb′ Region of the Human Cytomegalovirus Genome Confers an Increased Requirement for the Viral Protein Kinase UL97

Depeng Wang ^a, Gang Li ^a, Martin Schauflinger ^b, Christopher C Nguyen ^a, Ellie D Hall ^a, Andrew D Yurochko ^a, Jens von Einem ^b, Jeremy P Kamil ^a,^✉

PMCID: PMC3648124 PMID: 23536674

Abstract

We report a requirement for the viral protein kinase UL97 in human cytomegalovirus (HCMV) replication that maps to the ULb′ region of the viral genome. A UL97-null (Δ97) mutant of strain TB40/E, which encodes a full-length ULb′ region, exhibited replication defects, particularly in production of cell-free virus, that were more severe than those seen with a Δ97 mutant of laboratory strain AD169, which harbors extensive deletions in its ULb′ region. These differences were recapitulated with additional HCMV strains by treatment with a UL97 kinase inhibitor, 1-(β-l-ribofuranosyl)-2-isopropylamino-5,6-dichlorobenzimidazole (maribavir). We observed lower levels of viral DNA synthesis and an increased requirement for UL97 in viral late gene expression in strains with full-length ULb′ regions. Analysis of UL97-deficient TB40/E infections by electron microscopy revealed fewer C-capsids in nuclei, unusual viral particles in the cytoplasmic assembly compartment, and defective viral nuclear egress. Partial inhibition of viral DNA synthesis caused defects in production of cell-free virus that were up to ∼100-fold greater than those seen with cell-associated virus in strains TB40/E and TR, suggesting that UL97-dependent defects in cell-free virus production in strains with full-length ULb′ regions were secondary to DNA synthesis defects. Accordingly, a chimeric virus in which the ULb′ region of TB40/E was replaced with that of AD169 showed reduced effects of UL97 inhibition on viral DNA synthesis, late gene expression, and production of cell-free virus compared to parental TB40/E. Together, these results argue that the ULb′ region encodes a factor(s) which invokes an increased requirement for UL97 during viral DNA synthesis.

INTRODUCTION

Human cytomegalovirus (HCMV) is the prototypical member of the betaherpesvirus family (reviewed in reference 1). HCMV establishes lifelong, typically asymptomatic infections in healthy adults and children. However, HCMV is among the leading causes of congenital defects in newborns and of devastating opportunistic infections in immunocompromised patients. Moreover, HCMV promotes accelerated allograft rejection and is arguably the most common viral complication following organ transplantation (2, 3). The antiviral drug of first recourse to combat HCMV infection is the nucleoside analog ganciclovir (reviewed in reference 4). UL97, a viral protein kinase encoded by HCMV, phosphorylates ganciclovir, which activates the drug's potential to serve as a chain terminator during viral DNA synthesis while also providing specificity for infected cells. Nonetheless, ganciclovir shows undesirable levels of toxicity, and drug-resistant mutants can emerge during long-term treatment. Therefore, new drugs are being evaluated to combat HCMV infection. Among these is 1-(β-l-ribofuranosyl)-2-isopropylamino-5,6-dichlorobenzimidazole (maribavir [MBV]), a UL97 kinase inhibitor (reviewed in reference 4).

UL97 is required for efficient viral replication, and moderate to severe viral replication defects are seen during infection of cultured cells with UL97 mutants or when UL97 kinase activity is inhibited (5–11). However, the extent to which UL97 kinase activity is required for infection and pathogenesis in vivo is unknown and the therapeutic potential of MBV has yet to be firmly established (12–17). Regardless, the efficacy of UL97 inhibitors to combat HCMV disease in patients would depend upon the roles of this viral protein kinase during infection.

The current understanding of the biological roles of UL97 comes from studies of infections in cultured cells using UL97-null deletion mutants (Δ97), kinase-deficient point mutants, or wild-type (wt) viruses in the presence of UL97 inhibitors (5, 7–9, 18–26). However, these studies have been conducted almost exclusively with extensively passaged, “laboratory strains” of HCMV, such as AD169 and Towne. UL97-deficient infections with such strains exhibit defects in myriad viral processes during replication, including nuclear egress, viral DNA synthesis, virion morphogenesis, and cytoplasmic egress of enveloped virions, such that UL97 has been hypothesized to play roles in each of these processes (7, 9, 18–23, 27). Identification of the retinoblastoma tumor suppressor protein (pRb) and nuclear A-type lamins as cellular substrates of UL97 kinase activity has suggested mechanisms by which UL97 might contribute to viral DNA synthesis and nuclear egress, respectively (21, 23, 27, 28). Cdh1 (Fzr), a component of the anaphase-promoting complex/cyclosome, is also a substrate of UL97 (24), and a recent report indicates that its phosphorylation by UL97 is physiologically relevant during viral replication (25).

We are presently unaware of any reports of UL97 mutants in endotheliotropic HCMV strains, sometimes referred to as “clinical strain surrogates,” such as TR, TB40/E, VR1814 (FIX), and Merlin, even though these viruses have been cloned as infectious bacterial artificial chromosomes (BACs), which facilitate generation of mutant viruses (29–32). These strains display a number of biological properties that are lost following extensive passage in fibroblasts, including the potential to establish latency and the ability to productively infect several clinically relevant cell types, such as leukocytes, epithelial cells, and endothelial cells (33) (reviewed in references 34 and 35). Many of these properties depend on genes located in the ULb′ region (ULb′) of the viral genome, which accumulates large deletions during tissue culture adaptation (31, 36). Since HCMV encodes only one conventional protein kinase, UL97, we hypothesized that aspects of viral biology lost from extensively passaged, laboratory-adapted strains might encompass processes regulated by its viral protein kinase activity. Therefore, we set out to examine the role of UL97 during viral replication in two HCMV strains that preserve many genetic and phenotypic features of low-passage-number clinical isolates.

MATERIALS AND METHODS

Cells and virus.

BAC clones of HCMV strain AD169, AD169rv, a gift of Ulrich Koszinowski, and its UL97-null derivative, Δ97 AD169rv, have been described elsewhere (6, 37). A BAC clone of HCMV strain TB40/E, TB40-BAC4 (TB40/E) (38), was a gift of Christian Sinzger (University of Ulm, Ulm, Germany). A BAC clone of HCMV strain TR (31, 39), TRgfp repair 2, which expresses the Aequorea victoria green fluorescent protein from a cassette inserted between US7 and US8, was a gift of Dong Yu (Washington University, St. Louis, MO) and was used with permission from Jay Nelson (Oregon Health Sciences University, Portland, OR). A BAC clone of HCMV strain AD169 bearing a repaired UL131A gene, BAD r131-Y4 (BAD r131), which has been described elsewhere (40), was kindly provided by Tom Shenk (Princeton University, Princeton, NJ). Viruses used in experiments were passaged no more than once following reconstitution of infectious virus from BAC DNA, at a multiplicity of infection (MOI) of 0.01 or lower; in the case of ATCC Towne HCMV (a gift of Lee Fortunato, University of Idaho, Moscow, ID), virus was passaged only once following receipt of the virus. All experiments were performed using virus stocks that had been concentrated by ultracentrifugation through a 20% (wt/vol) sorbitol cushion and resuspended in Dulbecco's modification of Eagle's medium with l-glutamine, pyruvate, and phenol red (DMEM) (Cellgro, Manassas, VA), containing 0.1% fetal bovine serum (FBS; Atlanta Biologicals, Inc., Lawrenceville, GA), as described previously (20).

Primary human foreskin fibroblasts (HFF), a gift of Jennifer Spangle (Harvard Medical School, Boston, MA) and Karl Münger (Harvard Medical School), and HEK 293T cells (Genhunter Corp.) were maintained in complete medium, which was comprised of DMEM supplemented with 10% FBS, 20 μg/ml gentamicin (Life Technologies, Grand Island, NY), and 10 μg/ml ciprofloxacin (Genhunter Corp., Nashville, TN). For experiments involving serum starvation (Fig. 1), HFF were seeded in complete medium, allowed to attach for 2 h, washed twice with Dulbecco's phosphate-buffered saline lacking calcium and magnesium (DPBS; Cellgro, Inc.), and then maintained for 72 h in DMEM supplemented with 0.1% FBS plus gentamicin and ciprofloxacin at the concentrations described above.

Fig 1 — Generation and characterization of human foreskin fibroblasts stably expressing UL97. (A) Human foreskin fibroblast (HFF) cells were transduced with a lentiviral vector expressing UL97 (Lenti-97) or with a control lentiviral vector (Lenti-C). Cells that grew out following puromycin selection were analyzed for expression of UL97 by Western blotting. (B) The enzymatic activity of the UL97 expressed in Lenti-97 HFF was monitored by evaluating the phosphorylation status of the retinoblastoma tumor suppressor protein (pRb), a UL97 substrate, in the presence or absence of 2 μM maribavir (MBV), under conditions of serum starvation (0.1% FBS). pRb phosphorylated at Ser807/811 (P-pRb^S807) was detected using a phosphospecific antibody; total pRb levels (pRb, total) were detected using monoclonal antibody 4H1. In both panels, levels of beta-actin (actin) were also monitored to control for protein loading. (C and D) Cell-free (C) and cell-associated (D) titers of Δ97 TB40/E (Δ97) were compared to those of wild-type parental TB40/E (wt) in a replication kinetics experiment conducted at MOI = 1 on Lenti-97 HFF. Error bars represent standard deviations. For certain data points, error bars are too small to be visible.

Viral replication kinetics and yield experiments were conducted in 24-well cluster plates (Corning, Inc., Corning, NY) which had been seeded 1 day prior to infection with 8 × 10⁴ HFF cells per well, in complete medium. Infections for assays of viral protein expression and viral DNA synthesis were conducted in 6-well cluster plates (Corning, Inc.) that had been seeded with 4 × 10⁵ HFF per well, in complete medium, 1 day prior to infection. For all experimental infections, virus was applied to cells in complete medium supplemented with FBS at 2% (instead of 10%) (2% FBS–DMEM). After 2 h, inocula were aspirated and cells were washed in DPBS and maintained in 2% FBS–DMEM for the remainder of the experiment.

Growth analysis and virus titration.

For viral replication and yield experiments, duplicate wells were harvested for each condition at the indicated time points, and quantities of infectious virus were determined as follows. Infected-cell supernatants, comprised of 1 ml per well of a 24-well cluster plate, were collected and set aside. Cells were then scraped with a 1,000 μl pipette tip and collected separately in 1 ml of fresh 2% FBS–DMEM per well. Infected cell supernatant and scraped cell samples were then stored at −80°C until analysis.

Virus titers were determined by immunological detection of viral immediate early protein IE1-72, as previously described (41), with minor modifications. Infected-cell supernatants were thawed, subjected to a vortexing procedure, and used to determine titers of cell-free virus. Scraped cell samples were thawed, subjected to a vortexing procedure, spun for 5 min at 1,500 × g to pellet cell debris, and used to determine titers of cell-associated virus. Five-fold serial dilutions of samples were performed in 2% FBS–DMEM, and 100 μl of each dilution was applied to duplicate wells of a 96-well cluster plate (Corning, Inc.) containing freshly confluent HFF, incubated for 24 h at 37°C in a humidified environment containing 5% CO₂, and then fixed in cold methanol (−20°C) for 15 min. Following fixation, cells were washed twice with 3.2 mM Na₂HPO₄–0.5 mM KH₂PO₄–1.3 mM KCl–135 mM NaCl (pH 7.4) (PBS) and stained overnight at 4°C with a 1:200 dilution in antibody diluent solution (ADS; Life Technologies) of monoclonal antibody (MAb) 1B12 hybridoma supernatant (a gift of Tom Shenk, Princeton University, Princeton, NJ). Cells were then washed twice in PBS, incubated at 37°C for 1 h in a 1:500 solution of biotin-labeled goat anti-mouse antibody (Life Technologies)–ADS, washed twice in PBS, incubated 1 h at 37°C in a 1:1,000 dilution of horseradish peroxidase (HRP)-streptavidin conjugate (Life Technologies)–PBS containing 0.5% bovine serum albumin (EMD Millipore, Billericia, MA), 0.1% Tween 20 (Sigma-Aldrich, St. Louis, MO), and 0.01% thimerosal (Enzo Life Sciences, Farmingdale, NY), washed twice in PBS, and stained using an aminoethyl carbazole substrate kit (Life Technologies), according to the manufacturer's instructions. Cell nuclei staining positive for IE1-72 expression were enumerated in duplicate wells from dilutions that produced 50 to 100 positive cells per well. When titers were too low to result in 50 positive-testing cell nuclei per well, wells inoculated with neat sample were counted. Infectious units (IU) of virus determined by this method represent IE1-72-positive nuclei per ml of virus stock.

Pharmacological inhibitors.

A UL97 inhibitor, 1-(β-l-ribofuranosyl)-2-isopropylamino-5,6-dichlorobenzimidazole (maribavir [MBV]), was a gift of John Drach (University of Michigan, Ann Arbor, MI). Where indicated, MBV was applied to cell cultures at a final concentration of 2 μM and was diluted into tissue culture medium from a 2 mM (1,000×) stock solution in dimethyl sulfoxide (DMSO) (Sigma-Aldrich). DMSO (0.1% [vol/vol]) was used to control for effects of the carrier. For experiments in which phosphonoformic acid (PFA) (Sigma-Aldrich) was used to directly inhibit viral DNA synthesis (see Fig. 7), stock solutions (1,000×) were prepared in distilled water, filter sterilized, and stored at −20°C until use. Because MBV treatments were often conducted in parallel, 0.1% DMSO was included in all PFA conditions and controls.

Fig 7 — Partial inhibition of viral DNA synthesis has particularly drastic effects on cell-free virus production. (A and B) HFF were infected with HCMV strain TB40/E (A) or TR (B) at MOI = 1 and incubated in the presence of increasing concentrations of the viral DNA synthesis inhibitor phosphonoformic acid (PFA), from 0 to 200 μM, in 50 μM increments. Infections were harvested at 96 hpi for analysis of viral DNA levels and at 120 hpi for determination of cell-free and cell-associated virus titers. The x axis shows viral DNA synthesis as the number of viral *UL123* gene DNA copies, normalized to the number of copies of the cellular 18S rRNA gene (*18S*), a multicopy allele, as determined by qPCR. The y axis indicates the viral titer in infectious units per ml. Cell-free titers are indicated by hollow circles, and cell-associated titers are indicated by filled black squares. Linear regression analysis is shown as a dashed line for cell-free virus titers and a solid line for cell-associated titers. A two-tailed analysis was used to compare the slopes of the regression lines. In panel A, the r² values of the regression lines were 0.9722 for cell-associated virus and 0.9359 for cell-free virus. In panel B, the r² values were 0.9887 for cell-associated virus and 0.9400 for cell-free virus. Horizontal error bars for triplicate measurements of viral DNA synthesis indicate standard deviations. Variations in measurements of titers were in most cases too small for vertical error bars to be visible.

BAC mutagenesis.

Procedures for “en passant” two-step Red mutagenesis of BACs (“recombineering”) have been described elsewhere (42, 43) and were carried out as previously described (6, 20), with minor modifications. Briefly, genetic manipulations were performed in Escherichia coli strain GS1783 (a gift of Greg Smith, Northwestern University, Chicago, IL). Oligonucleotide primers used for recombineering procedures were custom synthesized and purified by polyacrylamide gel electrophoresis (PAGE) by Integrated DNA Technologies, Inc. (Coralville, IA). Procedures to incorporate a UL97-null mutation (Δ97) in BAC clones TRgfp (TR) and TB40/E-BAC4 (TB40/E) by deleting 1,513 bp of viral sequences located between the BamHI and PstI sites of the UL97 allele were performed as previously described (6), except that the use of pBAD-ISce-I plasmid was not necessary, since Escherichia coli strain GS1783 produces an l-(+)-arabinose-inducible I-SceI homing endonuclease activity. To generate rescue viruses from Δ97 TB40/E and Δ97 TR, a wild-type (wt) UL97 allele from strain AD169, bearing an N-terminal FLAG epitope tag (DYKDDDDK), and an excisable kanamycin-resistant (Kan^r) marker, was released as an RsrII-PpuMI restriction fragment from pFLAG97-TSR (6) and electroporated into E. coli strain GS1783 harboring Δ97 TB40/E or Δ97 TR. Following resolution of Kan^r integrates, recombinant BACs were verified by restriction enzyme digestion analysis and by DNA sequencing of the modified region (Genewiz, Inc., South Plainfield, NJ) (not shown).

To replace the ULb′ region (ULb′) of strain TB40/E with that of AD169, the ULb′ of TB40/E was first replaced with an amplicillin resistance allele (bla), using an approach similar to a method previously described by another group (44); in this case, the approach taken was designed to prevent undesired recombination events during subsequent insertion of the ULb′ from strain AD169. Primers dUL128 bla Fw (5′-TCTCAAAACGCGTATTTCGGACAAACACACATTTTATTATGCGGAACCCCTATTTGTTTA-3′) and dUL150 bla Rv (5′-GAGGGAGAATTCTTACTCGGGGAACAGTTGGCGGCA-3′) were used to generate a PCR product containing the bla allele from plasmid pSP72 (Promega, Inc., Madison, WI). Following gel purification, this PCR product was used to obtain ampicillin-resistant (Amp^r) integrates from GS1783 E. coli harboring the TB40/E BAC, resulting in TB40/E ΔULb′.

In parallel, a PCR product containing an I-SceI-aphAI cassette (43) was produced using primers ULb′ Kan Fw (5′-TACCCGTTCGCCCTTACCTTCCCGTTGTCATGCACCTTTAGCGCGTACCCTCACCTCTTGTAGGGATAACAGGGTAATCGATTT-3′) and ULb′ Kan Rv (5′-TTGGACAACTTTGACGTGCTCAAGAGGTGAGGGTACGCGCTAAAGGTGCATGACAACGGGGCCAGTGTTACAACCAATTAACC-3′) and was used to obtain Kan^r integrates of GS1783 E. coli harboring the AD169rv BAC. These integrates incorporated within the AD169rv ULb′ an I-SceI-aphAI element that could later be excised without leaving behind any residual sequences, per the method of Tischer et al. (43). One of the latter integrates was used as the template, with primers TB40E.Ad.ULb_in Fw (5′-TCTCAAAACGCGTATTTCGGACAAACACACATTTTATTATTCACTGCAGCATATAGCCCATTTTAGC-3′) and TB40E.Ad.ULb_in Rv (5′-CTTAATCCCCCCGCTTCTACACGCAACGCTGCATACAGCTAGTAAGCAGCCTCTTCGTGGCCG-3′), to produce a PCR product, which was used to obtain Kan^r, ampicillin-sensitive (Amp^s) integrates from GS1783 E. coli harboring TB40/E ΔULb′. I-SceI endonuclease and bacteriophage λ Red recombinase activities were sequentially induced, resulting in kanamycin-sensitive colonies containing TB40/E_ULb′:AD169, a TB40/E BAC in which the ULb′ had been replaced with that of strain AD169. The TB40/E_ULb′:AD169 BAC was confirmed for its genetic integrity and sequence fidelity by restriction enzyme digestion (see Fig. 9C) (data not shown) and by DNA sequencing (Genewiz, Inc.; data not shown), respectively.

Fig 9 — Construction of a recombinant TB40/E containing the ULb′ region of strain AD169. (A) Schematic of the strategy used to replace the ULb′ region (ULb′) of HCMV strain TB40/E with that of strain AD169. (Top panel) The TB40/E ULb′ is shown at the top, with open reading frames (ORFs) indicated by hollow arrows; viral gene names for selected ORFs are provided for reference. The TB40/E coordinates marking the left and right flanks where an ampicillin resistance marker, *bla*, was temporarily used to replace the intervening sequences are indicated by black lines that converge on the middle panel, which represents a PCR product encoding the *bla* ORF, shown as an arrow with horizontal stripes. (Bottom panel) A second PCR product (AD169 ULb′ I-SceI-aphAI) encompassing the ULb′ region from AD169, disrupted by an excisable kanamycin resistance marker, *aphAI*, coupled to an I-SceI homing-endonuclease site (*I-SceI*), and flanked by direct repeats (DR1 and DR2), was used to replace the *bla* marker. Finally, a recombination event to remove the *aphAI* marker was induced, resulting in TB40/E_ULb′:AD169. (B) The ULb′ of TB40/E_ULb′:AD169, along with part of its unique short region, is shown, with ORFs indicated by white arrows. The sequences originating from strain AD169 are indicated. (C) KpnI restriction enzyme digestion of TB40/E and TB40/E_ULb′:AD169 BAC DNAs. BAC DNAs were digested, resolved overnight on an 0.7% agarose gel, stained with ethidum bromide, and imaged. One fragment predicted to be absent from parental TB40/E but present in TB40/E_ULb′:AD169 and three fragments predicted to be absent from TB40/E_ULb′:AD169 but present in parental TB40/E are indicated by black arrows. (D and E) Multicycle replication of TB40/E_ULb′:AD169 was compared to that of wild-type (wt) parental TB40/E. HFF were infected at MOI = 0.05, and titers of cell-free (D) and cell-associated (E) virus were determined for the indicated time points. Error bars represent standard deviations and in certain cases are too small to be visible. dpi, days postinfection.

Virus reconstitution.

Infectious virus was reconstituted from BAC DNA by cotransfection of HFF with 1 to 3 μg of BAC DNA, pp71 expression plasmid pSG5-pp71 (45) (a gift of Robert Kalejta, University of Wisconsin, Madison, WI), and Cre recombinase expression plasmid pCAGGS-nlsCre (46) (a gift of Michael I. Kotlikoff, Cornell University, Ithaca, NY), using Superfect (Qiagen, Inc., Valencia, CA), according the manufacturer's recommendations, or by electroporation, as described previously (6).

Generation of UL97-expressing cells.

A FLAG epitope-tagged UL97 open reading frame (ORF) was PCR amplified from BAC DNA of FLAG97 AD169rv (6), using primers EcoF97 Fw (5′-AAAAAAGAATTCACCATGGACTACAAGGATGACGACGATAAGTC-3′) and EcoF97 Rv (5′-GAGGGAGAATTCTTACTCGGGGAACAGTTGGCGGCA-3′) and KOD Hot Start DNA polymerase (EMD Millipore, Billerica, MA). Unless otherwise noted, all subsequent restriction enzymes and bacterial competent cells for routine plasmid subcloning were purchased from New England BioLabs, Inc., of Danvers, MA. The PCR product was digested with EcoRI and inserted into calf intestinal phosphatase-treated, EcoRI-cut, “pENTR1A no ccDB” plasmid (47) (a gift of Eric Campeau; Addgene plasmid 17398), by treatment with T4 DNA ligase, and transformed into NEB 5alpha E. coli. The resulting plasmid, pENTR1A-UL97, was sequenced (Genewiz, Inc.; not shown) to confirm the integrity of the inserted sequence and then included in a LR recombination reaction (LR clonase II enzyme mix; Life Technologies) with “pLenti PGK Puro DEST” plasmid (47) (a gift of Eric Campeau; Addgene plasmid 19068) and transformed into NEB 5alpha E. coli. The resulting pLenti PGK Puro UL97 plasmid (pLenti-97) was verified by restriction digestion (not shown).

Lentivirus vector particles were produced and used to transduce HFF by following the Addgene pLKO.1 protocol (http://www.addgene.org/tools/protocols/plko/). Briefly, 7 × 10⁵ HEK293T cells (Genhunter Corporation, Nashville, TN) were seeded in a 60-mm-diameter dish and cotransfected the following day with 1 μg of either pLenti-PGK UL97 or a puromycin-resistant control lentivirus, pLKO.1-TRC control (Addgene plasmid 10879; gift of David Root, Massachusetts Institute of Technology, Cambridge, MA), along with 250 μg pMD2.g (Addgene plasmid 12259) and 750 μg psPAX2 (Addgene plasmid 12260) using Fugene 6 transfection reagent (Roche Applied Science, Indianapolis, IN), according to the manufacturer's instructions. pMD2.g and psPAX2 were gifts of Didier Trono (École Polytechnique Fédérale de Lausanne, Switzerland). Transfection reagents were removed after overnight incubation and replaced with complete medium. For each lentivirus vector, 24-h and 48-h supernatants were collected, pooled, spun for 5 min at 1,250 rpm, filtered through a 0.45-μm-pore-size syringe filter (Corning, Inc.), and frozen at −80°C until use. To transduce HFF, 4 × 10⁵ HFF were seeded in a T25 flask (Corning, Inc.) and allowed to attach overnight. On the following day, 0.5 to 1 ml of lentivirus supernatant was applied in a final volume of 3 ml complete medium supplemented with 8 μg/ml hexadimethrine bromide (Polybrene; Santa Cruz Biotechnology, Santa Cruz, CA). After 24 h, supernatant was removed and replenished with complete medium supplemented with 0.8 μg/ml puromycin dihydrochloride (EMD Calbiochem). Lentivirus-transduced cells were subsequently passaged and maintained in complete medium supplemented with 0.8 μg/ml puromycin.

Western blotting.

Western blotting was performed as previously described (6, 20). Briefly, HFF were seeded at 4 × 10⁵ cells per well in 6-well cluster plates and allowed to attach overnight in complete medium. Cells were infected with the indicated viruses, or mock infected, for 2 h. Upon removal of inocula, medium was replaced with 2% FBS–DMEM. At the indicated time points postinfection (pi), cells were washed twice in DPBS and then lysed in 2× sodium dodecyl sulfate-polyacrylamide electrophoresis (SDS-PAGE) sample buffer (48) containing HALT phosphatase and protease inhibitor cocktail (Thermo Scientific Pierce, Rockford, IL) and 5% β-mercaptoethanol, heated for 5 min at 95°C, and resolved on denaturing SDS-PAGE gels (6, 20). Proteins were electrophoretically transferred to Whatman Protran BA85 nitrocellulose membranes (GE Healthcare, Inc., Waukesha, WI) using a Criterion Blotter (Bio-Rad, Inc., Hercules, CA) and blocked in PBS containing 0.1% Tween 20 (PBST) and 5% powdered milk (PM-PBST). Antibodies specific for beta-tubulin (rabbit monoclonal; Epitomics Inc., Burlingame, CA), beta-actin (mouse monoclonal; Li-Cor, Inc. Lincoln, NE), UL97 (6), UL44 (mouse monoclonal clone CH13; Virusys Corp., Taneytown, MD), pp28 (mouse monoclonal; clone 5C3), UL57 (mouse monoclonal [clone CH167]; Virusys), IE1-72 (mouse monoclonal [clone 1B12]; a gift of Tom Shenk, Princeton University, Princeton, NJ), retinoblastoma protein (pRb, mouse monoclonal [clone 4H1]; Cell Signaling Technology, Danvers, MA), pRb phosphorylated at serine positions 807 and 811 (rabbit polyclonal; Cell Signaling Technology), and pp150 (mouse monoclonal clone 36-14; a gift of Bill Britt, University of Alabama, Birmingham, AL) were diluted into PM-PBST and used to probe membranes overnight at 4°C on a platform rocker. Membranes were washed four times in PBST for 5 min per wash and probed with anti-rabbit, anti-goat, or anti-mouse horseradish peroxidase (HRP)-conjugated secondary antibodies (Southern Biotechnology, Inc., Birmingham, AL) diluted 1:5,000 in PM-PBST. Finally, membranes were washed four times and imaged by chemiluminescence (Supersignal Pico; Thermo Scientific Pierce). For quantification of protein expression, IRDye 800-CW-conjugated secondary antibodies (Li-Cor, Inc.) were used at 1:15,000 instead of HRP conjugates, and membranes were imaged on a Li-Cor Odyssey infrared imaging system and analyzed using Li-Cor Image Studio 2.0 software (Li-Cor, Inc.).

qPCR.

Real-time quantitative PCR (qPCR) to determine levels of viral DNA, in UL123 (IE1-72) gene copy numbers, normalized to levels of copies of the cellular 18S ribosomal DNA (rDNA) gene (18S), was performed as previously described (1), with modifications described elsewhere (49), except that absolute quantification was performed using three replicates of 1.0 ng of total DNA per sample and ranges of serial 10-fold dilutions for standard curves of TB40/E BAC DNA (30) and of pJK-18S (49) were each 10⁷ to 10³ copies/well. For UL123 qPCRs, PCR efficiency ranged from 91.2% to 102.7%, standard curve regression values (r²) were from 0.987 to 0.997, and the slopes of the calibration curve ranged from −3.259 to −3.553. For 18S reactions, PCR efficiency was 97.1% to 101.5%, r² values ranged from 0.991 to 0.998, and the slopes of the calibration curves ranged from −3.287 to −3.393.

Statistical analyses.

Statistical analyses were performed using Prism software versions 5.0d and 6.0b for Macintosh (GraphPad Software, Inc., La Jolla, CA).

Electron microscopy.

Electron microscopy procedures were performed as described previously (50), with a few minor modifications. Briefly, HFF cells were cultured on carbon-coated sapphire discs (Engineering Office, M. Wohlwend GmbH, Switzerland) (3 mm in diameter), infected at an MOI of 1, flash-frozen at 144 h postinfection (hpi) by high-pressure freezing with an HPF 01 apparatus (Engineering Office, M. Wohlwend GmbH), and freeze substituted in acetone containing 0.1% (weight/vol) uranyl acetate, 0.2% (weight/vol) osmium tetroxide, and 5% (vol/vol) water, as described previously (50–52). After embedment in Epon, ultrathin sections were mounted on Formvar-coated single-slot copper grids for transmission electron microscopy (TEM). The sections were imaged with a JEOL JEM-1400 transmission electron microscope equipped with a 2k charge-coupled-device (CCD) camera at an acceleration voltage of 120 kV. For the quantification of viral capsids in nuclei, two micrographs with a primary magnification of 50,000 were taken in arbitrarily chosen areas of each nucleus analyzed, and the amounts of A-, B-, and C-capsids were determined in 15 infected cells from two independent experiments for each condition. For the assessment of cytoplasmic virus particles, micrographs covering the entire area of the cytoplasmic viral assembly complex of each infected cell analyzed were taken with a primary magnification of 25,000, and virus particles were classified in at least 20 infected cells from two independent experiments for each condition.

RESULTS

We engineered a UL97-null (Δ97) mutation into a BAC clone of TB40/E, an endotheliotropic HCMV strain (30), and observed that infectious virus could not readily be reconstituted following transfection of permissive human foreskin fibroblasts (HFF). It took several weeks for even sparse levels of cytopathic effects (CPE) to appear in Δ97 TB40/E-transfected cells. These discrete foci of CPE failed to spread throughout the cell monolayer, even after months in culture, and accordingly failed to produce detectable titers of infectious virus (not shown). In contrast, Δ97 mutant BACs of laboratory strains, such as AD169 or Towne, readily reconstitute on transfected HFF and ultimately produce large amounts of infectious virus (6, 10, 11). Unlike strains AD169 and Towne, which harbor large deletions in the ULb′ region (ULb′) of the viral genome, strain TB40/E retains a ULb′ that resembles those seen in HCMV clinical isolates (30, 31, 36, 53). We found that the same Δ97 lesion, when engineered into TRgfp, a BAC clone of HCMV strain TR, which also encodes a full-length ULb′ (31, 39), likewise resulted in a virus that replicated too poorly to produce detectable titers of infectious virus (not shown). These observations suggested to us that HCMV strains with full-length ULb′s might have a greater requirement for UL97 than laboratory-adapted HCMV strains, which harbor numerous deletions and rearrangements in this region.

Generation of UL97-expressing cells to complement UL97-null HCMVs.

The UL97 lesion that we introduced into TB40/E and TRgfp is identical to one that we characterized previously in the context of laboratory strain AD169 (6, 20, 27). It shares the same coordinates as the first reported Δ97 mutant, RCMPΔ97, which was also derived from strain AD169 (8). Since wild-type (wt) AD169 and TB40/E replicate to comparable titers on fibroblasts (30, 37), we hypothesized that HCMV strains that carry full-length ULb′s have a greater requirement for UL97 than do laboratory strains. However, in order to evaluate the phenotypes of Δ97 mutants of strains such as TB40/E and TR, we first needed complementing cells that could support their replication.

We therefore constructed a lentiviral vector, pLenti-97, to express UL97, generated lentivirus particles, and transduced low-passage-number HFF. After selecting for cells that continued to proliferate for at least 3 weeks under conditions of puromycin selection, UL97 expression was assayed by Western blotting. A prominent band immunoreactive to anti-UL97 rabbit polyclonal serum was observed in lysates of pLenti-UL97-transduced cells but not in those of age-matched control HFF or in HFF that had been transduced with a control lentivirus (Fig. 1A). The predicted molecular weight (MW) of the UL97 species expressed from pLenti-97 is 79.2, and the relative mobility of the immunoreactive band, compared to a prestained molecular weight ladder (not shown), was ∼85. UL97 is known to autophosphorylate (54), which could explain the slight decrease in observed mobility of the band compared to its theoretical MW. Therefore, we interpreted this band to represent UL97.

To assay for the biological activity of the UL97 protein expressed in our lentivirus-transduced HFF cells, we subjected the cells to serum starvation in the presence or absence of a UL97 kinase inhibitor, maribavir (MBV), and performed Western blotting to monitor the phosphorylation status of the retinoblastoma tumor suppressor protein (pRb), an established substrate of UL97 kinase activity (23, 28). Serum-starved, pLenti-UL97-transduced HFF exhibited constitutive pRb phosphorylation at serine positions 807 and 811 (Ser 807/811) which was abolished in the presence of 2 μM MBV (Fig. 1B). In contrast, pRb phosphorylation was not detected in age-matched, control HFF that were transduced with a puromycin-resistant control lentivirus which expresses a noncoding RNA (55) (Fig. 1B). Since phosphorylation of pRb at Ser 807/811 depends on UL97 activity in HCMV-infected cells and since this pair of serines is directly phosphorylated by UL97 in vitro (28), we concluded that the UL97 expressed in our stably transduced cells was enzymatically active. We also detected increased levels of pRb in UL97-expressing cells (Fig. 1B), which is consistent with reports that pRb hyperphosphorylation (inactivation) is associated with increased pRb expression and globally improved mRNA translation efficiency (56–58). Importantly, the UL97-expressing HFF (Lenti-97 HFF) were able support reconstitution of Δ97 TR and Δ97 TB40/E BACs (not shown).

We assessed the growth of Δ97 TB40/E and wt TB40/E on our UL97-expressing cells in a single-cycle replication kinetics experiment performed at MOI = 1. At 120 to 144 hpi, Δ97 TB40/E produced titers of cell-free and cell-associated virus that were nearly equivalent to those of the wt strain. At earlier time points, Δ97 TB40/E showed mild defects in production of cell-free and cell-associated virus compared to wt TB40/E (Fig. 1C and D). Nonetheless, because both Δ97 TB40/E and wt TB40/E replicated efficiently on our pLenti-97-transduced HFF and ultimately produced very similar titers, we concluded that these cells were suitable for propagation of Δ97 TB40/E.

Δ97 TB40/E exhibits severe replication defects on noncomplementing HFF.

To evaluate whether strains with a full-length ULb′ possessed a greater requirement for UL97 than strains that lack most of this region, we compared growth of Δ97 TB40/E and Δ97 AD169 to that of their respective parental wt viruses in multicycle and single-cycle replication experiments in HFF at MOIs of 0.05 and 1.0 infectious units (IU) per cell, respectively. Additionally, replication was assayed for a Δ97 rescue virus (Δ97-R) derived from Δ97 TB40/E in which the UL97 lesion had been repaired.

In multicycle infections at MOI = 0.05, Δ97 TB40/E exhibited an extremely pronounced defect in production of cell-free virus, yielding titers that were 80,000-fold lower than those seen with parental wt TB40/E or rescue (Δ97-R) viruses at 15 days postinfection (dpi), while the wt and rescue viruses replicated indistinguishably from each other (Fig. 2A). The defect in cell-associated virus production for Δ97 TB40/E approached only 100-fold and was thus at least 2 orders of magnitude smaller than the defect seen in cell-free virus production (Fig. 2B). In contrast to Δ97 TB40/E, Δ97 AD169 exhibited defects of only ∼120-fold for cell-free virus production and ∼10-fold for cell-associated virus (Fig. 2C and D).

Fig 2 — Δ97 TB40/E shows more severe replication defects than does Δ97 AD169. (A to D) Cell-free (A and C) and cell-associated (B and D) titers of Δ97 mutants of AD169 and TB40/E, containing identical lesions in the *UL97* gene, were compared to wild-type (wt) controls in a multicycle replication kinetics experiment following infection (MOI = 0.05) of HFF. (E and F) Δ97 TB40/E (Δ97) was compared to wild-type TB40/E (wt) and rescue virus (Δ97-R) for single-cycle production of cell-free (E) and cell-associated (F) virus following infection of HFF at MOI = 1. (G and H) Wild-type AD169 (wt) was compared to Δ97 AD169 (Δ97) for cell-free (G) and cell-associated (H) virus, at MOI = 1, in parallel to the experiment whose results are shown in panels E and F. For all panels, error bars represent standard deviations and in some cases are too small to be visible.

At MOI = 1, Δ97 TB40/E showed an ∼1,000-fold defect in cell-free virus production compared to the parental wt and rescue viruses, which replicated indistinguishably from each other (Fig. 2E). The peak defect of Δ97 TB40/E in cell-associated virus production, on the other hand, was only ∼20-fold (Fig. 2F). Thus, the single-cycle defect of Δ97 TB40/E in production of cell-free virus was ∼50-fold greater than its defect in production of cell-associated virus. Δ97 AD169, on the other hand, showed defects in cell-free virus production of only ∼10 to 20-fold at 120 to 144 hpi and a cell-associated defect of only ∼4-fold at 120 hpi (Fig. 2G and H). From these results, we concluded that Δ97 TB40/E showed replication defects that were more severe at MOI = 0.05 and MOI = 1 than did Δ97 AD169, especially in cell-free virus production.

Pharmacological inhibition of UL97 recapitulates especially severe replication defects for two HCMV strains that harbor full-length ULb′.

We next wanted to address whether pharmacological inhibition of UL97 kinase activity would produce single-cycle replication defects for TB40/E similar to those we had observed in Δ97 TB40/E at MOI = 1 (Fig. 2E and F). We also wished to interrogate the requirement for UL97 kinase activity in single-cycle replication of strain TR, as we had been unable to obtain sufficient titers of Δ97 TR to perform experiments at MOI = 1. We therefore assayed the effects of the UL97-specific kinase inhibitor maribavir (MBV) on replication of strains TB40/E and TR, each of which contains full-length ULb′, and on laboratory-adapted strains AD169 and Towne, which harbor extensive deletions in their ULb′s (31, 36). During infection of HFF at MOI = 1, we found that MBV treatment led to defects in cell-free virus production of ∼500-fold for TB40/E and of ∼2,500-fold for TR and cell-associated defects of ∼30-fold for TB40/E and 20-fold for TR (Fig. 3A to D). In contrast, MBV treatment of strains AD169 and Towne caused defects of only 10-fold in cell-free virus production and of 2- to 3-fold in production of cell-associated virus (Fig. 3E to H). The cell-free and cell-associated viral replication defects induced by MBV treatment of HCMV Towne strain infections were thus similar to the single-cycle defects we had observed for Δ97 AD169 as shown in Fig. 2G and H and in our earlier studies (6, 20).

Fig 3 — Strain-specific differences in effects of a UL97 inhibitor on single-cycle replication kinetics. HFF cells were infected at MOI = 1 with the indicated viruses. Starting at 2 hpi, infections were incubated in the presence of either 0.1% dimethyl sulfoxide (DMSO; filled circles connected by solid lines) or 2 μM maribavir (MBV; empty circles connected by dashed lines). At the indicated time points, samples were collected for quantification of titers of infectious cell-free and cell-associated virus produced during infection. Error bars represent standard deviations.

The single-cycle replication defects seen during MBV treatment of strain TR were comparable in magnitude to those of Δ97 TB40/E and of MBV-treated TB40/E (Fig. 2E and F and Fig. 3A to D). Thus, strains TR and TB40/E appeared to exhibit similar requirements for UL97, including a particularly strong requirement in cell-free virus production not seen with strains AD169 or Towne. Pharmacological inhibition of UL97 thus recapitulated the differing replication defects we had observed for Δ97 mutants: laboratory-adapted strains AD169 and Towne, which harbor extensive deletions in their ULb′s, showed replication defects in the presence of MBV that were less pronounced than those seen with strains TR and TB40/E, which possess full-length ULb′s.

Differing consequences of UL97-deficient infection for viral protein expression.

We wondered whether the striking inability of strains TR and TB40/E to produce cell-free virus under conditions in which UL97 was absent or inhibited (Fig. 2 and 3) might correlate with exacerbated defects in viral protein expression. We therefore compared strains TB40/E, TR, and AD169 for effects of Δ97 mutations and MBV inhibition on viral protein expression. Serum-fed HFF were infected at an MOI of 1, and cell lysates collected over a series of time points were analyzed by Western blotting for expression of viral proteins from each kinetic class. Δ97 AD169 and Δ97 TB40/E were compared, in parallel, to wt controls (Fig. 4A), and each of strains TR and TB40/E was compared to AD169, for determinations of effects of UL97 inhibition using MBV (Fig. 4C and data not shown).

Fig 4 — Analysis of viral protein expression in *UL97*-null versus wild-type virus and in the presence or absence of a UL97 inhibitor. (A) HFF were infected with wild-type (wt) or Δ97 mutants of strains AD169 and TB40/E at MOI = 1. A series of protein lysates were collected at the indicated time points, and expression of the indicated viral proteins and expression of beta-actin (actin) were compared by Western blotting. (B) Fold differences between wt and Δ97 strains in expression of viral late proteins pp28 and pp150 at 96 hpi were quantified on an infrared imaging system using the samples shown in panel A in four independent blots. Signal from detection of viral protein was normalized to beta-actin signal prior to calculation of fold differences. Error bars represent standard errors of the means. (C) HFF were infected with strain AD169 or TR at MOI = 1. Starting at 2 h postinfection (hpi), infections were maintained in the presence of 2 μM maribavir (MBV) or 0.1% dimethyl sulfoxide (DMSO), and viral protein expression was monitored as described for panel A. (D) Fold differences between DMSO and MBV treatments in expression of viral late proteins pp28 and pp150 at 96 hpi were quantified as described for panel B using lysates from the experiment whose results are shown in panel C and lysates of a similar experiment performed with TB40/E (not shown).

Genetic ablation or MBV inhibition of UL97 was not associated with large differences in viral immediate early or early gene expression as indicated by levels of IE1 (IE1-72) or levels of UL44 or UL57, respectively (Fig. 4A and C). Unexpectedly, MBV treatment appeared to cause a slight reduction in UL97 expression during infections with strains TR (Fig. 4C) and TB40/E (not shown). The lower level of discrete mobility of the UL97 species in the absence of MBV, due to autophosphorylation (54), leads to a more diffuse band, which may exaggerate underlying differences in protein expression.

Nonetheless, expression of other early gene products, i.e., UL44 and UL57, also appeared to be slightly reduced in MBV-treated infections and in Δ97 infections compared to controls (Fig. 4A and C). These differences were most notable at early times, just as the proteins became detectable (Fig. 4A and C), and might be explained by less-efficient protein translation in UL97-deficient infections. UL97 phosphorylates and inactivates pRb (23, 28), and one function of (active) pRb is to restrict mRNA translation by limiting the expression of ribosomal RNAs (57, 58). Thus, the efficiency of protein biosynthesis may be globally decreased to some degree in UL97-deficient infections relative to wt controls. Indeed, similar effects on UL57 expression were observed and yet not remarked upon in our previous study (20).

Notably, strain TB40/E showed basal levels of viral late gene expression that were lower than those seen with strain AD169, regardless of whether UL97 was present. This difference was especially striking with regard to pp28, a tegument phosphoprotein (Fig. 4A). Strain TR, on the other hand, showed basal pp28 expression that was only modestly lower than that of AD169 and expression of pp150 that was roughly similar (Fig. 4C).

The viral phosphoprotein pp150 was detected at early time points in infections with TR and with Δ97 TB40/E (Fig. 4A and C). Similarly, pp28 was detected at early times from TR infections (Fig. 4C). pp150 and pp28 are constituents of the HCMV virion tegument layer and are encoded by viral genes that exhibit “true late” (γ₂) kinetics (59, 60). Since pp150 and pp28 are not expressed de novo until late times, we interpreted our detection of these proteins at early times to reflect an increased particle-to-infectious unit (IU) ratio in our viral stocks of Δ97 TB40/E and TR, as this could explain increased delivery of tegument proteins. Regardless, the levels of pp150 and pp28 present at early times became undetectable by 24 to 48 hpi and thus did not likely affect our results concerning their de novo expression (72 to 96 hpi).

Infrared dye-labeled secondary antibodies were used to quantify differences in pp28 and pp150 expression at 96 hpi (Fig. 4B and D). We found that Δ97 TB40/E-infected cells expressed levels of these proteins that were ∼3-fold lower than those seen with wt TB40/E infections, while Δ97 AD169 showed levels that were reduced only ∼1.5-fold compared to wt AD169 levels (Fig. 4B). MBV inhibition of UL97 produced similar differences between laboratory strain AD169 and “clinical” strains TR and TB40/E (Fig. 4D). These findings suggested that strains TB40/E and TR possess a greater requirement for UL97 in expression of viral late genes than does strain AD169.

Comparison of roles of UL97 in viral DNA synthesis.

Optimal expression of viral late genes in herpesviruses requires efficient viral DNA synthesis. Therefore, we hypothesized that the increased requirement for UL97 in viral late gene expression in strains TB40/E and TR reflected an increased requirement for UL97 in viral DNA synthesis. To test this possibility, we used real-time quantitative PCR (qPCR) to compare the effects of UL97 inhibition on viral DNA synthesis in strains TR, TB40/E, AD169, and Towne and also compared the viral DNA synthesis defects of Δ97 mutants of TB40/E and AD169.

We found that laboratory-adapted strains AD169 and Towne each showed basal levels of viral DNA synthesis at 96 hpi that were approximately 2-fold higher than those seen with TB40/E or TR (Fig. 5A). This finding is consistent with the lower basal levels of late gene expression that we observed for strains TR and TB40/E compared to AD169 (Fig. 4). Moreover, TB40/E and TR showed increased defects in viral DNA synthesis when UL97 was inhibited using MBV. We found that MBV caused DNA synthesis defects of 1.8- and 1.6-fold for strains AD169 and Towne, respectively, while strains TB40/E and TR showed defects of ∼3-fold (Fig. 5B). The differences in viral DNA synthesis defects between laboratory strains AD169 and Towne and clinical-surrogate strains TR and TB40/E were statistically significant (Fig. 5B). Accordingly, Δ97 TB40/E showed a significantly larger defect in viral DNA synthesis than Δ97 AD169 in comparisons of each Δ97 mutant to its isogenic parental wt virus, i.e., 3-fold versus 1.6-fold, respectively (Fig. 5C). Thus, strains TR and TB40/E exhibited reduced basal levels of viral DNA synthesis, and an increased requirement for UL97 in viral DNA synthesis, compared to laboratory-adapted strains Towne and AD169.

Fig 5 — Viral DNA synthesis occurs at lower basal levels and exhibits a greater requirement for UL97 in strains TR and TB40/E than in laboratory strains Towne and AD169. (A and B) HFF were infected with the indicated virus strains at MOI = 1. Maribavir (MBV) (2 μM) or 0.1% dimethyl sulfoxide (DMSO) was added at 2 hpi. Total DNA was isolated from infected cells at 96 hpi, and triplicate 1-ng samples were analyzed by quantitative PCR to determine the number of copies of the viral gene *UL123* normalized to copies of cellular 18S rDNA (*18S*). (A) The basal level of viral DNA detected under DMSO control conditions (i.e., basal DNA synthesis) in a representative experiment. (B) Analysis of the effect of MBV on viral DNA levels at 96 hpi in multiple independent biological replicates (TB40/E, n = 9; AD169, n = 5; Towne, n = 3; TR, n = 4). Log₂(DMSO/MBV) values for each virus strain were compared to each other in a one-way analysis of variance (ANOVA); P values were calculated using Tukey's posttest. (C) HFF were infected with wild-type (wt) or Δ97 mutants of strains AD169 or TB40/E at MOI = 1, and viral DNA synthesis was measured as described for panel A. Differences between the wt and Δ97 mutant strains in viral DNA synthesis were compared as described for panel B. The results shown represent data from three independent biological replicates for each wt/Δ97 mutant comparison. For all panels, error bars indicate standard errors of the means.

Ultrastructural analyses of UL97-deficient TB40/E infections.

UL97-deficient infections led to more severe replication defects for strains TB40/E and TR than for strains AD169 and Towne (Fig. 2 and 3), and these differences appeared to involve a particularly drastic loss of cell-free virus production in strains encoding full-length ULb′s. To gain a more detailed understanding of this phenotype, we analyzed cells infected with Δ97 TB40/E and cells infected with wt TB40/E in the presence or absence of the UL97 inhibitor, MBV, by electron microscopy. Because electron microscopy analyses of Δ97 AD169 have been previously reported (7, 9), we did not include strain AD169 in these experiments. Following infection at MOI = 1, infected cells were harvested at 144 hpi and processed for imaging. Capsid forms from 15 nuclei, and viral particles from approximately 20 cytoplasmic assembly compartments, were quantified for each condition in two independent experiments.

Cells infected with Δ97 TB40/E or with wt TB40/E in the presence of MBV, compared to wt TB40/E control infections, showed an increase of ∼1.7-fold on average in the number of viral particles observed in nuclei (Table 1). The observation of a buildup of particles in infected cell nuclei would be consistent with reports suggesting a role for UL97 in viral nuclear egress (7, 21, 27). In nuclei in Δ97 and MBV-treated TB40/E infections, we also observed decreases of 3.2-fold and 4.4-fold, respectively, in the level of C-capsids as a percentage of total capsids (Table 1) (Fig. 6B and C versus 6A). C-capsids were classified as those containing electron-dense material, which was interpreted to represent packaged viral DNA. This decrease in the level of C-capsids was accompanied by an increase in the level of B-capsids, which contain viral scaffold proteins and which presumably represent immature capsids that have not yet packaged viral DNA (Fig. 6A to C and Table 1). A-capsids, which showed cores completely lacking electron density, were present at roughly similar percentages under all conditions (Fig. 6A to C and Table 1); these are thought to be dead end products of abortive DNA packaging.

Table 1.

Quantification of TB40/E viral capsids in cell nuclei

TB40/E strain and treatment	No. of capsids				% of capsids
TB40/E strain and treatment	B	C	A	Total	B	C	A
Δ97	2,303	148	339	2,790	82.54	5.30	12.15
wt	1,051	364	152	1,567	67.07	23.23	9.70
wt + MBV	1,975	182	376	2,533	77.97	7.19	14.84

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Fig 6 — Electron microscopy analysis of UL97-deficient infections with strain TB40/E. (A to C) Representative micrographs of arbitrarily chosen areas within the nuclei of infected HFF taken with a primary magnification of ×50,000. The amounts of A-, B-, and C-capsids were assessed (see Table 1) in HFF infected with wt TB40/E (A), Δ97 TB40/E (B), and wt TB40/E in the presence of MBV (C). Bars: 500 nm. (D to F) Representative micrographs of three types of cytoplasmic virus particles (see Table 2). (D) Virus particle with a dense (DNA-filled) core. (E) Noninfectious virus particle (NIEP) with a low-density core. (F) Particle which was presumably virus derived but could not be classified with absolute certainty (and hence is categorized as “unclassified” in Table 2). (G) Dense body. Bars: 100 nm.

Infections with Δ97 and MBV-treated wt TB40/E infections showed an overall decrease in viral particle numbers in cytoplasmic viral assembly compartments (Table 2). We also observed a paucity of enveloped DNA-containing capsids (virions; Fig. 6D) in the cytoplasm. There were approximately 10-fold-fewer virions in MBV-treated wt TB40/E and Δ97 TB40/E infections than under the wt TB40/E control condition (Table 2). This result is consistent with reports that have documented defects in numbers of cytoplasmic virions of ∼6.5-fold for a UL97 kinase-mutant based on strain AD169 and from ∼20- to 65-fold for Δ97 AD169 (7, 19). The scarcity of virions we observed in the cytosol of UL97-deficient infections was accompanied by a modest, ∼2-fold increase in the percentage of noninfectious enveloped particles (NIEPs; Fig. 6E) and a larger, ∼6-fold increase in the amount of enveloped particles that could not be classified as conventional NIEPs (Table 2 and Fig. 6F [1, 61]). Unlike dense bodies (Fig. 6G), which are pleomorphic with respect to size, these unusual particles were consistent in size, matching the ∼200-nm diameter seen for virions and NIEPs (Fig. 6D to G). Although these unusual particles did show a central region of decreased electron density (Fig. 6F) and thus possibly represented enveloped, tegumented A-capsids, we could not be certain that any steps during their morphogenesis had occurred in the nucleus. When these unusual cytoplasmic particles lacking evident capsids are excluded from calculations, the overall reductions—presumably due to nuclear egress defects—in cytoplasmic particle numbers in UL97-deficient infections were ∼4-fold and ∼9-fold for infections with Δ97 TB40/E and MBV-treated wt TB40/E, respectively (Table 2). Nonetheless, we did not find evidence of a specific defect in cytoplasmic egress. Hence, these findings could not readily account for why we had observed large decreases in production of cell-free virus but not cell-associated virus during UL97-deficient infections with strain TB40/E (Fig. 2 and 3) or, by extension, strain TR.

Table 2.

Quantification of TB40/E viral particles in cytoplasm

TB40/E strain and treatment	No. of cells/ACs^c	No. of particles counted^b				% of particles^b
TB40/E strain and treatment	No. of cells/ACs^c	NIEP^a	Nucleocapsid	Unclassified	Total no. of particles	NIEP^a	Nucleocapsid	Unclassified
Δ97	21	157	51	387	595	26.39	8.57	65.04
wt	20	205	671	122	998	20.54	67.23	12.22
wt + MBV	20	48	53	280	381	12.60	13.91	73.49

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NIEP values include a small number of nonenveloped cytoplasmic B-capsids.

For all conditions, fewer than 10% of the viral particles found in the cytoplasm were nonenveloped.

ACs, assembly compartments.

Inhibition of viral DNA synthesis disproportionately impacts cell-free virus.

Taken together, our results thus far were consistent with reported defects in viral nuclear egress (7) and viral DNA synthesis (5, 9) during UL97-deficient infections. Therefore, we wondered whether secondary effects of viral DNA synthesis defects could account for the very large (>2-log) defects in cell-free virus production that we had observed (Fig. 2 and 3). To address this possibility, we partially inhibited viral DNA synthesis using phosphonoformic acid (PFA), an antagonist of herpesvirus-encoded DNA polymerases (62) and measured for effects on accumulation of viral DNA and on levels of cell-free and cell-associated virus produced during infection. HFF cells were infected at MOI = 1 with strains TB40/E or TR and, following removal of the inoculum, were incubated with concentrations of PFA increasing from 0 to 200 μM (Fig. 7). Infections were harvested for total DNA at 96 hpi and for titration of cell-free and cell-associated virus at 120 hpi.

We performed regression analyses, plotting cell-free and cell-associated virus titers against levels of viral DNA synthesized during infection (Fig. 7). The slopes of the regression lines for titers of cell-associated and cell-free virus were strikingly different, and these differences were highly significant: P = 0.0017 for strain TB40/E and P < 0.0001 for strain TR (Fig. 7). Essentially, partial inhibition of viral DNA synthesis caused a far greater reduction in cell-free virus levels than in those of cell-associated virus (Fig. 7). For strain TB40/E, a 2.7-fold reduction in viral DNA synthesis (150 μM PFA) caused the titer of cell-associated virus to fall by 4.3-fold while causing the titer of cell-free virus to decrease by 57-fold (Fig. 7A). Similarly, a 4.2-fold reduction in DNA synthesis, seen at 200 μM PFA, caused cell-associated titers to drop by 10-fold, while cell-free titers plummeted 360-fold (Fig. 7A). For strain TR, a 2.6-fold reduction in viral DNA synthesis (150 μM PFA) led to a 2.2-fold decrease in production of cell-associated virus and a 100-fold reduction in production of cell-free virus. More remarkably, a 4.7-fold reduction in viral DNA synthesis (200 μM PFA) caused a 3.5-fold decrease in the cell-associated virus titer and a 420-fold decrease in the cell-free virus titer (Fig. 7B). Therefore, direct inhibition of viral DNA synthesis was sufficient to drastically decrease production of cell-free virus, but not cell-associated virus, in strains TB40/E and TR.

Similar results were found during treatment of TB40/E with ganciclovir, another inhibitor of viral DNA synthesis (data not shown). However, PFA was deemed superior to ganciclovir for these experiments because (i) TR is resistant to ganciclovir (31, 39) and (ii) ganciclovir, being a substrate of UL97 kinase activity, might conceivably interfere with UL97-mediated phosphorylation of its physiological substrates, confounding data interpretation. We also evaluated strain AD169 in our PFA experiments; however, effects of PFA on cell-free versus cell-associated virus in AD169 did not differ so dramatically, and accordingly, the regression lines for AD169 failed to show statistically significant differences (data not shown).

From the results of our PFA experiments, we concluded that modest (∼2- to 5-fold) decreases in viral DNA synthesis were sufficient to account for disproportionately large, ∼60- to 400-fold decreases in cell-free virus relative to cell-associated virus (∼2- to 10-fold) (Fig. 7). Furthermore, we noted that we had observed similarly divergent effects on production of cell-free virus versus cell-associated virus during UL97-deficient infections with strains TR and TB40/E which were likewise associated with modest, ∼3-fold defects in viral DNA synthesis (Fig. 2, 3, and 5).

Expression of the pentameric viral glycoprotein complex gH/gL/UL128-131A is not sufficient to cause an increased requirement for UL97.

The results of our PFA inhibition experiments (Fig. 7) suggested that the dramatic loss of cell-free virus production during UL97-deficient infections with strains TB40/E and TR likely resulted from inefficient viral DNA synthesis. Of interest, previous reports have suggested that the viral pentameric glycoprotein complex gH/gL/UL128-UL131A promotes increased retention of cell-associated virus during infection of fibroblasts (63, 64). Incorporation of the pentameric complex into progeny virions requires expression of UL128, UL130, and UL131A, which are encoded within the ULb′ (44, 74). UL128, UL130, and UL131A associate with gH and gL, which are products of UL75 and UL115, respectively. If any one of the three ULb′-encoded components is not expressed, then only the gH/gL/gO (or gH/gL) complex is expressed on virions. This feature of the pentameric complex allowed us to ask whether the presence of the pentameric complex was sufficient to cause an increased requirement for UL97 in viral replication.

To address this issue, we made use of an AD169-derived virus, BAD r131. Unlike parental AD169, which contains a frameshift mutation that prevents expression of UL131A protein, BAD r131 expresses a functional pentameric complex by virtue of a repaired UL131A gene (40). Compared under conditions of MBV inhibition for viral yield at 144 hpi, following an infection of HFF at MOI = 1, AD169 and BAD r131 showed roughly similar defects in cell-free virus production of 14-fold and 5-fold, respectively (Fig. 8). Moreover, the defects in production of cell-associated virus were virtually indistinguishable: 2-fold for AD169 and 2.5-fold for BAD r131 (Fig. 8). In contrast, TB40/E showed a 615-fold defect in cell-free virus production but only a 32-fold defect for cell-associated virus, which is consistent with the result shown in Fig. 3. Therefore, expression of the pentameric complex by an AD169-derived virus, BAD r131, failed to recapitulate the increased requirement for UL97 kinase activity we had observed in strains TB40/E and TR. These results argued against the possibility that this increased requirement for UL97 can be explained simply by a bias against release of cell-free virus conferred by expression of the pentameric gH/gL/UL128-131A complex.

Fig 8 — UL131A is not sufficient to cause an increased requirement for UL97 in cell-free virus production. HFF were infected with TB40/E, AD169, or BAD rUL131, an AD169 derivative restored for UL131A expression. Starting at 2 h postinfection (hpi), infections were maintained in the presence of 2 μM maribavir (MBV) or 0.1% (vol/vol) dimethyl sulfoxide (DMSO). At 144 hpi, samples were collected for determination of titers of cell-free and cell-associated virus. Error bars represent standard deviations. Numbers above pairs of bars indicate fold differences.

Generation and analysis of a chimeric TB40/E containing the ULb′ region of AD169.

The presence of a full-length ULb′ correlated with an increased requirement for UL97 in strains TR and TB40/E; conversely, the presence of a highly deleted ULb′ in Towne and AD169 correlated with a less pronounced UL97 requirement (Fig. 2 and 3). We therefore hypothesized that (i) sequences within the ULb′ of strains TB40/E and TR were required for an increased UL97 requirement and (ii) the loss during laboratory adaptation of ULb′ sequences in strains AD169 and Towne contributed to their decreased requirement for UL97. To test this hypothesis, we constructed a chimeric virus, TB40/E_ULb′:AD169, in which the ULb′ of strain TB40/E was replaced with that of AD169 (Fig. 9A and B). Essentially, 18,024 bp of the TB40/E ULb′, encompassing UL128 through UL150, was replaced with 3,435 bp, encompassing UL128 through UL132 of the AD169 ULb′ (Fig. 9A and B).

The integrity of the resulting recombinant virus was verified by DNA sequencing of the modified region (not shown) and by restriction digestion (Fig. 9C). Following digestion of BAC DNA with KpnI restriction enzyme and agarose gel electrophoresis, a new band, with an expected size of 15,770 bp, appeared in the TB40/E_ULb′:AD169 digest, as predicted (Fig. 9C). Furthermore, bands migrating in accordance with the expected relative mobility of predicted KpnI restriction fragments of 10,645 bp, 9,491 bp, and 7,388 bp were observed in the KpnI digest of parental TB40/E BAC DNA but not in that of TB40/E_ULb′:AD169, also as predicted. Finally, no unexpected bands were observed in KpnI (Fig. 9C) or in BamHI (not shown) digests of TB40/E_ULb′:AD169 BAC DNA. From these results, we concluded that we had successfully replaced the ULb′ of TB40/E with that of AD169 and that the resulting TB40/E_ULb′:AD169 BAC had not undergone any overt genetic rearrangements and did not contain spurious mutations within the modified region and was therefore suitable to generate virus for use in our studies.

After reconstituting infectious TB40/E_ULb′:AD169 virus, we compared multicycle replication of TB40/E_ULb:AD169 to that of parental wt TB40/E in HFF at MOI = 0.05. We observed that TB40/E_ULb′:AD169 exhibited a slight lag in production compared to parental wt virus (Fig. 9D and E). Nonetheless, TB40/E_ULb′:AD169 ultimately replicated to produce levels of cell-free and cell-associated virus similar to those seen with wt parental TB40/E, as evidenced by the lack of any appreciable differences in titers between wt TB40/E and TB40/E_ULb′:AD169 at from 12 to 18 dpi (Fig. 9D and E).

We had found differences between strains AD169 and Towne, compared to TR and TB40/E, in (i) basal levels of both viral DNA synthesis and viral late gene expression and (ii) the requirement for UL97 in these processes (Fig. 4 and 5). To test whether replacement of the TB40/E ULb′ with that of AD169 would be sufficient to confer to TB40/E any of these phenotypic aspects of strain AD169, we infected HFF at MOI = 1 in the presence or absence of MBV and compared viral DNA synthesis results at 96 hpi and viral late gene expression results over a series of time points from 24 hpi to 96 hpi (Fig. 10).

In the presence of MBV, TB40/E_ULb′:AD169 produced significantly higher levels of viral DNA than did parental wt TB40/E (Fig. 10A). MBV also caused a lower fold defect in viral DNA synthesis in TB40/E_ULb′:AD169 than in wt TB40/E: 3.1-fold versus 2.4-fold, respectively (Fig. 10B). Although the latter difference was small, TB40/E_ULb′:AD169 showed smaller fold defects than wt TB40/E in each of the five independent experiments we conducted for this study, and this difference was significant (Fig. 10B). In the absence of MBV, TB40/E_ULb′:AD169 synthesized slightly higher (basal) levels of viral DNA than did wt TB40/E; however, this effect was not uniformly reproducible (not shown) and was not statistically significant (Fig. 10A). We thus concluded that TB40/E_ULb′:AD169 exhibited a reduced requirement for UL97 in viral DNA synthesis compared to its parental virus, wt TB40/E.

In Western blotting experiments, TB40/E_ULb′:AD169 and wt TB40/E expressed the viral immediate early protein IE1 (also known as IE1-72) at similar levels, and no IE1 signal was observed from mock-infected cell samples, as expected (Fig. 10C). However, parental wt TB40/E strongly expressed the viral tegument phosphoprotein pp150 only in the absence of the UL97 inhibitor MBV, while TB40/E_ULb′:AD169 showed robust pp150 expression in both the presence and the absence of MBV (Fig. 10C). Although MBV caused slightly reduced pp150 expression at 72 hpi in TB40/E_ULb′:AD169-infected cells, its overall effects on pp150 expression in TB40/E_ULb′:AD169 were vastly eclipsed by those seen with wt TB40/E (Fig. 10C). In quantitative Western blotting experiments performed on lysates from 96 hpi, we found that MBV caused, on average, an ∼3-fold reduction in pp150 expression in wt TB40/E but only a 1.6-fold reduction in TB40/E_ULb′:AD169 (data not shown). Herpesviruses, such as HCMV, require efficient viral DNA synthesis for expression of true late (γ₂) genes, such as pp150 (59). Thus, the reduced impact of MBV on pp150 expression in TB40/E_ULb′:AD169 most likely reflects decreased effects of UL97 inhibition on viral DNA synthesis (Fig. 10).

A chimeric TB40/E containing the ULb′ region of AD169 shows improved viral replication in the presence of a UL97 inhibitor.

To address whether the differences we observed in viral DNA synthesis and late gene expression for TB40/E_ULb′:AD169 were associated with improved viral replication, we performed single-cycle replication kinetics assays comparing TB40/E_ULb′:AD169 to parental wt TB40/E in the presence and absence of the UL97 inhibitor MBV. In several independent biological replicate experiments, we found that MBV treatment consistently had a smaller effect on cell-free virus production in TB40/E_ULb′:AD169. In a representative result, shown in Fig. 11, MBV treatment of wt TB40/E led to defects in production of cell-free virus that ranged from 1,320-fold at 72 hpi to 568-fold at 144 hpi (Fig. 11A). However, in TB40/E_ULb′:AD169, the defect at 72 hpi was only 45-fold and at 144 hpi was only 89-fold (Fig. 11B). Notably, the viruses replicated to similar cell-free titers in the absence of MBV (Fig. 11A and B). Furthermore, cell-associated titers in the absence of MBV and the effects of MBV on cell-associated titers were similar in parental TB40/E and TB40/E_ULb′:AD169 (Fig. 11C and D). These findings indicated to us that replacement of the ULb′ from TB40/E with the highly deleted version from laboratory strain AD169 resulted in a reduced requirement for UL97 kinase activity in viral replication.

Fig 11 — The ULb′ contains a genetic determinant that invokes an increased requirement for UL97 in viral replication. (A to D) TB40/E and TB40/E_ULb′:AD169 were compared for viral replication kinetics at MOI = 1 in HFF in the presence and absence of maribavir (MBV). Cell-free (A and B) and cell-associated (C and D) virus titers were determined for samples collected at the indicated time points. Results from MBV-treated infections are shown as open circles with a dashed line, while results from DMSO (carrier alone) control conditions are shown as solid circles connected by a solid line. To facilitate comparison of differences, fold defects are shown in the space between lines representing MBV and DMSO treatments in panels A and B for time points from 96 to 144 hpi. For all panels, error bars represent standard deviations.

Taken together, our results argue that the ULb′ region encodes one or more factors that invoke an increased requirement for UL97 in viral DNA synthesis and, hence, viral replication.

DISCUSSION

In this study, we have demonstrated that two HCMV strains, TR and TB40/E, which retain full-length ULb′s, exhibit especially severe replication defects, particularly in cell-free virus production, when UL97 is either genetically ablated or pharmacologically inhibited (Fig. 2 and 3). Furthermore, we have mapped a genetic determinant of this increased requirement for UL97 in TB40/E to the ULb′ (Fig. 9 to 11). We initiated this work to investigate what appeared to be an increased requirement for UL97 in production of cell-free virus (e.g., cytoplasmic egress) in these strains. However, our findings argue that the profound defects in cell-free virus production of UL97-deficient infections are secondary to defects in viral DNA synthesis (Fig. 2, 3, 5, and 7). Indeed, we observed that modest reductions of ∼2- to 4-fold in viral DNA synthesis, due to phosphonoformate inhibition of viral DNA polymerase, caused decreases in production of cell-free virus that were up to ∼100-fold greater than those seen with cell-associated virus (Fig. 7). Therefore, our results support the notion that a key role of UL97 is to promote efficient viral DNA synthesis (5, 9, 20). We also encountered evidence that the ULb′ encodes one or more factors that invoke an increased requirement for UL97 in viral DNA synthesis (Fig. 10), which provides one mechanism to explain the importance of UL97 for cell-free virus production in strains TR and TB40/E.

In herpesviruses such as HCMV, late gene expression depends on viral DNA synthesis (1, 65–68). We speculate that (i) high-level expression of one or more viral late gene products is required for efficient cytoplasmic egress of virions and (ii) in UL97-deficient infections with strains such as TR and TB40/E, viral DNA synthesis—and hence late gene expression—is insufficient to promote cytoplasmic egress. Unlike strains TR and TB40/E, laboratory strain AD169 showed robust levels of late gene expression and cell-free virus production even under conditions where UL97 was absent or inhibited (Fig. 2 to 4). Accordingly, in AD169, basal levels of viral DNA synthesis were about 2-fold higher than those in TB40/E or TR, and the effect of genetic ablation or pharmacological inhibition of UL97 on viral DNA synthesis was significantly smaller (Fig. 5).

A virus, TB40/E_ULb′:AD169, in which the ULb′ of TB40/E was replaced with that of AD169 showed smaller effects of UL97 inhibition on viral DNA synthesis, late gene expression, and cell-free virus production (Fig. 10 and 11). We have also found preliminary evidence of a similarly decreased requirement for UL97 with a UL97-null TB40/E in which the ULb′ was exchanged with that of AD169 (G. Li, D. Wang, and J. P. Kamil, unpublished results). Nonetheless, the chimeric virus did not show significantly increased basal levels of viral DNA synthesis (Fig. 10) and hence did not appear to fully recapitulate the differences we observed between laboratory strains and strains TB40/E and TR (Fig. 5). These findings argue that an increased requirement for UL97 in viral DNA synthesis, invoked by the ULb′ region, together with other, as-yet-unmapped determinants that influence basal levels of viral DNA synthesis, accounts for the differing requirements for UL97 in laboratory-adapted strains AD169 and Towne versus strains TB40/E and TR, which might serve as models for clinical isolates.

Notably, we found it very difficult to obtain cell-free Δ97 TR virus, even from UL97-expressing cells that produced high titers of Δ97 TB40/E (not shown). A Δ97 TR rescue virus replicated indistinguishably from parental TR (not shown), which would argue against the possibility that spurious mutations in Δ97 TR contributed to our difficulty in cultivating it. Therefore, we relied on experiments with an UL97 inhibitor, MBV, to study UL97-deficient infections with strain TR (Fig. 3 to 5). It is likely that our UL97-expressing HFF failed to fully complement the replication defect of Δ97 TR. Nonetheless, we also observed that parental TR virus produced much lower titers of cell-free virus during infection at a low MOI than wt TB40/E (not shown), which may also have contributed to our difficulties in obtaining cell-free Δ97 TR virus.

Although we are unaware of previous studies of UL97 mutants in HCMV strains that encode full-length ULb′s, at least two reports have examined the effects of MBV on clinical isolates (5, 69). The use of infected cells as inocula may explain why the authors did not find an increased requirement for UL97 in their assays with clinical isolates. In contrast, we used cell-free virus for inocula in our experiments. It seems likely that the infected cells used as inocula in the previous studies contained infectious virions that had matured in the presence of UL97 activity, i.e., prior to exposure to drug and, if so, that any putative requirement for UL97 might have already been met. Indeed, we have preliminarily observed that MBV fails to show a dramatic effect on strains TR and TB40/E unless the drug is added at early times during infection (C. C. Nguyen and J. P. Kamil, unpublished data).

Our electron microscopy analyses indicated that UL97-deficient TB40/E infections showed a defect in viral nuclear egress (Fig. 6 and Tables 1 and 2), which has been previously reported for Δ97 AD169 (7). However, we found no evidence of defects in cytoplasmic egress that could account for the extremely large decreases we observed in production of cell-free but not cell-associated virus (Fig. 2 and 3). Nonetheless, disproportionately large decreases in cell-free virus production occur in other mutant viruses, such as UL71 null mutants, and are likewise difficult to account for by electron microscopy (50). Notably, we did observe decreased numbers of C-capsids, which contain packaged viral DNA, both in the nuclei and in the cytoplasm. The paucity of C-capsids in infected cell nuclei was associated with increased numbers of B-capsids, which contain scaffold and are presumably competent for DNA packaging, while numbers of A-capsids, which are products of abortive—possibly defective—DNA packaging, were relatively unaffected (Table 1 and Fig. 6A to C). We thus interpret the lack of C-capsids in Δ97 TB40/E infected-cell nuclei to likely reflect defects in viral DNA synthesis, although others have reported DNA encapsidation defects in Δ97 AD169 (9).

Our observation of increased levels of noninfectious enveloped particles (NIEPs) and other morphologically unusual particles in the cytoplasm of UL97-deficient TB40/E infections is consistent with reports that inhibition of DNA synthesis in laboratory strain AD169 and in simian cytomegalovirus strain Colburn is associated with increased levels of NIEPs and/or other unusual particles (Table 2) (70, 71). The unusual viral particles we observed may thus reflect nonspecific effects of impaired viral DNA synthesis (70, 71) or, instead, may reflect roles of UL97 during virion morphogenesis, possibly in regulating the solubility or trafficking of the pp65 tegument protein (22, 72). Meanwhile, the lack of enveloped C-capsids in the cytoplasm in UL97-deficient TB40/E infections is consistent with the reported nuclear egress defects of Δ97 AD169 (7).

During HCMV infection, UL97 inactivates the tumor suppressor function of the cellular retinoblastoma protein (pRb), and possibly other pRb family members (p107, p130), by phosphorylation at sites that regulate dissociation of E2F family transcription factors (23, 28). The notion of the physiological relevance of pRb as a UL97 substrate is supported by the observation that the defects in viral DNA synthesis and late gene expression seen for Δ97 AD169 in quiescent cells were reversed in a Δ97 virus that expresses human papillomavirus-16 E7, a protein that destabilizes pRb family proteins (20). We previously reported that Δ97 laboratory strain AD169 exhibited profound defects in viral DNA synthesis, late gene expression, and viral replication during infection of quiescent, serum-starved cells but not during infection of serum-fed, asynchronously dividing cells (20). In this study, however, we observed during infection of serum-fed, asynchronously dividing cells substantial defects in viral DNA synthesis, late gene expression, and viral replication for UL97-deficient infections with strains TB40/E and TR but not strain AD169 (Fig. 2 to 5). Given the importance of pRb as a UL97 substrate, it will be of interest to evaluate whether the profound defects of UL97-deficient infections with virus strains that preserve full-length ULb′s, such as TR and TB40/E, can also be complemented by E7-mediated destabilization of pRb family proteins.

Because the requirement for UL97 appears to be greater in HCMVs containing intact or largely intact ULb′s, UL97 is likely more pertinent during natural infection than has been previously appreciated from studies with laboratory strains (5, 8–11). Although we cannot exclude the possibility that the ULb′s of AD169 and Towne have independently accumulated gain-of-function mutations that make viral DNA synthesis less dependent on UL97, this seems unlikely, since these viruses have lost most of the genetic content of ULb′. Instead, the large deletions in the ULb′s of these two laboratory strains have most likely ablated one or more viral genes that impose an increased requirement for UL97 in viral DNA synthesis.

It seems plausible that UL97 may regulate ULb′-encoded functions that restrict viral DNA synthesis. Alternatively, UL97 may be required for viral DNA synthesis to benefit, directly or indirectly, from a ULb′-encoded factor(s). Regardless, the observation that TB40/E_ULb′:AD169 and laboratory strains harboring extensively deleted ULb′s showed smaller defects in the presence of a UL97 inhibitor than did viruses with full-length ULb′s implies that viral DNA synthesis in clinical isolates is influenced by UL97 in a more complex manner than it is in laboratory strains (Fig. 5 and 10). Future studies will be needed to distinguish between these and other possibilities and to dissect the mechanisms at play.

The specific viral gene(s) within the ULb′ that invokes an increased requirement for UL97 in viral DNA synthesis remains to be identified. Nonetheless, our results suggest that expression of gH/gL/UL128-131A, the pentameric viral glycoprotein complex, is not sufficient to invoke the increased requirement for UL97. Putting aside UL132, which is important for replication (73) and is conserved in both AD169 and TB40/E, there remain approximately 22 open reading frames that might be involved. However, it seems equally important to ask why HCMV strains such as TR and TB40/E might encode genes that impose an additional requirement for UL97 in viral DNA synthesis.

HCMV is a highly complex and ancient virus which establishes a lifelong persistent infection of a long-lived host. The fact that HCMV infection is typically asymptomatic likely belies sophisticated viral strategies to minimize negative impacts of infection on host fitness. Replication to levels that cripple the host would be detrimental not only to the host but also to the virus, as an incapacitated host would be less competent to disseminate virus over large distances and would be less likely to survive to do so over long periods of time. Interestingly, our results suggest that natural isolates synthesize DNA at levels very close to the threshold below which production of cell-free virus—and possibly cytoplasmic egress itself—is highly inefficient (Fig. 7). Therefore, the existence of ULb′-encoded functions that increase the requirement for UL97 in viral DNA synthesis may hint at mechanisms by which this virus tempers its own replication to promote persistence in vivo.

ACKNOWLEDGMENTS

We thank Christian Sinzger (Institute for Virology, Ulm, Germany), Ulrich Koszinowski (Max von Pettenkofer Institute, Munich, Germany), Dong Yu (Washington University, St. Louis, MO), Jay Nelson (Oregon Health Sciences University, Portland, OR), Greg Smith (Northwestern University, Chicago, IL), Lee Fortunato (University of Idaho, Moscow, ID), Rob Kalejta (Univeristy of Wisconsin, Madison, WI), Tom Shenk (Princeton University, Princeton, NJ), Michael Nevels (University of Regensburg, Regensburg, Germany), Karl Münger (Harvard Medical School, Boston, MA), Jennifer Spangle (Harvard Medical School), John Drach (University of Michigan, Ann Arbor, MI), Bill Britt (University of Alabama, Birmingham, AL), Didier Trono (École Polytechnique Fédérale de Lausanne, Switzerland), David Root (Massachusetts Institute of Technology, Cambridge, MA), and Eric Campeau (Resverlogix Corp., Calgary, Canada) for reagents. We are also grateful to the anonymous reviewers for constructive criticism, to Jean M. Pesola (Harvard Medical School) for statistical advice, and to Lindsey Hutt-Fletcher (LSU Health Sciences Center, Shreveport, LA), Maciej Nogalski (LSU Health Sciences Center, Shreveport, LA), Don Coen (Harvard Medical School), and Wade Gibson (Johns Hopkins Medicine, Baltimore, MD) for helpful discussions.

This project was supported by National Institutes of Health grants 8P20GM103433 (J.P.K.), AI050677 (A.D.Y.), and HD051998 (A.D.Y), a grant from the American Heart Association, 12GRNT11890012 (J.P.K.), and Deutsche Forschungsgesellschaft SPP1175 grant ME 1740/2-1 (J.V.E.).

Footnotes

Published ahead of print 27 March 2013

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PERMALINK

The ULb′ Region of the Human Cytomegalovirus Genome Confers an Increased Requirement for the Viral Protein Kinase UL97

Depeng Wang

Gang Li

Martin Schauflinger

Christopher C Nguyen

Ellie D Hall

Andrew D Yurochko

Jens von Einem

Jeremy P Kamil

Abstract

INTRODUCTION

MATERIALS AND METHODS

Cells and virus.

Fig 1.

Growth analysis and virus titration.

Pharmacological inhibitors.

Fig 7.

BAC mutagenesis.

Fig 9.

Virus reconstitution.

Generation of UL97-expressing cells.

Western blotting.

qPCR.

Statistical analyses.

Electron microscopy.

RESULTS

Generation of UL97-expressing cells to complement UL97-null HCMVs.

Δ97 TB40/E exhibits severe replication defects on noncomplementing HFF.

Fig 2.

Pharmacological inhibition of UL97 recapitulates especially severe replication defects for two HCMV strains that harbor full-length ULb′.

Fig 3.

Differing consequences of UL97-deficient infection for viral protein expression.

Fig 4.

Comparison of roles of UL97 in viral DNA synthesis.

Fig 5.

Ultrastructural analyses of UL97-deficient TB40/E infections.

Table 1.

Fig 6.

Table 2.

Inhibition of viral DNA synthesis disproportionately impacts cell-free virus.

Expression of the pentameric viral glycoprotein complex gH/gL/UL128-131A is not sufficient to cause an increased requirement for UL97.

Fig 8.

Generation and analysis of a chimeric TB40/E containing the ULb′ region of AD169.

Fig 10.

A chimeric TB40/E containing the ULb′ region of AD169 shows improved viral replication in the presence of a UL97 inhibitor.

Fig 11.

DISCUSSION

ACKNOWLEDGMENTS

Footnotes

REFERENCES

ACTIONS

PERMALINK

RESOURCES

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