Human Cytomegalovirus Protein Kinase UL97 Forms a Complex with the Tegument Phosphoprotein pp65

Abstract

UL97 is a protein kinase encoded by human cytomegalovirus (HCMV) and is an important target for antiviral drugs against this ubiquitous herpesvirus, which is a major cause of life-threatening opportunistic infections in the immunocompromised host. In an effort to better understand the function(s) of UL97 during HCMV replication, a recombinant HCMV, NTAP97, which expresses a tandem affinity purification (TAP) tag at the amino terminus of UL97, was used to obtain UL97 protein complexes from infected cells. pp65 (also known as UL83), the 65-kDa virion tegument phosphoprotein, specifically copurified with UL97 during TAP, as shown by mass spectrometry and Western blot analyses. Reciprocal coimmunoprecipitation experiments using lysates of infected cells also indicated an interaction between UL97 and pp65. Moreover, in a glutathione S-transferase (GST) pull-down experiment, purified GST-pp65 fusion protein specifically bound in vitro-translated UL97, suggesting that UL97 and pp65 do not require other viral proteins to form a complex and may directly interact. Notably, pp65 has been previously reported to form unusual aggregates during viral replication when UL97 is pharmacologically inhibited or genetically ablated, and a pp65 deletion mutant was observed to exhibit modest resistance to a UL97 inhibitor (M. N. Prichard, W. J. Britt, S. L. Daily, C. B. Hartline, and E. R. Kern, J. Virol. 79:15494-15502, 2005). A stable protein-protein interaction between pp65 and UL97 may be relevant to incorporation of these proteins into HCMV particles during virion morphogenesis, with potential implications for immunomodulation by HCMV, and may also be a mechanism by which UL97 is negatively regulated during HCMV replication.

Human cytomegalovirus (HCMV) is a ubiquitous, double-stranded DNA virus that is a major cause of serious disease in newborns and immunocompromised patients (37). HCMV UL97 was originally identified as a protein kinase homolog (14). UL97 was then shown to be the HCMV gene product responsible for the phosphorylation of the antiviral drug ganciclovir, a nucleoside analog, thus contributing to the susceptibility of HCMV to this compound (33, 51). Nonetheless, UL97 is a protein serine-threonine kinase (22). Maribavir, a specific inhibitor of UL97 protein kinase activity (6, 8), greatly inhibits viral replication and has shown promise as an anti-HCMV therapeutic agent in clinical trials (30, 56). Therefore, UL97 is an important antiviral drug target.

Recombinant HCMVs (rHCMVs) with deletions in UL97 (Δ97) are moderately to severely impaired for replication in cell culture (16, 28, 44, 59). Compared to wild-type virus yields, defects of between 1 and 3 logs have been reported for two isolates of RCΔ97, a Δ97 virus derived from HCMV strain AD169 (28, 44, 59), with the defects being less pronounced when RCΔ97 is grown on dividing cells (43). Cell culture conditions have also been reported to markedly influence the potency of maribavir in suppressing HCMV replication in vitro (15), which may reflect interplay between cellular kinases and UL97 (23). It has been reported that RCΔ97 is specifically defective at the stages of DNA replication and capsid assembly (59). However, our laboratory has found a role for UL97 at the stage of viral capsid egress from the nucleus during primary envelopment (nuclear egress) (28). Notably, two recent reports have each suggested a role for UL97 in later assembly events (4, 43). Specifically, RCΔ97-infected cells accumulate unusual refractile bodies, described as “vacuoles” or “aggregates,” in the nucleus and the perinuclear area (4, 43). Similar aggregates are observed when UL97 kinase activity is pharmacologically inhibited in wild-type AD169-infected cells (4, 43). Strikingly, the 65-kDa phosphoprotein (pp65), a major tegument protein of the HCMV virion (40, 47), was identified as a major component of these aggregates (43).

To better understand the role of UL97 in HCMV replication, we employed tandem affinity purification (TAP). There are several advantages to using a TAP strategy to identify protein-protein interactions. An open reading frame (ORF) can be tagged at its natural locus within the genome so that a tagged version of the protein will be expressed at physiological levels without competition from untagged protein. Moreover, because two separate affinity purification steps are performed, proteins found to coelute with the tagged protein of interest after TAP are more likely to reflect authentic protein-protein interactions than those found after one-step procedures (46). Using this strategy, we were able to identify pp65 as a major interacting partner of UL97. The pp65-UL97 interaction was also detected in reciprocal coimmunoprecipitation (co-IP) and in vitro glutathione S-transferase (GST) pull-down assays undertaken to validate the TAP findings. Taken together with data from other recent studies (4, 43), our results may indicate a mechanism by which UL97 and pp65 may influence each other's localization during assembly and whereby UL97 kinase activity may be negatively regulated during HCMV replication.

MATERIALS AND METHODS

Plasmids and BACs.

The AD169rv bacterial artificial chromosome (BAC) clone of HCMV strain AD169 (9, 24) was generously provided by Ulrich H. Koszinowski (Ludwig-Maximilians University, Munich, Germany). The Cre expression plasmid, pBRep-Cre (24), was kindly provided by W. Brune (Robert Koch Institute, Berlin, Germany). The pp71 expression plasmid, pCGN71 (7), was kindly provided by T. Shenk (Princeton University, Princeton, NJ). pT7-RRta, a pcDNA3.1 clone of the rhesus rhadinovirus Rta transcription factor (RRta), was a gift of Su-Fang Lin (National Health Research Institute, Zhunan, Taiwan) and Hsing-Jien Kung (UC Davis Medical Center, Sacramento, CA) (32). The luciferase (Luc) T7 control plasmid was supplied with a TnT quick-coupled transcription/translation kit (Promega, Madison, WI). Plasmids pEP-KanaS (53) and pBAD-I-SceI (53) were kindly provided by Nikolaus Osterrieder (Cornell University, Ithaca, NY) and B. Karsten Tischer (Universitaetsklinikum SH, Kiel, Germany).

UL97 and pp65 were each amplified by PCR from AD169rv (24) bacmid DNA with primers that added terminal CpoI sites by use of KOD Hot Start DNA polymerase (EMD Biosciences, Inc., San Diego, CA). Sequences of oligonucleotides used in this study are provided in Table S1 (posted at http://coen.med.harvard.edu). PCR products were digested with CpoI (Fermentas, Inc., Hanover, MD) and inserted into previously described pcDNA3.1 (Invitrogen, Carlsbad, CA)-based vectors that have been modified to incorporate a CpoI site at the polylinker and confer N-terminal hemagglutinin and T7 epitope tags and upstream T7 and CMV immediate-early promoters (25), resulting in the plasmids pHA-UL97 (hemagglutinin tag) and pT7-pp65 (T7 tag), respectively. For pp65, the same PCR product was also inserted into a previously described modified version of the pGEX2T vector (GE Healthcare, Piscataway, NJ) that contains a CpoI site in the polylinker (25), yielding pGEX-pp65. All plasmids constructed for this study were verified to contain no mutations by DNA sequencing.

The N-terminal TAP (NTAP) tag used in our studies, though modeled on the original (46), was custom synthesized (Geneart Inc., Toronto, Ontario, Canada) to better accommodate human codon bias and also to minimize the chance of intratag recombination events within the tag's tandem immunoglobulin G (IgG) binding domains during Red recombination steps in Escherichia coli. Per the example of another group (48), our TAP tag also employed a protein C epitope tag (pC) in place of the original calmodulin binding peptide (Fig. 1). The TAP tag was incorporated just after the start codon of UL97 in the AD169rv BAC by use of the two-step Red recombination method of Tischer et al. (53) and E. coli strain DY380 (31), which was kindly provided by Neal Copeland (Institute of Molecular and Cell Biology, Singapore). Procedures for use of DY380 and for two-step Red recombination were performed as previously described (31, 53). For preparation of an NTAP tag universal transfer construct, the I-SceI-AphAI element from pEP-KanaS (53) was inserted into a unique PstI site located within the TAP tag in the context of a plasmid (pJK-NTAP) such that the inserted element was flanked on either side with perfect 50-bp repeats of TAP tag sequence identical to that of the original context of the PstI site, yielding pNTAP-TSR. The PCR primers NTAP97 Fw and NTAP97 Rv (see Table S1 at http://coen.med.harvard.edu) were used to produce a PCR product from pNTAP-TSR for two-step Red recombination (53).

FIG. 1.

Construction of TAP tagged-UL97 in the context of a BAC cloned HCMV genome. (A) Incorporation of the TAP tag at the N terminus of UL97 in a BAC of AD169 HCMV. (Top panel) The organization of the unique and repeat regions of HCMV genome. Scale is indicated by a line representing 10 kb. TR_L, terminal repeat long; U_L, unique long; IR_L/S, internal repeat long and neighboring internal repeat short; U_S, unique short; TR_S, terminal repeat short; f, mini-F and associated sequences conferring growth as bacmid in E. coli. The UL97 region is indicated as a line expanded to show detail below. (Middle panel) The UL97 region of NTAP97 is drawn to scale, with UL97 shaded in gray, the TAP tag shown as a striped arrow, and portions of flanking genes represented as unfilled shapes. (Bottom panel) A detailed diagram of the TAP tag is drawn approximately to scale. Spacer regions are indicated by unfilled, unlabeled rectangles; regions of the TAP tag directly involved in purification procedures are labeled and illustrated as various shapes bearing horizontal stripes. IgG bd 1 and IgG bd 2, tandem IgG binding domains from S. aureus protein A; TEV, tobacco etch virus protease recognition site. (B) Restriction enzyme analysis of NTAP97. NTAP97 was compared to the parental AD169rv bacmid in BamHI, EcoRI, and XhoI restriction digests on a 0.7% agarose-Tris acetate EDTA gel run overnight at 65 V, with 1.25 μg of each bacmid per digest. M, Invitrogen 1-kb DNA marker. The arrow indicates an upward shifted band in the XhoI digest of NTAP97, reflecting decreased mobility due to presence of TAP tag sequences. Note that the band immediately under the arrow is a singlet in NTAP97 but appears in a doublet in AD169rv.

A markerless deletion mutant of UL97, Δ97, modeled on the original RCΔ97 deletion (44) but lacking the inserted lacZ and EcoGPT marker sequences, was also constructed from AD169rv by two-step Red recombination (53) using primers d97 Fw and d97 Rv (see Table S1 at http://coen.med.harvard.edu). In the Δ97 BAC, the unique BamHI and PstI sites within the original UL97 ORF were brought immediately adjacent to each other (GGATCCCTGCAG), resulting in a deletion of 1,513 bp of UL97 sequence. The remnant UL97 sequences in the Δ97 bacmid provide only the first 23 amino acids of native UL97 sequence, after which the polypeptide is predicted to terminate at a stop codon after reaching a final length of 37 amino acids. The Δ97 BAC was verified by restriction enzyme digestion and DNA sequencing of the affected region (data not shown).

Another recombinant bacmid, FLAG97, in which sequences encoding a FLAG epitope (DYKDDDDK) were incorporated at the N terminus of UL97, was also constructed using two-step Red recombination (53). Briefly, the primer pairs (i) UL97region Fw and FLAG97 OLE and (ii) UL97region Rv and FLAG97 OLE Fw (see Table S1 at http://coen.med.harvard.edu) were used to produce two separate, overlapping PCR products from the AD169rv BAC. These two PCR products were used as the template in a new PCR with UL97region Fw and UL97region Rv primers to produce an overlap extension PCR product of 2,909 bp containing a complete FLAG-tagged UL97 ORF, including 439 bp of HCMV sequence upstream of the UL97 start codon and 322 bp downstream of the UL97 stop codon. The PCR product was digested with XhoI and ligated to the cloning vector pSP72 (Promega), which had been cut with EcoRV and XhoI enzymes, resulting in pFLAG97. A PCR product containing an I-SceI-AphAI cassette from pEP-KanaS was prepared using the primer pair PstIAphAIUL97in Fw and PstIAphAIUL97in Rv (see Table S1 at http://coen.med.harvard.edu), digested with PstI, and ligated into pFLAG97 at a unique PstI site, yielding pFLAG97-TSR, which was verified to be free of mutations by DNA sequencing (data not shown). The viral DNA insert within pFLAG97-TSR was released by digestion with EcoRV and XhoI and used in two-step Red recombination procedures (53) to derive the FLAG97 BAC from the Δ97 BAC.

Cells and virus.

Primary human foreskin fibroblasts (HFF) of the Hs27 isolate (CRL-1634; American Type Culture Collection) were used to support the replication of HCMV in cell culture. For the experiment shown in Fig. 3A, telomerase-immortalized HFF (10), clone T12, a generous gift of Wade Bresnahan (University of Minnesota, Minneapolis, MN), were used. HFF were grown in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 20 μg/ml gentamicin sulfate (complete DMEM). Virus stocks were titrated using traditional plaque assays and serial dilutions in DMEM supplemented with 1% METHOCEL (Dow Chemical Co., Midland, MI), 2% FBS, and 20 μg/ml gentamicin sulfate in 24-well Costar dishes, and plaques from duplicate wells were enumerated using standard crystal violet staining procedures. rHCMV was reconstituted by electroporation of HFF with rHCMV BAC DNA, alongside plasmids pBRep-Cre (24) and pCGN71 (7), as previously described (60).

FIG. 3.

Coelution of pp65 and UL97 during TAP. (A) Western blot analysis of pp65 and UL97 during TAP of UL97. Parallel TAP procedures were performed on lysates of cells infected with either NTAP97 (lanes 2, 3, 6, 8, and 10) or, as a negative control, untagged parental virus (AD169rv; lanes 1, 4, 5, 7, and 9). Anti-pC polyclonal antibody was used to probe the upper blot, and anti-pp65 MAb was used in the lower blot. Lysate, 2 μl of clarified lysate (lanes 1 and 2); IgG FT, 2 μl unbound lysate fraction (“flowthrough”) after incubation with IgG-Sepharose (lanes 3 and 4); TEV eluate, 10 μl (1.0%) of TEV eluate from the step prior to loading on an anti-pC matrix (lanes 5 and 6); pC FT, 10 μl (∼0.2%) of anti-protein C epitope affinity matrix flowthrough (lanes 7 and 8); final eluate, 20 μl (2.2%) of final TAP eluate from an anti-pC matrix (lanes 9 and 10); TAP-97, full-length TAP-tagged UL97 expressed from NTAP97; pC-UL97, pC epitope-tagged UL97, formed from TAP-97 following removal of tandem IgG binding domains from a TAP tag by site-specific TEV protease cleavage. A shift in the mobility of the UL97 species in the anti-pC blot is observed, as ∼16 kDa of the TAP tag was removed from the N terminus of TAP-97 during TEV digestion to form pC-UL97. (B) Silver stain analysis of TAP from NTAP97-infected HFF. A silver-stained SDS-PAGE gel representing various steps during TAP from NTAP97 lysate is shown. Lysate, 2 μl of clarified lysate (lane 1); IgG beads, protein eluted from 5 μl (5%) of IgG-Sepharose beads following binding to NTAP97 lysate (lane 2; note the prominent IgG heavy and light chains); IgG FT, 2 μl unbound lysate fraction (“flowthrough”) after incubation with IgG-Sepharose (lane 3); TEV eluate, 10 μl (2%) of TEV eluate from the step prior to loading on an anti-pC matrix (lane 4); pC FT, anti-protein C epitope affinity matrix flowthrough (0.2%) and TCA precipitate of ∼1.4% of total pC FT were loaded (lanes 5 and 6); final eluate, final TAP eluate from anti-pC matrix (3%) and TCA precipitate of 54% of final eluate (500 μl) were loaded (lanes 7 and 8); M_r, Bio-Rad Precision Plus Prestained Protein MW markers.

For comparison of viral replication kinetics, 1 × 10⁵ HFF per well were seeded in 24-well Costar plates. The following day, inoculum was applied at a target multiplicity of infection (MOI) of 1 PFU/well, which was verified by back-titration. After 1 h incubation (37°C, 5% CO₂), the medium was aspirated and replaced with 1 ml of complete DMEM containing 5% FBS. To collect samples for time points, cells were dislodged by thoroughly scraping wells with a 1 ml pipette tip, and medium and dislodged cells were transferred to −80°C for storage until titration.

TAP.

Six 150-mm plates of HFF (∼7 × 10⁷cells) were infected with a passage 2 stock of NTAP97 virus or, as a negative control, with AD169rv, at an MOI of 1 PFU/cell, which was confirmed by back-titration of the inoculum, and harvested at 72 h postinfection. TAP procedures were carried out as previously described (45, 48) with the following modifications. Plates were washed three times with ice-cold Dulbecco's phosphate-buffered saline (PBS) and lysed at 4°C for 30 min in 1 ml of TAP lysis buffer (150 mM NaCl, 50 mM HEPES-KOH [pH 7.45], 0.1% NP-40, 0.25 mM sodium orthovanadate, 10 mM sodium fluoride, 10 mM β-glycerophosphate, 10% glycerol, 2 mM [DTT], 2 mM EDTA) containing two Complete-EDTA-free protease inhibitor tablets (Roche Applied Sciences, Indianapolis, IN) per 50 ml. Plates were then scraped, and lysate was collected and stored at −80°C until processing.

After being thawed, lysate was sonicated for 1 min using a 50% duty cycle and a micro-tip at a power setting of 6.5 on a Branson Sonifier 450 system (Branson Ultrasonics Inc., Danbury, CT) (unless otherwise noted, all following steps were performed at 4°C), clarified by centrifugation at 12,000 × g for 30 min, and then applied to 200 μl of IgG-Sepharose 6 Fast Flow resin (GE Healthcare) and processed as described previously (48) except that TAP lysis buffer was used in place of PA-150 (48). The resin was then washed with 10 ml of TEV buffer (150 mM NaCl, 25 mM Tris-Cl [pH 8.0], 3 mM MgCl₂, 0.5 mM EDTA, 1 mM DTT, 0.1% NP-40) and incubated overnight in 1 ml of TEV buffer containing 100 U of AcTEV protease (Invitrogen). TEV eluate was collected from the column and was later combined with 5 ml of buffer pC (150 mM NaCl, 25 mM Tris-Cl [pH 8.0], 3 mM MgCl, 1 mM CaCl₂, 1 mM DTT, 0.1% NP-40) containing one Complete-mini EDTA-free protease inhibitor tablet (Roche) per 20 ml and applied to rinse residual eluate from the column. TEV eluate was incubated with 200 μl of anti-protein C affinity matrix (Roche), after which the anti-pC matrix was then washed three times with 10 ml buffer pC containing protease inhibitors (1.6 μg/ml benzamidine-HCl, 1 μg/ml aprotinin, 1 μg/ml leupeptin, 1 μg/ml pepstatin A, 0.1 mM phenylmethylsulfonyl fluoride). The anti-pC matrix was then incubated in 300 μl of elution buffer (100 mM NaCl, 25 mM Tris-Cl [pH 7.4], 10 mM EDTA, 5 mM EGTA) for 10 min at room temperature. Two additional elution steps were performed, and the total eluate (∼900 μl) was pooled. The Wessel-Flügge method (57) was used to obtain precipitates from 400 μl eluate. For the TAP procedure shown (see Fig. 3B), a similar procedure was used; however, all steps were performed in batch format, the total eluate volume was 500 μl, and trichloroacetic acid (TCA) was used for protein precipitation (42).

MS.

Protein precipitates from TAP were analyzed at the Taplin Biological Mass Spectrometry (MS) Facility (Harvard Medical School) by microcapillary liquid chromatography-tandem MS with an LCQ DECA ion-trap mass spectrometer (ThermoFinnigan, San Jose, CA). Peptide sequences (and hence protein identity) were determined by matching protein or translated nucleotide databases with the acquired fragmentation pattern by the Sequest software program (ThermoFinnigan) (17).

SDS-PAGE and Western blotting.

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting procedures were carried out as previously described (26). To probe immunoblots, anti-pC rabbit polyclonal antibody (Delta Biolabs, Gilroy, CA) was used at 1:1,000; anti-pp65 mouse monoclonal antibody (MAb) (Fitzgerald Industries, Concord, MA) and anti-beta-actin mouse MAb (Abcam Inc., Cambridge, MA) were each used at 0.2 μg/ml. Anti-gB mouse MAb (Abcam) was used at 0.25 μg/ml, and mouse anti-UL44 MAb (Virusys Corp., Sykesville, MD) was used at 1:1,000. A custom rabbit antiserum was prepared commercially (Open Biosystems, Huntsville, AL) against GST-UL97 purified from a baculovirus expression system as described previously (5, 22) and was used at a dilution of 1:10,000 or 1:3,000, as indicated. For quantitative estimates, dilutions of known amounts of input lysate were analyzed alongside samples of interest, and Western blot images were quantified by densitometry using Quantity One version 4.5 for Macintosh (Bio-Rad). In some experiments, pp65 expression was analyzed using an Odyssey infrared imaging system and Odyssey 2.1 software (Li-Cor Biosciences, Lincoln, NE), in which case Alexa Fluor 680 goat anti-mouse IgG antibody (Invitrogen) was used as the secondary antibody at 0.4 μg/ml. In order to facilitate comparison of UL97 expression levels among rHCMVs used in this study, NTAP97-infected cell lysate was pretreated with AcTEV protease (Invitrogen) to remove the protein A component of the TAP tag, which binds IgGs in a manner independent of their antigen specificity (18).

Co-IP.

Procedures for co-IP were performed as described elsewhere (21), with modifications. Briefly, 2.5 × 10⁶ HFF were infected in 100-mm dishes with mock inoculum (medium lacking virus), AD169rv, Δ97, or FLAG97 at a target MOI of 1 PFU/cell, which was verified by back-titration. At 72 h postinfection, plates were washed twice with ice-cold PBS and lysed in 1 ml lysis buffer (50 mM HEPES-KOH [pH 7.4], 1% Triton X-100, 150 mM NaCl, 10% glycerol, 0.1 mM DTT, 2 mM EDTA) containing one Complete EDTA-free protease inhibitor tablet (Roche) per 50 ml. Two hundred microliters of clarified lysate was used per immunoprecipitation (IP) reaction. For pp65 IP reactions, 1.7 μg of pp65 MAb (Fitzgerald) was used; for anti-FLAG IPs, 40 μl of settled EZ-view anti-FLAG M2 affinity resin (Sigma Aldrich, St. Louis, MO) was used. Anti-pp65 antibody was reacted with lysates at 4°C with rotation for 1 h before 10 μl ImmunoPure protein A/G beads (Pierce) was added. IPs were washed five times with 750 μl of lysis buffer for 20 min with rotation at 4°C before beads were incubated in 75 μl of 2× SDS-PAGE sample buffer (21) containing 5% β-mercaptoethanol at 85°C for 10 min to elute proteins.

Recombinant proteins.

Purified GST-US11 was a gift of Jennifer Baltz (Harvard Medical School). GST-pp65 was expressed from pGEX-pp65 in Rosetta2 E. coli (EMD Biosciences, Inc.) from a 250 ml Luria broth culture which had been induced at an optical density at 600 nm of ∼0.6 with 0.2 mM isopropyl-β-d-thiogalactopyranoside for 24 h at 16°C. E. coli cells were collected by centrifugation and frozen at −80°C in 10 ml of PBS containing 10% glycerol. GST affinity purification was performed using a previously published procedure (19) with modifications. Lysate was loaded on 0.5 ml glutathione-Sepharose 4 Fast Flow resin (GE Healthcare) and washed with 50 ml of high-salt buffer (1 M NaCl, 50 mM Tris-HCl [pH 8.0], 1 mM EDTA, 2 mM DTT, 10% glycerol, 0.1% NP-40) containing one-half of a Complete EDTA-free protease inhibitor tablet. GST-pp65 was eluted in 5 ml 300 mM NaCl-50 mM Tris (pH 7.0)-10% glycerol containing 5 mM glutathione. Eluate was diluted with 25 ml buffer A (50 mM Tris [pH 8.0], 2 mM DTT, 10% glycerol) to achieve a final salt concentration of ∼50 mM NaCl and loaded on a 1 ml Q-Sepharose anion exchange column (GE Healthcare) on a BioLogic LP system (Bio-Rad). A 60-ml linear salt gradient (50 mM to 1 M NaCl) in 50 mM Tris-Cl [pH 8.0]-10% glycerol-2 mM DTT was applied, and 1 ml fractions were collected. Fractions containing purified GST-pp65 were pooled and dialyzed into HEPES-buffered saline (100 mM NaCl, 25 mM HEPES-KOH [pH 7.4], 10% glycerol, 2 mM DTT, 0.5 mM EDTA). Protein concentrations of GST-US11 and GST-pp65 were determined by amino acid analysis.

GST pull-down assays.

A TnTT7 quick-coupled transcription/translation system (Promega) was used to obtain in vitro-translated ³⁵S-labeled UL97, RRta, and Luc from rabbit reticulocyte lysates per the manufacturer's instructions. GST pull-down assays were carried out as described previously (34) with the following modifications: 10 μl of in vitro translation reaction mixture was incubated overnight with rotation at 4°C with 4 μg of GST-pp65 or GST-US11 in 400 μl of binding buffer (200 mM NaCl, 25 mM HEPES [pH 7.45], 0.5 mM EDTA, 0.5% NP-40, 10% glycerol, 2 mM DTT, 100 μg/ml bovine serum albumin) containing one Complete-mini EDTA-free protease inhibitor tablet (Roche) per 20 ml. Glutathione beads were washed with 1 ml of binding buffer for 10 min at 4°C with rotation, then in 1 ml binding buffer without bovine serum albumin for 3 h, and finally in 1 ml of binding buffer containing 400 mM NaCl for 5 min at 25°C. Radioactivity was imaged in dried SDS-PAGE gels on a phosphor screen (Kodak) and quantified using a Personal Molecular Imager FX system (Bio-Rad) and Quantity One software (Bio-Rad).

GenBank accession numbers.

The complete DNA sequences of pJK-NTAP, pNTAP-TSR, and pFLAG97-TSR have been submitted to GenBank and assigned the following nucleotide sequence accession numbers EV040031, EV040032, and EV043160, respectively.

RESULTS

Construction and characterization of a recombinant HCMV bearing a TAP-tagged UL97 gene.

To assess the interaction of UL97 with other proteins from HCMV-infected cells, we used a TAP strategy. As depicted in Fig. 1A, NTAP97, an rHCMV bacmid in which the UL97 gene has been modified to incorporate sequences encoding a modified version of the original TAP tag, was prepared from a parental wild-type bacmid clone of HCMV strain AD169 (AD169rv) (46, 48). Because the kinase domain of UL97 is located in the C-terminal half of the protein, and because N-terminal GST fusions of UL97 are enzymatically active (22), the TAP tag was inserted between the first and second codon of UL97, conferring an additional 474 bp to the AD169rv bacmid.

As shown in Fig. 1B, the NTAP97 bacmid was compared to parental AD169rv by restriction enzyme analysis. An 8,455-bp XhoI fragment that contains the UL97 gene, which is seen as part of a doublet in the digest of AD169rv, was missing in the XhoI digest of NTAP97. Instead, a new band approximately 0.5 kb larger was observed, reflecting the incorporation of the TAP tag at the N terminus of UL97 (Fig. 1B). Digests with BamHI and EcoRI were also performed to verify that spurious rearrangements had not occurred during the recombination procedures. (Differences between NTAP97 and parental DNA could not be readily discerned in restriction patterns generated by these enzymes, as the largest EcoRI fragment, which was greater than 16 kb in size, and a BamHI band less than 3 kb in size would be affected). Proper insertion and sequence integrity of the TAP tag were further confirmed by DNA sequencing (data not shown). These results showed that the TAP tag was successfully incorporated at the N terminus of UL97.

NTAP97 virus was reconstituted in HFF, and a single-step growth kinetics experiment was performed in order to assess whether the growth phenotype of NTAP97 was affected by the insertion of the TAP tag at the N terminus of UL97. As shown in Fig. 2, NTAP97 was compared alongside three other BAC-based viruses which were also reconstituted for use in this study: parental AD169rv (wild type); Δ97, a UL97 deletion virus based on AD169rv; and FLAG97, a derivative of Δ97 in which a UL97 ORF bearing an N-terminal FLAG tag was used to rescue the Δ97 deletion. As seen in Fig. 2, NTAP97 and FLAG97 were found to replicate indistinguishably from parental AD169rv, while a defect of ∼1 log was observed for Δ97, which is within the range of what has been observed with RCΔ97 (reference 28 and data not shown), especially in dividing cells (43) such as those used in our assay. Moreover, an AD169rv derivative bearing a K355Q point mutation in the UL97 ORF, which greatly impairs UL97 kinase activity (22), also exhibited a defect of ∼1 log (J. P. Kamil and D. M. Coen, unpublished results). In addition, the expression level of UL97 in TEV protease-treated NTAP97-infected cell lysate was found to be similar to that of parental AD169rv in Western blotting experiments (see Fig. S1 at http://coen.med.harvard.edu). We concluded that the NTAP-tagged version of UL97 expressed by NTAP97 was able to function like wild-type UL97 to promote viral replication and was suitable for use in TAP experiments.

FIG. 2.

Single-cycle growth kinetics of NTAP97, FLAG97, and Δ97 versus parental AD169rv. Viruses reconstituted from NTAP97, FLAG97, Δ97, and AD169rv BACs were applied at an MOI of 1 PFU/cell to 10⁵ HFF/well in a 24-well cluster plate format. For each data point, two wells were pooled and titers were calculated by averaging plaque counts from duplicate titrations. Because counts from duplicate titrations differed less than twofold for all data points, error bars are not shown.

Identification of a UL97-pp65 complex by TAP.

A TAP experiment was performed in which AD169rv and NTAP97 were each used to infect parallel cultures of ∼7 × 10⁷ HFF at an MOI of 1 PFU/cell. Infected cells were harvested at 72 h postinfection and processed by a TAP procedure, and eluates of NTAP97 and, as a negative control, AD169rv were each precipitated and submitted for analysis by MS.

In MS analysis UL97 and pp65 were well represented in the NTAP97 eluate, as indicated by the number of peptides detected (Table 1) and by amino acid coverage (see Tables S2 and S3 at http://coen.med.harvard.edu). While common antibody contaminants were observed in both NTAP97 and AD169rv eluates, no viral proteins were detected in the AD169rv eluate (Tables 1 and 2). pp65 was also observed in an MS analysis of an NTAP97 eluate prepared in a separate TAP experiment (see Table S4 at http://coen.med.harvard.edu); this eluate was also subjected to SDS-PAGE and visualized by silver staining (Fig. 3B, lane 9). These data indicated that pp65 specifically and reproducibly copurified with UL97 during TAP.

TABLE 1.

MS results obtained with NTAP97 TAP eluate

Protein description	Nominal database hit	No. of peptides detected
HCMV UL97	GCVK_HCMVA	18
HCMV pp65 (UL83)	PP65_HCMVA	11
Ig kappa chain, c region	KAC_HUMAN	4
Apolipoprotein A-IV precursor	APOA4_MOUSE	4
IgG1 chain, c region	IGH1M_MOUSE	2
IgG1 chain, c region	IGHG1_HUMAN	2
70-kDa heat shock protein 1A	HS70A_BOVINa	1
IgG2 chain, c region	IGHG2_HUMAN	1
Ig lambda chain	LAC_HUMAN	1
70-kDa heat shock protein 6	HSP76_HUMAN	1
HCMV RL11, viral Fc-gamma receptor-like protein	IR11_HCMVAb	1

^aBovine 70-kDa heat shock protein 1A is 100% identical to the human homolog in the peptide sequence detected.

^bDatabase hit was found only when the viral database was searched, but the general database nomenclature for RL11 is provided here for name conformity.

TABLE 2.

MS results obtained with AD169rv TAP eluate

Protein description	Nominal database hit	No. of peptides detected
Ig kappa chain, c region	KAC_HUMAN	4
IgG1 chain, c region	IGHG1_HUMAN	4
IgG1 chain, c region	IGH1M_MOUSE	3
Ig lambda chain	LAC_HUMAN	2
Ig kappa chain, V-II region	KV2A_HUMAN	2
Vimentin 1/2a	VIM1_XENLA	2
Ig kappa chain, c region	KAC_MOUSE	2
Apolipoprotein A-IV precursor	APOA4_MOUSE	2

^aThe vimentin peptides detected match human sequence with 100% identity (though the database hit is from the Xenopus laevis database).

We detected a single tryptic peptide, indicating that RL11, an HCMV glycoprotein, may have been present in the NTAP97 eluate (Table 1). Detection of only a single peptide was considered insufficient to establish with certainty that a particular protein species was present. Moreover, this protein has been reported to be a viral Fcγ receptor (3). As the first affinity step of TAP involves binding to human IgG-coated Sepharose beads, RL11 might well be expected as a nonspecific contaminant during the initial stages of TAP of HCMV-infected cells.

Notably, we did not detect cellular p32 or HCMV UL44 in our eluates. These two proteins have been reported to interact with UL97 (29, 35, 36). p32 has been reported to recruit UL97 to redistribute the nuclear lamina (36). However, p32 is a mitochondrial protein (39, 54) which can so promiscuously interact with diverse viral, cellular, and bacterial proteins that these interactions have been suspected to be artifactual (39, 54). HCMV UL44, which is a viral DNA polymerase subunit, is a substrate for UL97 in vitro (29, 35), and its phosphorylation status in infected cells depends on UL97 (29). However, evidence of stable interactions between UL97 and UL44 has mainly been observed in vitro and in Saccharomyces cerevisiae two-hybrid experiments, where the concentrations of these proteins may be artificially high (29, 35). Nevertheless, it may be that assays with greater sensitivity to UL97 interactions in infected cells, perhaps under conditions where pp65 is absent, would detect these two proteins.

Confirmation of pp65 copurification with UL97 during TAP by Western blot analysis.

To further investigate whether pp65 was specifically present in TAP eluate from NTAP97-infected HFF but not from AD169rv-infected cells, we performed parallel anti-pp65 and anti-pC Western blot analyses using samples taken from various steps during the parallel NTAP97 and AD169rv TAP procedures. As opposed to samples processed for MS analysis (Tables 1 and 2) or for visualization on silver-stained gels (Fig. 3B), none of these samples were precipitated or manipulated in any way other than addition of sample buffer and heating prior to loading on SDS-PAGE gels.

As seen in Fig. 3A, pp65 was abundant in lysates and IgG-Sepharose flowthrough samples prepared from AD169rv and NTAP97-infected cells (bottom panel, lanes 1 to 4). However, a polypeptide (TAP-97) of ∼100 kDa that was immunoreactive with anti-pC antibody was only detected in NTAP97 samples (top panel, lanes 2 to 3), reflecting the specific presence of the protein C epitope on the TAP tag in NTAP97. A band of M_r ∼35 kDa, which was observed in AD169rv and NTAP97 lysate and flowthrough steps (Fig. 3A, top panel, lanes 1 to 4), is attributed to nonspecific reactivity of the rabbit polyclonal anti-pC antibody preparation. While the IgG-Sepharose matrix did not capture all of the TAP-tagged UL97 (recovery rates of ∼40% are routine for this step) (13), a significant depletion of the TAP-UL97 band was observed in the flowthrough volume (Fig. 3A, top panel, lane 3) compared to the original NTAP97 lysate results (lane 2).

As expected, a slightly faster-migrating immunoreactive polypeptide (∼75 kDa; pC-UL97) was readily observed in the TEV eluate in the anti-pC blot in lane 6 of Fig. 3A (top panel), reflecting the loss of 144 amino acids (∼16.3 kDa) from the TAP tag on TAP-UL97 (lanes 2 to 3) after site-specific TEV protease cleavage. Importantly, pp65 was seen to specifically coelute with UL97 in the TEV eluate, as indicated by the pp65 band seen in the NTAP97 TEV eluate sample in the anti-pp65 blot (Fig. 3A, bottom panel, lane 6). Meanwhile, in the corresponding AD169rv control TEV eluate sample (lane 5), pp65 was not detected. Ultimately, pp65 was observed to specifically coelute with pC-tagged UL97 (pC-UL97) in the final eluate from NTAP97, as evidenced by the presence of the prominent pC-UL97 band in lane 10 of the anti-pC blot (Fig. 3A, top panel) and by the presence of a pp65 band in lane 10 of the anti-pp65 blot (bottom panel). In additional Western blot analyses, UL44 and gB were each readily detected in NTAP97 and AD169rv lysates but not in final TAP eluates (data not shown), further suggesting that other viral proteins present in the original lysates did not nonspecifically contaminate TAP eluates. From this experiment, and the MS results, we conclude that UL97 and pp65 copurify during the TAP procedure.

Figure 3B shows a silver-stained gel resulting from a TAP procedure using NTAP97-infected cells. Polypeptides the size of pC-UL97 and pp65 can be readily observed in the final eluate (lane 9) along with two other smaller species. We infer that the band of ∼50 kDa is an IgG heavy chain, as it has also been detected as a contaminant in TAP eluates by other groups (48, 58) and was detected in our TAP eluates by MS. We interpret the band of ∼25 kDa to be an IgG light chain, which we also observed as a nonspecific contaminant in our TAP eluates by MS (Tables 1 and 2). The IgG-Sepharose, which is coupled to human IgG, and the anti-pC matrix, which is coupled to a mouse monoclonal IgG, were the likely sources of nonspecific human and mouse IgG contamination in our TAP eluates, respectively. The relatively intense staining of the pp65 band in the eluate suggests that the interaction with UL97 was strong (Fig. 3B).

Co-IP of UL97 and pp65 from lysates of HCMV-infected cells.

To ensure that the interaction of pp65 with UL97 was not an anomaly specific to the NTAP97 virus, we performed experiments to assess whether antibodies against pp65 would co-IP UL97. Reciprocally, we tested whether anti-FLAG antibodies, used to IP a FLAG-tagged UL97 expressed from FLAG97, could co-IP pp65. In addition to lysates from AD169rv-, FLAG97-, and mock-infected cells, lysate from cells infected with Δ97, which was rescued to construct FLAG97, was included to provide a control where UL97 is absent and in which pp65 is reported to form unusual aggregates (43). UL97 was detected in Western blot analyses of IPs using a polyclonal rabbit serum raised against GST-UL97 fusion protein, while pp65 was detected using the same pp65-specific MAb used to IP pp65 (Fig. 4).

FIG. 4.

Co-IP of pp65 and UL97 from HCMV-infected cell lysates. (A) Detection of UL97 in IPs of pp65. Lysates of mock-infected HFF (mock) and HFF infected with a Δ97 rHCMV (Δ97), parental AD169rv (AD169rv), and an rHCMV expressing an N-terminal FLAG tag on UL97 (FLAG97) were subjected to IP with an anti-pp65 MAb. Proteins were resolved on 8% acrylamide Precise protein gels (Pierce) and transferred to polyvinylidene difluoride membranes. Bound proteins were detected by anti-UL97 rabbit polyclonal serum at 1:3,000 (top panel) or anti-pp65 MAb at 1:5,000 (bottom panel). Relative mobility (M_r) values of Precision Plus (Bio-Rad) prestained molecular-weight markers are displayed to the left of each immunoblot. (B) Detection of pp65 in anti-FLAG immunoprecipitate of FLAG epitope-tagged UL97 expressed from FLAG97 rHCMV. Lysates of mock-, Δ97-, AD169rv-, and FLAG97-infected HFF were reacted with anti-FLAG M2 MAb-conjugated agarose beads (Sigma), and IPs were resolved by SDS-PAGE as described above. pp65 was detected using anti-pp65 MAb at 1:5,000 (top panel), and UL97 was detected using rabbit anti-UL97 polyclonal serum at 1:10,000 (bottom panel). An arrow indicates the presence of pp65 detected in anti-FLAG IP from FLAG97-infected HFF (top panel). Note that the prominent nonspecific band of ∼50 kDa detected in the anti-pp65 blot is attributed to the presence of a mouse IgG heavy chain. (C) Analysis of input proteins in lysates subjected to IP. Approximately 1.25% of the total input for each IP reaction was resolved by SDS-PAGE as described above and subjected to immunoblot analysis with anti-pp65 MAb (1:5,000; top panel), anti-UL97 rabbit polyclonal serum (1:3,000; middle panel), or anti-beta-actin MAb (1:5,000; bottom panel). An arrow indicates the specific UL97 immunoreactive band in the anti-UL97 blot (middle panel). Note the lack of pp65 expression in mock-infected cells, the lack of UL97 expression in mock- and Δ97-infected cells, and the approximately equal levels of beta-actin across all four lysates.

As seen in Fig. 4A (left panels), UL97 was specifically detected in anti-pp65 IPs from AD169rv- and FLAG97-infected HFF but not in those from mock-infected cells or from cells infected with Δ97. Furthermore, pp65 was specifically detected in the anti-FLAG IP from FLAG97-infected cells but not in those from cells infected with wild-type AD169rv or Δ97 or mock infected (Fig. 4B, middle panels). Levels of beta-actin appeared to be consistent among all four lysates (Fig. 4C, bottom panel), and levels of pp65 were also observed to be roughly equivalent among Δ97, AD169rv, and FLAG97 lysates (Fig. 4C, top panel). Western blotting experiments that were more quantitative (see Fig. S2 at http://coen.med.harvard.edu) showed that pp65 was approximately 35% to 45% less abundant in lysates of Δ97-infected cells than in those from cells infected with AD169rv or FLAG97, which may have been due to decreased solubility of pp65 in the absence of UL97 (43), to lower expression resulting from decreased DNA synthesis in Δ97-infected cells (59), or to a requirement for UL97 for pp65 stability. Roughly similar levels of an approximately 75-kDa polypeptide were detected in Western blots of lysates probed with the anti-UL97 polyclonal antibody (Fig. 4C, middle panel) (see Fig. S1 at http://coen.med.harvard.edu). This band was not observed in mock-infected lysate or a Δ97-infected lysate, and its mobility was slightly less in FLAG97-infected lysates. Therefore, we conclude that this band represents UL97. The bands with higher mobility appeared to be nonspecific. Densitometry was used to estimate that approximately 5% of UL97 expressed in AD169rv-infected cells coimmunoprecipitated with pp65 (see Fig. S3 at http://coen.med.harvard.edu), although it should be noted that not all the pp65 was immunoprecipitated from the lysate (not shown).

From this experiment we concluded that UL97 and pp65 co-IP from infected cells, including cells infected with parental wild-type AD169rv in which neither UL97 nor pp65 bears exogenous affinity tags. Therefore, our detection of a pp65-UL97 interaction in TAP experiments was likely not an artifact specific to NTAP97.

Detection of UL97-pp65 interaction in a GST pulldown experiment.

To investigate the interaction between UL97 and pp65 further, we performed an experiment to assess whether GST-pp65 was capable of specifically pulling down in vitro-translated UL97 in a GST pull-down assay. A GST fusion protein in which GST was fused to herpes simplex virus type 1 US11 (GST-US11) was used as a control for nonspecific binding. Meanwhile, in addition to UL97, two other in vitro-translated control proteins were used: firefly Luc and Rta, the rhesus rhadinovirus immediate-early transactivator. Radiolabeled in vitro translation reaction mixtures were each incubated overnight with either GST-pp65 or the GST-US11 control before glutathione beads were used to pull down GST fusion proteins. After wash steps and elution with a buffer containing excess free glutathione, eluates were resolved by SDS-PAGE and subjected to Coomassie staining and autoradiography.

GST-pp65 was observed to pull down UL97 but not Luc or RRta (Fig. 4A, left panel). While faint binding of in vitro-translated UL97 to GST-US11 was observed, the signal intensity of the UL97 band in the GST-pp65 eluate was more than eightfold more intense than that of the UL97 band seen in the GST-US11 eluate, as measured using a phosphorimager (see Fig. S4 at http://coen.med.harvard.edu). GST-pp65 was determined to bind approximately 5% of radiolabeled UL97. This percentage compared favorably to the percentage (3.5%) of in vitro-translated UL54, the catalytic subunit of the HCMV DNA polymerase, observed to bind a GST fusion of the polymerase accessory subunit, UL44 (data not shown), which was used as a positive control (34). Amounts of GST fusion proteins (Fig. 4A, right panel) and radiolabeled input proteins (left panel, lanes 1 to 3) were approximately equivalent, as were levels of the proteins in the flowthrough fractions from each pull down (Fig. 4B). Therefore, UL97 can bind specifically to pp65 in vitro and no other viral proteins are necessary for this interaction. Because rabbit reticulocyte lysates do contain a significant number of cellular proteins, we cannot entirely rule out the possibility that the pp65-UL97 interaction may require cellular factors. However, the simplest explanation of the data is a direct interaction between UL97 and pp65.

DISCUSSION

In this study, we have shown that pp65 and UL97 coeluted when lysates from cells infected with rHCMV bearing an N-terminal TAP tag on the UL97 ORF were purified by TAP. We have also shown that the pp65-UL97 interaction could be detected by reciprocal co-IP of lysates from HCMV-infected cells. Furthermore, we have demonstrated in an in vitro pull down that pp65 specifically interacts with UL97 in the absence of other viral proteins. Taken together, these data suggest that UL97 and pp65 may form a protein complex in HCMV-infected cells.

Caution compels one to consider the possibility that as one of the most highly expressed viral proteins during HCMV replication (50), pp65 might simply be a nonspecific contaminant in TAP eluates from HCMV-infected cells. Moreover, pp65 is reportedly prone to form aggregates (43) which might nonspecifically adhere to UL97 or otherwise copurify with it. However, we did not detect pp65 in TAP eluates from a negative-control lysate of AD169rv-infected cells either by MS or by Western blotting, which argues against the notion that pp65 was a nonspecific contaminant during TAP (Tables 1 and 2; Fig. 3A). Also, pp65 was not observed to nonspecifically contaminate IPs (Fig. 4B), even those from cells infected with a Δ97 virus, under which conditions pp65 reportedly forms unusual aggregates (43). Finally, pp65 specifically interacted with UL97 in an in vitro pulldown experiment (Fig. 5), further supporting the notion that UL97 and pp65 might genuinely interact during viral replication.

FIG. 5.

Detection of UL97-pp65 interaction in an in vitro pull-down experiment. (A) GST-pp65 specifically interacts with UL97 in an in vitro pull-down assay. GST-pp65 and GST-US11 (as a negative control) were used in parallel GST pull-down assays with in vitro translation reaction mixtures containing one of the following radiolabeled proteins: UL97, RRta (rhesus rhadinovirus Rta transactivator protein), or Luc (firefly luciferase). The left panel shows a phosphorimage of the radioactivity detected in the input and eluate samples, as detected following SDS-PAGE, and the right panel shows the same gel following Coomassie blue staining. The results for 10% of input in vitro translation reaction mixtures and 10% of eluates are shown. Arrows on the left indicate radiolabeled UL97, Rta, and Luc in vitro translation products in the phosphorimage; arrows on the right indicate GST fusion proteins in the Coomassie-stained gel. M, Bio-Rad Precision Plus Prestained MW markers. A digitally enhanced image in which the relative amounts of radiolabeled UL97 in the eluate fractions are made more readily visible is shown immediately below the left panel. (B) Proteins in flowthrough after GST pull down. Levels of radiolabeled proteins are indicated by autoradiography in the left panel; levels of total protein, as detected by Coomassie-staining of the same gel, are shown in the right panel. Approximately equivalent levels of protein loading can be observed across the samples.

Additional support for the notion of a physical interaction between UL97 and pp65 can be found in the literature. As early as 1986—6 years before UL97 was identified as a biologically active protein (33, 51)—Britt and Auger reported that a protein serine-threonine kinase activity was associated with IPs of pp65 from infected cells and virions (11). The authors specifically described the presence of an “as-yet-uncharacterized” phosphorylated polypeptide of “70 to 80 kDa” in their IPs. UL97 has a nominal molecular weight (MW) of ∼78 and is known to autophosphorylate (22). Therefore, the serine-threonine kinase activity observed by Britt and Auger might well be attributed to co-IP of UL97.

Two published observations support the notion of a biological interaction between UL97 and pp65. First, when UL97 is genetically ablated or pharmacologically inhibited, pp65 localizes in unusual refractile bodies (4, 43). Second, UL83 deletion mutants exhibit resistance to maribavir, a UL97 inhibitor (43). These observations suggest two non-mutually exclusive roles for the UL97-pp65 interaction that we have detected. In one role, UL97 may direct localization of pp65 during assembly and/or vice versa. Since pp65 localizes to the nucleus early in infection and is incorporated into progeny virions during acquisition of tegument, site-specific phosphorylation by UL97 might regulate localization of pp65 to the nucleus, as in the example of nuclear localization of Pho4 in budding yeast (27, 41) or its incorporation into virions. We have preliminarily observed phosphorylation of recombinant pp65 by UL97 in vitro (Kamil and Coen, unpublished results). Moreover, since UL97 is an HCMV virion component (55), an interaction between pp65 and UL97 may be relevant to incorporation of UL97 during virion morphogenesis. Interestingly, it has been reported that virions of a UL83 deletion mutant are deficient in virion-associated kinase activity (49).

The second possible role for the UL97-pp65 interaction is that pp65 may negatively regulate UL97 by sequestering kinase that might otherwise be available to promote viral replication. Such a model might explain why higher concentrations of maribavir are required to inhibit viral replication in the absence of pp65 expression (43). Because pp65 is completely dispensable for HCMV replication in cultured cells, despite its abundant expression (16, 47, 49), UL97 appears capable of promoting HCMV replication via mechanisms that do not require its interaction with pp65. Therefore, a relationship between UL97 and pp65 cannot, per se, explain the considerable growth defects described for Δ97 viruses in culture that have been attributed to roles of UL97 in viral DNA replication and encapsidation (59) or in nuclear egress (28).

Although an interaction of UL97 and pp65 may not be important to the yield of infectious virus in cultured cells, it may be germane in vivo. In particular, pp65 may be required for viral persistence in the face of the host immune system. When the murine CMV homolog of pp65 (M83) was disrupted, the resulting viruses replicated indistinguishably from wild-type virus in cultured cells but were severely attenuated for growth and spread in vivo (38, 61). In HCMV experiments, pp65 has been reported to block expression of host antiviral genes (1, 12). However, some of the reported effects of pp65 on antiviral gene expression (1, 12) may have been an artifact of delayed IE86 expression due to effects on UL82 in the HCMV UL83 deletion mutant studied (52). Nonetheless, pp65 has also been reported to bind and disable NKp30 (2), a signaling receptor of natural killer cells, and to block processing and presentation of viral immediate-early proteins to T cells (20). Interestingly, the latter effect was associated with phosphorylation of the proteins (20). Therefore, a stable interaction between pp65 and UL97 could play a role in pp65-mediated immune evasion.