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Journal of Virology, January 2008, p. 96-104, Vol. 82, No. 1
0022-538X/08/$08.00+0     doi:10.1128/JVI.01559-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Identification of Human Cytomegalovirus UL84 Virus- and Cell-Encoded Binding Partners by Using Proteomics Analysis

Yang Gao, Kelly Colletti, and Gregory S. Pari^*

University of Nevada—Reno, School of Medicine, Department of Microbiology and Immunology and Cell and Molecular Biology Graduate Program, Reno, Nevada 89557

Received 17 July 2007/ Accepted 10 October 2007

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Human cytomegalovirus (HCMV) UL84 is a phosphoprotein that shuttles from the nucleus to the cytoplasm and is required for oriLyt-dependent DNA replication and viral growth. UL84 was previously shown to interact with IE2 (IE86) in infected cells, and this interaction down-regulates IE2-mediated transcriptional activation in transient assays. UL84 and IE2 were also shown to cooperatively activate a promoter within HCMV oriLyt. UL84 alone can interact with an RNA stem-loop within oriLyt and is bound to this structure within the virion. In an effort to investigate the binding partners for UL84 in infected cells, we pulled down UL84 from protein lysates prepared from HCMV-infected human fibroblasts by using a UL84-specific antibody and resolved the immunoprecipitated protein complexes by two-dimensional gel electrophoresis. We subsequently identified individual proteins by matrix-assisted laser desorption ionization-tandem time of flight analysis. This analysis revealed that UL84 interacts with viral proteins UL44, pp65, and IE2. In addition, a number of cell-encoded proteins were identified, including ubiquitin-conjugating enzyme E2, casein kinase II (CKII), and the multifunctional protein p32. We also confirmed the interaction between UL84 and IE2 as well as the interaction of UL84 with importin . UL44, pp65, and CKII interactions were confirmed to occur in infected and cotransfected cells by coimmunoprecipitation assays followed by Western blotting. Ubiquitination of UL84 occurred in the presence and absence of the proteasome activity inhibitor MG132 in infected cells. The identification of UL84 binding partners is a significant step toward the understanding of the function of this significant replication protein.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Human cytomegalovirus (HCMV) lytic DNA replication requires the cis-acting oriLyt sequence, six core replication proteins, and, in human fibroblasts, the immediate-early protein IE2 and the multifunctional protein UL84 (4, 29, 30). Recent evidence indicates that UL84 may have several roles in the virus life cycle. Also, a number of activities have been associated with UL84. These include a shuttling function, inhibition of IE2-mediated transcriptional activation, RNA binding, and UTPase activity (9, 12, 17, 43). The UL84-IE2 protein complex can activate a strong bidirectional promoter within oriLyt, presumably to initiate a transcription event that triggers initiation of DNA synthesis. Evidence that UL84 is the initiator protein for lytic DNA synthesis comes from recent data demonstrating that UL84 binds to a stem-loop RNA structure within oriLyt (7). In vitro binding assays suggest that UL84 can change the conformation of RNA stem-loop structures and possibly allow for the assembly of the replication complex. UL84 was shown to be a component of the virion (40) and is bound to viral DNA at the lytic origin within isolated virions (7). This suggests that UL84 can play a positive or a negative role in initiation of lytic DNA synthesis. Both the wide range of activities associated with UL84 and analysis of the protein-coding sequence point to UL84 being a member of the DExD/H-box family of proteins. These proteins have been implicated as RNA helicases that have nucleocytoplasmic shuttling properties and the ability to up-regulate or repress transcription (37).

Much of our current knowledge about UL84 was ascertained using transient or in vitro assays. However, it is apparent that UL84 plays a key role in the virus life cycle and its activity may center on the interaction of UL84 with cellular and other virus-encoded factors. In an effort to gain more understanding of the role of UL84 in viral replication, it is essential to identify the binding partners of UL84 in the infected-cell environment. Due to the complex and broad activities associated with UL84, it can be assumed that many of these functions are attributed to the interaction of UL84 with other proteins, comprising a reactome that enhances or determines the role of UL84 in regulation and DNA replication.

In this report, we employed a mass spectrometry (MS)-based proteomic approach by first immunoprecipitating UL84 from infected cells and evaluating the proteome/reactome by two-dimensional (2-D) gel electrophoresis, followed by computer analysis and subsequent elucidation of peptide sequence by matrix-assisted laser desorption ionization-tandem time of flight (MALDI-TOF/TOF) analysis. This analysis revealed that the UL84 interactome consists of the virus-encoded proteins UL44 and UL83 (pp65) and the previously identified IE2 transactivator (36). As for cellular proteins associated with UL84, they include casein kinase II (CKII), the multifunctional protein p32, ubiquitin-conjugating enzyme E2, and annexin A2, along with several others and the previously identified importin alpha-3 subunit (18).

On the basis of the findings from the proteomics analysis, we confirmed the interactions of the virus-encoded proteins and several of the cellular proteins by coimmunoprecipitation followed by Western blot analysis. The results show an interaction between UL84 and both UL44 and pp65 in infected cells, as well as demonstrating an interaction between UL84 and these proteins in the absence of any other HCMV-encoded proteins. We also confirmed the interaction between UL84 and CKII, the catalytic subunit of this kinase, in infected cells and in cells cotransfected with CKII expression plasmids and a UL84 expression plasmid. Monoubiquitination was also confirmed with and without the proteasome inhibitor MG132, followed by immunoprecipitation using a ubiquitin-specific antibody. In addition, we show that UL84 interacts with p32, a protein that binds to several other herpesvirus regulatory proteins, including herpes simplex virus type 1 ICP27, Epstein-Barr virus EBNA-1, and herpesvirus Saimiri open reading frame (ORF) 73 (6, 13, 39, 41). More recently, p32 was shown to interact with HCMV UL97 and facilitate the redistribution of the nuclear lamina (23). This proteomics study is the first to identify novel protein binding partners for UL84 in an infected-cell environment. This is a major leap toward the understanding of the multifunctional role of this key factor in the virus growth cycle.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Cells and virus. Human fibroblasts were propagated as previously described (8). HCMV strain AD169 was maintained as stocks and propagated as described previously (42). HEK293FT cells were purchased from Invitrogen and maintained on Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum and 500 µg/ml G418 (Invivogen). Cos-7 cells were propagated and used in transfection experiments as previously described (8).

Plasmids. The CKII expression plasmid pRc/CMV-CKII-HA was a gift from David Litchfield (University of Western Ontario). The p32 expression plasmid pcDNAp32 (1-282) was a gift from Peter O'Hare (Marie Curie Research Institute). UL84 expression plasmid pCMV-UL84TAG was described previously (8). pUL44-dsRed was generated by amplification of the UL44 ORF from AD169 DNA by using primers UL44SacI FW (5'-GCGCGAGCTCGTCCGGGATGGATCGCAAG-3'; restriction sites are underlined) and UL44KpnI RV (5'-GCGCGGTACCGTGCCGCACTTTTGCTTCTTGG-3'; restriction sites are underlined), which was subsequently ligated into the SacI-KpnI-cleaved pDsRed-Monomer-N1 vector (Clontech). The UL83-hemagglutinin (HA) expression plasmid was made by PCR amplification of AD169 DNA with the primers 5'-CGCGCAGGCAGCATGGAGTCGCGCGGTCGCCGTT-3' and 5'-TTATCAAGCGTAGTCTGGGACGTCGTATGGGTAACCTCGGTGCTTTTTG-3', which was subsequently ligated into pcDNA3.1 (Invitrogen).

Antibodies. CKII-specific antibody was purchased from Sigma (catalog no. C5367). The pp65 antibody was purchased from Virusys (catalog no. CA003-100). The UL44-specific antibody was a gift from William Britt. A p32-specific antibody was a gift from Janice Hearing (Cold Spring Harbor). The UL84-specific antibody MAb84 has been previously described (8). The dsRed antibody was purchased from SantaCruz (catalog no. sc-33353). The anti-ubiquitin antibody was purchased from Santa Cruz Biotechnologies (catalog no. sc-8017).

Immunoprecipitation of UL84 from infected cells for proteomic analysis. HF cells (6 x 10⁷) were infected with HCMV (AD169) by using a multiplicity of infection of 5. At 3 days postinfection, cells were washed twice with cold phosphate-buffered saline (PBS; pH 7.4) and lysed in 3 ml lysis buffer (50 mM Tris-HCl [pH 7.4], 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% Tween 20, 10 µl protease inhibitor cocktail/ml). Cells were removed from the flask by scraping and passed through a 22-gauge needle three times to shear DNA. Cellular debris was removed by centrifugation at 4,000 x g for 10 min. The supernatant was transferred to a new 15-ml conical tube. Normal mouse immunoglobulin G (5ul/ml) was added to the supernatant, and the mixture was rotated for 30 min at 4°C. Lysates were incubated with anti-UL84 antibody (5 µg/ml) for 1 h at 4°C, at which time 50 µl/ml of protein G plus agarose beads was added and incubated at 4°C overnight. The complexes were washed with ice-cold PBS three times. The protein complexes were removed from the beads by the addition of 2x Laemmli sample buffer (Bio-Rad) containing 2-mercaptoethanol and then heated to 95°C for 5 min. Samples were analyzed by Western blotting before use for 2-D gel analysis.

For coimmunoprecipitations followed by analysis by Western blotting, the same procedure as that described above was followed, except pulled-down complexes from infected or cotransfected cells were separated through a sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel. Gels were subsequently transferred to a polyvinylidene difluoride (PVDF) membrane and reacted with antibodies as described in Results and Materials and Methods. For electrophoreses, 15 µl of lysate (out of a total of 800 µl) was used for the Lysate lanes and the remaining lysate was used for immunoprecipitations. In the final step of the immunoprecipitation, beads were resuspended in 100 µl of gel loading buffer and 30 µl was loaded in each well.

Preparation of immunoprecipitated protein complexes for proteomics analysis. All reagents and solutions for 2-D electrophoresis, IPG strips, and SDS polyacrylamide gels were obtained from Bio-Rad (Hercules, CA) except as noted. The EZQ protein assay kit was purchased from Invitrogen. DeStreak rehydration solution was a product of GE Healthcare. Protein complexes from immunoprecipitations were precipitated from Laemmli sample buffer by the addition of 4 volumes of –20°C acetone. The samples were kept at –20°C overnight and spun at 16,000 x g for 10 minutes at 0°C. The supernatants were discarded, and the pellets were washed twice with ice-cold acetone-water (4:1). The final pellets were dried in a Speed Vac for 15 min and then resuspended in 240 µl DeStreak rehydration solution. The protein content of the resultant supernatants was determined using the EZQ protein assay. After the addition of 3/10 ampholytes to a final concentration of 0.2%, the extracts were spun at 16,000 x g at room temperature for 10 min. Samples were analyzed by Western blotting for the presence of UL84 and IE2 before use for 2-D gel analysis.

2-D gel electrophoresis. Two hundred microliters of each extract was loaded onto a pH 3 to 10 11-cm IPG strip by overnight passive rehydration. Isoelectric focusing was carried out on a Bio-Rad Protean isoelectric focusing cell by using a program as follows: 250-V linear ramp for 20 min, 8,000-V linear ramp for 2 h 30 min, and 8,000 V for a total of 20,000 V·h (all steps with a maximum current of 50 µA per gel). Strips were stored at –80°C overnight and then thawed on the next day and incubated twice for 10 min each in 8 M urea, 2% SDS, 0.05 M Tris-HCl, pH 8.8, 20% glycerol. The first incubation mixture contained 2% dithiothreitol, and the second contained 2.5% iodoacetamide. The strips were then layered on 8 to 16% Criterion Tris-HCl gradient gels and embedded in place with 0.5% agarose, along with Invitrogen BenchMark protein ladder molecular-weight markers. Electrophoresis was performed at a constant current of 200 mA until the dye front ran to the bottom of the gel. Gels were stained overnight as per directions with Sypro Ruby stain.

Imaging and comparison of protein spots. Stained gels were imaged on a Bio-Rad VersaDoc imager. Images of gels were compared using Bio-Rad PDQuest version 8.0 software, and spot sets were created. The spots in these sets were then cut using a Bio-Rad ExQuest spot cutter.

Protein digestion and MS. Protein digestion and MS were performed by the Nevada Proteomics Center according to the following protocol. Spots were digested using the Investigator Proprep (Genomic Solutions, Ann Arbor, MI), using a previously described protocol (33a) with some modifications. Samples were washed twice with 25 mM ammonium bicarbonate and 100% acetonitrile, reduced and alkylated using 10 mM dithiothreitol and 100 mM iodoacetamide and incubated with 75 ng trypsin in 25 mM ammonium bicarbonate for 6 h at 37°C.

Samples were prepared and spotted onto a MALDI target with ZipTip µ-C18 tips (Millipore, Billerica, MA). Samples were aspirated and dispensed three times; eluted with 70% acetonitrile, 0.2% formic acid; and overlaid with 0.5 µl of 5 mg/ml MALDI matrix (-cyano-4-hydroxycinnamic acid) and 10 mM ammonium phosphate.

All MS data was collected using an ABI 4700 MALDI-TOF/TOF (Applied Biosystems, Foster City, CA). The data were acquired in reflector mode from a mass range of 700 to 4,000 Da, and 1,250 laser shots were averaged for each mass spectrum. Each sample was internally calibrated on trypsin's autolysis peaks. The eight most intense ions from the MS analysis were subjected to MS/MS. The data were stored in an Oracle database.

The data were extracted from the Oracle database, and a peak list was created by GPS Explorer software (Applied Biosystems, Foster City, CA) from the raw data generated from the ABI 4700. This peak list was based on signal-to-noise filtering and included deisotoping. The resulting file was then searched by Mascot (Matrix Science, Boston, MA). A tolerance of 20 ppm was used as the samples were internally calibrated. The database search parameters included one missed cleavage, oxidation of methionines, and carbamidomethylation of cysteines.

Ubiquitinated UL84 pulldown. For infection-based assays, HFs cells (1 x 10⁶) were plated into a 10-cm dish and infected with HCMV (AD169). After 72 h, the medium was changed and 50 µM MG132 was added, or in the case of control cells, dimethyl sulfoxide was added to cells. Cells were incubated for 6 h and harvested in 1 ml lysis buffer (50 mM Tris-HCI [pH 7.4], 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% Tween 20, 10 µl protease inhibitor cocktail/ml). Lysates were incubated with an anti-ubiquitin-specific antibody for 1 h at 4°C, after which 50 µl/ml of protein G plus agarose beads was added and incubated at 4°C overnight. The complexes were washed with ice-cold PBS three times. The protein complexes were removed from the beads by the addition of 2x Laemmli sample buffer (Bio-Rad) containing 2-mercaptoethanol and then heated to 95°C for 5 min.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Proteomics analysis of UL84 binding partners from infected cells. After comparing the 2-D gel data between control (nonspecific antibody) and UL84-specific-antibody-immunoprecipitated protein, we selected and analyzed 20 distinct protein spots found in the gel containing infected cell proteins that were not present in control 2-D gels. Table 1 shows the results of protein sequencing and the protein annotations, predicted molecular weights, and ion count indexes. We report only proteins that had ion counts greater than 98%. The analysis revealed that 4 virus- and 15 cell-encoded proteins were immunoprecipitated along with UL84. We decided to confirm the interactions of UL84 and all of the identified novel virus-encoded proteins by coimmunoprecipitation with infected and cotransfected cells. Also, we selected several cellular binding partners identified and further investigated these interactions.

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TABLE 1. Proteins identified by MALDI-TOF/TOF analysis of UL84 binding partners

Interaction of UL84 with UL44 and pp65 occurs in the absence of any other viral protein. Our proteomics analysis identified UL44, UL83 (pp65), and IE2 as binding partners for UL84 in infected cells. IE2 was previously identified as a binding partner for UL84 and was shown to suppress IE2-mediated transcriptional activation of the UL112/113 promoter in transient reporter assays (12). UL44, which had not previously been implicated as a UL84 binding partner, encodes the polymerase accessory protein or processivity factor and was identified as one of the six core proteins required for oriLyt-dependent DNA replication (29, 30). The gene product of UL83, pp65, encodes the lower matrix phosphoprotein and is localized to the nucleus shortly after infection (34).

Our first step was to confirm the UL44-UL84 interaction in infected cells and then determine if any other virus-encoded protein(s) was required for this interaction. We infected HFs with HCMV (AD169) and performed coimmunoprecipitation assays using anti-UL84- and anti-UL44-specific antibodies. Before immunoprecipitation, samples were treated with DNase and RNase. This was done to eliminate the possibility that UL44 and UL84 (both of which bind nucleic acid) are tethered by either DNA or RNA and are not actually interacting via protein domains. Protein samples were separated using SDS-PAGE, and subsequently, Western blot analyses were performed on immunoprecipitated protein by using UL44- and UL84-specific antibodies. Coimmunoprecipitation assays confirmed that UL84 and UL44 interacted in infected cells when either anti-UL84 or anti-UL44 antibodies were used to pull down protein (Fig. 1A). Immunoprecipitations using an anti-UL44-specific antibody efficiently pulled down UL84 in infected cells. (Fig. 1A, lane IP:UL44). This result confirmed the accuracy of the proteomics data with respect to a bona fide UL44-UL84 interaction.

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FIG. 1. UL44 interacts with UL84. (A) Protein lysates were prepared from HCMV-infected HFs, and protein complexes were immunoprecipitated using anti-UL44 or anti-UL84 monoclonal antibodies. Precipitated protein was analyzed by Western blot (WB) detection using anti-UL84 MAb. Lanes: Lysate, cellular protein extract before immunoprecipitation (IP); Control, protein lysates immunoprecipitated with protein G-beads; IP:UL84, protein lysates immunoprecipitated using MAb84; IP:UL44, protein lysates immunoprecipitated using anti-UL44 monoclonal antibody. (B and E) The UL44-UL84 interaction requires no other HCMV viral protein. HEK293 and Cos7 cells were cotransfected with UL44 and UL84 expression vectors, protein lysates were prepared, and protein complexes were immunoprecipitated with anti-UL44- or anti-UL84-specific antibodies. Lanes: Lysate, cellular protein extract before immunoprecipitation; Control, protein lysates from cells transfected with pCMV-UL84TAG and immunoprecipitated with anti-UL44 antibody; IP:UL84, protein lysates immunoprecipitated using MAb84; IP:UL44, protein lysates immunoprecipitated using anti-UL44 monoclonal antibody. (C and G) Detection of UL44 by immunoprecipitation with anti-UL84 antibody. HEK293 and Cos7 cells were cotransfected with UL84 and UL44-dsRed fusion expression plasmids. Protein lysates were prepared, and protein complexes were immunoprecipitated with MAb84. Lanes: Lysate-1, protein lysate prepared from cells cotransfected with pCMVUL84-FLAG and pUL44-dsRed; Lysate-2, protein lysate prepared from cells cotransfected with a plasmid that expresses FLAG-tagged luciferase (TAG-control) and pUL44-dsRed; IP:UL84-1, protein lysates from lane Lysate-2 immunoprecipitated with anti-UL84 antibody; IP:UL84-2, protein lysates from lane Lysate-1 immunoprecipitated with anti-UL84 antibody. (D and F) Coimmunoprecipitation of UL44 and UL84. HEK293 or Cos7 cells were cotransfected with pUL44-dsRed and pCMV-UL84TAG, and protein complexes were immunoprecipitated with MAb84. Lanes: Lysate-1, protein lysate prepared from cells transfected with pUL44-dsRed; Control, protein lysate from cells transfected with pUL44-dsRed and immunoprecipitated with MAb84; Lysate-2, protein lysate from cells cotransfected with pUL44-dsRed and pCMV-UL84TAG; IP:UL84, protein lysate from lane Lysate-2 immunoprecipitated with MAb84. The antibodies used for Western blot detection are shown below each figure.

In an effort to show that UL44 can interact with UL84 in transfected cells, we cotransfected HEK293FT and Cos7 cells with a UL44-dsRed fusion expression plasmid along with a UL84-FLAG-tagged expression plasmid, prepared protein lysates, and immunoprecipitated protein complexes with anti-UL84 or anti-UL44 antibodies. UL44-dsRed expresses a fusion protein composed of the UL44 ORF and the red fluorescent protein dsRed. This fusion expression plasmid was used because the molecular weight of UL44 is approximately the same as that of the immunoglobulin G antibody heavy chain, and hence, it was difficult to detect UL44 by Western blot analysis when performing immunoprecipitations. Immunoprecipitated protein was analyzed by Western blot detection using an anti-dsRed monoclonal antibody. Cells were cotransfected with UL44-dsRed and pCMV-UL84TAG, and protein complexes were immunoprecipitated with an anti-UL44 antibody. Western blot analyses were performed using MAb84. UL84 was efficiently pulled down by the immunoprecipitation of UL44 in both HEK293 and Cos7 cells (Fig. 1B and E, lane IP:UL44). When cotransfected cell extracts were used to immunoprecipitate protein complexes with an anti-UL84 antibody, dsRed-UL44 was detected by Western blot analysis, confirming the interaction between the two proteins (Fig. 1C and G, lane IP:UL84-2). The Lysate-1 lane contains protein that was prepared from cells cotransfected with a FLAG-tagged luciferase expression plasmid and pUL44-dsRed (Fig. 1C and G, lane Lysate-1). The Lysate-2 lane contains protein that was prepared from cells cotransfected with pUL44-dsRed and a pCMV-UL84TAG (Fig. 1C and G, lane Lysate-2). For a control, we cotransfected a luciferase expression plasmid expressing FLAG-tagged luciferase protein along with UL44-dsRed. Protein complexes were immunoprecipitated with MAb84, and Western blots of precipitated proteins were reacted with anti-dsRed. No band was detected in this control sample (Fig. 1C and G, lane IP:UL84-1). A faint band can be detected in the control lane of Fig. 1C, which could be due to some nonspecific interactions; however, this band was not observed upon confirmation in Cos7 cells or in the infected-cell coimmunoprecipitations (Fig. 1G and A). The same cotransfection was performed with pUL44-dsRed and pCMV-UL84TAG, except that we reacted Western blots with an anti-UL44 antibody (Fig. 1D and F). Again, we were able to detect UL44 (dsRed fusion) when the immunoprecipitation was done using MAb84 (Fig. 1D and F, lane IP:UL84). Lanes Lysate-1 and Lysate-2 show the efficient expression of the dsRed fusion of UL44. Also, the control lane showed no nonspecific interaction between MAb84 and UL44 dsRed fusion (Fig. 1D and F, Control).

These experiments demonstrated that UL44 interacts with UL84 and that this interaction can occur in the absence of other virus-encoded factors.

UL84 binds to pp65. The interaction of UL84 with the viral capsid protein pp65 was also investigated, in an experiment similar to that performed with UL44. HFs were infected with HCMV, and at 7 days postinfection, protein lysates were prepared and protein complexes were immunoprecipitated with either UL84- or pp65 (UL83)-specific antibodies, followed by Western blotting. Again, we confirmed that UL84 coimmunoprecipitated pp65 in infected cells (Fig. 2). Protein complexes from infected cells immunoprecipitated with a UL83-specific antibody revealed that UL84 was able to bind and be pulled down by anti-UL83 (Fig. 2A, lane IP:UL83). Also, when MAb84 was used to immunoprecipitate protein complexes from infected cells, pp65 was detected in Western blots (Fig. 2B, lane IP:UL84).

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FIG. 2. Interaction of UL84 with pp65. (A and B) Protein lysates were prepared from HCMV-infected HFs, and protein complexes were immunoprecipitated (IP) with either anti-UL84 or anti-pp65 antibodies. Immunoprecipitated protein was detected by Western blot (WB) analysis using pp65- or UL84-specific monoclonal antibodies. Lanes: Lysate, preimmunoprecipitation protein lysates prepared from infected HFs; Control, infected cell protein lysates immunoprecipitated with protein G-plus beads; IP:UL84, protein lysates immunoprecipitated with MAb84; IP:UL83, protein lysates immunoprecipitated with an anti-pp65-specific antibody. (C and E) UL84 and pp65 interaction in transfected cells. HEK293 and Cos7 cells were cotransfected with UL84 and pp65-HA fusion expression plasmids, respectively. Protein lysates were prepared, and complexes were immunoprecipitated using an anti-HA-specific monoclonal antibody. Immunoprecipitated protein was detected by Western blot analysis using MAb84. Lanes: Lysate, preimmunoprecipitation protein lysates prepared from cotransfected cells; Control, protein lysates produced from cells transfected with pCMV-UL84TAG and immunoprecipitated with anti-HA antibody; IP:UL84, protein lysates immunoprecipitated with MAb84; IP:HA, protein lysates immunoprecipitated with an HA-specific antibody. (D and F) Immunoprecipitation of pp65 by a UL84-specific antibody. Lanes: Lysate, preimmunoprecipitation protein lysates prepared from cotransfected cells; Control, protein lysates produced from cells transfected with an HA-tagged pp65 expression plasmid and immunoprecipitated with MAb84. The antibodies used for Western blot detection are shown below each figure.

HEK293 and Cos7 cells were cotransfected with a pp65-HA-tagged expression plasmid and the UL84-FLAG-tagged expression plasmid. Protein lysates were incubated with either anti-pp65 or anti-UL84 antibodies, immunoprecipitated protein was resolved by PAGE gel, and Western blot analyses were performed. Western blots from the cotransfection experiments also showed that the interaction between pp65 and UL84 took place in the absence of infecting virus and any other viral protein when immunoprecipitations were done using an anti-HA-specific antibody (Fig. 2C and E, lane IP:HA) or when MAb84 was used and Western blots were reacted with the anti-pp65 antibody (Fig. 2D and F, lane IP:UL84).

These experiments showed that the UL84 binding partners identified by the proteomics screen were genuine, and we confirmed that these interactions occur in infected cells. In addition, cotransfection studies showed that UL84 binds to UL44 and pp65 in the absence of any other viral proteins.

UL84 is ubiquinated. The identification of ubiquitin E2-conjugating enzyme from the proteomic analysis suggested that UL84 is ubiquinated. This would also be consistent with the fact that the two protein spots that were identified as UL84 differed only slightly with respect to molecular weight but had distinct isoelectric focusing points (pI) (data not shown). We investigated the possibility that UL84 is ubiquinated in infected and transfected cells by treating them with the proteasome inhibitor MG132 and then performing immunoprecipitation experiments using an anti-ubiquitin antibody. Immunoprecipitated protein was resolved through an SDS-PAGE gel, and protein samples were transferred to an Immobilon-P PVDF membrane and reacted with MAb84. Immunoprecipitation experiments using a ubiquitin-specific antibody would ensure that only the ubiquinated UL84 species would be identified by subsequent Western blotting. For molecular-weight comparison, we performed immunoprecipitations using MAb84 and separated this protein sample next to the lane containing protein from the anti-ubiquitin immunoprecipitation.

Western blots reacted with MAb84 from MG132-treated, infected cell lysates immunoprecipitated with an anti-ubiquitin antibody detected a protein species with an apparent molecular weight that was slightly higher than that of UL84 immunoprecipitated with MAb84 and not treated with MG132 (Fig. 3A, compare lanes 1 and 2 to lane 3). In order to confirm this result, we repeated the experiments, but this time we loaded more protein and used a lower-percentage gel to better resolve the ubiquinated species from the unubiquinated UL84 protein. This repeated experiment clearly demonstrated that immunoprecipitation of UL84 by use of an anti-ubiquitin-specific antibody results in the detection of a species with a high molecular weight compared to that of UL84 immunoprecipitated from similarly infected, untreated cells (Fig. 3A, compare lanes 6 and 7). We were also able to detect a slight UL84 ubiquinated species in the absence of MG132 treatment, again suggesting that the monoubiquinated form is present in infected cells (Fig. 3A, lane 4).

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FIG. 3. UL84 is ubiquinated in infected and transfected cells. HF cells were infected and treated with MG132 or left untreated. Protein lysates were prepared and incubated with either a ubiquitin-specific antibody or MAb84. (A) Immunoprecipitated protein was resolved through an SDS-PAGE gel and transferred to PVDF, and Western blot analysess were performed using MAb84. Lanes: 1, Cell lysate treated with MG132; 2, cell lysate untreated; 3, protein immunoprecipitated from infected cells treated with MG132 by using an anti-ubiquitin-specific antibody; 4, protein immunoprecipitated from untreated infected HF cells by using an anti-ubiquitin-specific antibody; 5, protein immunoprecipitated from infected cells treated with MG132 by using an anti-UL84-specific antibody; 6, protein immunoprecipitated from infected cells treated with MG132 by using an anti-ubiquitin antibody; 7, protein immunoprecipitated from infected cells treated with MG132 by using an anti-UL84 antibody. All blots were reacted with MAb84. *, ubiquinated UL84 species; hc, antibody heavy chain. (B) Western blot of immunoprecipitated UL84 from infected cells reacted with an anti-ubiquitin antibody.

To further characterize the ubiquitination of UL84, we performed pulldown experiments using MAb84 from HCMV-infected cells that were not treated with MG132 and analyzed the immunoprecipitated product by Western blot detection using an anti-ubiquitin antibody. This analysis reveled that UL84 can be polyubiquinated in the absence of proteasome inhibition, as evidenced by the detection of several ubiquinated species of UL84 (Fig. 3B).

These experiments identify UL84 as a ubiquinated protein, and the data suggest that monoubiquitinated and polyubiquitinated protein species are present in infected cells. The data further suggest that UL84 ubiquitination may play a role other than or in addition to targeting the protein for degradation.

Interaction of UL84 with CKII. Proteomics analysis identified CKII as a potential binding partner of UL84 in infected cells. CKII is a serine/threonine-specific kinase composed of two parts of each subunit ( and β) to form a tetramer where the subunits are the catalytic kinase components of the enzyme (19, 28). To confirm that CKII interacts with UL84, we performed coimmunoprecipitation assays using both infected cell protein lysates and lysates from cells cotransfected with plasmids expressing and β CKII subunits. Protein complexes were immunoprecipitated using antibodies specific for either the CKII or β subunit. Western blots were reacted with antibodies specific for UL84 or the CKII or β subunit. Immunoprecipitations from infected cell lysates using a CKII-specific antibody showed that UL84 can be pulled down when this antibody is used (Fig. 4A, lane IP:CK2). Likewise, when infected cell protein lysates were immunoprecipitated with MAb84, a CKII-specific band was detected on Western blots of immunoprecipitated protein complexes (Fig. 4B, lane IP:UL84). Control immunoprecipitations failed to show any specific binding or detectable signal on the Western blots (Fig. 4A and B, lanes Control). Western blots failed to detect any specific interaction with the CKIIβ subunit (data not shown).

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FIG. 4. Interaction of CKII subunit with UL84. (A and B) Protein lysates were prepared from HCMV-infected HFs, and protein complexes were immunoprecipitated (IP) with either anti-UL84 or anti-CKII antibodies. Immunoprecipitated protein was detected by Western blot (WB) analysis using CKII-or UL84-specific monoclonal antibodies. Lanes: Lysate, preimmunoprecipitation protein lysates prepared from infected HFs; Control, infected cell protein lysates immunoprecipitated with G-plus beads; IP:UL84, protein lysates immunoprecipitated with MAb84; IP:CKII, protein lysates immunoprecipitated with anti-CKII antibody. (C and E) UL84 and CKII interaction in transfected cells. HEK293 and Cos7 cells were cotransfected with UL84 and CKII-HA fusion expression plasmids, respectively. Protein lysates were prepared, and complexes were immunoprecipitated using an anti-HA monoclonal antibody. Immunoprecipitated protein was detected by Western blot analysis using MAb84. Lanes: Lysate, preimmunoprecipitation protein lysates prepared from cells cotransfected with pCMV-UL84TAG and pRc/CMV-CK2-HA; Control, protein lysates from cells transfected with pCMV-UL84TAG and immunoprecipitated with anti-HA antibody; IP:UL84, protein lysates immunoprecipitated with MAb84; IP:HA, protein lysates immunoprecipitated with an HA-specific antibody. (D and F) HEK293 and Cos7 cells were cotransfected with plasmids expressing UL84 and CK2, respectively. Lanes: Lysate, preimmunoprecipitation protein lysates prepared from cells cotransfected with pCMV-UL84TAG and pRc/CMV-CK2-HA; Control, protein lysates from cells transfected with pCKII-HA and immunoprecipitated with anti-FLAG antibody; IP:FLAG, cells cotransfected with pRc/CMV-CK2-HA and pCMV-UL84TAG, with protein complexes immunoprecipitated with anti-FLAG antibody. The antibodies used for Western blot detection are shown below each figure.

Protein lysates from HEK293 and Cos7 cotransfected cells were also used for immunoprecipitation assays with UL84-and CKII-specific antibodies. Cells were cotransfected with UL84-FLAG and a CKII-HA-tagged expression plasmid. Protein lysates from cotransfected cells were reacted with HA-specific antibody (pulling down CKII). Protein complexes were resolved, and subsequent Western blots were reacted with MAb84 to show that UL84 can be efficiently coimmunoprecipitated by pulling down CKII (Fig. 4C and E, lane IP:HA). In the reverse experiment, the pulling down of UL84 from cotransfected protein lysates resulted in the detection of CKII (Fig. 4D and F, lane IP:FLAG). These experiments indicated that CKII interacts with UL84 in infected and cotransfected cells and suggests that UL84 is a substrate for CKII.

UL84 binds to p32. The cellular protein p32 is known to interact with a wide array of host and virus-encoded proteins (14). One of the most intriguing activities assigned to p32 is its ability to associate with the nuclear splicing factor SF-2 (25, 31). We sought to confirm the interaction between UL84 and p32 in infected and cotransfected cells by performing coimmunoprecipitation experiments in the same manner as for those carried out with other identified cellular proteins from the proteomics analysis.

Infected cell protein lysates were incubated with UL84- or p32-specific antibodies, and immunoprecipitated protein complexes were separated by SDS-PAGE and subsequently analyzed by Western blot detections. Blots containing protein complexes immunoprecipitated using the anti-UL84 antibody were reacted with an antibody specific for p32 to show that indeed p32 can interact with UL84 in infected cells (Fig. 5A, lane IP:UL84). In addition, blots containing protein complexes from infected cell samples in cases where a p32-specific antibody was used in the immunoprecipitation assay reacted with MAb84, confirmed that UL84 was present, and indicated an interaction between the two proteins (Fig. 5B, lane IP:P32).

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FIG. 5. UL84 interacts with p32. (A and B) Protein lysates were prepared from HCMV-infected HFs, and protein complexes were immunoprecipitated with either anti-UL84 or anti-p32 antibodies. Lanes: Lysate, preimmunoprecipitation protein lysates; Control, protein lysates from infected cells immunoprecipitated with protein G-plus beads; IP:UL84, protein complexes immunoprecipitated from infected cells with MAb84; IP:p32, protein complexes immunoprecipitated from infected cells with anti-p32-specific antibody. (C to F) HEK293 (C and D) and Cos7 (E and F) cells were cotransfected with pCMV-TAG-UL84 and pcDNA-p32 expression plasmids, and protein complexes were immunoprecipitated using MAb84 or anti-p32-specific antibodies. Lanes: Lysate, protein lysates prepared from cells cotransfected with pFLAG-84 and a p32 expression plasmid; Control, cells cotransfected with pCMV-UL84TAG and a pcDNA p32 expression plasmid, with immunoprecipitation performed using protein G-plus beads; IP:UL84, protein complexes immunoprecipitated with MAb84; IP:p32, protein complexes immunoprecipitated with an anti-p32 antibody. Western blots were reacted with either MAb84 or a p32-specific antibody, as indicated below the blots.

To determine that UL84 can interact with p32 in the absence of other virus-encoded proteins, we cotransfected HEK293FT cells with a p32 expression plasmid and pCMV-UL84TAG. Protein complexes were immunoprecipitated with either MAb84 or an anti-p32-specific antibody. Protein complexes were evaluated in the same manner as infected cell lysates. Immunoprecipitation assays using the p32 antibody showed that protein complexes contained UL84 (Fig. 5C and E, lane IP:p32). Pulldown assays using MAb84 also indicated that p32 was present in the complex (Fig. 5D and F, lane IP:UL84). These results confirmed that p32 and UL84 interact in infected and cotransfected cellular environments.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
HCMV UL84 has emerged as a key protein involved in DNA replication and regulation of gene expression. We employed a proteomics approach as a first step in an attempt to comprehend how the interaction of UL84 with cellular and/or viral binding partners influences or determines the function of UL84. Our current understanding of how UL84 fits into the replication scheme of HCMV is mostly from transient assays. Even with the limitations of these assays, several activities that appear to comprise small pieces of the growing puzzle concerning the complex regulation of lytic DNA synthesis have been assigned to UL84. Here, we report a comprehensive examination of the binding partners for UL84 in infected cells. As a control for nonspecific interactions, we used an unrelated antibody in infected cell lysates and used this sample of immunoprecipitated protein to pick distinct spots on a 2-D gel. This method was done to ensure that we identified bona fide protein-protein interactions. Lastly, we performed the analysis three separate times. Each time, the analysis identified the exact same proteins interacting with UL84. For our cotransfection studies, we used two different cell lines to confirm an interaction between proteins identified in the proteomics screen. We acknowledge that HEK293 cells express adenovirus proteins and Cos7 cells produce T antigen. It is possible that these other viral proteins could interact with HCMV proteins and possibly bridge an interaction. This possibility is slight, however, since infected primary human fibroblast cells were also used for pulldown experiments and many other herpesvirus coimmunoprecipitation experiments have been performed with these cell types.

Many of the properties associated with UL84 have hinted at the possibility that other viral and cellular proteins may interact with UL84 in the infected-cell environment. For example, it is known that UL84 is phosphorylated in vivo but to date no potential cellular kinase has been identified. Our data now show that UL84 interacts with CKII and strongly suggest that UL84 is a substrate for this kinase. An examination of the UL84 protein sequence identified seven potential CKII phosphorylation sites (amino acids 64, 65, 74, 148, 157, 292, and 293). Although the consequence of phosphorylation of UL84 is unknown, we now have the tools to test the activity of UL84 in the context of DNA replication and regulation of gene expression in the presence or absence of CKII phosphorylation. Phosphorylation of UL84 by CKII is consistent with our previously published results showing that UL84 is phosphorylated at serine residues (9). Recently, it was shown that CKII is a component of the virion, again implicating this kinase as playing a regulatory role in HCMV replication (26). Also, HCMV UL44 (polymerase accessory protein) and Epstein-Barr virus Zta are phosphorylated by CKII (2, 3, 15). In both cases, phosphorylation serves to regulate the activities of these proteins.

We acknowledge that CKII may not be the only kinase involved in phosphorylation of UL84; nevertheless, it is a major step forward in an attempt to understand the regulation of UL84 activities.

Another interesting modification discovered from the proteomics analysis was the observation that UL84 interacted with ubiquitin-conjugating enzyme E2. This led us to investigate whether UL84 is ubiquinated in infected cells. It appears that the main species of ubiquinated UL84 is the monoubiquinated form. We did detect at least two other ubiquinated species of UL84, suggesting that polyubiquitination is possible. This is also consistent with a slight increase in ubiquinated UL84 in the presence of MG132. However, we were able to detect several ubiquinated UL84 species in the absence of MG132 treatment.

Although polyubiquitination is linked to protein degradation by a proteasome-dependent pathway, monoubiquitination appears to affect the regulatory properties of proteins and is associated with diverse proteasome-independent cellular functions, including intracellular protein movement (1, 24). It was recently demonstrated that UL84 is able to shuttle from the nucleus to the cytoplasm (17). Although this process was attributed to two regions of the protein, it cannot be ruled out that protein modification such as ubiquitination could influence protein localization. Monoubiquitination is also implicated in regulation of transcription (10). This would also be consistent with the fact that UL84 was shown to repress IE2-mediated transcriptional activation, an activity that could be regulated by ubiquitination. Monoubiquitination does not preclude UL84 degradation through a proteasome-dependent pathway. There are cases in which monoubiquitination serves as a precursor to subsequent addition of other ubiquitin molecules, leading to degradation of the protein. The effects of ubiquitination of UL84 will be the subject of further investigation.

We also confirmed that UL84 interacts with the mitochondrial protein p32. p32 is an enigmatic factor that appears to have many purported activities within the cell and has been implicated in regulation of gene expression and replication in viral systems, including HCMV. p32 was originally identified as a protein associated with the cellular splicing factor ASF/SF2 (16). In cells infected with adenovirus, p32 was shown to repress major late gene transcription (27). Also, it was shown that human immunodeficiency virus type 1 Tat protein regulates splicing via an interaction with p32 (5). p32 appears to be a multifunctional protein that is localized to the cell surface, nucleus, and mitochondria. For HCMV, p32 was shown to recruit pUL97 to the nuclear lamina (23). For other herpesviruses, p32 is involved in regulation of splicing, transcriptional activation, and possible maintenance of latent viral DNA episomes (6, 13, 39, 41). Speculation as to the role of p32 in regard to an interaction with UL84 could include regulation of transcriptional activation of the oriLyt promoter (43) and/or regulation of host splicing and RNA transport. Although to date no inhibition of host RNA processing or splicing has been attributed to UL84, there are data showing that UL84 binds to RNA and has nucleocytoplasmic shuttling activity (7).

The finding that UL84 interacts with UL44 demonstrates for the first time that there is an interaction between a component of the replication complex and the proposed lytic DNA synthesis initiator protein UL84. HCMV UL44 is the polymerase accessory protein and was shown to interact with UL54, an HCMV-encoded polymerase (20-22, 32). The evidence presented here suggests that UL44 may play a role in initiation of lytic DNA synthesis. Recently, UL84 was shown to bind to an RNA stem-loop structure within oriLyt (7). Although this interaction occurred in vitro in the absence of any other viral protein, it is possible that UL44 may serve to stabilize this interaction or facilitate unwinding of the RNA-DNA hybrid duplex within the context of the viral genome. The chromatin immunoprecipitation assay demonstrated that UL84 interacts with oriLyt. UL44 could exist in a distinct complex with UL84 that does not involve the HCMV polymerase. UL84 and UL44 could function as an initiation complex early in the replication cycle, before the recruitment of the other replication factors. Other possible roles for UL44 could include aiding in some as-yet-unidentified enzymatic activity provided by UL84. We will explore these possibilities as part of future studies.

The interaction of UL84 with pp65 is unexpected; however, this interaction may serve to anchor UL84 within the virion. pp65 is the lower matrix phosphoprotein, and a large amount of the protein targets to the nucleus early after infection by two nuclear localization domains (34). The protein was also found to be associated with condensed chromatin (11). UL84 is a component of the virion and is bound to viral DNA packaged within the virus particle (7, 40).

Although not confirmed in this report, several other cell-encoded proteins that interact with UL84 were identified in the proteomics screen. A number of other identified proteins are implicated in nucleic acid binding or repair. For example, ATP-dependent DNA helicase II is a nuclear complex consisting of two subunits with molecular masses of approximately 70 and 80 kDa. The complex functions as a single-stranded-DNA-dependent ATP-dependent helicase that is active in DNA repair. The complex may be involved in the repair of nonhomologous DNA ends, such as that required for double-strand break repair, transposition, and V(D)J recombination (33, 35).

Ribosomal protein P0 was also identified in the proteomics screen. This protein, along with other ribosomal binding proteins, interacts with rRNA and aids in the assembly of the ribosome. This protein is the primary component that comprises the ribosomal stalk structure. P0 appears to be a cytoplasmic as well as nuclear-localized protein (38) and, in Drosophila, has an apurinic/apyrimidinic endonuclease activity (44).

Proteins associated with the cytoskeleton, for example, actin-like protein 2, actin-related protein 2/3 complex subunit 2, annexin A2, and vimentin, were also identified. These proteins could aid in the migration of UL84 from the cytoplasm to the nucleus and back. These interactions could also be significant and warrant further attention.

Taken together, the data shown here reveal several novel interaction partners that comprise the UL84 interactome/reactome. These identified interactions set the groundwork for a new direction in the study of HCMV lytic DNA replication. The present study establishes that UL84, the key component in initiation and regulation of lytic DNA replication, acts by interacting with various factors that may each or in combination facilitate its role in virus propagation.

 
This work was supported by NIH PHS AI45096. The proteomics analysis was performed by the Nevada Proteomics Center at the University of Nevada and is supported by P20 RR-016464 from the INBRE Program of the National Center for Research Resources.

We thank David Litchfield for the CKII expression plasmids, Janet Hearing and Peter O'Hare for the p32 antibody and expression plasmids, and William Britt for the UL44 antibody.

 
* Corresponding author. Mailing address: University of Nevada—Reno, Department of Microbiology, Howard Bldg. 210, Reno, NV 89557. Phone: (775) 784-4824. Fax: (775) 327-2332. E-mail: gpari{at}medicine.nevada.edu

Published ahead of print on 24 October 2007.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 

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Journal of Virology, January 2008, p. 96-104, Vol. 82, No. 1
0022-538X/08/$08.00+0     doi:10.1128/JVI.01559-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

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