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. 2010 Sep;84(17):8409-21.
doi: 10.1128/JVI.00189-10. Epub 2010 Jun 10.

Role of the specific interaction of UL112-113 p84 with UL44 DNA polymerase processivity factor in promoting DNA replication of human cytomegalovirus

Young-Eui Kim  1 , Jin-Hyun Ahn
Affiliations

Role of the specific interaction of UL112-113 p84 with UL44 DNA polymerase processivity factor in promoting DNA replication of human cytomegalovirus

Young-Eui Kim et al. J Virol. 2010 Sep.

Abstract

The human cytomegalovirus (HCMV) UL112-113 region encodes four phosphoproteins with common amino termini (p34, p43, p50, and p84) via alternative splicing and is thought to be required for efficient viral DNA replication. We have previously shown that interactions among the four UL112-113 proteins regulate their intranuclear targeting and enable the recruitment of the UL44 DNA polymerase processivity factor to viral prereplication foci. Here, we show that in virus-infected cells, the UL112-113 proteins form a complex with UL44 and other replication proteins, such as UL84 and IE2. In vitro assays showed that all four phosphoproteins interacted with UL44. Interestingly, p84 required both the shared amino-terminal region and the specific near-carboxy-terminal region for UL44 binding. UL44 required both the carboxy-terminal region and the central region, including the dimerization domain for p84 binding. The production of recombinant virus from mutant Towne bacterial artificial chromosome (BAC) DNA, which encodes intact p34, p43, and p50 and a carboxy-terminally truncated p84 defective in UL44 binding, was severely impaired compared to wild-type BAC DNA. A similar defect was observed when mutant BAC DNA encoded a carboxy-terminally truncated UL44 defective in p84 binding. In cotransfection replication assays using six replication core proteins, UL84, IE2, and UL112-113, the efficient replication of an HCMV oriLyt-containing plasmid required the regions of p84 and UL44 necessary for their interaction. Our data suggest that the UL112-113 proteins form a complex with other replication proteins such as UL44, UL84, and IE2 and that the specific interaction of UL112-113 p84 with UL44 is necessary for efficient viral DNA replication.

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Figures

FIG. 1.
FIG. 1.
CoIP assays demonstrating the formation of a complex containing UL112-113, UL44, UL84, and IE2 in virus-infected cells. (A to D) HF cells were infected with HCMV (Towne) at an MOI of 2.0. At 48 h, total cell lysates were prepared and immunoprecipitated with anti-UL44 (αUL44) (A), anti-UL112-113 (M23) (B), anti-UL84 (C), and anti-IE2 (D) Abs. Immunoprecipitation with mouse IgG was used as a negative control. The immunoprecipitated samples were subjected to SDS-PAGE, followed by immunoblotting with Abs specific for UL112-113 p84, UL84, IE1/IE2, and UL44. Total cell lysates were also subjected to SDS-PAGE, and immunoblot analysis was performed to confirm the protein expression levels. (E) Immunoprecipitation was performed, as described above (A), with total cell lysate untreated or treated with 100 U/ml DNase (Roche) plus 10 μg/ml RNase (Sigma) at 4°C for 12 h. To confirm the removal of nucleic acids, DNA was recovered from total cell lysates by phenol and chloroform extraction and then analyzed on a 1% agarose gel containing ethidium bromide (EtBr).
FIG. 2.
FIG. 2.
Specific interaction of UL112-113 p84 with UL44 among the six replication core proteins. (A) X-Gal filter assays of yeast cells expressing both the GAL4-DB/UL112-113 (p34, p43, p50, and p84) fusion proteins and the GAL4-A/replication core (UL44, UL54, UL57, UL70, UL102, and UL105) fusion proteins. The cells expressing the GAL4-A proteins alone were used as a control. Green indicates a positive interaction. (B) 293T cells were cotransfected with plasmids encoding HA-tagged UL112-113 proteins and Myc-tagged replication core proteins (UL44, UL54, UL57, UL70, UL102, or UL105). (Top) At 48 h, total cell lysates were prepared and immunoprecipitated with anti-Myc Ab, followed by immunoblotting with anti-HA Ab. (Middle and bottom) Total cell lysates were also immunoblotted with anti-Myc or anti-HA Abs. (C) HF cells were infected with HCMV Towne at an MOI of 2.0. Total cell lysates were prepared 2 days postinfection and immunoprecipitated with M23 Ab specific for the UL112-113 proteins, followed by immunoblotting with anti-PCNA or anti-p84 Abs. Total cell lysates were also immunoblotted with anti-p84 or anti-PCNA Abs to show the protein expression levels.
FIG. 3.
FIG. 3.
Interaction of the UL112-113 proteins with UL44 in vitro. (A) Structures of the p34, p43, p50, and p84 proteins and the ΔΝ252 and ΔC347 mutants. The ATG start codons of the cDNA are indicated. The 35S-labeled p34, p43, p50, and p84 proteins and ΔN25 and ΔC347 mutants were synthesized individually by in vitro transcription/translation reactions as described in Materials and Methods. (B and C) In vitro binding assays of the in vitro-translated UL112-113 proteins with bacterially produced GST or GST-UL44. One-sixth of the GST or GST-UL44 proteins used in each reaction was visualized with Coomassie blue staining (B). The GST or GST-UL44 proteins immobilized to glutathione-Sepharose beads were incubated with 35S-labeled UL112-113 proteins (p34, p43, p50, p84, ΔN252, or ΔC347). One-tenth of the labeled UL112-113 proteins used in each binding reaction was loaded as input controls. The bound proteins were fractionated on an SDS-8% PAGE gel and visualized by autoradiography (C).
FIG. 4.
FIG. 4.
CoIP assays to determine the region of UL112-113 p84 required for UL44 binding. (A) Diagram indicating the splice donor and splice acceptor sites used for the production of p43 from p84 cDNA. A cDNA expressing only p84 was generated by a thymine-to-cytosine substitution at nucleotide 1038 (top). 293T cells were transfected with the indicated plasmids encoding Myc-tagged p43, p84 plus p43 (from the original p84 cDNA), or p84 only. At 48 h, total cell lysates were prepared and subjected to SDS-PAGE, followed by immunoblotting with anti-Myc Ab (bottom). (B) Structures of intact p84 and its truncation mutants that were used. The location of the reported NLS (13, 15, 19) is indicated by open circles. The results (shown in C) are summarized (+++, strong positive interaction; +, positive interaction; −, negative interaction). aa, amino acids. (C) Interaction of UL44 with p84 (wild type or mutants). 293T cells were cotransfected with HA-tagged UL44 and Myc-tagged p84 (wild-type or mutant versions). At 48 h, total cell lysates were prepared and immunoprecipitated with anti-Myc Ab, followed by immunoblotting (IB) with anti-HA Ab (top). The expression levels of each protein are shown by immunoblotting of total cell lysates with anti-HA Ab (middle) or anti-Myc Ab (bottom). (D) Localization patterns of p84 and its mutants. HF cells were transfected with plasmids encoding Myc-tagged wild-type or indicated mutant p84. After 48 h, the cells were fixed with methanol, followed by IFA with anti-Myc Ab and rhodamine/redX-labeled anti-mouse IgG.
FIG. 5.
FIG. 5.
CoIP assays to map the region of UL44 required for dimerization and UL112-113 p84 binding. (A) Interaction of p84 with UL44 (wild type or mutants). 293T cells were cotransfected with the indicated plasmids, and total cell lysates were prepared and immunoprecipitated with anti-Myc Ab. (Top) The precipitated proteins were subjected to SDS-PAGE and immunoblotting with anti-HA Ab. (Middle and bottom) The expression levels of the HA-p84 and Myc-UL44 proteins are shown by immunoblotting with anti-HA Ab (middle) and anti-Myc Ab (bottom). Note that the 6-Myc-UL44(ΔN290) level in total cell lysates is very low compared to those of other proteins. (B) Dimerization of UL44 proteins. 293T cells were cotransfected with the plasmids encoding HA-UL44 or Myc-UL44 (wild type or mutants), as indicated, and CoIP assays were performed as described above (A). (C) Diagram showing the structure of the wild-type and mutant UL44 proteins used and summary of the results in A and B. The abilities of the wild type and UL44 truncation mutants to dimerize with each other and interact with p84 are indicated as + (positive) or − (negative). The positions of the reported two potential NLSs (6) are indicated by open circles. (D) Localization patterns of wild-type and mutant UL44 are shown by IFA as described in the legend of Fig. 4D.
FIG. 6.
FIG. 6.
Colocalization interactions between the UL112-113 and UL44 proteins. (A) HF cells were transfected with a plasmid encoding Flag-UL44 or Flag-UL44(ΔC290) alone or cotransfected with a plasmid carrying the UL112-113 genomic gene, which encodes the p34, p43, p50, and p84 proteins (as HA-tagged forms). After 48 h, the cells were fixed with paraformaldehyde, followed by double-label IFA with anti-Flag (red) and anti-HA (green) Abs. For cotransfected cells, two side-by-side panels of single-labeled IFA images and a third panel of merged images are shown. (B) Quantitation of the relocalization of UL44 by the UL112-113 proteins. HF cells were cotransfected with plasmids encoding wild-type or mutant forms of Flag-UL44 and HA-UL112-113 proteins as indicated. Double-label IFA was performed as described above (A). The percentages of cotransfected cells exhibiting only the nuclear diffuse pattern of UL44 are indicated as gray bars, whereas those displaying both the nuclear diffuse and nuclear focus patterns of UL44, which represents the relocalization of UL44 to the PML-NB-associated sites by the UL112-113 proteins, are indicated as black bars. For each cotransfection, more than 100 cotransfected cells were counted.
FIG. 7.
FIG. 7.
Generation of recombinant T-BAC UL112-113(ΔC560) and UL44(ΔC290) clones. (A) Construction of the mutant T-BAC UL112-113(ΔC560) clone. The C-terminal region of UL113 was replaced by a marker cassette (rpsL-neo) containing the 50-nucleotide homologous arm, which increases sensitivity to streptomycin, and the kanamycin resistance marker. Intermediate BAC clones were isolated based on resistance to kanamycin (see Materials and Methods). In a second round of homologous recombination, the rpsL-neo cassette was replaced by the mutated DNA fragment containing a stop codon at amino acid position 561 of UL112-113 p84. To construct the revertant T-BAC clone, the mutant gene was replaced with the rpsL-neo cassette, and the wild-type T-BAC clone was then rescued by homologous recombination with the wild-type DNA fragment. (B) Construction of the mutant T-BAC UL44(ΔC290) clone. Insertion of a marker cassette and replacement by the mutant or wild-type DNA fragment were performed as described above (A). (C) Restriction fragment DNA patterns obtained following EcoRI/BamHI digestion of the wild-type, mutant, and revertant T-BAC DNAs were analyzed by agarose gel electrophoresis. The sizes of λ-HindIII are shown.
FIG. 8.
FIG. 8.
Infectivity of the mutant T-BAC clones carrying the UL112-113(ΔC560) or UL44(ΔC290) gene. (A) HF cells were transfected with wild-type, UL112-113(ΔC560), or revertant T-BAC clones via electroporation (see Materials and Methods) and monitored for the propagation of green fluorescent protein (GFP) signals. The GFP and phase-contrast images were taken 15 days after electroporation. (B) HF cells were transfected with wild-type, UL44(ΔC290), or revertant T-BAC clones via electroporation, and the images were taken 21 days later. (C) Total RNAs were prepared in cells at 9 days after electroporation, and the amounts of IE1, IE2, UL112-113, and UL44 mRNAs were measured by RT-PCR.
FIG. 9.
FIG. 9.
Requirements of the regions of UL112-113 p84 and UL44 responsible for their interaction for oriLyt-dependent DNA replication. (A) HF cells were cotransfected with plasmids encoding the HCMV replication origin (pSP38), six replication core proteins (UL54, UL44, UL57, UL105, UL70, and UL102), UL84, IE2, or UL112-113 (wild type or mutant), as indicated. At 5 days, total cellular DNAs were isolated and digested with XbaI and DpnI. DNA fragments were separated by electrophoresis, and Southern blot analysis was performed with the 32P-labeled KpnI-digested pSP38 DNA as a probe. (B) HF cells were cotransfected with plasmids encoding the HCMV replication origin (pSP38), five replication core proteins (UL54, UL57, UL105, UL70, and UL102), UL84, IE2, UL112-113, and UL44 (wild type or mutant), as indicated. The replicated oriLyt-containing plasmid DNAs were detected as described above (A).
FIG. 10.
FIG. 10.
Requirements of the interaction between UL112-113 p84 and UL44 for their efficient association with the oriLyt region. HF cells were cotransfected with plasmids containing the HCMV replication origin (pSP38), five replication core proteins (UL54, UL57, UL105, UL70, and UL102), UL84, IE2, Flag-UL44 (wild type or mutant), and HA-UL112-113 (wild type or mutant), as indicated. At 5 days, the cells were harvested and analyzed by ChIP assays using anti-HA, anti-Flag, and anti-IE2 Abs or using a control IgG. The immunoprecipitated oriLyt-containing DNAs were amplified by PCR. The total amounts of input plasmid DNAs were also amplified by PCR.
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