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. 2010 Sep;91(Pt 9):2167-75.
doi: 10.1099/vir.0.022640-0. Epub 2010 May 5.

Association of human cytomegalovirus proteins IRS1 and TRS1 with the viral DNA polymerase accessory subunit UL44

Blair L Strang  1 , Adam P Geballe, Donald M Coen
Affiliations

Association of human cytomegalovirus proteins IRS1 and TRS1 with the viral DNA polymerase accessory subunit UL44

Blair L Strang et al. J Gen Virol. 2010 Sep.

Abstract

Multiple proteins interacting with DNA polymerases orchestrate DNA replication. Human cytomegalovirus (HCMV) encodes a DNA polymerase that includes the presumptive processivity factor UL44. UL44 is structurally homologous to the eukaryotic DNA polymerase processivity factor proliferating cell nuclear antigen (PCNA), which interacts with numerous proteins. Previous proteomic analysis has identified the HCMV protein IRS1 as a candidate protein interacting with UL44. Nuclease-resistant reciprocal co-immunoprecipitation of UL44 with IRS1 and with TRS1, which has an amino terminus identical to that of IRS1, was observed from lysate of cells infected with viruses expressing epitope-tagged UL44, epitope-tagged IRS1 or epitope-tagged TRS1. Western blotting of protein immunoprecipitated from infected cell lysate indicated that epitope-tagged IRS1 and TRS1 do not associate simultaneously with UL44. Glutathione S-transferase pull-down experiments indicated that IRS1 and TRS1 interact with UL44 via a region that is identical in both proteins. Taken together, these data suggest that IRS1 and TRS1 may compete for association with UL44 and may affect UL44 function differentially.

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Figures

Fig. 1.
Fig. 1.
Positions of IRS1, IRS1263 and TRS1 open reading frames in the HCMV genome. The diagram at the top of the figure represents the entire HCMV genome. The terminal and internal repeat regions of the long unique (UL) segment of the genome (TRL and IRL, respectively) are represented as black boxes. The terminal and internal repeat regions of the short unique (US) segment of the genome (TRS and IRS, respectively) are represented as grey boxes. Dashed lines from the TRS and IRS regions of US lead to expanded areas showing the relative positions of the IRS1, IRS1263 and TRS1 coding sequences in the HCMV genome. Grey lines represent genome sequences in repeat regions; black lines represent genome sequences in the US segment. Arrows show the position and direction of transcription of the coding sequences. IRS1263 is the product of an alternative IRS1 transcript encoding the carboxyl terminus of IRS1 (aa 583–846) (Romanowski & Shenk, 1997).
Fig. 2.
Fig. 2.
Detection of protein immunoprecipitated from FLAG–44-infected cell lysate by Western blotting. Lysate (Lys.) from AD169rv-infected (lane 1) and FLAG–44-infected (lane 2) cells and protein immunoprecipitated (IP) using an anti-FLAG antibody from those lysates (lanes 3 and 4, respectively) were separated on a 10 % polyacrylamide gel. Proteins in each lane were examined by Western blotting for the presence of UL44, FLAG, IRS1, TRS1 and β-actin using antibodies recognizing these proteins, as indicated to the right of the figure. The positions of IRS1 and TRS1 are indicated by arrows. The positions of molecular mass markers (in kDa) are indicated to the left of the figure.
Fig. 3.
Fig. 3.
Characterization of recombinant virus. (a) Replication of AD169rv (•), IRSF (▪) and TRSF (▴) viruses. HFF cells were infected at an m.o.i. of 1 and virus supernatant was harvested at the indicated time points. Virus titre is represented as p.f.u. ml−1 on HFF cells. Data are representative of two experiments. (b) Western blotting of infected cells. Cell lysates of uninfected HFF cells (lane 1) or HFF cells infected at an m.o.i. of 1 with AD169rv (lane 2), IRSF (lane 3) or TRSF (lane 4) viruses were prepared 72 h p.i. Proteins in each lane were examined by Western blotting for the presence of FLAG-tagged IRS1 and FLAG-tagged TRS1 (top panel), IRS1 and TRS1 (middle panel) and β-actin (bottom panel) using antibodies recognizing these proteins or FLAG, as indicated to the right of the figure. Note that, in the middle panel, a combination of antibodies recognizing IRS1 or TRS1 was utilized and that FLAG-tagged versions of each protein migrate more slowly than wild-type proteins. The positions of molecular mass markers (in kDa) are indicated to the left of the figure.
Fig. 4.
Fig. 4.
Detection of protein immunoprecipitated from IRSF- and TRSF-infected cell lysate by Western blotting. (a, c) Proteins immunoprecipitated (IP) from lysate of AD169rv- and IRSF-infected cells (a) or AD169rv- and TRSF-infected cells (c) prepared 72 h p.i. were separated on a 10 % polyacrylamide gel and examined by Western blotting using antibodies recognizing FLAG (top panels) or UL44 (bottom panels). Immunoprecipitated proteins are examined in lanes 1–4. Lysates precleared with control antibody before IP (IRSF/Ig or TRSF/Ig) are shown in lane 3 of both figures. Lysates treated with Benzonase (IRS+B or TRS+B) are shown in lane 4 of both figures. Lysates (Lys.) used in the IP are examined in lanes 5 and 6. The positions of molecular mass markers (in kDa) are indicated to the left of the figure. The novel IRS1 band discussed in the text is marked with an asterisk. (b, d) Cell lysate used in the IP in lanes 2 and 4 of (a) and (c) was run out on an ethidium bromide-stained 0.8 % agarose gel [(b) and (d), respectively]. Lanes: 1, no sample; 2, IP in the absence of Benzonase (Ben.); 3, IP in the presence of Ben.
Fig. 5.
Fig. 5.
Detection by Western blotting of IRS1 and TRS1 in protein immunoprecipitated from IRSF- and TRSF-infected cell lysate. Lysate from AD169rv-infected (lane 4), IRSF-infected (lane 5) and TRSF-infected (lane 6) cells and protein immunoprecipitated using an anti-FLAG antibody from those lysates (lanes 1–3, respectively) were separated on a 10 % polyacrylamide gel. Proteins in each lane were examined by Western blotting for the presence of FLAG-tagged protein (top panel), IRS1 or TRS1 (middle panel) or UL44 (bottom panel) using antibodies recognizing these proteins, as indicated to the right of the figure. Note that, in the middle panel, a combination of antibodies recognizing IRS1 or TRS1 was utilized. The positions of molecular mass markers (in kDa) are indicated to the left of the figure.
Fig. 6.
Fig. 6.
Binding of IRS1 and TRS1 mutants to UL44 in vitro. (a) Schematic of IRS1 and TRS1 proteins used. Full-length (FL) IRS1 and TRS1 proteins are shown, as are mutants of these proteins. Each mutation is noted to the left of the figure. The position of certain amino acids in each protein in the diagram above both IRS1 and TRS1 proteins is noted. Black regions represent the amino terminus common to IRS1 and TRS1; white and grey regions represent the unique carboxyl termini of IRS1 and TRS1, respectively. (b) GST or GST–UL44ΔC290 was incubated with radiolabelled IRS1 or TRS1 proteins and passed over a glutathione column. The GST protein used in each reaction is noted below the figure; the radiolabelled protein used in each reaction is noted above the figure. The top panel of the figure shows the proteins used in each reaction (Input) and the bottom panel shows the proteins eluted from the glutathione column (Elution). The positions of molecular mass markers (in kDa) are indicated to the left of each figure.
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