Interaction network of proteins associated with human cytomegalovirus IE2-p86 protein during infection: a proteomic analysis

Abstract

Human cytomegalovirus protein IE2-p86 exerts its functions through interaction with other viral and cellular proteins. To further delineate its protein interaction network, we generated a recombinant virus expressing SG-tagged IE2-p86 and used tandem affinity purification coupled with mass spectrometry. A total of 9 viral proteins and 75 cellular proteins were found to associate with IE2-p86 protein during the first 48 hours of infection. The protein profile at 8, 24, and 48 h post infection revealed that UL84 tightly associated with IE2-p86, and more viral and cellular proteins came into association with IE2-p86 with the progression of virus infection. A computational analysis of the protein-protein interaction network indicated that all of the 9 viral proteins and most of the cellular proteins identified in the study are interconnected to varying degrees. Of the cellular proteins that were confirmed to associate with IE2-p86 by immunoprecipitation, C1QBP was further shown to be upregulated by HCMV infection and colocalized with IE2-p86, UL84 and UL44 in the virus replication compartment of the nucleus. The IE2-p86 interactome network demonstrated the temporal development of stable and abundant protein complexes that associate with IE2-p86 and provided a framework to benefit future studies of various protein complexes during HCMV infection.

Figure 1. Recombinant HCMVs expressing IE2-p86 protein with a Carboxyl-terminal SG-tag.

(A) At the amino terminus (N) of the tag is the streptavidin binding peptide (SBP) followed by the Tobacco Etch Virus (TEV) protease cleavage site and at the carboxyl terminus (C) is the protein G peptide (ProtG) that binds to IgG. (B) Schematic representation of the HCMV MIE gene locus downstream of the MIE promoter (MIEP) of wt Towne BAC. During the first recombination step, the Rspl-Neo cassette was inserted at the 3′ end of MIE Exon 5 to render DY380 E. coli kanamycin resistant and streptomycin sensitive. The Rspl-Neo was replaced by the SG-tag during the second recombination step as described in the Materials and Methods. (C) Recombinant BAC DNAs digested with restriction endonucleases EcoRI or HindIII and compared to wt HCMV BAC DNA after agarose gel electrophoresis.

Figure 2. Growth property of HCMV recombinant virus IE2-p86SG.

Expression of viral proteins in HFF cells infected with either wt Towne or IE2SG (A and B). HFF cells were infected at high (1 PFU/cell) or low (0.1 PFU/cell) MOI and analyzed by Western blot analysis. MIE viral proteins IE1-p72, IE2-p86, IE2-p86SG, early viral proteins UL84 and UL44, early/late viral proteins UL83 and late viral protein UL99, and cellular protein GAPDH were detected using specific antibodies as described in the Materials and Methods. (C and D) Growth curves of wt HCMV BAC Towne and Towne-IE2SG after high (1 PFU/cell) or low (0.1 PFU/cell) MOI. Cell culture supernatants were harvested at various d p.i. for viral titers via a GFP-based plaque assay. The standard deviations indicated for each data point on the graphs were derived from three independent experiments.

Figure 3. TAP of protein complexes associated with IE2-p86SG.

1×10⁸ HFF cells were infected in parallel with wt HCMV Towne or IE2SG at an MOI of 2 PFU/cell, and harvested at 8 (A), 24 (B), and 48 (C) h p.i. TAP with IgG sepharose resin and Strepavidin Sepharose resin was as described in the Materials and Methods. One tenth of the purified eluate was fractionated by SDS-PAGE, and visualized by silver staining. IE2-SBP indicates the tagged IE2-p86 protein with the IgG binding moiety removed by TEV protease. The position of viral protein UL84 and cellular protein C1QBP in the gel are also designated. The protein standard (Std) represents approximately 50 ng of protein per band. The asterisk marks the sole protein band (beta-actin) in the Towne wt sample that is visible on the silver staining gel.

Figure 4. An interaction network of IE2-p86 associated proteins identified in HCMV IE2SG- infected HFF cells.

To map the landscape of the IE2-p86 protein interactions, in silico networking analysis of identified proteins in both Table 2 and 3 using STRING database combined with published experimental data were schematically presented by Cytoscape software (version 2.8.3). Solid lines show physical protein–protein interactions based on experimental evidence from published reports and STRING search results. Dashed lines indicate the predicted protein–protein association by STRING database using predictive methods, such as textmining, coexpression, neighborhood, and co-occurance . To simplify the map, only representative ribosomal proteins were shown.

Figure 5. Association of cellular proteins with viral proteins IE2-p86, UL84 and UL44 in co-immunoprecipitation.

HFF cells were infected with wt Towne at an MOI of 2/cell, harvested at 48 h p.i. for immunoprecipitation with specific antibodies against C1QBP (A, B and C), IE2-p86 (A and B), UL84 (C), UL44 (C), or isotype antibody control (ctrlAb). The precipitates were fractionated by 10% SDS-PAGE. Proteins were detected by Western blot with indicated specific antibodies (PTRF, NPM1, YXB1, and C1QBP) as described in the Materials and Methods. Since the heavy chain of the antibody used in co-immunoprecipitation migrates to the same location as UL44, and overshadows the signal of UL44, the protein UL84 was examined in anti-UL44 precipitates (C).

Figure 6. HCMV infection up-regulates the expression of cellular protein C1QBP, which associates with the IE2-p86/UL84/UL44 complex in virus replication compartments.

(A) HFF cells, infected with wt HCMV (V) at an MOI of 2 PFU/cell and were harvested at the indicated time-points for western blot analysis of proteins IE1-p72, IE2-p86, UL84, C1QBP, and GAPDH by using specific antibodies described in the Materials and Methods. Cellular protein GAPDH serves as sample loading control (C). (B) Cytoplasmic and nuclear fractions of mock-infected or HCMV-infected cells were analyzed for C1QBP and IE2-p86. Lamin A and GAPDH were used as makers for the nuclear (Nu) and cytoplasm (Cy) fractions, respectively. (C and D) Subcellular localization of cellular C1QBP and viral UL84, IE2-p86, and UL44 proteins in HFF cells infected with wt HCMV at MOI of 2 PFU/cell for 48 h. Uninfected cells and infected cells were fixed before (C, whole-cell view) or after (D, nuclear view) permeabilization with 0.3% Triton X-100 and immunostained with specific antibodies against C1QBP, IE2-p86, UL84, or UL44 as described in the Materials and Methods. Cellular DNA was stained by TO-PRO. The 63× objective lense was used. Pearson's correlation (R) for colocalizaion of fluorescent signals was determined for indicated images by Image J software (ver 1.46) and are shown on the right of each panel.