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. 2014 Aug;13(8):2132-46.
doi: 10.1074/mcp.M113.035782. Epub 2014 Jun 26.

Proteomic analysis of the multimeric nuclear egress complex of human cytomegalovirus

Jens Milbradt  1 , Alexandra Kraut  2 , Corina Hutterer  1 , Eric Sonntag  1 , Cathrin Schmeiser  1 , Myriam Ferro  2 , Sabrina Wagner  1 , Tihana Lenac  3 , Claudia Claus  4 , Sandra Pinkert  5 , Stuart T Hamilton  6 , William D Rawlinson  6 , Heinrich Sticht  7 , Yohann Couté  8 , Manfred Marschall  9
Affiliations

Proteomic analysis of the multimeric nuclear egress complex of human cytomegalovirus

Jens Milbradt et al. Mol Cell Proteomics. 2014 Aug.

Abstract

Herpesviral capsids are assembled in the host cell nucleus before being translocated into the cytoplasm for further maturation. The crossing of the nuclear envelope represents a major event that requires the formation of the nuclear egress complex (NEC). Previous studies demonstrated that human cytomegalovirus (HCMV) proteins pUL50 and pUL53, as well as their homologs in all members of Herpesviridae, interact with each other at the nuclear envelope and form the heterodimeric core of the NEC. In order to characterize further the viral and cellular protein content of the multimeric NEC, the native complex was isolated from HCMV-infected human primary fibroblasts at various time points and analyzed using quantitative proteomics. Previously postulated components of the HCMV-specific NEC, as well as novel potential NEC-associated proteins such as emerin, were identified. In this regard, interaction and colocalization between emerin and pUL50 were confirmed by coimmunoprecipitation and confocal microscopy analyses, respectively. A functional validation of viral and cellular NEC constituents was achieved through siRNA-mediated knockdown experiments. The important role of emerin in NEC functionality was demonstrated by a reduction of viral replication when emerin expression was down-regulated. Moreover, under such conditions, reduced production of viral proteins and deregulation of viral late cytoplasmic maturation were observed. Combined, these data prove the functional importance of emerin as an NEC component, associated with pUL50, pUL53, pUL97, p32/gC1qR, and further regulatory proteins. Summarized, our findings provide the first proteomics-based characterization and functional validation of the HCMV-specific multimeric NEC.

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Figures

Fig. 1.
Fig. 1.
Essential role of the E56 and Y57 residues of pUL50 in HCMV core NEC assembly. HFFs were transfected with recombinant HCMV BACmid DNA carrying the mutations indicated and expressing GFP as a marker of virus-positive cells. A, 10 to 15 days post-transfection, cells were fixed and used for confocal immunofluorescence analysis to detect the recruitment of core NEC pUL50–pUL53 to the nuclear rim (pAb-HA for the detection of pUL50-HA, mAb-UL53 for pUL53). Note the lack of both rim recruitment and colocalization of pUL50 and pUL53 with pUL50 mutations E56A and E56A/Y57A. DAPI, 4′,6-diamidino-2-phenylindole; scale bars, 10 μm. B, number of GFP-positive cells per well of a six-well plate transfected with mutant BACmids at different days post-transfection. -, none; +, >10; ++, 10–100; +++, 100–1000; ++++, >1000.
Fig. 2.
Fig. 2.
Schematic depiction of the purification and proteomic analysis of the multimeric NEC from HCMV-infected HFFs. A, core NEC formation in HCMV-infected HFFs. HFFs were infected with HCMV-GFP UL50-HA, fixed at 7 dpi, and stained with pAb-HA and pAb-UL53 as indicated. Scale bar, 10 μm. B, proteomics workflow for purification and analysis of the native NEC using recombinant HCMVs carrying tagged versions of pUL50 and pUL53. C–E, quality controls. CoIP samples of the pUL50–pUL53 core NEC were analyzed via Wb analysis using detection antibodies pAb-UL53 and pAb-UL50 (C). Differences in the precipitated proteomes of specific NEC samples relative to controls were examined by means of Coomassie Blue (D) and silver staining (E) of SDS-PAGE gels. Quality control samples are only depicted for NEC sample 1 and its direct control c1, but Wb analysis and Coomassie Blue and silver stainings were conducted similarly for all other NEC and control samples.
Fig. 3.
Fig. 3.
Specific interaction between endogenous emerin and pUL50 in transfected and HCMV-infected cells. A–F, FLAG-/HA-tagged HCMV proteins, pUL50 deletion mutants, or pcDNA3.1 (vector) were transiently expressed in 293T cells as indicated. Cells were lysed at 2 days post-transfection, and this was followed by immunoprecipitation of the FLAG-tagged viral proteins and the HA-tagged pUL50 deletion mutants using mouse mAb-FLAG (A) and rabbit pAb-HA (C and E), respectively. A mouse Fab fragment was used as a control for specificity (C and E, lane 1). Coimmunoprecipitates and expression control samples were subjected to Wb analysis using tag-specific antibodies or mAb-emerin. G, H, HFFs were infected with HCMV strain AD169 at an MOI of 0.1 or remained uninfected (mock). At 3 dpi, cells were lysed and used for CoIP analysis with mAb-emerin. Detection of coimmunoprecipitates and of expression controls was performed by means of Western blotting using mAb-UL50, mAb-emerin, and mAb-β-actin. Ig-HC, cross-reactive band for immunoglobulin heavy chain.
Fig. 4.
Fig. 4.
Intracellular localization of NEC proteins in HCMV-infected primary fibroblasts. HFFs were infected with recombinant HCMV-GFP pUL50-HA (panels a–e and l–p) or recombinant HCMV UL32-GFP (panels v–z) or remained uninfected (mock; panels f–k and q–u). At 7 dpi (panels a–u) or 3 dpi (panels v–z), cells were fixed and coimmunostained with the indicated antibodies. Arrows point to yellow dots of merged signals of antibody staining indicating a partial colocalization of the respective proteins. Scale bars, 7.5 μm.
Fig. 5.
Fig. 5.
Knockdown analysis with HCMV-infected primary fibroblasts to verify the functional role of the established NEC proteins pUL50, pUL97, and p32/gC1qR. HFFs were transfected with synthetic siRNAs directed to the viral UL97 or UL50 genes (A) or transduced with adenoviral knockdown particles expressing siRNA directed to the cellular p32/gC1qR or a scrambled sequence as a control (B, C). In each case, transfected/transduced cells were additionally infected with the reporter virus HCMV-GFP. At 7 days (A) or 6 days (B, C) post-HCMV infection, the production of viral proteins was monitored by means of Wb analysis (A, B) and the formation of GFP-positive viral plaques was determined via wide-field microscopy (C). For A, transfection of siRNA targeting HCMV pUL54 strictly inhibiting HCMV replication was used as a positive control (39). For C, Student's t test was used to calculate statistical significance (a mean of four values with standard deviations is depicted for each sample).
Fig. 6.
Fig. 6.
Effect of emerin knockdown on HCMV replication, viral protein expression, and viral late maturation. HFFs were transiently transfected with emerin-specific siRNAs or with a scrambled siRNA as a control. In addition, transfected cells were infected with the reporter virus HCMV-GFP (A), HCMV strain AD169 (B), or recombinant HCMV UL32-GFP (C–E) as indicated. At 7 dpi, the production of viral proteins was monitored by means of Wb analysis (A), and HCMV replication efficiency was determined via automated GFP fluorometry (B). For A, Student's t test was used to calculate statistical significance (samples were performed in quadruplicate, and GFP signals were measured in duplicate; a mean of eight values and standard deviations are depicted). C–E, at 5 dpi, cells were fixed and immunostained with mAb-emerin. Representative confocal images (C) or examples of corresponding z-series with higher magnification (D) illustrate emerin immunostaining (red) and the distribution of GFP-labeled viral capsids (green) under emerin knockdown conditions or in control cells as indicated. Solid arrows, distribution of viral capsids in a cVAC-like fashion in emerin-expressing cells; open arrows, distribution of viral capsids throughout the cytoplasm of cells with no detectable emerin staining; solid triangle, distribution of viral capsids in a cVAC-like fashion in cells with slightly reduced levels of emerin staining; dashed lines/arrowheads, optical sections through the z stack (xy) or the focal plane (xz and yz); dotted line, nuclear margin deduced from the z-series of the corresponding DAPI images. E, quantitation of cVAC formation in emerin knockdown cells relative to control cells. The percentage of infected cells, showing GFP-labeled viral capsids in cVAC-like fashion, was determined by scoring 15 microscopic fields (>330 cells) for each setting. Student's t test was used to calculate statistical significance.
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