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. 2009 Nov;83(22):11421-8.
doi: 10.1128/JVI.00762-09. Epub 2009 Sep 9.

The endoplasmic reticulum chaperone BiP/GRP78 is important in the structure and function of the human cytomegalovirus assembly compartment

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The endoplasmic reticulum chaperone BiP/GRP78 is important in the structure and function of the human cytomegalovirus assembly compartment

Nicholas J Buchkovich et al. J Virol. 2009 Nov.

Abstract

We previously demonstrated that the endoplasmic reticulum (ER) chaperone BiP functions in human cytomegalovirus (HCMV) assembly and egress. Here, we show that BiP localizes in two cytoplasmic structures in infected cells. Antibodies to the extreme C terminus, which includes BiP's KDEL ER localization sequence, detect BiP in regions of condensed ER near the periphery of the cell. Antibodies to the full length, N terminus, or larger portion of the C terminus detect BiP in the assembly compartment. This inability of C-terminal antibodies to detect BiP in the assembly compartment suggests that BiP's KDEL sequence is occluded in the assembly compartment. Depletion of BiP causes the condensed ER and assembly compartments to dissociate, indicating that BiP is important for their integrity. BiP and pp28 are in association in the assembly compartment, since antibodies that detect BiP in the assembly compartment coimmunoprecipitate pp28 and vice versa. In addition, BiP and pp28 copurify with other assembly compartment components on sucrose gradients. BiP also coimmunoprecipitates TRS1. Previous data show that cells infected with a TRS1-deficient virus have cytoplasmic and assembly compartment defects like those seen when BiP is depleted. We show that a fraction of TRS1 purifies with the assembly compartment. These findings suggest that BiP and TRS1 share a function in assembly compartment maintenance. In summary, BiP is diverted from the ER to associate with pp28 and TRS1, contributing to the integrity and function of the assembly compartment.

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Figures

FIG. 1.
FIG. 1.
Immunofluorescence analysis of BiP shows that it is localized in two distinct locations during HCMV infection. Mock- or HCMV-infected cells were prepared for immunofluorescence microscopy at 96 hpi. BiP (red) was detected using the following antibodies described in Materials and Methods and Table 1: BiPA (A), BiPH (H), BiPR (R), BiPS (S), BiPY (Y), BiPC (C), and KDEL (K). Normal rabbit IgG (IgG) was used as a control. Nuclei were stained with DAPI (blue).
FIG. 2.
FIG. 2.
BiP colocalizes with assembly compartment markers. (A) Mock- and HCMV-infected cells were prepared for immunofluorescence at 96 hpi and stained for BiPD (red), a mouse monoclonal antibody. (B) HCMV-infected cells were prepared at 96 hpi and stained for BiP (red) using BiPS and BiPR antibodies (Table 1) and either pp28 or gB (green). Colocalization of BiP and pp28 is indicated in yellow. Nuclei shown in panels A and B were stained with DAPI (blue). Comp., composite.
FIG. 3.
FIG. 3.
BiP cosediments with assembly compartment markers in a sucrose gradient. Lysates were prepared from infected cells treated with SubAA272B (mutant toxin) (A) or SubAB (WT toxin) (B) and sedimented on a discontinuous sucrose gradient as previously described (36). The gradient was fractionated from the top (300 μl/fraction) and analyzed by Western analysis. AC, assembly compartment.
FIG. 4.
FIG. 4.
Cleavage of BiP disrupts the assembly compartment. At 60 hpi, HCMV-infected cells were treated with SubAB (Toxin) or SubAA272B (Mutant Toxin) or left untreated (No Toxin). At 72 hpi, the cells were prepared for immunofluorescence microscopy, and assembly compartment integrity was examined by staining for gB and pp28. Nuclei were stained with DAPI (blue).
FIG. 5.
FIG. 5.
The condensed cytoplasmic BiP-containing regions colocalize with ER markers. Samples were prepared as described for Fig. 4 except that they were examined for the ER marker PDI (green) and BiP (red) using the BiPC antibody, which detects BiP in the condensed regions and not in the assembly compartment. Mut Toxin, mutant toxin.
FIG. 6.
FIG. 6.
BiP associates with pp28 and TRS1 during infection. (A) Lysates from mock (M)- and HCMV (V)-infected HFs were prepared at 96 hpi for immunoprecipitation with BiPY, an antibody that detects BiP in the assembly compartment, and the KDEL antibody, which does not detect BiP in the assembly compartment. Following immunoprecipitation, reactions were analyzed by Western blotting using antibodies against BiP, TRS1, and pp28. Western analysis of 20% of the input is also shown (Input). (B and C) Similar lysates from mock- and HCMV-infected cells were immunoprecipitated with anti-pp28, and Western blots were probed with anti-BiP (B) or anti-TRS1 (C). A cross-reaction with light chains by the pp28 antibody is shown (LC) in panels B and C.

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