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. 2010 Jul;84(14):7005-17.
doi: 10.1128/JVI.00719-10. Epub 2010 May 19.

Role of the endoplasmic reticulum chaperone BiP, SUN domain proteins, and dynein in altering nuclear morphology during human cytomegalovirus infection

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Role of the endoplasmic reticulum chaperone BiP, SUN domain proteins, and dynein in altering nuclear morphology during human cytomegalovirus infection

Nicholas J Buchkovich et al. J Virol. 2010 Jul.

Abstract

The process of assembly and egress of human cytomegalovirus (HCMV) virions requires significant morphological alterations of the nuclear and cytoplasmic architecture. In the studies presented we show that the nuclear periphery is dramatically altered, especially near the cytoplasmic assembly compartment, where the nuclear lamina is specifically rearranged, the outer nuclear membrane is altered, and the nucleus becomes permeable to large molecules. In addition, the tethering of the inner and outer nuclear membranes is lost during infection due to a decrease in levels of the SUN domain proteins. We previously demonstrated that the endoplasmic reticulum protein BiP functions as a component of the assembly compartment and disruption of BiP causes the loss of assembly compartment integrity. In this study we show that the depletion of BiP, and the loss of assembly compartment integrity, results in the loss of virally induced lamina rearrangement and morphology of the nucleus that is characteristic of HCMV infection. BiP functions in lamina rearrangement through its ability to affect lamin phosphorylation. Depletion of BiP and disruption of the assembly compartment result in the loss of lamin phosphorylation. The dependency of lamin phosphorylation on BiP correlates with an interaction between BiP and UL50. Finally, we confirm previous data (S. V. Indran, M. E. Ballestas, and W. J. Britt, J. Virol. 84:3162-3177, 2010) suggesting an involvement of dynein in assembly compartment formation and extend this observation by showing that when dynein is inhibited, the nuclear morphology characteristic of an HCMV infection is lost. Our data suggest a highly integrated assembly-egress continuum.

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Figures

FIG. 1.
FIG. 1.
Models of the nuclear periphery in normal human fibroblasts (A) and HCMV-infected human fibroblasts (B). See the introduction for details.
FIG. 2.
FIG. 2.
Electron micrographs of mock- or HCMV-infected human fibroblasts. (A) A mock-infected cell highlighting the nuclear periphery. (B) An infected cell (96 hpi) highlighting the thin nuclear membrane (NM; arrow) adjacent to the assembly compartment (AC). (C) A mock-infected cell highlighting the inner and outer nuclear membranes (INM and ONM, respectively). (D) An infected cell (96 hpi) highlighting the INM and ONM, showing disruptions in the ONM, e.g., between the two thin arrows. (E) An infected cell (96 hpi) showing the irregularity of the separation between the inner and outer nuclear membranes, indicative of the loss of SUN domain proteins.
FIG. 3.
FIG. 3.
The distance between the inner and outer nuclear membranes is increased in infected cells. Mock- or HCMV-infected HFFs were prepared for EM analysis at 96 hpi. The two HCMV samples are from similar regions of two different infected nuclei. Thirty measurements of the perinuclear space were digitally taken on the micrographs shown using ImagePro 6.3 software.
FIG. 4.
FIG. 4.
The levels of SUN proteins are reduced during HCMV infection. (A and B) Proteins were harvested from mock- and HCMV-infected life-extended (LE) HFFs at the indicated hours postinfection (hpi) and assessed by Western analysis using antibodies that detect SUN1 (A) and SUN2 (B). Actin was used as a loading control. (C) A nucleocapsid in the perinuclear space causing distension of the outer nuclear membrane (white arrow).
FIG. 5.
FIG. 5.
Nuclear lamina rearrangement is disrupted when BiP is depleted. (A) Mock- or HCMV-infected human fibroblasts were treated with SubAB (Toxin) or SubAA272B (Mut.Toxin) or left untreated (No Toxin) at 84 hpi and prepared for immunofluorescence analysis at 96 hpi by staining with antibodies that detect pp28 (green) and lamin B (red). (B) HCMV-infected human fibroblasts were stained as in panel A at 96 hpi and examined by confocal microscopy. Maximum projections of a z-axis series are shown; the far right panel was enlarged slightly and rotated as indicated by the white arrow.
FIG. 6.
FIG. 6.
Depletion of BiP alters nuclear lamin phosphorylation. (A) Proteins were harvested from mock- and HCMV-infected life-extended (LE) HFFs at the indicated times (hours) postinfection and analyzed by Western blot analysis using antibodies that detect lamins A and C phosphorylated at serine 22 (P-lam A and P-lam C) and total lamins A and C. Actin was used as a loading control. (B) Proteins were harvested from mock- and HCMV-infected samples treated with SubAB (Toxin) or NGIC-1 and analyzed by Western analysis using antibodies that detect lamins A and C phosphorylated at serine 22 (P-lam A/C), total lamins A and C, BiP, and pUL50. Antibodies directed against the major immediate-early proteins (MIEP); an early protein, p52; and a late protein, pp28, were used to monitor viral protein expression. Actin was used as a loading control. The asterisks indicate that, for the 96-hpi pUL50 and MIEP samples, exposures are shown that are less than that for the corresponding 24-hpi samples; this is because equal exposure time resulted in uninterpretable overexposure for these samples. (C) Immunofluorescence analysis of BiP after mock infection and at 24 and 96 hpi.
FIG. 7.
FIG. 7.
BiP interacts with nuclear egress factor pUL50. (A) Proteins were harvested from mock- and HCMV-infected human fibroblasts at the indicated times (hours) postinfection and analyzed by Western analysis using the anti-UL50 antibody described in the text. Actin was used as a loading control. (B) Mock (M)- and HCMV-infected lysates, harvested at 96 hpi, were subjected to immunoprecipitation (IP) using antibodies that detect BiP or UL50. The precipitates were assessed by Western (W) analysis using anti-BiP and anti-UL50. Input represents 20% of protein lysates used for IP-Western analyses. (C) Western analysis of BiP in immunoprecipitates of gB and pp65.
FIG. 8.
FIG. 8.
High-molecular-weight dextran permeates infected cell nuclei. (A and B) Mock- (A) and HCMV-infected (B) human fibroblasts were loaded with dextran-TRITC (green) at 48 hpi as described in Materials and Methods. Cells were examined for the presence of nuclear dextran at 72 (B, right two panels) and 96 hpi (B, left two panels). The position of the assembly compartment (AC) is indicated in the lower left panel of B. (C) Using the cell in the lower left panel of B, a z-axis series was generated, and slices through the cell from top to bottom are shown. The DAPI staining of the nucleus is included (blue).
FIG. 9.
FIG. 9.
Inhibition of dynein causes disruption of assembly compartment formation and loss of HCMV-mediated nuclear shape alterations. Human fibroblasts were infected for 24 h and then electroporated with a plasmid that expresses CC1-mCherry. The cells were plated on coverslips and incubated for an additional 48 h (72 hpi) and then prepared for immunofluorescence. CC1-expressing cells are indicated by the mCherry (red). Anti-pp28 was used to stain infected cells and indicate the status of the assembly compartments (green or yellow where it overlaps CC1). The shape and size of the nuclei were visualized with DAPI. The arrows show an uninfected cell expressing CC1 (CC1 Uninfected), an infected cell expressing CC1 (CC1 Infected), and an infected cell not expressing CC1 (Infected).
FIG. 10.
FIG. 10.
The assembly-egress continuum: a model of HCMV-mediated nuclear restructuring and assembly compartment formation/function as it relates to nucleocapsid egress and virion formation. The model is based on the data presented, previous models (12), and the Discussion. Refer also to Fig. 1, and see the text for details.

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References

    1. Azzeh, M., A. Honigman, A. Taraboulos, A. Rouvinski, and D. G. Wolf. 2006. Structural changes in human cytomegalovirus cytoplasmic assembly sites in the absence of UL97 kinase activity. Virology 354:69-79. - PubMed
    1. Beaudouin, J., D. Gerlich, N. Daigle, R. Eils, and J. Ellenberg. 2002. Nuclear envelope breakdown proceeds by microtubule-induced tearing of the lamina. Cell 108:83-96. - PubMed
    1. Beck, K. A. 2005. Spectrins and the Golgi. Biochim. Biophys. Acta 1744:374-382. - PubMed
    1. Bresnahan, W. A., G. E. Hultman, and T. Shenk. 2000. Replication of wild type and mutant human cytomegalovirus in life-extended human diploid fibroblasts. J. Virol. 74:10816-10818. - PMC - PubMed
    1. Buchkovich, N. J., T. G. Maguire, A. W. Paton, J. C. Paton, and J. C. Alwine. 2009. The endoplasmic reticulum chaperone BiP/GRP78 is important in the structure and function of the HCMV assembly compartment. J. Virol. 83:11421-11428. - PMC - PubMed

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