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. 2016 Feb:489:223-32.
doi: 10.1016/j.virol.2015.12.019. Epub 2016 Jan 13.

Epstein-Barr virus glycoprotein gM can interact with the cellular protein p32 and knockdown of p32 impairs virus

Harish Changotra  1 , Susan M Turk  1 , Antonio Artigues  2 , Nagendra Thakur  1 , Mindy Gore  1 , Martin I Muggeridge  1 , Lindsey M Hutt-Fletcher  3
Affiliations

Epstein-Barr virus glycoprotein gM can interact with the cellular protein p32 and knockdown of p32 impairs virus

Harish Changotra et al. Virology. 2016 Feb.

Abstract

The Epstein-Barr virus glycoprotein complex gMgN has been implicated in assembly and release of fully enveloped virus, although the precise role that it plays has not been elucidated. We report here that the long predicted cytoplasmic tail of gM is not required for complex formation and that it interacts with the cellular protein p32, which has been reported to be involved in nuclear egress of human cytomegalovirus and herpes simplex virus. Although redistribution of p32 and colocalization with gM was not observed in virus infected cells, knockdown of p32 expression by siRNA or lentivirus-delivered shRNA recapitulated the phenotype of a virus lacking expression of gNgM. A proportion of virus released from cells sedimented with characteristics of virus lacking an intact envelope and there was an increase in virus trapped in nuclear condensed chromatin. The observations suggest the possibility that p32 may also be involved in nuclear egress of Epstein-Barr virus.

Keywords: Epstein–Barr virus; Glycoprotein gM; Glycoprotein gN; Virus egress; p32.

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Figures

Figure 1
Figure 1
Interaction of gN and truncated gM. SDS-PAGE and autoradiography of extracts of CV-1 cells infected with vaccinia virus expressing T7 polymerase, transfected with pTM1 plasmids encoding HA-gM, gN, or HA-gMΔ79 as indicated, labeled with [3H]-leucine and immunoprecipitated with antibody to peptides in the ectodomain of gN or with antibodies to HA. Arrows indicated the positions of gM and gN.
Figure 2
Figure 2
Proteins pulled down by GST or GST-gM. SDS-PAGE and autoradiography of extracts of induced (I) or uninduced (U) Akata cells labeled with [3H]-leucine and pulled down with GST or GST-gM bound to glutathione-Sepharose.
Figure 3
Figure 3
Proteins pulled down by MBP or MBP-gM. SDS-PAGE and autoradiography of extracts of uninduced Akata cells labeled with [3H]-leucine and pulled down with MBP or MBP-gM bound to amylose resin.
Figure 4
Figure 4
Interaction of gM with p32. A. Coomassie-stained SDS-PAGE analysis of proteins pulled down by MBP or MBP-gM from uninduced Akata cell lysates. MW = molecular weight markers. Arrow indicates protein excised for analysis. B. Tryptic peptides (bold) identified by mass spectroscopy of the excised protein which correspond to the sequence of p32/gC1-q-R. C. Western blot analysis with antibody to p32 of proteins pulled down from Akata cell lysate by MBP or MBP-gM.
Figure 5
Figure 5
Interaction of full-length gM with p32. Upper panels: SDS-PAGE and western blot analysis of lysates of AGS epithelial cells nuceloporated with vector pCAGGS (lanes 1 and 3), pCAGGS expressing full length HA-tagged gM and pCAGGS expressing p32 (lanes 2 and 4). Lysates in lanes 3 and 4 were either immunoprecipitated (IP) with antibody to p32 and western blotted with antibody to HA, or immunoprecipitated with antibody to HA and western blotted with antibody to p32 as indicated. Lower panels: SDS-PAGE and western blot analysis of lysates of AGS cells nucleoporated with pCAGGS-HA-BFLF2 and pCAGGS-p32. Lysates in lanes 3 were immunoprecipitated (IP) with anti-HA. Lysates in lanes 2 and 3 were western blotted with antibody to p32; lysates in lane 1 were western blotted with anti-HA.
Figure 6
Figure 6
Confocal images of cellular localization of p32 after nucleoporation or virus induction. Top panel: cells were nucleoporated with pCAGGS-p32, pCAGGS-HA-gM, pCAGGS-gN and stained with DAPI, antibody to p32 (red) and antibody to HA (green). Bottom panel: Akata cells were induced to make virus and cells were stained with DAPI, antibody to gB (red) and antibody to p32 (green).
Figure 7
Figure 7
Sedimentation analysis of virus produced by cells treated with siRNA to p32. A. Western blot of virus-producing Akata cell lysates transfected with siRNA to p32 (lanes 1) or non-targeting siRNA (lanes 2) with antibody to p32 or tubulin as indicated. B. Sedimentation profiles in 24–42% Nycodenz of virus produced from cells transfected with non-targeting siRNA (upper panel) or siRNA targeting p32 (lower panel). Virus DNA in each fraction was measured by QPCR.
Figure 8
Figure 8
Sedimentation profile of virus produced by cells transduced with lentivirus expressing shRNA to p32. A. Western blot of uninduced Akata cells (lane 1), induced Akata cells (lane 2), induced Akata cells transduced with non-targeting lentivirus (lane 3) or induced Akata cells transduced with lentivirus targeting p32 (lane 4). Blots were probed with antibodies to tubulin (upper bands) and p32 (lower bands). B. Sedimentation profiles in 24–42% Nycodenz of virus produced from cells transduced with non-targeting lentivirus (upper panel) or lentivirus targeting p32 (lower panel). Virus DNA in each fraction was measured by QPCR.
Figure 9
Figure 9
Electron micrographs of induced Akata cells which had been transduced with lentivirus targeting p32 (left panel) or non-targeting lentivirus (right panel). Cells were harvested after being induced for 24 h.
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