The varicella-zoster virus (VZV) ORF9 protein interacts with the IE62 major VZV transactivator

Abstract

The varicella-zoster virus (VZV) ORF9 protein is a member of the herpesvirus UL49 gene family but shares limited identity and similarity with the UL49 prototype, herpes simplex virus type 1 VP22. ORF9 mRNA is the most abundantly expressed message during VZV infection; however, little is known concerning the functions of the ORF9 protein. We have found that the VZV major transactivator IE62 and the ORF9 protein can be coprecipitated from infected cells. Yeast two-hybrid analysis localized the region of the ORF9 protein required for interaction with IE62 to the middle third of the protein encompassing amino acids 117 to 186. Protein pull-down assays with GST-IE62 fusion proteins containing N-terminal IE62 sequences showed that amino acids 1 to 43 of the acidic transcriptional activation domain of IE62 can bind recombinant ORF9 protein. Confocal microscopy of transiently transfected cells showed that in the absence of other viral proteins, the ORF9 protein was localized in the cytoplasm while IE62 was localized in the nucleus. In VZV-infected cells, the ORF9 protein was localized to the cytoplasm whereas IE62 exhibited both nuclear and cytoplasmic localization. Cotransfection of plasmids expressing ORF9, IE62, and the viral ORF66 kinase resulted in significant colocalization of ORF9 and IE62 in the cytoplasm. Coimmunoprecipitation experiments with antitubulin antibodies indicate the presence of ORF9-IE62-tubulin complexes in infected cells. Colocalization of ORF9 and tubulin in transfected cells was visualized by confocal microscopy. These data suggest a model for ORF9 protein function involving complex formation with IE62 and possibly other tegument proteins in the cytoplasm at late times in infection.

FIG. 1.

Sequence comparison of the VZV ORF9 protein and HSV-1 UL49 (VP22). (A) Alignment of the complete ORF9 and VP22 amino acid sequences. (B) Alignment of the UL49 homology domains of ORF9 and VP22.

FIG. 2.

Coimmunoprecipitation of the ORF9 protein and IE62. (A) Coimmunoprecipitation of the ORF9 protein and IE62 using the monoclonal H6 anti-IE62 antibody. Lane 1, infected cell extract; lane 2, material bound to protein G-Sepharose beads coupled with anti-IE62 antibody; lane 3, beads alone. Upper panel, ORF9 protein; lower panel, IE62. (B) Coimmunoprecipitation of IE62 using polyclonal anti-ORF9 protein antibody. The lane designations are identical to those in panel A.

FIG. 3.

Results of yeast two-hybrid analysis of the potential ORF9 protein/IE62 interaction. (A) Filter colony lift β-galactosidase assay results using the intact ORF9 protein and an IE62 fragment containing the IE62 acidic activation domain (aa 1 to 201). The upper line shows yeast growth; the lower line shows β-galactosidase expression. The table indicates the selective growth conditions using the indicated SD medium to select and test for specific phenotypes (described in Materials and Methods). As a positive control for interaction, the plasmids p53, which expresses the murine p53 protein fused to the GAL4 DNA binding domain, and pSV40, which expresses the SV40 large T antigen fused to the GAL4 activation domain, were used. As a negative control, pLaminC, which expresses the human lamin C protein fused to the GAL4 binding domain, was used in conjunction with the pSV40 plasmid. (B) Yeast two-hybrid analysis using fragments of the ORF9 protein-coding sequences and the N-terminal IE62 fragment.

FIG. 4.

Mapping of the minimal region of IE62 that interacts with the ORF9 protein. Results from protein pull-down assays using GST-IE62 fusions showing the presence or absence of the ORF9 protein in samples eluted from glutathione-Sepharose beads. (A) N-terminal IE62 fragments. (B) C-terminal IE62 fragments. The lower portion of each panel is a Coomassie gel showing the levels of the GST-IE62 fusions and GST bound to the beads in these assays. Arrows in upper panels indicate the position of the intact ORF9 protein.

FIG. 5.

The ORF9 protein does not affect IE62 transactivation activity in the absence of other viral proteins. The results of luciferase reporter assays showing IE62 activation of the VZV ORF29 promoter in the presence and absence of the ORF9 protein are shown.

FIG. 6.

Cellular localization of the ORF9 protein and IE62. (A) Confocal microscopy of HeLa cells transfected with ORF9- and IE62-expressing plasmids at 24 h posttransfection. TOTO-3 was used as a nuclear marker. (B) Confocal microscopy of MeWo cells at 24 h post-VZV infection.

FIG. 7.

Effect of the ORF66 viral kinase on IE62 transactivation and cellular distribution of the ORF9 protein and IE62. (A) Luciferase reporter assay results from transient transfections of MeWo cells using constant amounts of the pCMV62 expression plasmid and increasing amounts of a plasmid expressing the VZV ORF66 kinase.

FIG. 8.

Effect of the ORF66 viral kinase on the intracellular distribution of the ORF9 protein and IE62. Confocal microscopy results of HeLa cells transfected with plasmids expressing the ORF9 protein, IE62, and the ORF66 kinase at 24 h posttransfection are shown. The presence of the kinase results in a redistribution of IE62 from the nucleus to the cytoplasm.

FIG. 9.

Effect of the VZV ORF47 kinase on the cellular localization of the ORF9 protein and IE62. Confocal microscopy results of MeWo cells transfected with plasmids expressing the ORF9 protein, IE62, and the ORF47.12 kinase are shown. IE62 is confined to the nucleus. The ORF9 protein and the ORF47 kinase are both cytoplasmic but show different distributions.

FIG. 10.

ORF9 and IE62 are coprecipitated by anti-β-tubulin antibodies. (A) Immunoblot analysis of coimmunoprecipitation experiments using anti-β-tubulin antibodies. Lane 1, input from whole-cell extract; lane 2, eluate from antibody-bound beads; lane 3, eluate from beads alone without antibody. Arrows indicate the positions of the ORF9 protein, IE62, gE, and β-tubulin. (B) Results of control experiments using monoclonal IgG directed against the Xpress peptide and analyzed for the presence of the ORF9 protein and IE62. The lane designations are as described for panel A.

FIG.11.

Results of confocal microscopy visualizing intracellular tubulin, the ORF9 protein, and IE62. (A) MeWo cell transfected with plasmids expressing the ORF9 protein and IE62. The IE62 signal is confined to the nucleus. The ORF9 protein and tubulin colocalize in a long filamentous structure. (B) MeWo cell transfected with plasmids expressing the ORF9 protein, IE62, and the ORF66 and ORF47 kinases. IE62 is present in both the nucleus and the cytoplasm. ORF9 and tubulin signals overlap with the IE62 signal in the cytoplasm.

FIG.11.

Results of confocal microscopy visualizing intracellular tubulin, the ORF9 protein, and IE62. (A) MeWo cell transfected with plasmids expressing the ORF9 protein and IE62. The IE62 signal is confined to the nucleus. The ORF9 protein and tubulin colocalize in a long filamentous structure. (B) MeWo cell transfected with plasmids expressing the ORF9 protein, IE62, and the ORF66 and ORF47 kinases. IE62 is present in both the nucleus and the cytoplasm. ORF9 and tubulin signals overlap with the IE62 signal in the cytoplasm.

FIG. 12.

Model of ORF9 protein/IE62 interactions. In this model, IE62 is phosphorylated by the ORF66 kinase either in the nucleus or in the cytoplasm late in infection, resulting in its exclusion from the nucleus. IE62 can then interact with the ORF9 protein and potentially, either simultaneously or sequentially, with the ORF47 kinase. This complex then binds to the microtubules via the ORF9 protein. An alternative mechanism could involve initial binding of ORF9 to microtubules, followed by recruitment of the other proteins.