Characterization of the Varicella-zoster virus ORF25 gene product: pORF25 interacts with multiple DNA encapsidation proteins

Abstract

The Herpesviridae contain a group of highly conserved proteins designated the Herpes UL33 Superfamily (pfam03581). The Varicella-zoster virus (VZV) homolog, encoded by the ORF25 gene, was used to generate a GST-ORF25 fusion protein. Purified GST-ORF25 was used to generate a polyclonal rabbit antiserum that detected the 17.5 kDa ORF25 protein (pORF25) in VZV infected cells. In pull-down assays, GST-ORF25 interacted with a number of encapsidation proteins including ORF30, ORF42 (the second exon of ORF45/42) and itself. The self-interaction was confirmed via a yeast two-hybrid assay. Additionally, pORF25 and pORF30 were shown to co-immunoprecipitate from VZV infected cells. Our results suggest that pORF25 is part of the trimeric terminase complex for VZV. However, combined with data from previous studies on HSV-1 and Kaposi's sarcoma associated herpesvirus (KSVH), we hypothesize that VZV pORF25 and the Herpes UL33 Superfamily homologs are not encapsidation proteins per se but instead work to bring viral proteins together to form functional complexes.

Figure 1

The Herpes UL33 Superfamily. HHV-1 (Human herpesvirus 1 or Herpes simplex virus type 1), HHV-2 (Human herpesvirus 2 or Herpes simplex virus type 2), HHV-3 (Human herpesvirus 3 or Varicella-zoster virus), HHV-4 (Human herpesvirus 4 or Epstein-Barr virus), HHV-5 (Human herpesvirus 5 or Human cytomegalovirus), HHV-6 (Human herpesvirus 6), HHV-7 (Human herpesvirus 7), and HHV-8 (Human herpesvirus 8 or Kaposi’s sarcoma associated virus). ClustalW2 alignment showing identical (*), conserved (:), or semi-conserved (.) amino acids within pfam 03581 for the eight human herpesviruses.

Figure 2

Expression of VZV ORF25. (A) Immunoblot analysis was performed with the ORF25 rabbit polyclonal antiserum on mock infected or VZV infected IMR90 cell extracts. (B) Indirect immunofluorescence analysis of pORF25 expressed in transiently transfected cells. Cells were transfected with pcDNA3.1D/V5-ORF25 and 48 hr post-transfection fixed, stained with DAPI and incubated with an anti-V5 primary antibody followed by a FITC conjugated secondary antibody. (C) Indirect immunofluorescence analysis of pORF25 expressed in HSV-2 infected cells. Cells were transfected with pcDNA3.1D/V5-ORF25 and infected with HSV-2 24 hr post-transfection. Transfected, infected cells were fixed at 10 hr post-infection, stained with DAPI, and incubated with an anti-V5 rabbit and anti-HSV-2 ICP8 mouse antibodies followed by FITC anti-rabbit and rhodamine red-X conjugated anti-mouse secondary antibodies. pORF25 expressing cells appear green, HSV-2 infected cell nuclei appear red and DAPI stained nuclei appear blue.

Figure 3

GST pull-down assays were performed to detect potential protein-protein interactions. (A) GST or GST-ORF25 were purified on glutathione beads. Proteins were analyzed on Coomassie blue stained SDS-polyacrylamide gels to assess purity prior to performing pull-down assays. (B) pcDNA3.1D/V5–lacZ (β-gal), -ORF43, -ORF42, -ORF30, or -ORF25 were used in in vitro transcription/translation reactions to synthesize the respective protein products containing a C-terminal V5 epitope. Equal concentrations of GST or GST-ORF25 immobilized on beads were resuspended in binding buffer containing various pORF-V5s or β-gal-V5. Beads were washed, fractionated by SDS-PAGE and subjected to immunoblot analysis using an anti-V5 monoclonal antibody.

Figure 4

Two-hybrid analysis of pORF25 self-interaction. Target and bait vectors containing a DNA binding domain (BD) or transcriptional activation domain (AD) fused to the full length ORF25 gene were co-transformed into yeast. (A) Immunoblot analysis of yeast transformed with: pGBKT7 +pGADT7pORF25 (lanes 1 and 4), pGADT7+pGBKT7pORF25 (lanes 2 and 5) or pGADT7pORF25+pGBKT7pORF25 (lanes 3 and 6). Lanes 1–3 and lanes 4–6 were incubated with antibodies to the GAL4 activation (anti-HA) or binding (anti-GAL4 DNA BD) domains respectively. (B) Relative α-gal activity detected in the supernatants of yeast co-transformed with: 1-pGADT7 + pGBKT7pORF25, 2-pGBKT7 + pGADT7pORF25, 3-pGADT7pORF25 + pGBKT7pORF25, 4-pGBKT7-lam + pGADT7-T, or 5-pGADT7 + pGBKT7. A positive control using two known interacting partners, pGBKT7-53 and pGADT7-T, consistently yielded activity greater than 35,000 fold over baseline (data not shown).

Figure 5

Co-immunoprecipitation of pORF25 and pORF30. Uninfected or VZV infected MeWo cell extracts were immunoprecipitated using an ORF25 specific antiserum or a V5 epitope specific antibody. Precipitated proteins were fractionated by SDS-PAGE and analyzed by western blot with an ORF30 specific antiserum. The arrow indicates the position of the 87 kDa pORF30.