Article Figures & Data
Figures
- Fig. 1.
Binding of HAUSP to C-terminal fragments of Vmw110 in GST pull-down assays. Extracts of cellular proteins labelled with [35S]methionine were incubated with glutathione-agarose beads charged with GST or Vmw110 fusion protein derivatives as described in Materials and Methods. Bound proteins were eluted with reduced glutathione in two sequential steps (labelled 1 and 2 in panel A) and analyzed by SDS–7.5% polyacrylamide gel electrophoresis and autoradiography. Each set of experiments included a negative control with GST alone (GST) and the positive control fusion protein expressed by GEXE52 (594–775). The left-hand track in each panel is a sample of the extract, and the arrow points to the position of the HAUSP band. The identification of the HAUSP band at approximately 130 kDa has been described in detail previously (29, 30). (A) The results obtained with pGEXE4 (615–775) and pGEXE9 (618–775). (B, C, and D) Only the relevant parts of the gels obtained by using proteins expressed by the construct pGEXE52PmlI (594–713) and pGEXE52RsaI (594–680), the construct pGEXE52AvaI (594–646), and the constructs pGEXE23X (594–638) and pGEXE58X (594–633), respectively.
- Fig. 2.
Mutagenesis of selected charged residues within the minimal HAUSP binding region of Vmw110. The upper three lines show the amino acid (aa) and coding sequences (seq.) of residues 616 through 629 of Vmw110. The next three lines show the relevant changes in the mutagenic oligonucleotides (oligo4, oligo5, and oligo6). Uppercase letters denote base changes which were present invariably, and lowercase letters indicate positions where equal proportions of normal and mutant nucleotide precursors were included during synthesis, so that individual single and double mutants could be isolated in the same mutagenesis experiment. The actual mutagenic oligonucleotides included greater lengths of flanking sequence than shown here, but these regions did not contain any substitutions. The line labelled Replace indicates the expected amino acid substitution if the mutagenic nucleotide change is present and also the presence of the FspI site (FspI) in residues 621 and 622. Below are shown the actual amino acid sequences of mutants that were isolated. The presence of the mutations was detected in the M13 isolates, then confirmed by DNA sequencing after transfer of the mutant fragment to plasmids of the p110 series (see Materials and Methods).
- Fig. 3.
Binding of HAUSP to GST-Vmw110 fusion proteins with substitution mutations with the minimal HAUSP binding region. GST pull-down experiments were conducted as described in the legend to Fig.1 and in Materials and Methods by using unlabelled extracts of cellular proteins. Proteins remaining bound to the beads were separated by SDS–7.5% polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes by Western blotting. Bound HAUSP was detected by probing with rabbit serum r201 (which detects a number of other bands in addition to the major band of HAUSP). (A) The left-hand track contains a sample of the extract, and the adjacent lane shows the result obtained by using the fusion protein expressed by pGEXE52. The results obtained with the mutants of the M series, whose details are given in Fig. 2, are shown with the position of HAUSP indicated by the arrow. (B) The relevant portion of the same blot reprobed with MAb 10503 to compare the quantities of the fusion protein used.
- Fig. 4.
Coimmune precipitation of HAUSP with Vmw110 from extracts of cells infected with wild-type and Vmw110 mutant viruses. HeLa cells were mock infected (mock) or infected with wild-type virus (1–775) and with the deletion-carrying mutant viruses A78 (del 592–647) and A8X (1–646) (A) and with the deletion mutants A8X and E58X (1–632) (B). Extracts were prepared and used for immune precipitation of Vmw110 as described in Materials and Methods. Panel A shows precipitated proteins (IP) analyzed alongside samples from the corresponding extracts (ex) by Western blotting. In panel B, only the precipitated proteins are shown. The upper part of each panel shows the relevant portion of the filter probed with anti-HAUSP serum r201, while the lower part shows the same filter probed with anti-Vmw110 serum r95. The arrows point to HAUSP.
- Fig. 5.
Coimmune precipitation of HAUSP with Vmw110 from extracts of cells infected with wild-type and Vmw110 substitution mutant viruses. An experiment similar to that illustrated in Fig. 5 was conducted with wild-type virus positive control (1–775), the mutant E52X negative control (1–593), and the substitution-carrying mutant viruses M1, M2, and M4. The upper part of the figure shows proteins detected by Western blotting by using anti-HAUSP serum r201 in a sample of cell extract and in anti-Vmw110 immune precipitates from mock-infected (mock) and virus-infected cells as indicated. The arrow indicates the position of HAUSP. The lower part shows the presence of Vmw110 proteins in the immune precipitates after reprobing of the filter with anti-Vmw110 serum r95.
- Fig. 6.
Activation of gene expression by Vmw110 and derivatives with mutations in the C-terminal region. Cos7 cells were cotransfected with reporter plasmid pSS80 (ICP6 promoter linked to CAT) and Vmw110 expression plasmids. The negative control is the vector pCIneo, and all CAT activities are given as fold activation over this basal level. The data are averages for at least four independent transfection assays. The nature of the mutations carried by E52X, E58X, A8X, D12, A78, M1, M2, M4, and FXE is shown in Table 2. The deletion carried by D13 (deletion of residues 633 through 680) affects the multimerization and ND10 binding of Vmw110 but not its ability to bind to HAUSP (29). The lower part of the figure shows a Western blot of total proteins of cells, transfected in parallel with the same plasmids, probed with anti-Vmw110 MAb 11060.
- Fig. 7.
Distribution of wild-type and mutant Vmw110 proteins in HEp-2 cells 2 h after the start of infection. Cells on coverslips were infected with the HSV-1 strain 17+ (A and B) and with mutant derivatives FXE (C and D), A8X (E and F), D12 (G and H), A78 (I and J), M1 (K and L), M2 (M and N), and M4 (O and P). Each pair of panels shows the same field of cells stained for Vmw110 (MAb 11060) (left) and PML (r8, to indicate ND10) (right). The bar in P corresponds to 10 μm.
- Fig. 8.
Disruption of ND10 by wild-type and mutant Vmw110 proteins. Coverslips were processed for immunofluorescence 4 h after the start of infection. All other details are exactly as described for Fig. 7.
- Fig. 9.
Growth curves of the HSV-1 strain 17+ and derivatives with lesions in Vmw110. BHK cells were infected with the viruses at 1 PFU per cell, replicate plates were harvested 4, 8, 16, and 24 h later, and progeny virus was titrated on BHK cells. Panels A and C show the results obtained with the viruses constructed for this study, with wild-type virus, and with the Vmw110 RING finger deletion mutant virus FXE. For comparison and completeness, panel B shows the results obtained with the complete C-terminal region deletion mutant, E52X, and the HAUSP-binding-region-deletion mutant, D12 (taken from reference30). The genotypes of the viruses are given in Table2. The data are averages of two independent experiments.
Tables
- Table 1.
Characteristics of the GST fusion proteins including C-terminal portions of Vmw110 used in this study
Plasmid Vmw110 amino acid residuesa HAUSP bindingb pGEXE52 594–775 + pGEXE4 615–775 + pGEXE9 618–775 + pGEXE58 633–775 − pGEXE52Pm1I 594–713 + pGEXE52RsaI 594–680 + pGEXE52AvaI 594–646 + pGEXE23X 594–638 Weak pGEXE58X 594–633 − - Table 2.
Vmw110 mutant viruses used in this study
Virus strain Vmw110 residues, deletions, or substitution(s) HAUSP bindinga ND10 colocali-zationb ND10 disruptionc Multimerd 17+ 1–775 (wild type) + + + + FXE Δ106–149 + + − + E52X 1–593 − − − − E58X 1–632 − − − (−) A8X 1–646 − − − (−) D12 Δ594–632 − + + + A78 Δ592–647 − + + (+) M1 R623L/K624I − + + (+) M2 R623L Reduced + + (+) M4 K620I − + + (+) ↵a The ability to bind HAUSP in coimmunoprecipitation experiments from virus-infected cell extracts as shown in Fig. 4. The data for viruses FXE and D12 were taken from references 26 and 27.
↵b The ability of the mutant protein to colocalize with PML in ND10 as shown in Fig. 6. The data for viruses not shown in Fig. 6 were taken from references 11,24, and 27.
↵c Disruption of ND10 in HEp-2 cells 5 h after the start of infection. See Fig. 6.
↵d The ability of Vmw110 to multimerize and the effects of mutations of strains FXE, E52X, and D12 have been determined experimentally (30). Parentheses indicate that the mutations in these proteins affect sequences in the mapped multimerization domain (4, 30).
