1 Department of Veterinary and Biomedical Sciences, Nebraska Center for Virology, University of Nebraska, Lincoln, Fair Street at East Campus Loop, Lincoln, NE 68583-0905, USA
2 Center for Biotechnology, University of Nebraska, Lincoln, Fair Street at East Campus Loop, Lincoln, NE 68583-0905, USA
Correspondence
Clinton Jones
cjones{at}unlnotes.unl.edu
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The ICP0 homologues encoded by BoHV-1 and HSV-1 contain a well-conserved C3HC4 zinc RING finger near their respective N termini. Mutational analysis has demonstrated the importance of the C3HC4 zinc RING finger domain of bICP0 and ICP0 (Everett, 1987, 1988
; Everett et al., 1993
; Inman et al., 2001
). ICP0 (Everett et al., 1997
, 1999a
, b
; Maul & Everett, 1994
; Maul et al., 1993
) and bICP0 (Inman et al., 2001
; Parkinson & Everett, 2000
) co-localize with and disrupt the proto-oncogene promyelocytic leukaemia protein-containing nuclear domains (ND10 or PODS). ICP0 regulates steady-state levels of cellular and viral proteins because ICP0 has E3 ubiquitin ligase activity (Boutell et al., 2002
; Van Sant et al., 2001
), and it interacts with protein degradation machinery (Everett et al., 1997
, 1999a
). The E3 ubiquitin ligase activity of ICP0 disrupts the cell cycle and alters cellular gene expression (Hobbs & DeLuca, 1999
; Lomonte & Everett, 1999
). Apart from the zinc RING finger, bICP0 and ICP0 share little amino acid similarity.
In transient transfection assays, bICP0 is a potent transactivator of viral promoters, and can regulate certain cellular promoters. For example, bICP0 relieves mad/max mediated transcriptional repression through its association with histone deacetylase 1 (Zhang & Jones, 2001). bICP0 also inhibits the human interferon-
promoter, in part, by sequestering the co-activator p300 (Y. Zhang, G. Henderson & C. Jones, unpublished data). In the absence of other viral genes, overexpression of bICP0 is toxic (Inman et al., 2001
), and in transiently transfected mouse neuroblastoma cells bICP0 activates caspase 3 by an indirect mechanism that ultimately leads to apoptosis (Henderson et al., 2004
). Deletion of amino acids spanning 356676 of bICP0 altered subcellular localization of bICP0 and prevented transactivation of the TK promoter (Inman et al., 2001
).
In this study, we generated a panel of bICP0 mutants by random transposon insertion. We identified two domains that were important for transcriptional activation, aa 78256 and amino acids at or near position 457. Insertion of a transposon into aa 91W reduced the stability of the bICP0 protein in transiently transfected cells. C-terminal amino acids spanning 607676 contain a nuclear localization signal (NLS). Deletion of the NLS altered the cellular localization of bICP0 and reduced its ability to activate the TK promoter. These results indicate that bICP0 contains at least three functional domains that are important for transactivating viral gene expression, and one domain near the C terminus that promotes nuclear localization.
![]() |
METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The E2.6 plasmid (a gift from M. Schwyzer; Zurich, Switzerland) contains bICP0 coding sequence controlled by the human CMV promoter. Mutagenesis of the bICP0 zinc RING finger was described previously (Inman et al., 2001). The coding regions of the wild-type (wt) bICP0 and the zinc RING finger mutant 13G/51A were inserted into a Flag-tagged expression vector pCMV2C (Stratagene). The resulting plasmids were designated bICP0 or 13G/51A (see Fig. 1b
). A C-terminal deletion of bICP0 (
bICP0) was constructed by deleting the SalIXhoI fragment (aa 356676) from the Flag-tagged bICP0 construct (see Fig. 1b
). Another C-terminal deletion mutant, designated the
NcoI mutant was generated by deleting the NcoIXhoI fragment from the Flag-tagged bICP0 construct (see Fig. 4a
).
|
|
Transient expression and Western blot.
293 cells were transfected with 20 µg bICP0 expression plasmid by calcium phosphate precipitation method (Zhang & Jones, 2001). At 40 h after transfection, cells were collected and suspended in 250 µl lysis buffer (20 mM HEPES pH 7·9, 400 mM KCl, 1·5 mM MgCl2, 0·2 mM EDTA, 20 % glycerol, 0·5 mM DTT and complete proteinase inhibitors in one tablet per 10 ml). The lysate was kept on a rotating device for 1 h at 4 °C and centrifuged for 10 min at 4 °C, 15 000 r.p.m. (12 500 g). To the supernatant, 50 µl 5x sample buffer (250 mM Tris/HCl pH 6·8, 10 % SDS, 25 % mercaptoethanol) was added and the solution boiled for 5 min. The lysate was used for SDS-PAGE. Immunodetection of bICP0 and its mutants was performed with an anti-Flag antibody (Stratagene; catalogue #200471-21).
Transient transfection and CAT assays.
The pMinCAT reporter construct and bICP0 expression plasmids were co-transfected into CV-1 cells by the calcium phosphate precipitation method. For each transfection, 15 µg reporter plasmid and 1 µg bICP0 expression plasmid were used to form DNAcalcium phosphate co-precipitates. This solution was incubated with CV-1 cells for 12 h and then replaced with fresh medium. After 24 h, cell lysate was prepared by three freezethaw cycles in 0·25 M Tris pH 8·0. CAT activity was measured in the presence of 0·2 µCi (7·4 kBq) [14C]chloramphenicol and 0·5 mM acetyl coenzyme A. All forms of chloramphenicol were separated by thin layer chromatography. CAT activity was measured by using a Bio-Rad Molecular Imager FX. The levels of CAT activity were expressed as fold induction relative to the vector control.
Generation of bICP0 mutant by EZ : : TN in-frame linker insertion.
In-frame linker insertion was performed according to the manufacturer's manual (Epicentre; catalogue #EZ104KN) with some modifications. The BamHISalI fragment of bICP0 from pCMV2C-bICP0 was released and cloned into the same restriction sites of pUC19. The resulting plasmid was used as target DNA for transposon insertion. The transposon insertion reaction contains 1 µl EZ : : TN 10x reaction buffer, 250 ng target DNA, 1 µl EZ : : TN<NotI/KAN-3>transposon, 1 µl EZ : : TN transposase, 4·5 µl dH2O. The reaction mixture was incubated for 2 h at 37 °C. EZ : : TN 10x stop buffer (1 µl) was added and the reaction mixture was heated for 10 min at 70 °C. Two microlitres of the reaction was used to transform competent E. coli cells (Transfer Max EC100, Epicentre; catalogue #CC02810). One-third of a 300 µl transformation reaction was plated on three separate dishes containing different antibiotics (ampicillin, kanamycin or ampicillin/kanamycin). Colonies from the ampicillin/kanamycin plate were randomly picked to extract target DNA for mapping the transposon insertion sites. Mapping was initially performed by digestion with SalI and confirmed by BamHI digestion. Certain clones were then sequenced to determine the precise insertion sites. A panel of BamHISalI fragments carrying transposons was religated with the remaining sequences of bICP0 within the Flag-tagged vector.
Confocal microscopy to examine subcellular localization of bICP0.
In brief, neuro-2A cells in six-well culture dishes were transfected with 2·5 µg of the designated Flag-tagged bICP0 constructs by Superfect (Qiagen; catalogue #301305). After 16 h, cultures were split into eight-well Lab-Tek culture slides. After incubating for 24 h, cultures were fixed in 4 % paraformadehyde, and then incubated in cold 100 % ethanol at 20 °C for 2 min. After washing three times with PBS, the slides were blocked in 4 % BSA in PBS for 30 min, then incubated with the anti-Flag antibody (1 : 100) for 2 h at room temperature. The secondary antibody, Cy2 goat anti-mouse IgG (Jackson ImmunResearch Laboratories) (1 : 100) was added and incubated for 1 h at room temperature in the dark. The images were collected by using a Bio-Rad confocal laser-scanning microscope (MRC-1024ES) with excitation/emission at 488/520 nm.
Construction of GFPbICP0 fusions.
The bICP0 expression vector E2.6 was digested with NcoI and then blunted with the Klenow enzyme. The digested plasmid was purified and subjected to a second digestion with SacII. The NcoISacII fragment containing amino acids spanning 607676 was recovered from an agarose gel. A GFP expression vector, phr-GFP-N1 (Stratagene; catalogue #240059), contains a copy of the hrGFP that lacks a translational termination codon inserted upstream of a versatile multiple cloning site, which allows fusion of hrGFP to the N terminus of protein. The NcoISacII fragment was cloned into SalI and SacII digested GFP expression vector (the SalI site was first filled-in with Klenow enzyme). The resulting plasmid was designated as GFPNLSbICP0. Five micrograms of each GFP vector or GFPNLSbICP0 expression plasmid was transfected into neuro-2A cells by Superfect. At 40 h after transfection, cells were fixed in 4 % paraformaldehyde for 10 min and washed with PBS. Subcellular localization of GFP was visualized by using a fluorescence microscope.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
DNA fragments containing the respective transposon insertions were reintroduced into the Flag-tagged CMV expression vector. To test for expression of the respective bICP0 mutant proteins, human 293 cells were transfected with 15 µg of each mutant expression vector, and at 40 h after transfection Western blots were performed using the anti-Flag antibody (Fig. 1c). The 293 cell line was used for this study because we can readily achieve greater than 60 % transfection efficiency, and these cells are resistant to the toxic effects of bICP0. Except for mutant B, the rest of the mutants expressed wt levels of the bICP0 protein. Mutant L migrated faster than wt bICP0 when high salt was used to extract bICP0 from transfected cells. As expected, similar levels of
-actin were present in 293 cells after transfection.
Transactivation potential of the bICP0 transposon mutants
To test whether the transposon insertion affected the ability of bICP0 to activate a minimal HSV-1 TK promoter, pMinCAT and each transposon mutant (A to O) were co-transfected into CV-1 cells (Fig. 2). bICP0 can reproducibly activate the TK promoter more than 10-fold (Inman et al., 2001
; Zhang & Jones, 2001
), and as such the TK promoter is a simple model promoter for assessing the transactivation potential of bICP0. At 40 h after transfection, CAT activity was measured. Mutant M, which efficiently expressed the mutant bICP0 protein (Fig. 1c
), was not able to activate the minimal TK promoter above basal levels (Fig. 2
). Mutants A to H, and N transactivated the TK promoter two- to threefold less than wt bICP0. Mutant B did not activate the TK promoter, but this was expected because this mutant expressed low levels of protein. Mutants I to L, and O did not dramatically reduce transactivation of the TK promoter. Mutant K and L contained transposon insertions in the acidic domain (Fig. 1a
), but these mutants activated the TK promoter nearly as effectively as wt bICP0. These results suggested that in addition to the zinc RING finger domain, amino acid sequences between 78 and 265, and 457 and 470 were important for maximal transcriptional activation of the minimal TK promoter. Similar results were obtained by using a mouse neuroblastoma cell line (data not shown).
|
|
Confocal microscopy demonstrated that the protein expressed by the NcoI mutant was primarily expressed in the cytoplasm (Fig. 5a
). The
C terminus protein (
bICP0) was expressed in the cytoplasm of transfected cells whereas wt bICP0 or the 13G/51A protein was expressed in the nucleus, which was consistent with previous studies (Inman et al., 2001
). Within sequences deleted from the NcoI mutant, amino acids spanning 622625 contain a NLS-like core sequence (KRRR) suggesting this region plays a role in nuclear localization.
|
Transactivation potential of the NcoI mutant
Additional studies were performed to test whether the NcoI mutant could activate the minimal TK reporter construct (pMinCAT). CV-1 cells were co-transfected with pMinCAT and the designated bICP0 mutants, and cell lysate was prepared 40 h after transfection. Fig. 6
demonstrated that wt bICP0 activated pMinCAT promoter activity eight- to ninefold, which was expected. The
NcoI mutant had reduced levels of transcriptional activation relative to wt bICP0. The
bICP0 or the 13G/51A mutant had little or no transactivation activity on this promoter, which is consistent with previous studies (Inman et al., 2001
). This result demonstrates that nuclear localization was important for maximal activation of the minimal TK promoter.
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Transposon insertion within the N-terminal residues of bICP0 (mutants A to H; aa 78265) had a dramatic effect on transactivation of the TK promoter. Some of the transposon insertions within this domain (A, E, F and G for example) led to altered subnuclear localization, suggesting these mutations altered the secondary structure of the zinc RING finger, or prevented interactions with cellular proteins. The N terminus of HSV-1 ICP0 also contains a promoter-specific transactivation domain (Lium et al., 1998). Mutant B, which is also within this large domain, did not express high levels of protein suggesting these sequences regulate protein stability. Another transcriptional activation domain was identified at or near aa 457 (mutant M). Although mutant M was abundantly expressed in the nucleus of transfected cells, and had punctate staining similar to wt bICP0, it did not transactivate the TK promoter.
Adjacent to the N-terminal transcriptional activation domain is an acidic domain (residues 280330) that is rich in acidic (Asp and Glu) and hydroxyl (Ser and Thr) amino acids (Fig. 1a) (Wirth et al., 1992
). Two transposon insertions (mutant K and L) disrupt the acidic domain, but had no dramatic effect on protein stability or transactivation of the TK promoter. However, the nuclear localization of mutant K or L appeared to be different from wt bICP0, and mutant protein L migrated faster than wt bICP0. Other proteins, GCN4 and GAL4 for example, contain acidic domains that are necessary for stimulating transcription (Mitchell & Tjian, 1989
). An analogous acidic domain is not readily apparent in ICP0 coding sequences suggesting this domain has novel functions unique to bICP0. It is also possible that the acidic domain plays a role in activating certain promoters, but not the minimal TK promoter used in this study, or it is only important in certain cell types.
Amino acid sequences located near the C terminus of bICP0 (NcoISacII fragment; Fig. 1a) were necessary for localization of bICP0 to the nucleus in transiently transfected cells. Although this plasmid did not transactivate the TK promoter as effectively as wt bICP0, the level of transactivation was higher than the
bICP0 construct. We hypothesize that cytoplasmic bICP0 can activate transcription by an unknown mechanism, or that low levels of the protein enter the nucleus. When the NcoISacII fragment was fused in-frame with GFP, the GFP signal localized to the nucleus confirming this fragment contained a NLS. An amino acid motif, KRRR, is located between aa 622 and 625, which resembles other basic motifs known to regulate nuclear localization. Classical NLS are categorized as either monopartite, containing a single cluster of basic amino acid residues (K/R), or bipartite, containing two clusters of basic amino acid residues separated by a linker of 1012 unconserved amino acids (Kalderon et al., 1984
; Lanford & Butel, 1984
; Richter et al., 1985
; Robbins et al., 1991
). The SV40 large T antigen NLS (PKKKRKV) is the prototype NLS. Although the KRRR motif does not match the SV40 T NLS, it does resemble a monopartite NLS. There is another NLS-like core motif, RRRRRT (aa 462467), within bICP0. However, a fragment containing this motif was unable to alter the GFP localization in transfected cells (data not shown). HSV-1 ICP0 also contains a domain that is necessary for nuclear localization, and this region contains a basic NLS core sequence (PRLRR) between residues 501 and 506 (Everett, 1988
). ICP0 subcellular localization is influenced by two other HSV IE proteins, ICP4 and ICP27 (Zhu et al., 1994
). Future studies will test whether the KRRR motif is important for NLS function, and if viral proteins influence the subcellular localization of bICP0.
In summary, our studies have identified four separate domains that play a role in activating a simple promoter: (i) the zinc RING finger located between aa 13 and 51 (Inman et al., 2001), (ii) a large domain that spans aa 78265, (iii) sequences at or near aa 457, and (iv) an NLS that is located near the C terminus of bICP0. We have recently developed a bICP0 null mutant virus that grows very poorly in bovine cells (V. Geiser, Y. Zhang & C. Jones, unpublished data). This mutant will be useful to develop additional mutants in the four functional domains that were identified in this study. Since bICP0 is considered to be the major regulatory protein encoded by BoHV-1 (Wirth et al., 1992
), understanding the role that bICP0 plays during productive infection and the latency-reactivation cycle is important.
![]() |
ACKNOWLEDGEMENTS |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Everett, R. D. (1987). A detailed mutational analysis of Vmw110, a trans-acting transcriptional activator encoded by herpes simplex virus type 1. EMBO J 6, 20692076.[Abstract]
Everett, R. D. (1988). Analysis of the functional domains of herpes simplex virus type 1 immediate-early polypeptide Vmw110. J Mol Biol 202, 8796.[CrossRef][Medline]
Everett, R. D., Barlow, P., Milner, A., Luisi, B., Orr, A., Hope, G. & Lyon, D. (1993). A novel arrangement of zinc-binding residues and secondary structure in the C3HC4 motif of an alpha herpes virus protein family. J Mol Biol 234, 10381047.[CrossRef][Medline]
Everett, R. D., Meredith, M., Orr, A., Cross, A., Kathoria, M. & Parkinson, J. (1997). A novel ubiquitin-specific protease is dynamically associated with the PML nuclear domain and binds to a herpesvirus regulatory protein. EMBO J 16, 15191530.
Everett, R. D., Earnshaw, W. C., Findlay, J. & Lomonte, P. (1999a). Specific destruction of kinetochore protein CENP-C and disruption of cell division by herpes simplex virus immediate-early protein Vmw110. EMBO J 18, 15261538.
Everett, R. D., Lomonte, P., Sternsdorf, T., van Driel, R. & Orr, A. (1999b). Cell cycle regulation of PML modification and ND10 composition. J Cell Sci 112, 45814588.
Henderson, G., Zhang, Y., Inman, M., Jones, D. & Jones, C. (2004). Infected cell protein 0 encoded by bovine herpes virus 1 can activate caspase 3 when overexpressed in transfected cells. J Gen Virol 85, 35113516.
Hobbs, W. E., II & DeLuca, N. A. (1999). Perturbation of cell cycle progression and cellular gene expression as a function of herpes simplex virus ICP0. J Virol 73, 82458255.
Inman, M., Zhang, Y., Geiser, V. & Jones, C. (2001). The zinc ring finger in the bICP0 protein encoded by bovine herpesvirus-1 mediates toxicity and activates productive infection. J Gen Virol 82, 483492.
Jones, C. (1998). Alphaherpesvirus latency: its role in disease and survival of the virus in nature. Adv Virus Res 51, 81133.[Medline]
Jones, C. (2003). Herpes simplex virus type 1 and bovine herpesvirus 1 latency. Clin Microbiol Rev 16, 7995.
Kalderon, D., Roberts, B. L., Richardson, W. D. & Smith, A. E. (1984). A short amino acid sequence able to specify nuclear location. Cell 39, 499509.[CrossRef][Medline]
Lanford, R. E. & Butel, J. S. (1984). Construction and characterization of an SV40 mutant defective in nuclear transport of T antigen. Cell 37, 801813.[Medline]
Lium, E. K., Panagiotidis, C. A., Wen, X. & Silverstein, S. J. (1998). The NH2 terminus of the herpes simplex virus type 1 regulatory protein ICP0 contains a promoter-specific transcription activation domain. J Virol 72, 77857795.
Lomonte, P. & Everett, R. D. (1999). Herpes simplex virus type 1 immediate-early protein Vmw110 inhibits progression of cells through mitosis and from G1 into S phase of the cell cycle. J Virol 73, 94569467.
Maul, G. G. & Everett, R. D. (1994). The nuclear location of PML, a cellular member of the C3HC4 zinc-binding domain protein family, is rearranged during herpes simplex virus infection by the C3HC4 viral protein ICP0. J Gen Virol 75, 12231233.[Abstract]
Maul, G. G., Guldner, H. H. & Spivack, J. G. (1993). Modification of discrete nuclear domains induced by herpes simplex virus type 1 immediate early gene 1 product (ICP0). J Gen Virol 74, 26792690.[Abstract]
Misra, V., Bratanich, A. C., Carpenter, D. & O'Hare, P. (1994). Protein and DNA elements involved in transactivation of the promoter of the bovine herpesvirus (BHV) 1 IE-1 transcription unit by the BHV alpha gene trans-inducing factor. J Virol 68, 48984909.[Abstract]
Misra, V., Walker, S., Hayes, S. & O'Hare, P. (1995). The bovine herpesvirus alpha gene trans-inducing factor activates transcription by mechanisms different from those of its herpes simplex virus type 1 counterpart VP16. J Virol 69, 52095216.[Abstract]
Mitchell, P. J. & Tjian, R. (1989). Transcriptional regulation in mammalian cells by sequence-specific DNA binding proteins. Science 245, 371378.[Medline]
Parkinson, J. & Everett, R. D. (2000). Alphaherpesvirus proteins related to herpes simplex virus type 1 ICP0 affect cellular structures and proteins. J Virol 74, 1000610017.
Parkinson, J. & Everett, R. D. (2001). Alphaherpesvirus proteins related to herpes simplex virus type 1 ICP0 induce the formation of colocalizing, conjugated ubiquitin. J Virol 75, 53575362.
Richter, J. D., Young, P., Jones, N. C., Krippl, B., Rosenberg, M. & Ferguson, B. (1985). A first exon-encoded domain of E1A sufficient for posttranslational modification, nuclear localization, and induction of adenovirus E3 promoter expression in Xenopus oocytes. Proc Natl Acad Sci U S A 82, 84348438.[Abstract]
Robbins, J., Dilworth, S. M., Laskey, R. A. & Dingwall, C. (1991). Two independent basic domains in nucleoplasmin nuclear targeting sequences: identification of a class of bipartite nuclear targeting sequence. Cell 64, 615623.[Medline]
Van Sant, C., Hagglund, R., Lopez, P. & Roizman, B. (2001). The infected cell protein 0 of herpes simplex virus 1 dynamically interacts with proteasomes, binds and activates the cdc34 E2 ubiquitin-conjugating enzyme, and possesses in vitro E3 ubiquitin ligase activity. Proc Natl Acad Sci U S A 98, 88158820.
Wirth, U. V., Fraefel, C., Vogt, B., Vlcek, C., Paces, V. & Schwyzer, M. (1992). Immediate-early RNA 2·9 and early RNA 2·6 of bovine herpesvirus 1 are 3' coterminal and encode a putative zinc finger transactivator protein. J Virol 66, 27632772.[Abstract]
Zhang, Y. & Jones, C. (2001). The bovine herpesvirus 1 immediate-early protein (bICP0) associates with histone deacetylase 1 to activate transcription. J Virol 75, 95719578.
Zhu, Z., Cai, W. & Schaffer, P. A. (1994). Cooperativity among herpes simplex virus type 1 immediate-early regulatory proteins: ICP4 and ICP27 affect the intracellular localization of ICP0. J Virol 68, 30273040.[Abstract]
Received 15 October 2004;
accepted 18 December 2004.