Multimerization of ICP0, a herpes simplex virus immediate-early protein.

ICP0, a herpes simplex virus immediate-early gene product, is a highly phosphorylated nuclear protein that is a potent activator of virus and host genes. Using biochemical and genetic assays employing plasmids encoding mutant forms of ICP0 and a recombinant adenovirus that expresses ICP0, we mutant forms of ICP0 and a recombinant adenovirus that expresses ICP0, we provide evidence that the protein multimerizes. Some mutant forms of ICP0 were transdominant and interfered with activation of a target reporter gene or with complementation of an ICP0-minus virus.     

Phosphorylation site mutations affect herpes simplex virus type 1 ICP0 function

The herpes simplex virus type 1 (HSV-1) immediate-early (IE) regulatory protein infected-cell protein 0 (ICP0) is a strong and global transactivator of both viral and cellular genes. In a previous study, we reported that ICP0 is highly phosphorylated and contains at least seven distinct phosphorylation signals as determined by phosphotryptic peptide mapping (D. J. Davido et al., J. Virol. 76:1077-1088, 2002). Since phosphorylation affects the activities of many viral regulatory proteins, we sought to determine whether the phosphorylation of ICP0 affects its functions. To address this question, it was first necessary to identify the regions of ICP0 that are phosphorylated. For this purpose, ICP0 was partially purified, and phosphorylation sites were mapped by microcapillary high-pressure liquid chromatography tandem mass spectrometry. Three phosphorylated regions containing 11 putative phosphorylation sites, all within or adjacent to domains important for the transactivating activity of ICP0, were identified. The 11 sites were mutated to alanine as clusters in each of the three regions by site-directed mutagenesis, generating plasmids expressing mutant forms of ICP0: Phos 1 (four mutated sites), Phos 2 (three mutated sites), and Phos 3 (four mutated sites). One-dimensional phosphotryptic peptide analysis confirmed that the phosphorylation state of each Phos mutant form of ICP0 is altered relative to that of wild-type ICP0. In functional assays, the ICP0 phosphorylation site mutations affected the subcellular and subnuclear localization of ICP0, its ability to alter the staining pattern of the nuclear domain 10 (ND10)-associated protein PML, and/or its transactivating activity in Vero cells. Only mutations in Phos 1, however, impaired the ability of ICP0 to complement the replication of an ICP0 null mutant in Vero cells. This study thus suggests that phosphorylation is an important regulator of ICP0 function.     

Physical interaction between the herpes simplex virus type 1 immediate-early regulatory proteins ICP0 and ICP4.

The herpes simplex virus type 1 immediate-early protein ICP0 enhances expression of a spectrum of viral genes alone and synergistically with ICP4. To test whether ICP0 and ICP4 interact physically, we performed far-Western blotting analysis of proteins from mock-, wild-type-, and ICP4 mutant virus-infected cells with in vitro-synthesized [35S]Met-labeled ICP0 and ICP4 as probes. The ICP4 and ICP0 polypeptides synthesized in vitro exhibited molecular weights similar to those of their counterparts in herpes simplex virus type 1-infected cells, and the in vitro-synthesized ICP4 was able to bind to a probe containing the ICP4 consensus binding site. Far-Western blotting experiments demonstrated that ICP0 interacts directly and specifically with ICP4 and with itself. To further define the interaction between ICP0 and ICP4, we generated a set of glutathione S-transferase (GST)-ICP0 fusion proteins that contain GST and either ICP0 N-terminal amino acids 1 to 244 or 1 to 394 or C-terminal amino acids 395 to 616 or 395 to 775. Using GST-ICP0 fusion protein affinity chromatography and in vitro-synthesized [35S]Met-labeled ICP0 and ICP4, ICP4 was shown to interact preferentially with the fusion protein containing ICP0 C-terminal amino acids 395 to 775, whereas ICP0 interacted efficiently with both the N-terminal GST-ICP0 fusion proteins and the C-terminal GST-ICP0 fusion proteins containing amino acids 395 to 775. Fusion protein affinity chromatography also demonstrated that the C-terminal 235 amino acid residues of ICP4 are important for efficient interaction with ICP0. Collectively, these results reveal a direct and specific physical interaction between ICP0 and ICP4.     

Association of ICP0 but not ICP27 with purified virions of herpes simplex virus type 1.

Recent studies have shown that ICP4, one of the major immediate-early proteins of herpes simplex virus type 1 is present within the tegument region of the virion (F. Yao and R. J. Courtney, J. Virol. 63:3338-3344, 1989). With monoclonal antibodies to two additional immediate-early proteins, ICP0 and ICP27, and Western blot (immunoblot) analysis, ICP0, but not ICP27, was also found to be associated with purified virus particles. In an effort to localize the ICP0 within the virion, purified virions were treated with trypsin in the presence and absence of detergent. The data suggest that ICP0 is located within the tegument region of the virion and is not localized in the envelope or within the nucleocapsid. The number of molecules of ICP0 per virion was estimated to be approximately 150.     

Identification of dominant-negative mutants of the herpes simplex virus type 1 immediate-early protein ICP0.

ICP0 is a 110,000-molecular-weight immediate-early protein of herpes simplex virus type 1 (HSV-1) which is encoded by three exons. It has been shown to function as a promiscuous transactivator of a variety of different HSV-1 and non-HSV-1 promoters in transient expression assays. Analysis of mutations which truncated the carboxy-terminal end of this 775-amino-acid (aa) protein demonstrated that a polypeptide which contained only aa 1 to 553 still possessed significant transactivation potential. Additional carboxy-terminal truncations which sequentially removed aa 245 to 553 and thus the remainder of the third exon resulted in the eventual loss of transactivation capability in these mutants. However, further analysis of these truncated derivatives demonstrated that they behaved as dominant-negative mutants to the wild-type polypeptide. Moreover, one of the mutants was found to act as a promiscuous repressor, in that it could dramatically inhibit a variety of HSV-1 promoters, non-HSV-1 promoters, and heterologous transactivator proteins in transient expression assays, despite having lost almost the entire third exon. These results indicate that a domain encoded by the first two exons probably interacts with, and can effectively titrate, the unknown cellular factor(s) through which ICP0 mediates transactivation.     

Interaction of herpes simplex virus 1 alpha regulatory protein ICP0 with elongation factor 1delta: ICP0 affects translational machinery.

The herpes simplex virus 1 (HSV-1)-infected cell protein 0 (ICP0) is a promiscuous transactivator, and by necessity, its functions must be mediated through cellular gene products. In an attempt to identify cellular factors interacting with ICP0, we used the carboxyl-terminal domain of ICP0 as "bait" in the yeast (Saccharomyces cerevisiae) two-hybrid system. Our results were as follows: (i) All 43 cDNAs in positive yeast colonies were found to encode the same translation factor, elongation factor delta-1 (EF-1delta). (ii) Purified chimeric protein consisting of glutathione S-transferase (GST) fused to EF-1delta specifically formed complexes with ICP0 contained in HSV-1-infected cell lysate. (iii) Fractionation of infected HEp-2 cells and immunofluorescence studies revealed that ICP0 was localized both in the nucleus and in the cytoplasm. In primary human foreskin fibroblasts, ICP0 was localized predominantly in the cytoplasm throughout HSV-1 infection even early in infection. (iv) Addition of the chimeric protein GST-carboxyl-terminal domain of ICP0 to the rabbit reticulocyte lysate in vitro translation system resulted in a dose-dependent decrease in protein synthesis. In contrast, GST alone or GST fused to the amino-terminal domain of ICP0 had no effect on the in vitro translation system. (v) The predominant forms of EF-1delta on electrophoresis in denaturing gels have apparent Mrs of 38,000 and 40,000. The higher-Mr form is a minor species in mock-infected cells, whereas in human fibroblasts and Vero cells infected with HSV-1, this isoform becomes dominant. These results indicate that ICP0 is present and may have a significant role in the cytoplasm of infected cells, possibly by altering the efficiency of translation of viral mRNAs.     

Varicella-zoster virus open reading frame 61 protein is functionally homologous to herpes simplex virus type 1 ICP0.

The varicella-zoster virus (VZV) open reading frame 61 (ORF61) protein is thought to be the homolog of herpes simplex virus type 1 (HSV-1) ICP0, based on gene location and limited amino acid homology. However, HSV-1 ICP0 trans activates HSV-1 genes, while VZV ORF61 protein trans represses the function of VZV trans activators on VZV promoters in transient expression assays. To investigate the functional relatedness of HSV-1 ICP0 and VZV ORF61 protein, we established Vero and MeWo cell lines which stably express VZV ORF61 under the control of a metallothionein promoter and performed complementation studies with an HSV-1 ICP0 deletion mutant (7134). Mutant 7134 is impaired for plaque formation and replication at a low multiplicity of infection in cell culture, but these defects were complemented by up to 200-fold in Vero cell lines expressing VZV ORF61. Likewise, the efficiency of plaque formation was improved by up to 100-fold in MeWo cell lines expressing VZV ORF61. A cell line expressing another VZV immediate-early gene product (ORF62) was unable to complement mutant 7134. HSV-1 mutants which are deleted for other HSV-1 immediate-early gene products (ICP4, ICP27) were unable to grow in VZV ORF61-expressing cell lines. These results indicate that, despite marked differences in their sequences and in effects on their cognate promoters in transient expression assays, VZV ORF61 protein is the functional homolog of HSV-1 ICP0.     

Cooperativity among herpes simplex virus type 1 immediate-early regulatory proteins: ICP4 and ICP27 affect the intracellular localization of ICP0.

The results of transient expression assays and studies of viral mutants have shown that three of the five immediate-early proteins of herpes simplex virus type 1 (HSV-1) perform regulatory functions, individually and cooperatively. As part of efforts designed to explore the molecular basis for the functional cooperativity among ICP0, ICP4, and ICP27 in the regulation of HSV gene expression, we have examined the intracellular localization of ICP0 in cells infected with ICP4 and ICP27 null mutant viruses by indirect immunofluorescence. Although ICP0 was localized predominantly to the nuclei of wild-type virus-infected cells, it was found exclusively in the nuclei of ICP27 mutant-infected cells and in both the cytoplasm and nuclei of ICP4 mutant-infected cells, the cytoplasmic component being especially strong. These observations indicate that both ICP4 and ICP27 can affect the intracellular localization of ICP0. Transient expression assays with plasmids that express wild-type and mutant forms of ICP0, ICP4, and ICP27 confirmed that ICP4 promotes and that ICP27 inhibits the nuclear localization of ICP0. These results confirm the observations made for mutant virus-infected cells and indicate that the localization pattern seen in infected cells can be established by these three immediate-early proteins exclusive of other viral proteins. The C-terminal half of ICP27 was shown to be required to achieve its inhibitory effect on the nuclear localization of ICP0. The region of ICP0 responsive to ICP27 was mapped to the C terminus of the molecule between amino acid residues 720 and 769. In addition, the concentration of ICP27 was shown to have a significant effect on the intracellular localization of ICP0. Because the major regulatory activities of ICP0, ICP4, and ICP27 are expressed in the nucleus, the ability of these three proteins collectively to determine their own localization patterns within cells adds a new dimension to the complex process of viral gene regulation in HSV.     

ICP0 prevents RNase L-independent rRNA cleavage in herpes simplex virus type 1-infected cells

The classical interferon (IFN)-dependent antiviral response to viral infection involves the regulation of IFN-stimulated genes (ISGs), one being the gene encoding cellular endoribonuclease RNase L, which arrests protein synthesis and induces apoptosis by nonspecifically cleaving rRNA. Recently, the herpes simplex virus type 1 (HSV-1) protein ICP0 has been shown to block the induction of ISGs by subverting the IFN pathway upstream of the 2'-5'-oligoadenylate synthetase (OAS)/RNase L pathway. We report that ICP0 also prevents rRNA degradation at late stages of HSV-1 infection, independent of its E3 ubiquitin ligase activity, and that the resultant rRNA degradation is independent of the classical RNase L antiviral pathway. Moreover, the degradation is independent of the viral RNase vhs and is independent of IFN response factor 3. These studies indicate the existence of another, previously unidentified, RNase that is part of the host antiviral response to viral infection.     

A pre-immediate-early role for tegument ICP0 in the proteasome-dependent entry of herpes simplex virus

Herpes simplex virus (HSV) entry requires host cell 26S proteasomal degradation activity at a postpenetration step. When expressed in the infected cell, the HSV immediate-early protein ICP0 has E3 ubiquitin ligase activity and interacts with the proteasome. The cell is first exposed to ICP0 during viral entry, since ICP0 is a component of the inner tegument layer of the virion. The function of tegument ICP0 is unknown. Deletion of ICP0 or mutations in the N-terminal RING finger domain of ICP0 results in the absence of ICP0 from the tegument. We show here that these mutations negatively influenced the targeting of incoming capsids to the nucleus. Inhibitors of the chymotrypsin-like activity of the proteasome the blocked entry of virions containing tegument ICP0, including ICP0 mutants that are defective in USP7 binding. However, ICP0-deficient virions were not blocked by proteasomal inhibitors and entered cells via a proteasome-independent mechanism. ICP0 appeared to play a postpenetration role in cells that supported either endocytosis or nonendosomal entry pathways for HSV. The results suggest that ICP0 mutant virions are defective upstream of viral gene expression at a pre-immediate-early step in infection. We propose that proteasome-mediated degradation of a virion or host protein is regulated by ICP0 to allow efficient delivery of entering HSV capsids to the nuclear periphery.     

Intragenic complementation of herpes simplex virus ICP8 DNA-binding protein mutants.

The major DNA-binding protein, or infected-cell protein 8 (ICP8), of herpes simplex virus is required for viral DNA synthesis and normal regulation of viral gene expression. Previous genetic analysis has indicated that the carboxyl-terminal 28 residues are the only portion of ICP8 capable of acting independently as a nuclear localization signal. In this study, we constructed a mutant virus (n11SV) in which the carboxyl-terminal 28 residues of ICP8 were replaced by the simian virus 40 large-T-antigen nuclear localization signal. The n11SV ICP8 localized into the nucleus and bound to single-stranded DNA in vitro as tightly as wild-type ICP8 did but was defective for viral DNA synthesis and viral growth in Vero cells. Two mutant ICP8 proteins (TL4 and TL5) containing amino-terminal alterations could complement the n11SV mutant but not ICP8 gene deletion mutants. Cell lines expressing TL4 and TL5 ICP8 were isolated, and in these cells, complementation of n11SV was observed at the levels of both viral DNA replication and viral growth. Therefore, complementation between n11SV ICP8 and TL4 or TL5 ICP8 reconstituted wild-type ICP8 functions. Our results demonstrate that (i) the carboxyl-terminal 28 residues of ICP8 are required for a function(s) involved in viral DNA replication, (ii) this function can be supplied in trans by another mutant ICP8, and (iii) ICP8 has multiple domains possessing different functions, and at least some of these functions can complement in trans.