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.     

The herpes simplex virus type 1 alpha protein ICP27 can act as a trans-repressor or a trans-activator in combination with ICP4 and ICP0

The herpes simplex virus type 1 (HSV-1) alpha proteins ICP4, ICP0, and ICP27 are trans-acting proteins which affect HSV-1 gene expression. To investigate potential interactions between these alpha products and to determine the specificity of action of the alpha proteins in combination with each other compared with their activities individually, we performed a series of transient-expression assays. In these assays we used plasmids containing the alpha genes encoding ICP4, ICP0, and ICP27 either singly or in combination as effectors and HSV-1 genes of different kinetic classes and heterologous genes as targets. The HSV-1 targets consisted of promoter-regulatory domains from alpha (ICP0 and ICP27), beta (thymidine kinase and alkaline exonuclease), beta-gamma (glycoprotein D, glycoprotein B, and VP5), and gamma (glycoprotein C) genes, each fused to the chloramphenicol acetyltransferase (CAT) gene. The heterologous target genes consisted of the simian virus 40 early promoter with enhancer and the Rous sarcoma virus long terminal repeat promoter and enhancer each fused to the CAT gene. Target promoter activity was measured by the assay of CAT activity in extracts of transfected cells and by Northern (RNA) blot hybridization of CAT mRNA. The results of these experiments showed that ICP4 activated only HSV-1 target genes, whereas ICP0 activated all of the targets and ICP27 had little effect on any of the targets. ICP4 and ICP0 had a synergistic effect when inducing HSV-1 targets, but they did not have this effect on the heterologous targets pSV2-CAT or pRSV-CAT. In fact, lower levels of CAT activity and CAT mRNA were found in the presence of both effectors than with ICP0 alone. Most interestingly, although the effector plasmid containing the ICP27 gene had little effect on its own, two different and marked effects depending on the target were observed when ICP27 was combined with ICP4 or ICP0 or both. A trans-repression of the induction seen with ICP4 and ICP0 was found when ICP27 was present in the transfections with pSV2-CAT, pRSV-CAT, pICP0-CAT, pICP27-CAT, pTK-CAT, pgD-CAT, pgB-CAT, and pgC-CAT. This resulted in CAT activity levels which were similar to or lower than the basal level of expression of the target genes in the absence of effector plasmids. This trans-repression occurred over a wide range of concentrations of input ICP27 plasmid. In contrast to this repressive effect of ICP27, a trans-activation was seen when ICP4, ICP0, and ICP27 plasmids were combined in transfections with pAE-CAT and pVP5-CAT as targets. This trans-activation also occurred over a 10-fold range of input ICP27 plasmid. These results suggest that ICP27 can facilitate both down     

Overexpression of the herpes simplex virus type 1 immediate-early regulatory protein, ICP27, is responsible for the aberrant localization of ICP0 and mutant forms of ICP4 in ICP4 mutant virus-infected cells.

ICP0 and ICP4 are immediate-early regulatory proteins of herpes simplex virus type 1. Previous studies by Knipe and Smith demonstrated that these two proteins are characteristically observed in the nuclei of wild-type virus-infected cells but predominantly in the cytoplasms of cells infected with several ICP4 temperature-sensitive (ts) mutant viruses at the nonpermissive temperature (NPT) (D. M. Knipe and J. L. Smith, Mol. Cell. Biol. 6:2371-2381, 1986). Consistent with this observation, it has been shown previously that ICP0 is present predominantly in the cytoplasms of cells infected with an ICP4 null mutant virus (n12) at high multiplicities of infection and that the level of ICP27, a third viral regulatory protein, plays an important role in determining the intracellular localization of ICP0 (Z. Zhu, W. Cai, and P. A. Schaffer, J. Virol. 68:3027-3040, 1994). To address whether the cytoplasmic localization of ICP0 is a common feature of cells infected with all ICP4 mutant viruses or whether mutant ICP4 polypeptides, together with ICP27, determine the intracellular localization of ICP0, we used double-staining immunofluorescence tests to examine the intracellular staining patterns of ICP0 and ICP4 in cells infected with an extensive series of ICP4 mutant viruses. In these tests, compared with the localization pattern of ICP0 in wild-type virus-infected cells, more ICP0 was detected in the cytoplasms of cells infected with all ICP4 mutants tested at high multiplicities of infection. Each of the mutant forms of ICP4 exhibiting predominantly cytoplasmic staining contains both the nuclear localization signal and the previously mapped ICP27-responsive region (Z. Zhu and P. A. Schaffer, J. Virol. 69:49-59, 1995). No correlation between the intracellular staining patterns of ICP0 and mutant forms of ICP4 was demonstrated, suggesting that mutant ICP4 polypeptides per se are not responsible for retention of ICP0 in the cytoplasm. This observation was confirmed in studies of cells cotransfected with plasmids expressing ICP0 and mutant forms of ICP4, in which the staining pattern of ICP0 was not changed in the presence of mutant ICP4 proteins. Studies of cells infected at low multiplicities with a variety of ICP4 ts mutant viruses at the NPT showed that both ICP0 and ts forms of ICP4 were localized predominantly within the nucleus. These observations are a further indication that the aberrant localization of the ts forms of ICP4 at the NPT is not a direct result of specific mutations in the ICP4 gene. In the final series of tests, the localization of ICP0 in cells infected with a double-mutant virus unable to express either ICP4 or ICP27 was examined. In these tests, ICP0 was detected exclusively in the nuclei of Vero cells but in both the nuclei and the cytoplasms of ICP27-expressing cells infected with the double mutant. These results demonstrate that ICP27, rather than the absence of functional ICP4, is responsible for the cytoplasmic localization of ICP0 in ICP4 mutant virus-infected cells. Taken together, these findings demonstrate that the aberrant localization of ICP0 and certain mutant forms of ICP4 in cells infected with ICP4 mutant viruses is mediated by high levels of ICP27 resulting from the inability of mutant forms of ICP4 to repress the expression of ICP27.     

Current and historical patterns of heavy metals pollution in Estonia as reflected in natural media of different ages: ICP Vegetation,ICP Forests and ICP Integrated Monitoring data

In order to characterise current and historical pattern of heavy metal (HM) pollution in Estonia, this article will compare the concentrations and stocks of Cd, Cr, Cu, Ni, Pb and Zn represented in current deposition (data from 18 local precipitation stations) with natural media of three different ages: 1–3-year-old moss carpet (ICP Vegetation moss survey data from 99 open area plots), 3–5-year-old litter layer, and several-decades-old organic layer (mor humus) of coniferous forest, in mostly podzolic soils (ICP Forest soil survey data, 75 stands).Objectives of this study are (1) to assess differences in HM retention and accumulation in various aged media of coniferous stands (2) to estimate territorial differences in current HM distribution and previously accumulated concentrations and stores of HM (3) to compare territorial distribution of HM concentration in Estonia between five different regions: N-W; N-E; S-W; S-E and Western insular region, whereas the local oil shale industry in N-E part of Estonia has been the main source of HM pollution over a long period of time and therefore may have an effect on HM regional distribution.Comparing the studied media, three types of HM retention patterns were detected: (1) for Cu, Ni, Cr (2) for Cd, Pb, (3) for Zn. The mean current level of HM deposition in Estonia is low comparison to previous decades, especially the 1980s. The effect of the previously significantly higher exposure of HM emissions and deposition is preserved in older part of soil organics (OF), where the highest stocks and concentrations of HMs (with the exception of Zn) are currently found. The HM proportions in fly ash of oil shale and in OF layer of soil were very similar with regards to Ni and Cr—indicating their origin from the oil shale industry in the N-E region. According to spatial distribution analysis, the greatest accumulated storages of Ni and Cr in OF layer of coniferous forest soils are characteristic to S-W Estonia.     

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.     

Prolonged gene expression and cell survival after infection by a herpes simplex virus mutant defective in the immediate-early genes encoding ICP4, ICP27, and ICP22.

Very early in infection, herpes simplex virus (HSV) expresses four immediate-early (IE) regulatory proteins, ICP4, ICP0, ICP22, and ICP27. The systematic inactivation of sets of the IE proteins in cis, and the subsequent phenotypic analysis of the resulting mutants, should provide insights into how these proteins function in the HSV life cycle and also into the specific macromolecular events that are altered or perturbed in cells infected with virus strains blocked very early in infection. This approach may also provide a rational basis to assess the efficacy and safety of HSV mutants for use in gene transfer experiments. In this study, we generated and examined the phenotype of an HSV mutant simultaneously mutated in the ICP4, ICP27, and ICP22 genes of HSV. Unlike mutants deficient in ICP4 (d120), ICP4 and ICP27 (d92), and ICP4 and ICP22 (d96), mutants defective in ICP4, ICP27, and ICP22 (d95) were visually much less toxic to Vero and human embryonic lung cells. Cells infected with d95 at a multiplicity of infection of 10 PFU per cell retained a relatively normal morphology and expressed genes from the viral and cellular genomes for at least 3 days postinfection. The other mutant backgrounds were too toxic to allow examination of gene expression past 1 day postinfection. However, when cell survival was measured by the capacity of the infected cells to form colonies, d95 inhibited colony formation similarly to d92. This apparent paradox was reconciled by the observation that host cell DNA synthesis was inhibited in cells infected with d120, d92, d96, and d95. In addition, all of the mutants exhibited pronounced and distinctive alterations in nuclear morphology, as determined by electron microscopy. The appearance of d95-infected cells deviated from that of uninfected cells in that large circular structures formed in the nucleus. d95-infected cells abundantly expressed ICP0, which accumulated in fine punctate structures in the nucleus at early times postinfection and coalesced or grew to the large circular objects that were revealed by electron microscopy. Therefore, while the abundant accumulation of ICPO in the absence of ICP4, ICP22, and ICP27 may allow for prolonged gene expression, cell survival is impaired, in part, as a result of the inhibition of cellular DNA synthesis.