No abstract

The biology and pathogenesis of hepatitis E virus are poorly understood due to the lack of an in vitro culture or infection models. The viral Orf3 protein activates the cellular mitogenactivated protein kinase pathway and is likely to modulate the host cell environment for efficient viral replication. We screened for cellular genes whose transcription was differentially up-regulated in an Orf3-expressing stable cell line (ORF3/ 4). The gene for mitochondrial voltage-dependent anion channel (VDAC) was one such candidate. The up-regulation of VDAC in ORF3/4 cells was confirmed by Northern and Western blotting in various cell lines. Transfection of ORF3/4 cells with an ORF3-specific small interfering RNA led to a reduction in VDAC protein levels. VDAC is a critical mitochondrial outer membrane protein, and its overexpression results in apoptosis. Surprisingly, Orf3-expressing cells were protected against staurosporine-induced cell death by preservation of mitochondrial potential and membrane integrity. A small interfering RNA-mediated reduction in Orf3 and VDAC levels also made cells sensitive to staurosporine. Chemical cross-linking showed Orf3-expressing cells to contain higher levels of oligomeric VDAC. These cells also contained higher levels of hexokinase I that directly interacted with VDAC. This interaction is known to preserve mitochondrial potential and prevent cytochrome c release. We report here the first instance of a viral protein promoting cell survival through such a mechanism.

The influence of Zn-complexes with biologically active ligands like aminoacids: picolinic acid as Zn(pic)2 and asparaginic acid as Zn(asp)2 on HSV-1 infection in vitro was investigated. Studies on kinetics of Zn-ions using labeled 65Zn(pic)2 as a marker, show that at the 45th min Zn-ions are exposed onto or into cells. When cells were infected 60min after the treatment with 65Zn(pic)2, 1h later the activity of Zn-ions in the cell fraction decreased while in the medium increased. This result shows that soon after the infection cells expelled part of their Zn. If Zn(pic)2 or Zn(asp)2 are added at the end of the adsorption period, ICP4 and ICP8 are localized in the cytoplasm but not in the nucleus of infected cells, while the synthesis of gH is decreased. A sharply increased number of nucleocapsids with low electron density cores was also found.

We have found that when copper, zinc or cobalt is bound to a suitable ligand, the appropriate complex exhibited a significant anti-HSV effect (Varadinova et al., 1993; 1996). Recently published data by Sagripanti et al. (1997) also show that the inhibition of HSV by copper was enhanced by reducing agents and that mechanism of the inactivation is similar as for copper-mediated DNA damage (Aruoma, et al. 1991; Dizdaroglu, et al., 1991; Toyokuni and Sagripanti, 1994). Therefore it was interesting to study the efect of Cu(ll) coordination compounds with acyclovir (ACV) on the replication of HSV in cultured cells. The experiments on cytotoxicity as well as on the activity of three different Cu-ACV complexes [Cu(ACV)2Cl2(H2O)2] = (A); [Cu(ACV)2(H2O)3](NO3)2.H2O = (B) and [Cu(ACV)2(H2O)2](NO3)2] = (C) towards virus replication, with special attention on the growth of ACV-resistant strain R-100 were performed on MDBK cells. ACV was used as a reference compound. The following results were obtained: 1) Increased cell’s viability in the presence of 20-40(g/ml ACV and decreased one in the presence of Cu-ACV complexes with relative level (A) >> (B) > (C); 2) Cu-ACV complexes are more cytotoxic than the ligand - ACV and the relative level is (C)>(B)>(A); 3) The anti-HSV effect of ACV can be modulated by copper at levels depending on the specificity of the particular virus strain: (i) for the ACV sensitive strain DA (HSV-1) - ACV ((A) > (C) > (B); (ii) for the ACV sensitive strain Bja (HSV-2) (A) > ACV > (C) > (B); (iii) for strain R-100 (ACVR, TKa) - (A) > ACV > (C) > (B). This findings are consistent with previously published data and undoubtedly show that Cu-ACV complexes could be useful in the treatment of HSV infections, especially when the causative agent is a resistant to ACV mutant.

Our previous results show that Zn(pic)2 and Zn(asp)2 inhibit key steps of the replication of HSV-1. Anti-HSV effect of complexes of Co(II) with aminoacids Lys and Ser was also found. In the present study we describe the effect of complexes of Zn(II), Co(II) and Cu(II) with D-aminosugars on the replication of HSV-1 and on the infectivity of free virions. The experiments were done using primary rabbit kidney cells (r.k.), diploid human embryonal fibroblasts (F) and Vero cells. No differences in the toxicity of metal complexes on diploid cells- r.k. and F, were found. Neither metal complexes, nor ligands-galactosoxime and glucosoxime, influenced the viral replication. During 1-4h prolonged contact only Cu(Gl.NOH)2 inactivated HSV-1 virions up to 90%. The results show that D-aminosugars are not suitable ligands for Zn(II), Cu(II) and Co(II) in respect of the inhibition of viral replication. However, only Cu(Gl.NOH)2 was able to inhibit the infectivity of free virions.