Effect of Complexes of Zinc, Cobalt and Copper With D-Aminosugars on the Replication of Herpes Simplex Virus Type 1 (HSV-1)

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.


Introduction
Among metals essential for life and according to the amount in the human body, z!nc is the second most abundant trace metal.after the iron (1_-4). The p_hysiological functions of zinc_ are primarily related to its presence in the actiye site of_up tO 200 metalloenzymes (5). Apart from the presence of cobaltas a c_onstituent Of vitamin B, this metal also influences many other physiologi_cal and enzymati_c functions (1)(2)(3)6). The copp.er, being an essential trace metal, s a part of the active site Of at least 60 metalloenzymes (2,3,7). A specific compartment within the cell predetermines the_ activity of the pa_rticular metal ion.
Thus, zinc acts ma!nly in the cytoplasm. Small amounts of zinc participate also !n .the nucleus as a constituent .Of proteins involved in th.e regulation and the expression of the genome (2,3). Cobalt realises its activity only in the cytoplasm, while copper acts in extracellular aCe (3). reover, during the regulation of a wide range of metabolic, proc_esses these metals interact with each other and their main characteristic, as well as that of their compounds, is their extremely low toxicity, for humans (6,8).
Because of the significant role of the ligand in the expression of the activity of a particular metal ion, we deciled to study the effect of aminoderivatives of D-glucose and D-galactose, as. well as of their complexes with Zn(ll), Cu(ll) and Co.(ll) on HSV-1 infection in cell cultures. These. experiments are a part of a large =nvestigation on the role of complexes of the above metal ions with different I:iioligands on HSV-1 replication (9-11).

Materials and Methods
Virus and cells. HSV-1, strain Victoria; primary rabb!t kidney cells (r.k.), diploid human embryonal fibroblasts (F), and Vero cells were used in the experiments. Ligand$. Aminoderivatives of D-glucose glucosoxime (GI.NOH), and of D-galactose galactosoxime (Ga.NOH), were used as ligands of Zn(ll), Co(ll) and Cu(ll). Maximal nontoxic concentration (MNC). Cells from mon_olayers were washed and covered with media modified with different concentrations of metal complexes or ligands. Microscopically, degeneration of monolayers and changes of cell morphology were investigated from the 24th till the 96th h. Samples of cells grown in nonmodified medium servedas a control. Each exp.eriment was duplicated. The highest concentration in the presence of which and during the whole period of investigation the morphology of the cells and monolayers was similar to that in the control, is recognsed as MNC.
Vol. 4, No. 1,1997 Effect of Complexes of Zinc, Cobalt and Copper with D-Aminosugars on the Replication of Herpes Simplex v Virus Type 1 (HSV-1) Infectious virus titre. Cells from confluent monolayers grown in 96 well plates were infected with ten-fold dilutions of HSV-I. Infectious virus titre was determined at the 48th h after culturing at 37 C by Reed and Muench (12). Effect of. metal complexes on the replication of HSV-1. Experiments were done in mu!ticycle growth conditions. Confluent cell monolayers from 96 well plates were washed and infected with ten-fold dilutions of HSV-I. After lh for adsorption, cells were covered with m_edia modified with different concentrations of metal complexes and of ligands. One set of infected cells served as untreated control. The effect on the replication of HSV-1 was determined at the 48th h after culturing at 37_C by reduction of nfectious virus titres as compared to that in the control.
Effect of metal complexes on extracellular (free) HSV-1 virions virucidal effect.
Equal volumes of HSV-1 stock containing 100 pfu/0.1 ml and medium modified with the appropriate metal complex or the li.gand, in MNC, were incubated at 37 C for_ 15min, 30min, lh, 2h, 4h, 6h and 12h. The infectious titre of each sample was determined on the 48th h.
Data were compared to those of viral control equal volumes of HSV-1 stock and nonmodified medium incubated as described above. Results the data presented in table 1. show that the metal complexes are 1000 times more toxic an the ligand. It is interesting to note a specific response of Vero cells after the action with Co(ll) complexes. Thus, Co(Ga.NOH) is 10 times more toxic (MNC 0,01 IM) than Co(GI.NOH) (MNC 0,1 li). Table 1. Summarised data on the toxicity of complexes of Zn(ll), Co(ll) and Cu(ll) and of Daminosugars in r.k., F and Vero cells.. No differences in the toxicity of the metal complexes on diploid cells -_ r.k. and F, were found.
In order to obtain comparative data, experiments on the effect of metal complexes and !igands on HSV-1 infection were done on diploid cells. Neither metal complexes, nor ligands Ga.NOH and GI.NOH, influenced the replication of HSV-1 in r.k. and in F cells (data not shown).

Discussion
In 1970 Spear et al. (13,14) have shown that within 4-5 h after HSV infection the only apparent protein glycosilation taking place in_the infected cells is that of virus-specific p.roteins. The _result is the deposition in infected cell membranes of virus-specific g!ycoproteins. On the .other hand, in 1976 Brennan et al. (15) have_ reported that HSV. g!ycoproteins presented in the !nfected cells are not suitable substrates for the attachment of glucose units..They suggest that in HSV infected cells enhanced glycolysis or abnormal compartmentalisation _ofnucleot!de sugar pools isprobably involved. On the other hand, 2deoxy-D-glucose interfere with the synthesis of HSV glycoproteins. As a result,production of infectious virus progeny and the fusion between cells are markedly inhibited (12,(16)(17)(18)(19). The present data show that aminoderivat!ves .of D-glucose (GI.NOH) and of D-galactose (Ga.NOH) are _1000 times less toxic than the appropriate metal complex MNC 10 M. There is no doubt that the low toxicity of GI.NOH and of Ga.NOH is due to the fact tha.t these sugars, being natural products, drectly participate in the normal cell metabolic pathways and must be well tolerated by cells in vi_tro, as well.  (11). However, in order to inhibit HSV-1 replication within the host cell, complexes of 3?
Vol. 4, No. 1,1997 Effect of Complexes of Zinc, Cobalt and Copper with D-Aminosugars on the Replication of Herpes Simplex v Virus Type 1 (HSV-1) Zn(ll) with picolinic and with asp_artic acids are recommended (9).. In addition, the latter complexes.posses virucidal activity towards VZV virions, which is due to the virus-specific properties (11 ).