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In vitro inhibition of human herpesvirus-6 by phosphonoformate
Journal of Virological Methods, 21 (1988) 301-304 Elsevicr
30 1
JVM 00785
In vitro inhibition of human herpesvirusphosphonoformate H.Z. Streicher’,
by
C.L. Hung’, D.V. Ablashi’, K. Hellman’, C. Saxinger’, J. Fullen’ and S.Z. Salahuddin’
‘National Camw Insrituie, Nationul Institures of Health, BerheAda. Maryland. U.S. A. atld ‘Elecwonucleonics IIIC.. Silvc~rSprirlg. Marylatld. U.S. A.
Summary HSB-2 cell cultures productively infected with human herpesviruswere treated with the antiviral drugs phosphonoformic acid (PFA), acyclovir (ACV), and gancyclovir (DHPG). ACV and DHPG showed significant toxic effects on uninfected HSB-2 cells. yet only incompletely inhibited viral expression upon infection of the cells. PFA, however, showed little direct toxicity on HSB-2 cells while viral replication was inhibited significantly. Human clovir
herpesvirus-6;
Antiviral
drug;
Phosphonoformic
acid; Acyclovir;
Gancy-
Introduction Human B-lymphotropic virus was isolated from the peripheral blood of six patients with AIDS or lymphoproliferative diseases (Salahuddin et al., 1986). The virus neither crossreacts nor hybridizes with other human herpesviruses. Although the virus may share various biologic features with several of them and limited homology to CMV has been reported, it is a unique member of the herpes virus group. It is enveloped, approximately 200 nm in size with icosahedral capsid symmetry, demonstrates a nuclear maturation pattern, and contains a large size double-
Correspondence to: H.Z. Streicher. thesda. MD 20892. U.S.A.
National
Cancer
Institute.
National
Institutes
of Health.
Be-
302
stranded DNA gcnome (~iberf~ld et al.. 1987). In culture the virus is cytopathic with easily recognized CPE forming large rcfractile or ‘juicy’ appearing cefls prior to viral production and cell death. Viral genetic material has been identified in the tumor tissue of three patients with B-cell lymphomas. Recently in vitro systems have been established that support viral infection in fresh ‘T-lymphocytes as well as human T-cell, B-ceil, ghal, and me~akar~ocytic cefl lines (Abtashi et al.. 19X8; Lusso et at., IoN). One of these celi lines, HSB-2. has been used rout~neIy to efficiently propagate virus. Serologic surveys of normal populations indicate that antibody prevalence by immunofhtorescent assay (IFA) is 20~40% of healthy adults in the United States. Similar to other h~rpesvir~ses. infection in childh~~od is a likely possibility. The clinical syndromes of mild febrile illness and rose&form rash have been suggested to be associated with initial infection in childhood. A group of diseases with increased antiviral titers and broadly characterized as ‘I~mphoproliferative’ has been shown in serologic surveys of viral prevalence. The marked ahitity of the virus to infect and kill T-cells along with the frequency of infection suggest a possible role for this virus in the acquired immune deficiency syndrome. The broadened celi tropism and i~enti~c~~ti~?nas a herpesvirus has led to the proposai that the virus can be taxon~~rnic~~lf~idcntihed as human herpesvirus(HHV-A). Although a disease associated with HI-IV-6 has not yet been identified. we have begun testing a number of anti-viral drugs for in vivo inhibition of HHV-6 to characterize biologic features that may be useful as a guide to ph~~rmacologic imervention.
Methods
A productive viral infection was established in target cells utilizing cell free virus concentrated on a sucrose gradient. Fresh HSB-2 cells. Cells were cultured at a IO”:mt in 75 ml flasks with RPM1 and IO% fetal calf serum. penjci~lin. streptomycin, and g~~ltarninc at 37°C in 5% CO,. In order to test the protective effect of a broad spectrum antiherpes drug 20 @g/ml of phosphonoformic acid (PFA), Sigma, were added to one flask at the same time as virus. Additional cultures were established with HSB-2 cells with and without PFA. Cells were kept periodicalIy observed and counted on day 7 and 14. and examined by IFA for the presence of virus using human sera as previously described on day 14. Acyctovir (ACV). 3urroughs-welcome. and gancyciovir (DWPG). Syntex, were added to HSB-2 cells either 4 h or 3 days after the addition of virus. Each drug was added at the concentration indicated in Table 1 and included at the same concentration when cultures were fed with additional fresh media twice a week. Viral infection of HSB-2 cells was accomplished by incubation of 2x 10’ cells with HBLVcultured cell-free supernatant containing approximately 10” infectious virus particles/ml for 4 h at 37°C. Infected cells were spun down and resuspended in 5 ml RPM1 with 10% FCS. Viable cells/ml and % IFA-positive cells are reported.
303
1
TABLE Effect
of nucleoside
Acyclovir
Gancyclovir
analogues
on infection
Treatment
Control no virus
no drug 50 &ml 80 (*g/ml no drug no drug 80 kg/ml 1.50 kg/ml no drug
1.4x 106 4.7x 105 2.89x lo5 2x 106 7x104 8x10”
of HSB-2
cells
Infected
cells*
4 h post infection
3 days
3.6~ 1V 3x lo’ (10%) 3x 10’ 1.3XlO’(
(10%) (10%)
1.2x 10” 1 x 10s (80%)**
(80%) (80%)
1 x 105 (30%) 8~10~ (1%) 1.2~10~
* The number of viable cells and (%) positive by IFA are given for cells exposed to virus for either four hours or three days before addition of drug at the concentration indicated starting at 2~ 10s cells per ml. **The number and (%) IFA positive cells are given for cells exposed to virus alone (without drug).
Results and Discussion Acyclovir (ACV) and gancyclovir (DHPG) are purine nucleosides with anti-viral activity. In order to be active pharmacological agents, they must be in the triphosphate form. Some viruses such as herpes simplex types 1 and 2 and varicellazoster virus have a specific aviral thymidine kinase which effectively phosphorylates ACV. DHPG, in addition, is phosphorylated effectively in cells infected with all of the herpesviruses and in particular is effective in vitro in cytomegalovirusinfected cells (Hermans and Cockerill, 1987; Robins and Revankar, 1988). Under the experimental conditions described, both drugs showed significant toxic effects on uninfected HSB-2 cells (Table 1). Uninfected cells appeared to have a doubling time of 2-3 days. DHPG may significantly interfere with lymphocyte proliferation at the levels used in vitro in this experiment (Bowden et al., 1987). However, even at levels that are toxic to target cells, only incomplete inhibition of virus expression was achieved. The toxic effect of both virus and drug did not appear to be additive. While the drugs may have some protective effect at the highest concentrations, their action does not appear to be selective. ACV similarly appears to have little protective effect in this experimental system. It is possible that this virus lacks a tk gene, or that the tk gene is not efficient in phosphorylating the drugs, or the cell line is not efficient in utilizing the monophosphate. Alternatively, the viral polymerase may be relatively resistant in binding the drug. It will be of interest to retest these drugs in other systems and to consider possible combinations that may be more selective and less toxic. Phosphonoformate does not require metabolic modification in order to become an active drug. Its mechanism of action is similar to PAA (phosphonoacetic acid). Both inhibit the replication of various herpesviruses by selective inhibition of viral DNA polymerase (Roberts, 1988). PFA added to culture HSB-2 cells showed lit-
304 TABLE Effect
2 of PFA on infection
of HSB-2
tells by HBLV
Treatment
Cell number/ml*
IFA oobitive
Control
2x 10” 7 x IOi l.XXlO” 1.6X 10”
0 65 0 0
HBLV HBLV+PFA PFA * The number
of viable cells and Ci positive by IFA
were counted
on day
C;
14 following
exposure
IO
virus.
tle toxicity at the 20 pgiml concentration used in this experiment while providing a striking protective effect against both the cytopathic effect of the virus and the expression of viral antigens detected by the indirect IFA (Table 2). The effect of PFA on the cycle of viral infection will require further analysis in order to determine dose response and the time course of inhibition. In vitro, foscarnet inhibits HIV infection and directly inhibits retroviral reverse transcriptase in a cell free system (Sarin et al., 1985). The drug has been tested in several clinical trials and the serum levels attained by constant intravenous transfusion appears to be well above effective in vitro levels. However, bone marrow suppression may limit human use. Although the nucleoside analogues were not effective in this system, suggesting that the virus will not be susceptible to these drugs in vivo, further evaluation should be done for both these and other compounds.
References Ablashi. D., Lusso, P.. Hung, S. et al. (1988) Utilization of human hematopoietic cell line for the propagation. detection, and characterization of HBLV [human herpesvirus-6). Int. J. Cancer, in press. Biberfeld. P., Karmarsky, S., Salahuddin, S.Z. and Gallo, R.C. (1987) Ultrastructural characterization of a new human B lymphotropic DNA virus (HBLV) isolated from patients with iymph~proliferative disease. J. Natl. Cancer Inst. 79, 933-931. Bowden. R., Digcl, J., Reed. E. and Meyers, J. (1987) Immunosuppressive effects of gancyclovir on in vitro lymphocyte responses. J. Infect. Dis. 156, 899-903. Hermans, P. and Cockerill, F. (1987) Antiviral agents. Mayo Clin. Proc. 62. 1108-1115. Lusso, P., ~~arkham, P.. Tschachler. E. et al. (1988) In vitro cellular tropism of human B-lymphotropic virus (human herpesvirus-6). J. Exp. Med. 167. 1659-1670. Roberts. S. (1988) Design of anti-viral agents other than nucleoside analogues. In: De Clercq and Walker (Eds.), Antiviral Drug Development, Plenum Press. New York. Robins, R. and Revankar (198X) Design of nucleoside analogs as potential antiviral agents. In: De Clercq and R.T. Walker (Eds.). Antiviral Drug Development. Plenum Press. New York. Salahuddin. S.Z.. Ablashi, P.. Markham. P.D. et al. (1986) Isolation of a new virus. HBLV, in patients with lymphoproliferative disorders. Science 234. 596660. Sarin, P.S., Taguchi, Y., Sun. D. et al. (1985) Preliminary communication: inhibition of HTLV-IIIILAV replication by foscarnet. Biochem. Pharmacol. 34. 407S-Uf79.
30 1
JVM 00785
In vitro inhibition of human herpesvirusphosphonoformate H.Z. Streicher’,
by
C.L. Hung’, D.V. Ablashi’, K. Hellman’, C. Saxinger’, J. Fullen’ and S.Z. Salahuddin’
‘National Camw Insrituie, Nationul Institures of Health, BerheAda. Maryland. U.S. A. atld ‘Elecwonucleonics IIIC.. Silvc~rSprirlg. Marylatld. U.S. A.
Summary HSB-2 cell cultures productively infected with human herpesviruswere treated with the antiviral drugs phosphonoformic acid (PFA), acyclovir (ACV), and gancyclovir (DHPG). ACV and DHPG showed significant toxic effects on uninfected HSB-2 cells. yet only incompletely inhibited viral expression upon infection of the cells. PFA, however, showed little direct toxicity on HSB-2 cells while viral replication was inhibited significantly. Human clovir
herpesvirus-6;
Antiviral
drug;
Phosphonoformic
acid; Acyclovir;
Gancy-
Introduction Human B-lymphotropic virus was isolated from the peripheral blood of six patients with AIDS or lymphoproliferative diseases (Salahuddin et al., 1986). The virus neither crossreacts nor hybridizes with other human herpesviruses. Although the virus may share various biologic features with several of them and limited homology to CMV has been reported, it is a unique member of the herpes virus group. It is enveloped, approximately 200 nm in size with icosahedral capsid symmetry, demonstrates a nuclear maturation pattern, and contains a large size double-
Correspondence to: H.Z. Streicher. thesda. MD 20892. U.S.A.
National
Cancer
Institute.
National
Institutes
of Health.
Be-
302
stranded DNA gcnome (~iberf~ld et al.. 1987). In culture the virus is cytopathic with easily recognized CPE forming large rcfractile or ‘juicy’ appearing cefls prior to viral production and cell death. Viral genetic material has been identified in the tumor tissue of three patients with B-cell lymphomas. Recently in vitro systems have been established that support viral infection in fresh ‘T-lymphocytes as well as human T-cell, B-ceil, ghal, and me~akar~ocytic cefl lines (Abtashi et al.. 19X8; Lusso et at., IoN). One of these celi lines, HSB-2. has been used rout~neIy to efficiently propagate virus. Serologic surveys of normal populations indicate that antibody prevalence by immunofhtorescent assay (IFA) is 20~40% of healthy adults in the United States. Similar to other h~rpesvir~ses. infection in childh~~od is a likely possibility. The clinical syndromes of mild febrile illness and rose&form rash have been suggested to be associated with initial infection in childhood. A group of diseases with increased antiviral titers and broadly characterized as ‘I~mphoproliferative’ has been shown in serologic surveys of viral prevalence. The marked ahitity of the virus to infect and kill T-cells along with the frequency of infection suggest a possible role for this virus in the acquired immune deficiency syndrome. The broadened celi tropism and i~enti~c~~ti~?nas a herpesvirus has led to the proposai that the virus can be taxon~~rnic~~lf~idcntihed as human herpesvirus(HHV-A). Although a disease associated with HI-IV-6 has not yet been identified. we have begun testing a number of anti-viral drugs for in vivo inhibition of HHV-6 to characterize biologic features that may be useful as a guide to ph~~rmacologic imervention.
Methods
A productive viral infection was established in target cells utilizing cell free virus concentrated on a sucrose gradient. Fresh HSB-2 cells. Cells were cultured at a IO”:mt in 75 ml flasks with RPM1 and IO% fetal calf serum. penjci~lin. streptomycin, and g~~ltarninc at 37°C in 5% CO,. In order to test the protective effect of a broad spectrum antiherpes drug 20 @g/ml of phosphonoformic acid (PFA), Sigma, were added to one flask at the same time as virus. Additional cultures were established with HSB-2 cells with and without PFA. Cells were kept periodicalIy observed and counted on day 7 and 14. and examined by IFA for the presence of virus using human sera as previously described on day 14. Acyctovir (ACV). 3urroughs-welcome. and gancyciovir (DWPG). Syntex, were added to HSB-2 cells either 4 h or 3 days after the addition of virus. Each drug was added at the concentration indicated in Table 1 and included at the same concentration when cultures were fed with additional fresh media twice a week. Viral infection of HSB-2 cells was accomplished by incubation of 2x 10’ cells with HBLVcultured cell-free supernatant containing approximately 10” infectious virus particles/ml for 4 h at 37°C. Infected cells were spun down and resuspended in 5 ml RPM1 with 10% FCS. Viable cells/ml and % IFA-positive cells are reported.
303
1
TABLE Effect
of nucleoside
Acyclovir
Gancyclovir
analogues
on infection
Treatment
Control no virus
no drug 50 &ml 80 (*g/ml no drug no drug 80 kg/ml 1.50 kg/ml no drug
1.4x 106 4.7x 105 2.89x lo5 2x 106 7x104 8x10”
of HSB-2
cells
Infected
cells*
4 h post infection
3 days
3.6~ 1V 3x lo’ (10%) 3x 10’ 1.3XlO’(
(10%) (10%)
1.2x 10” 1 x 10s (80%)**
(80%) (80%)
1 x 105 (30%) 8~10~ (1%) 1.2~10~
* The number of viable cells and (%) positive by IFA are given for cells exposed to virus for either four hours or three days before addition of drug at the concentration indicated starting at 2~ 10s cells per ml. **The number and (%) IFA positive cells are given for cells exposed to virus alone (without drug).
Results and Discussion Acyclovir (ACV) and gancyclovir (DHPG) are purine nucleosides with anti-viral activity. In order to be active pharmacological agents, they must be in the triphosphate form. Some viruses such as herpes simplex types 1 and 2 and varicellazoster virus have a specific aviral thymidine kinase which effectively phosphorylates ACV. DHPG, in addition, is phosphorylated effectively in cells infected with all of the herpesviruses and in particular is effective in vitro in cytomegalovirusinfected cells (Hermans and Cockerill, 1987; Robins and Revankar, 1988). Under the experimental conditions described, both drugs showed significant toxic effects on uninfected HSB-2 cells (Table 1). Uninfected cells appeared to have a doubling time of 2-3 days. DHPG may significantly interfere with lymphocyte proliferation at the levels used in vitro in this experiment (Bowden et al., 1987). However, even at levels that are toxic to target cells, only incomplete inhibition of virus expression was achieved. The toxic effect of both virus and drug did not appear to be additive. While the drugs may have some protective effect at the highest concentrations, their action does not appear to be selective. ACV similarly appears to have little protective effect in this experimental system. It is possible that this virus lacks a tk gene, or that the tk gene is not efficient in phosphorylating the drugs, or the cell line is not efficient in utilizing the monophosphate. Alternatively, the viral polymerase may be relatively resistant in binding the drug. It will be of interest to retest these drugs in other systems and to consider possible combinations that may be more selective and less toxic. Phosphonoformate does not require metabolic modification in order to become an active drug. Its mechanism of action is similar to PAA (phosphonoacetic acid). Both inhibit the replication of various herpesviruses by selective inhibition of viral DNA polymerase (Roberts, 1988). PFA added to culture HSB-2 cells showed lit-
304 TABLE Effect
2 of PFA on infection
of HSB-2
tells by HBLV
Treatment
Cell number/ml*
IFA oobitive
Control
2x 10” 7 x IOi l.XXlO” 1.6X 10”
0 65 0 0
HBLV HBLV+PFA PFA * The number
of viable cells and Ci positive by IFA
were counted
on day
C;
14 following
exposure
IO
virus.
tle toxicity at the 20 pgiml concentration used in this experiment while providing a striking protective effect against both the cytopathic effect of the virus and the expression of viral antigens detected by the indirect IFA (Table 2). The effect of PFA on the cycle of viral infection will require further analysis in order to determine dose response and the time course of inhibition. In vitro, foscarnet inhibits HIV infection and directly inhibits retroviral reverse transcriptase in a cell free system (Sarin et al., 1985). The drug has been tested in several clinical trials and the serum levels attained by constant intravenous transfusion appears to be well above effective in vitro levels. However, bone marrow suppression may limit human use. Although the nucleoside analogues were not effective in this system, suggesting that the virus will not be susceptible to these drugs in vivo, further evaluation should be done for both these and other compounds.
References Ablashi. D., Lusso, P.. Hung, S. et al. (1988) Utilization of human hematopoietic cell line for the propagation. detection, and characterization of HBLV [human herpesvirus-6). Int. J. Cancer, in press. Biberfeld. P., Karmarsky, S., Salahuddin, S.Z. and Gallo, R.C. (1987) Ultrastructural characterization of a new human B lymphotropic DNA virus (HBLV) isolated from patients with iymph~proliferative disease. J. Natl. Cancer Inst. 79, 933-931. Bowden. R., Digcl, J., Reed. E. and Meyers, J. (1987) Immunosuppressive effects of gancyclovir on in vitro lymphocyte responses. J. Infect. Dis. 156, 899-903. Hermans, P. and Cockerill, F. (1987) Antiviral agents. Mayo Clin. Proc. 62. 1108-1115. Lusso, P., ~~arkham, P.. Tschachler. E. et al. (1988) In vitro cellular tropism of human B-lymphotropic virus (human herpesvirus-6). J. Exp. Med. 167. 1659-1670. Roberts. S. (1988) Design of anti-viral agents other than nucleoside analogues. In: De Clercq and Walker (Eds.), Antiviral Drug Development, Plenum Press. New York. Robins, R. and Revankar (198X) Design of nucleoside analogs as potential antiviral agents. In: De Clercq and R.T. Walker (Eds.). Antiviral Drug Development. Plenum Press. New York. Salahuddin. S.Z.. Ablashi, P.. Markham. P.D. et al. (1986) Isolation of a new virus. HBLV, in patients with lymphoproliferative disorders. Science 234. 596660. Sarin, P.S., Taguchi, Y., Sun. D. et al. (1985) Preliminary communication: inhibition of HTLV-IIIILAV replication by foscarnet. Biochem. Pharmacol. 34. 407S-Uf79.