- Email: [email protected]
Inhibitory activity of guanidine on poliovirus proteins
Life Sciences Vol. 10, Part Printed in Great Britain
II, pp. 81-89, 1971 .
Pergamon Press
INHTBITORY ACTIVITY OF GUANIDINE ON POIaIOYIRUS PROTBINS P. 7~e, Colla, C .De Giuli( °), 0 . Zuffardi,A .M . Cioglia, B . Zroddo Departments of Hygiene, Microbiology I and II, Pharmacology IIniveraity of Cagliari ; Department of Pharmacology, IIniveraity of Modeaa (Italy) . (Received in final form 7 December 1970) Previous researches (1,2) have shown that guanidine inhibits virus RNA replication in poliovirus infected cells, but
has
a
modest effect on virus protein synthesis . However, guanidine prevents the appearance in infected cultures of two virus protein dependent "products", i .e ., RNA polymerise activity and cytopathic effect (3,4) . Since both of these "products" could be sustained by a small percentage of viral proteins, the problem remains unsolved whether guanidine is capable of preventing the synthesis of the respective proteins, whether it induces synthesis
of
abnormal proteins or prevents activity by interacting with preformed proteins . Since capsids represent a good percentage of virus proteins, it was of interest to examine the effect of guanidine on precursors .
To solve this problem, we took advantage of the fact
capsid that
unless capsid precursors previously synthesized in a drug-free
medium are available, low concentrations of p-fluorophenylalanine (FPA), an inhibitor of proteins synthesis, prevent the incorporation of viral RNA in complete infectious virus (5,6) .This provides (°) Researcher of Consiglio Naziorale delle Ricerche at the Department of Pharmacology, University of Milano (Italy) .
S1
82
InhiMtory Action ad Guanidine
Vol . 10, No. 2
the possibility of establishing whether or not guanidine
acts
directly on capaid precursors . DHaterials and Methods Poliovirus 1 (Brunhenders), He1,a cells and most
of
the
substances employed were the same previously described (4) . The techniques adopted for evaluating the synthesis of
infectious
virus, RNA and virus proteins have been previously described(4) . p-Fluorophenylalanine (FPA) was obtained from Aldrich Chem . ; Sephadex G 100 from Pharmacia (Uppsala) . The following technique was adopted for the evaluation
of
mature virus particles or empty capsids . Cell monolayers (4 x 10 6 cells/petri dish)completely confluent in 16 hours at 37°C were treated with actino~yrcin D (5 ug/ml in Hank's BSS) for 30 min.a t 37°C and infected (30 plaque forming units =PFU/cell) at 4°C for 60 min . The cells were then washed 3 times and 5 ml of Hank's BSS containing 2 ug/ml of Actinomycin D (AMD) were added . FPA (25ug/ml), guanidine (100 ug/ml) and H3 leucine (Amersham 15,200 mCi/mmole~ 5uCi/ml) were added to
the
cultures at various intervals after infection (see results) . To neutralize FPA inhibition and block incorporation of the isotope, the cultures were washed 3 times and finally Hank's BSS containing 100 ug/ml respectively of
1-phenylalanine and 1-leucine was added .
Guanidine inhibition was neutralized by 3 washings and the
cells
were covered with normal Hank's BSS . At prefixed intervals the cultures were frozen and thawed "in toto" 3 times (-30°C : +20°C) . Then, they were dialyzed through cellophane against 100 volumes of Hank's (24 hours at 4°C) . Cell debris was removed by centrifugation at 24,000 g (15 min.a t +4°C) and the cultures were treated with sodium deoxycholate (0,1yb) for 90 min . at 37°C and with crystalline DNAase and RNAase (100ug/ml) for 15 min . at 37°C . The viral particles and empty capsids were separated
at
104,000 g (90 min. at + 4°C) and resuspended in 0,5 ml of TRIG-EDTA
Vol. 10, No . 2
89
Inhibitory Action c2 Guanidine
(0,01M and 0,05M, pH 7 .3) . After treatment with ethyl ether and
the addition of 10 9 PFU of unlabelled poliovirus 1 (0 .1 ml),
materials were fractionated through Sephadex G 100 columns (20 cm a 0 .9 cm, bed = 18 em3 ) in TRIS-EDTA buffer . 1 ml fractions were collected (8 ml/hour) ; of these, 0 .1 ml fractions were used
for
infectious virus titration (PF[T) and 0 .9 ml for radioactivity measurement in the scintillation counter (Packard ; scintillation medium : PPO 7 g., dimethylPOPOP 0.1 g ., Triton % 100 333 g" , Toluene 667 8. ) "
Parallel experiments were made in isotope-free media
to
evaluate the production of infectious virus (PFU) . Results Fig. 1 shows that, in the presence of 25 ug/ml of FPA viral RNA replication and virus protein synthesis were slightly reduced, while empty capsid formation was strongly diminished and
in
fectious virus production completely prevented. Fig. 2 shows that in-
when FPA was present in the cultures for only 150 min. after
fection and then removed, infectious virus were produced . However, only a small amount of the proteins synthesized in the presence of FPA entered mature vicia or empty capaida . When the cells were incubated for only 150 min. after infection in a drug-free medium and then incubated in the presence of FPA, complete infectious virus, which have been largely coated with proteins synthesized in the first 150 min., were formed.. These data show that virus proteins synthesized in
the
presence of FPA were incapable of coating viral RNA in complete infectious virus, and that capsid precursors synthesized within 150 min . after infection in a drug-free medium were capable
of
organizing RNA (synthesized in the preaeace of FPA) into complete infectious virus when FPA is present . Therefore, the synthesis of these capsid precursors represents a useful means for evaluating the effects of guanidine on poliovirus proteins .
Vo1 .10, No. 2
InhiMtory Action a~ Cruanidine
84
8
r O O
3 6
c
5
4
150
300
IIIIITES
450
5
10
FIACTIII 11~6Eß
FIG .
1
Effect of FPA (25 ug/ml) on poliovirus synthesis A) Incorporation of H3 uridine (1 uCi/ml from time 0) i~ virus RNA : in Hank's BSS (1) in FPA (2) ; incorporation of H leucine (1 uCi/ml from time 0) in virus antigens : in Hank's BSS (3), in FPA (4) ; development of infectious virus (PFU) in Hank's BSS (5), in FPA (6) . H) Incorporation of H3 leucine (5 uCi/ml added at 90 min, after infection) in virus particles or empty capside formed at 510 min. after infection in Hank's BSS (7) or in FPA (8) (Sephadex G 100 eluates) . Actinomycin D 2 ug/ml from time 0 . Arrow indicates maximum infectivity . Vertical bars indicate standard error (4 determin_a tions for sample) .
Vol. 10, No . 2
Inhibitory Acrion ad Cruanidine
IIFECTIOIIS YIAOS fIElO~ 1 = ~AT 150 m'~
g
2 =~AT510m~
3,2A10 8
3 -_
"
'
-
6,8
4-
"
"
"
5,1X10T
x
x
104 10 ~
5 10 fAACTIOA AUIIBER
FIG . 2 Tsffect of FPA (25 ug/ml .) on the ability of capsid proteins synthesized within 150 min . after infection to organize virus particles or empty capaids . Cell cultures were labelled with H3 leucine (5 uCi/ml) from min . up to 150 min after infection.
90
Sephadex eluates from infected cells maintained : in Hank's BSS x 150 min. (1), in Hank's BSS x 510 min . (2), in FPA a 150 min. and in Hank's BSS a 360 min . (3), in Hank's BSS x 150 min. and in FPA x 360 min. (4) . Actinomycin D 2 ug/m)_ from time 0 . Arrow indicates maximum fectivity .
in-
Inàibitory Action of. Guanidine
Vol. 10, No. 2
Fig. 3 shown that when guanidine was présent in cell ures for the entire period needed for virus growth, both
cultRNA
replication and infectious virus production were completely inhi b ited . The formation of empty capaids was likewise inhi bited though virus protein synthesis ~s not greatly reduced, at least for 180 min . after infection . YPhen guanidine was present for only 150 min . afteriafeotion sad then removed (fig . 4), the production of complete infectious virus was slightly inferior to that in untreated controls .It must be emphasized that a very small amount of the proteins eyatheaized in the presence of guanidine enters viral proger~y . Ylhen treatment with guanidine was limited to 150 min . after infection and
then
substituted by treatment with FPA, only viral RNA was synthesized. In this case the formation of viral particles or empty capaida was greatly reduced and the production of infectious virus completely inhibited. Discussion and Conclusions The results reported here, together with those of our previous research (4), can be summarized as follows : Guanidine inhibited poliovirus RNA replication even when preformed RNA polymerase was available . 2
Guanidine exerted a modest effect on the net synthesis of virus proteins . However virus proteins synthesized in the presence of guanidine were completely deprived of their specific activitie~, such as cytopathogenicity, RNA replication and
the
formation of capsids capable of organizing complete infectious virus . Using these data as a basis, we may hypothetically explain the antiviral mechanism of action of guanidine as follows: 1 - Guanidine acts upon viral proteins by inhibiting the assembling of polypeptides (synthesized in the polyribosomes)in. macro
87
InhiMtory Action ~ Guanidine
Vol. 10, No . 2
r O
M
~o K a
U
180
380
10
5
IIIMUTES
FAACTIIR
RUIIRER
FIG . 3 Fffect of guanidine HC1 (100 ug/ml added at time 0) on poliovirus synthesis A) Incorporation of H3 uridine (1 uCi/m1 from time 0) in viral RNA (1), and of H3 leucine (1 uCi/ml from time 0) in viral antigens (2) ; infectious virus production (3) . B) Incorporation of H3 leucine (5 uCi/ml, added at 90 min . after infection) in virus particles or empty capside produced 510 min . after infection (Sephadex eluatea) . Actinomycin D 2 ug/ml from time 0 . Arrow indicates maximum infectivity . Vertical beta indicate standard error (4 determinatic®a per sample) .
Inhibitory Action od Guanidine
88
Vol. 10, No. 2
~o a u
150
300 IIIYYTES
450
FRACTI011 AYIIeER
FIG . 4 Fffect of guanidine HCl (100 ug/ml) and FPA (25 ug/ml) subsequently added to infected cells on poliovirus synthesis . A - Incorporation of H3 uridine (1 uCi/ml from time 0) in virus RftA sinthesized in : Hank's BSS (1) ; guanidine x 150 min . and then Hank's BSS (2) ; guanidine x 150 min . and then FPA (3) . Infectious virus production in : Hank's BSS (4) ; guanidine x 150 min . and then Hank's $SS (5) ; guanidine x 150 min . and then FPA (6) . B - Incorporation of H3 leucine (5 uCi/ml from 90 min . up to 150 min . after infection) in virus particles or empty capside organized in : Hank's BSS x 510 min . (8) ; guanidine x 150 min . and FPA x 360 min. (9) (Sephadex eluates) . Actinomycin D 2 ug/ml from time 0 . Arrow indicates maximum infectivity . Vertical bars indicate standard error (4 determinations for sample) .
Vol . 10, No. 2
89
Inhibitory Action of Guuanidine
molecules, or by preventing the cleavage of these
macro-
molecules in active proteins .
2 - Guanidine interacts with viral RftA by inhibiting its template and messenger activities . As a result viral RftA replication is promptly arrested and anomalous proteins, which are
in
capable of carrying out their functions in viral metabolism and in the formation of complete infectious virus are formed . The latter hypothesis, which presupposes a direct interaction between guanidine and the viral genome, seems to be in agreement with the well-known capacity of guanidine to induce resist ence and dependence in Enterovirus sensitive to it (7) . References 1) L .A . CALIGUIRI and I . TA~ - Virolot~Y
3~, 408 (1968)
2) B . LODDO, B. SCARPA, F . PALMAS, P. LA COLLA, L . MA%IA Internat . Con~ess Chemother. Tokyo pag. 792,(1970) 3) B . LODDO, W. FERRARI, G . BRO~TZU, A. SPANEDDA - Rature ~, 97 (1962) 4) P. LA COLLA, C . DE GIULI, AM . CIOGLIA, 0 . ZUFFARDI, B . LODDO Life Sciences 9, 1351 (1970) 5) M.D . 3CHARFF, D .F . SUMHÛERS, L . LEVIftTOW - Ann. ft .Y . Acad . Sci . 130, 282 (19 65 ) 6) L . LEVINTOW, J .E . DARftELL Jr . - Federation Proc . 20 , 1 (1961) s 7) B . IADDO, W. FERRARI, A .~SPANEDDA, G . BROTZU - Saperieatia 1~8 158 (1962) Work supported by a grant of Coasiglio ftazionale delle cerche (Rome) .
Ri-
II, pp. 81-89, 1971 .
Pergamon Press
INHTBITORY ACTIVITY OF GUANIDINE ON POIaIOYIRUS PROTBINS P. 7~e, Colla, C .De Giuli( °), 0 . Zuffardi,A .M . Cioglia, B . Zroddo Departments of Hygiene, Microbiology I and II, Pharmacology IIniveraity of Cagliari ; Department of Pharmacology, IIniveraity of Modeaa (Italy) . (Received in final form 7 December 1970) Previous researches (1,2) have shown that guanidine inhibits virus RNA replication in poliovirus infected cells, but
has
a
modest effect on virus protein synthesis . However, guanidine prevents the appearance in infected cultures of two virus protein dependent "products", i .e ., RNA polymerise activity and cytopathic effect (3,4) . Since both of these "products" could be sustained by a small percentage of viral proteins, the problem remains unsolved whether guanidine is capable of preventing the synthesis of the respective proteins, whether it induces synthesis
of
abnormal proteins or prevents activity by interacting with preformed proteins . Since capsids represent a good percentage of virus proteins, it was of interest to examine the effect of guanidine on precursors .
To solve this problem, we took advantage of the fact
capsid that
unless capsid precursors previously synthesized in a drug-free
medium are available, low concentrations of p-fluorophenylalanine (FPA), an inhibitor of proteins synthesis, prevent the incorporation of viral RNA in complete infectious virus (5,6) .This provides (°) Researcher of Consiglio Naziorale delle Ricerche at the Department of Pharmacology, University of Milano (Italy) .
S1
82
InhiMtory Action ad Guanidine
Vol . 10, No. 2
the possibility of establishing whether or not guanidine
acts
directly on capaid precursors . DHaterials and Methods Poliovirus 1 (Brunhenders), He1,a cells and most
of
the
substances employed were the same previously described (4) . The techniques adopted for evaluating the synthesis of
infectious
virus, RNA and virus proteins have been previously described(4) . p-Fluorophenylalanine (FPA) was obtained from Aldrich Chem . ; Sephadex G 100 from Pharmacia (Uppsala) . The following technique was adopted for the evaluation
of
mature virus particles or empty capsids . Cell monolayers (4 x 10 6 cells/petri dish)completely confluent in 16 hours at 37°C were treated with actino~yrcin D (5 ug/ml in Hank's BSS) for 30 min.a t 37°C and infected (30 plaque forming units =PFU/cell) at 4°C for 60 min . The cells were then washed 3 times and 5 ml of Hank's BSS containing 2 ug/ml of Actinomycin D (AMD) were added . FPA (25ug/ml), guanidine (100 ug/ml) and H3 leucine (Amersham 15,200 mCi/mmole~ 5uCi/ml) were added to
the
cultures at various intervals after infection (see results) . To neutralize FPA inhibition and block incorporation of the isotope, the cultures were washed 3 times and finally Hank's BSS containing 100 ug/ml respectively of
1-phenylalanine and 1-leucine was added .
Guanidine inhibition was neutralized by 3 washings and the
cells
were covered with normal Hank's BSS . At prefixed intervals the cultures were frozen and thawed "in toto" 3 times (-30°C : +20°C) . Then, they were dialyzed through cellophane against 100 volumes of Hank's (24 hours at 4°C) . Cell debris was removed by centrifugation at 24,000 g (15 min.a t +4°C) and the cultures were treated with sodium deoxycholate (0,1yb) for 90 min . at 37°C and with crystalline DNAase and RNAase (100ug/ml) for 15 min . at 37°C . The viral particles and empty capsids were separated
at
104,000 g (90 min. at + 4°C) and resuspended in 0,5 ml of TRIG-EDTA
Vol. 10, No . 2
89
Inhibitory Action c2 Guanidine
(0,01M and 0,05M, pH 7 .3) . After treatment with ethyl ether and
the addition of 10 9 PFU of unlabelled poliovirus 1 (0 .1 ml),
materials were fractionated through Sephadex G 100 columns (20 cm a 0 .9 cm, bed = 18 em3 ) in TRIS-EDTA buffer . 1 ml fractions were collected (8 ml/hour) ; of these, 0 .1 ml fractions were used
for
infectious virus titration (PF[T) and 0 .9 ml for radioactivity measurement in the scintillation counter (Packard ; scintillation medium : PPO 7 g., dimethylPOPOP 0.1 g ., Triton % 100 333 g" , Toluene 667 8. ) "
Parallel experiments were made in isotope-free media
to
evaluate the production of infectious virus (PFU) . Results Fig. 1 shows that, in the presence of 25 ug/ml of FPA viral RNA replication and virus protein synthesis were slightly reduced, while empty capsid formation was strongly diminished and
in
fectious virus production completely prevented. Fig. 2 shows that in-
when FPA was present in the cultures for only 150 min. after
fection and then removed, infectious virus were produced . However, only a small amount of the proteins synthesized in the presence of FPA entered mature vicia or empty capaida . When the cells were incubated for only 150 min. after infection in a drug-free medium and then incubated in the presence of FPA, complete infectious virus, which have been largely coated with proteins synthesized in the first 150 min., were formed.. These data show that virus proteins synthesized in
the
presence of FPA were incapable of coating viral RNA in complete infectious virus, and that capsid precursors synthesized within 150 min . after infection in a drug-free medium were capable
of
organizing RNA (synthesized in the preaeace of FPA) into complete infectious virus when FPA is present . Therefore, the synthesis of these capsid precursors represents a useful means for evaluating the effects of guanidine on poliovirus proteins .
Vo1 .10, No. 2
InhiMtory Action a~ Cruanidine
84
8
r O O
3 6
c
5
4
150
300
IIIIITES
450
5
10
FIACTIII 11~6Eß
FIG .
1
Effect of FPA (25 ug/ml) on poliovirus synthesis A) Incorporation of H3 uridine (1 uCi/ml from time 0) i~ virus RNA : in Hank's BSS (1) in FPA (2) ; incorporation of H leucine (1 uCi/ml from time 0) in virus antigens : in Hank's BSS (3), in FPA (4) ; development of infectious virus (PFU) in Hank's BSS (5), in FPA (6) . H) Incorporation of H3 leucine (5 uCi/ml added at 90 min, after infection) in virus particles or empty capside formed at 510 min. after infection in Hank's BSS (7) or in FPA (8) (Sephadex G 100 eluates) . Actinomycin D 2 ug/ml from time 0 . Arrow indicates maximum infectivity . Vertical bars indicate standard error (4 determin_a tions for sample) .
Vol. 10, No . 2
Inhibitory Acrion ad Cruanidine
IIFECTIOIIS YIAOS fIElO~ 1 = ~AT 150 m'~
g
2 =~AT510m~
3,2A10 8
3 -_
"
'
-
6,8
4-
"
"
"
5,1X10T
x
x
104 10 ~
5 10 fAACTIOA AUIIBER
FIG . 2 Tsffect of FPA (25 ug/ml .) on the ability of capsid proteins synthesized within 150 min . after infection to organize virus particles or empty capaids . Cell cultures were labelled with H3 leucine (5 uCi/ml) from min . up to 150 min after infection.
90
Sephadex eluates from infected cells maintained : in Hank's BSS x 150 min. (1), in Hank's BSS x 510 min . (2), in FPA a 150 min. and in Hank's BSS a 360 min . (3), in Hank's BSS x 150 min. and in FPA x 360 min. (4) . Actinomycin D 2 ug/m)_ from time 0 . Arrow indicates maximum fectivity .
in-
Inàibitory Action of. Guanidine
Vol. 10, No. 2
Fig. 3 shown that when guanidine was présent in cell ures for the entire period needed for virus growth, both
cultRNA
replication and infectious virus production were completely inhi b ited . The formation of empty capaids was likewise inhi bited though virus protein synthesis ~s not greatly reduced, at least for 180 min . after infection . YPhen guanidine was present for only 150 min . afteriafeotion sad then removed (fig . 4), the production of complete infectious virus was slightly inferior to that in untreated controls .It must be emphasized that a very small amount of the proteins eyatheaized in the presence of guanidine enters viral proger~y . Ylhen treatment with guanidine was limited to 150 min . after infection and
then
substituted by treatment with FPA, only viral RNA was synthesized. In this case the formation of viral particles or empty capaida was greatly reduced and the production of infectious virus completely inhibited. Discussion and Conclusions The results reported here, together with those of our previous research (4), can be summarized as follows : Guanidine inhibited poliovirus RNA replication even when preformed RNA polymerase was available . 2
Guanidine exerted a modest effect on the net synthesis of virus proteins . However virus proteins synthesized in the presence of guanidine were completely deprived of their specific activitie~, such as cytopathogenicity, RNA replication and
the
formation of capsids capable of organizing complete infectious virus . Using these data as a basis, we may hypothetically explain the antiviral mechanism of action of guanidine as follows: 1 - Guanidine acts upon viral proteins by inhibiting the assembling of polypeptides (synthesized in the polyribosomes)in. macro
87
InhiMtory Action ~ Guanidine
Vol. 10, No . 2
r O
M
~o K a
U
180
380
10
5
IIIMUTES
FAACTIIR
RUIIRER
FIG . 3 Fffect of guanidine HC1 (100 ug/ml added at time 0) on poliovirus synthesis A) Incorporation of H3 uridine (1 uCi/m1 from time 0) in viral RNA (1), and of H3 leucine (1 uCi/ml from time 0) in viral antigens (2) ; infectious virus production (3) . B) Incorporation of H3 leucine (5 uCi/ml, added at 90 min . after infection) in virus particles or empty capside produced 510 min . after infection (Sephadex eluatea) . Actinomycin D 2 ug/ml from time 0 . Arrow indicates maximum infectivity . Vertical beta indicate standard error (4 determinatic®a per sample) .
Inhibitory Action od Guanidine
88
Vol. 10, No. 2
~o a u
150
300 IIIYYTES
450
FRACTI011 AYIIeER
FIG . 4 Fffect of guanidine HCl (100 ug/ml) and FPA (25 ug/ml) subsequently added to infected cells on poliovirus synthesis . A - Incorporation of H3 uridine (1 uCi/ml from time 0) in virus RftA sinthesized in : Hank's BSS (1) ; guanidine x 150 min . and then Hank's BSS (2) ; guanidine x 150 min . and then FPA (3) . Infectious virus production in : Hank's BSS (4) ; guanidine x 150 min . and then Hank's $SS (5) ; guanidine x 150 min . and then FPA (6) . B - Incorporation of H3 leucine (5 uCi/ml from 90 min . up to 150 min . after infection) in virus particles or empty capside organized in : Hank's BSS x 510 min . (8) ; guanidine x 150 min . and FPA x 360 min. (9) (Sephadex eluates) . Actinomycin D 2 ug/ml from time 0 . Arrow indicates maximum infectivity . Vertical bars indicate standard error (4 determinations for sample) .
Vol . 10, No. 2
89
Inhibitory Action of Guuanidine
molecules, or by preventing the cleavage of these
macro-
molecules in active proteins .
2 - Guanidine interacts with viral RftA by inhibiting its template and messenger activities . As a result viral RftA replication is promptly arrested and anomalous proteins, which are
in
capable of carrying out their functions in viral metabolism and in the formation of complete infectious virus are formed . The latter hypothesis, which presupposes a direct interaction between guanidine and the viral genome, seems to be in agreement with the well-known capacity of guanidine to induce resist ence and dependence in Enterovirus sensitive to it (7) . References 1) L .A . CALIGUIRI and I . TA~ - Virolot~Y
3~, 408 (1968)
2) B . LODDO, B. SCARPA, F . PALMAS, P. LA COLLA, L . MA%IA Internat . Con~ess Chemother. Tokyo pag. 792,(1970) 3) B . LODDO, W. FERRARI, G . BRO~TZU, A. SPANEDDA - Rature ~, 97 (1962) 4) P. LA COLLA, C . DE GIULI, AM . CIOGLIA, 0 . ZUFFARDI, B . LODDO Life Sciences 9, 1351 (1970) 5) M.D . 3CHARFF, D .F . SUMHÛERS, L . LEVIftTOW - Ann. ft .Y . Acad . Sci . 130, 282 (19 65 ) 6) L . LEVINTOW, J .E . DARftELL Jr . - Federation Proc . 20 , 1 (1961) s 7) B . IADDO, W. FERRARI, A .~SPANEDDA, G . BROTZU - Saperieatia 1~8 158 (1962) Work supported by a grant of Coasiglio ftazionale delle cerche (Rome) .
Ri-