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The role of active cellular sulfhydryl groups in virus reproduction
Life Scieaoes No . 8, pp" 5T9-583, 1963" United States .
~~ Saar ~c .
Printed in the
THE BALE OF ACTIVE CELLULAR SULFFIYDRYL GROUPS IN VIRUS REPRODUCTION J . Zemls Institute of Virology, Czechoslovak Academy of Sciences Bratislava, Czechoslovakia (Received 21~ June 1963) The inhibition of virus growth by agents blocking ce]lular aulfhydryl (SH) groups has been repeatedly demonstrated (1-4) .
It has been concluded
that the glycolytic pathway is essential for virus grarth .
However, the role
of glycolysia in virus multiplication in various cells, xhich char certain metabolic differences, xas not diati~~shed from other possible sites of action of Sd3 inhibitors . To study this problem ftu~ther, iodoacetate (NaIAc), xhich inhibits glycolysis, as xell ae three other SH reagents N-ethylmaleimide (N~), fornamidined avlfide (FDS) and o~iodoeobenzoate (IOB), xere used in ~o different types of cells : HeLa and fresh, trypsinized chick embryo (CE) cells.
The former cells
ahoy high aerobic glycolyais, xhereas the latter metabolize mainly using o=Cdative pathways (5) . Cells were infected xith type 2 poliovirus, strain I~F-1 (gram in HeLn ce]1s) and xith a variant of the Hypr strain of tick-borne encephalitis (TE) virus, cytopathic for HeLa cells (6) (gram in HeLa and CE cells) .
The inhibi
tion of virus grarth xae studied as follaras cells xere inoculated xith retively high dose of virus (1-lA TCID~ per cell), which xae left to absorb for l hr, at 37° on cells in suspension . into stationary tube cultures, cells .
The cells xere xaahed 3 times and seeded
The inhibitors xere added at the seeding of
Follaring incubation for 21~ hours at 37°, the frozen-thawed cultures
were titrated for total infectious virus in HeLa cel].s .
In other nxperimenta,
the effect of the inhibitors at the same concentration on aerobic glycolysia
579
c,~r~lnaR ~ m O svr~rn~ ~a ~aa~ m v~us ~AOncc~ a
actriv~
58o
+m~~Oô _+~ W Û,~ Omé oo 1ô am p ô +
.zoa
1
Oq N
,. .. 0
,. _M N
N
a
1
0
.. v ô O
O
M w
M v O N m O~
~
xo.
4y O m
N
F.
p a
N
md md 1
~
0
.., o~ M r-i
0
m c, â. ,-1s~ m +
â m qO mf. 0 Â
wO
II v
1
~o
a O0 4,O
_M
ô M
N
ri o.
a
,~ m ,iF,
~a m b m
O
_M
N M O
1
~1 v
O
a N
O
a.
4-1 O Â6 R
m _N ti o~
ô
a
~ Ô w
FO.
m O
E U O %
1
Pr
>r
m
v N O
O
IU ti +" m
0 â~ 1 r~i
É t m x
W E 1 Û
. >
O q
8
ACTIFS CBt.I~TLAß BtJLF~fL aADIIPB Za VIRUS RLPRODUCTIOB
80 . 8
581
and respiration was tested . Prs~i~ nary eaperimeats aho~red that with the exception of ICH, the SH inhi bitors did not directly iafluenoe the infectivity titres of polio and 'l'S viruses .
IOB (lÔ ~) la~rsred the TCID~ titre of T8 vitae after 24 hours in-
cubat3on at 37 " by 4 .17 log units ; the effect of 10 ~ IOH was, harever, negligible (-0 .34 log units ) .
The other agent, SatlÔ ~ aJ~S was reported to in-
activate ente~rovirusea, but the optimal p~ for this action was 8,6 and no effect was observed at pH 7 .0 (7) .
It can be concluded therefore, that the inhi bitory
effects of Na71o, NB~S, FD3 and the partial effect of IOB on virus growth (Table 1) are caused by changes in cellular metabolism, required for virus synthesis . NaIAc caused a marked inhibition of virus gro~h regardless of the type of metabolism of the cells used .
3i.nce the aerobd.c glycolysis was markedly in-
hibited (Table 2), the glyoolytic cleavage of glucose seams to bs essential for T1BL8 2 The effect of ~ Inhi bdtors on Aerobic Qlyoolyaia and Oxygen IIptake of Cells Tnhi bitor Conc .
(Ii)
Heïa Cells
C8 hells
air
air ~
4LA
~x
53 .6
0
71 .4
100 .2
35.6 11i.9
87 .3
93 .0
NaIAc
5aî10
0
a~
l0 - ~ l0"3
109 .7 81 .4
106 .3
FAS
l0" 4 l0" 3
83 .5
]A2 .3
IOH
l0"4 10 -3
20 .4 ]17 .4 32 .3
0
91 .3
-
x
q0 2 ~
51 .7 104 .0 46 .4
-
0
902
34.8
g1 .0
88 .8
0
14.0
Per cent values of metabolic quotients relate to control celle (=100 virus grarth and probably cannot be clroumve~ed by any other alternative metabolic pathway. NH?S and FDS also inhibited virus growth .
Their inhibitory effects were
often accompanied by a more or less pronounced suppression of aerobic glycolysis
582
acr~vs c
and respiration .
za
vzROS
R~o~x.-rro~
a
~o .
Nevertheless, certain concentrations of the inhibitors xhich
xere still effective to~rarda virus growth, ezerted no detectable effect on the This is the case for 10 -3 - 10 -Lt M N$!S
measured metabolic aotivitiea of cell . (Hem oslla) and 10 4 M FDS (CB cells) .
IOH inhi lrita virus grarth in a similar xay to N&d and FDS .
Moreover, it
influenced the infeotivity of TS virus itself, thus complicating the interpretation of the results xith this virus . hibition of virus growth to 2 .59
4
In fact, 10
-4
M IOH led to the in
of the control, xhereas the direct inactiva-
tion of virus under the name oonditione xas characterised by a decrease of the titre by 0 .3k log units only .
Thin reanlt supports the interpretation that in
addition to direct vitae inactivation inhibition of virus growth took p1aCe in this aa~periseat . ~e-mode oY action of the used agents is connected xith active ~i groups of en$ymee or some other importgut compounds .
~e suspected mode of action,
summarised on Scheme 1, is represented by substitution of the aulthydryl hydrogen by a small molecule 1n the case of NaIAc, H~ and FAS (reaction a), or by the wddation of parallel sH groups to disulfide bridges in the case of IOB (reaction b) . enayme -3H
en$yme-,SR
eng
enzy~ee
-R
-3
-C~I 2 COONa
IjN-C2H5 -~ ~
(a)
(b)
(NaIAc) (NHM)
2
-s-~
Ns
sa~
1
Zhe Reactiona of Sulfhydryl t}roupe of Nnaymes xith iodoacetate (NaIAn), N-ethylealeimide (NBi+i) and for~eamidinedianlfide (PDS) (reaction a) and xith o-iodoaobenaoate (reaction b) .
lfo . 8
Acriv$ a
a svr~s~aaL c
s n~ vn~s x~DQCria~
NaUc inhibits the enzyme trioaophoaphatedehydrogenaae, various other p~roteina having free SH groupa .
583
bnt it reacts also with
NBIS is reported to bdnd the NS
groups of histidine and of peptidic linkages (8) .
FDS inhibits papain, messe,
transamidinase and transaminaaes (9) . The inhibitory effect of NaIAc on virus groxth further supporta the visor that provided that glucose serves as a substrats, the glycolytic pathoray is important for virus reproduction regardless of the type of energy metabolism of the cell .
Hoorever, the inhibitory effects of the other SH reagents on virus
grarth xithovt demonstrable impairment of glycolyais suggest that is addition to glycolysia other, st present un]marn, metabolic activities requiring active SH groupa are involved in virus grwth . References l.
R .L . THCd~SON, J . Zmisunol . ~, 31S (19117) .
2.
Y . BEC~t, N . (Hi033QiICZ and H . ~7, 77 (1958) .
3.
J . POIdTNZCä and H . L . BICHRA~, Froc . Soc, eut . Biol . Died . 10
4.
D .N . PLANT~t03S,
5.
J . ZF~A, Life Sci , in press (1963) .
6.
H . LIBI~PA, Acta virol . ~, 387 (1961) .
7.
L. PHILIPSON and P . (~iOPPIN, J . e~p . Died, u 2
8,
D .Q . S?RTH, A . NA(~AMATSII and J .S . FRDTON, J . Amer . Chem . Soc . ~ 4600 (1960) . -
9.
J .H . iTAL~R and H .S . VAhâSR ., Arch . Biocham . Bionhus . s 8 6 , 80 (1960) .
BRIiNEOPF, Pros . Soc . eue . Hiol . Ked . 601 (1960) .
Biochim . Hiophsa . Acts ~, 186 (1961) .
455 (1960) .
~~ Saar ~c .
Printed in the
THE BALE OF ACTIVE CELLULAR SULFFIYDRYL GROUPS IN VIRUS REPRODUCTION J . Zemls Institute of Virology, Czechoslovak Academy of Sciences Bratislava, Czechoslovakia (Received 21~ June 1963) The inhibition of virus growth by agents blocking ce]lular aulfhydryl (SH) groups has been repeatedly demonstrated (1-4) .
It has been concluded
that the glycolytic pathway is essential for virus grarth .
However, the role
of glycolysia in virus multiplication in various cells, xhich char certain metabolic differences, xas not diati~~shed from other possible sites of action of Sd3 inhibitors . To study this problem ftu~ther, iodoacetate (NaIAc), xhich inhibits glycolysis, as xell ae three other SH reagents N-ethylmaleimide (N~), fornamidined avlfide (FDS) and o~iodoeobenzoate (IOB), xere used in ~o different types of cells : HeLa and fresh, trypsinized chick embryo (CE) cells.
The former cells
ahoy high aerobic glycolyais, xhereas the latter metabolize mainly using o=Cdative pathways (5) . Cells were infected xith type 2 poliovirus, strain I~F-1 (gram in HeLn ce]1s) and xith a variant of the Hypr strain of tick-borne encephalitis (TE) virus, cytopathic for HeLa cells (6) (gram in HeLa and CE cells) .
The inhibi
tion of virus grarth xae studied as follaras cells xere inoculated xith retively high dose of virus (1-lA TCID~ per cell), which xae left to absorb for l hr, at 37° on cells in suspension . into stationary tube cultures, cells .
The cells xere xaahed 3 times and seeded
The inhibitors xere added at the seeding of
Follaring incubation for 21~ hours at 37°, the frozen-thawed cultures
were titrated for total infectious virus in HeLa cel].s .
In other nxperimenta,
the effect of the inhibitors at the same concentration on aerobic glycolysia
579
c,~r~lnaR ~ m O svr~rn~ ~a ~aa~ m v~us ~AOncc~ a
actriv~
58o
+m~~Oô _+~ W Û,~ Omé oo 1ô am p ô +
.zoa
1
Oq N
,. .. 0
,. _M N
N
a
1
0
.. v ô O
O
M w
M v O N m O~
~
xo.
4y O m
N
F.
p a
N
md md 1
~
0
.., o~ M r-i
0
m c, â. ,-1s~ m +
â m qO mf. 0 Â
wO
II v
1
~o
a O0 4,O
_M
ô M
N
ri o.
a
,~ m ,iF,
~a m b m
O
_M
N M O
1
~1 v
O
a N
O
a.
4-1 O Â6 R
m _N ti o~
ô
a
~ Ô w
FO.
m O
E U O %
1
Pr
>r
m
v N O
O
IU ti +" m
0 â~ 1 r~i
É t m x
W E 1 Û
. >
O q
8
ACTIFS CBt.I~TLAß BtJLF~fL aADIIPB Za VIRUS RLPRODUCTIOB
80 . 8
581
and respiration was tested . Prs~i~ nary eaperimeats aho~red that with the exception of ICH, the SH inhi bitors did not directly iafluenoe the infectivity titres of polio and 'l'S viruses .
IOB (lÔ ~) la~rsred the TCID~ titre of T8 vitae after 24 hours in-
cubat3on at 37 " by 4 .17 log units ; the effect of 10 ~ IOH was, harever, negligible (-0 .34 log units ) .
The other agent, SatlÔ ~ aJ~S was reported to in-
activate ente~rovirusea, but the optimal p~ for this action was 8,6 and no effect was observed at pH 7 .0 (7) .
It can be concluded therefore, that the inhi bitory
effects of Na71o, NB~S, FD3 and the partial effect of IOB on virus growth (Table 1) are caused by changes in cellular metabolism, required for virus synthesis . NaIAc caused a marked inhibition of virus gro~h regardless of the type of metabolism of the cells used .
3i.nce the aerobd.c glycolysis was markedly in-
hibited (Table 2), the glyoolytic cleavage of glucose seams to bs essential for T1BL8 2 The effect of ~ Inhi bdtors on Aerobic Qlyoolyaia and Oxygen IIptake of Cells Tnhi bitor Conc .
(Ii)
Heïa Cells
C8 hells
air
air ~
4LA
~x
53 .6
0
71 .4
100 .2
35.6 11i.9
87 .3
93 .0
NaIAc
5aî10
0
a~
l0 - ~ l0"3
109 .7 81 .4
106 .3
FAS
l0" 4 l0" 3
83 .5
]A2 .3
IOH
l0"4 10 -3
20 .4 ]17 .4 32 .3
0
91 .3
-
x
q0 2 ~
51 .7 104 .0 46 .4
-
0
902
34.8
g1 .0
88 .8
0
14.0
Per cent values of metabolic quotients relate to control celle (=100 virus grarth and probably cannot be clroumve~ed by any other alternative metabolic pathway. NH?S and FDS also inhibited virus growth .
Their inhibitory effects were
often accompanied by a more or less pronounced suppression of aerobic glycolysis
582
acr~vs c
and respiration .
za
vzROS
R~o~x.-rro~
a
~o .
Nevertheless, certain concentrations of the inhibitors xhich
xere still effective to~rarda virus growth, ezerted no detectable effect on the This is the case for 10 -3 - 10 -Lt M N$!S
measured metabolic aotivitiea of cell . (Hem oslla) and 10 4 M FDS (CB cells) .
IOH inhi lrita virus grarth in a similar xay to N&d and FDS .
Moreover, it
influenced the infeotivity of TS virus itself, thus complicating the interpretation of the results xith this virus . hibition of virus growth to 2 .59
4
In fact, 10
-4
M IOH led to the in
of the control, xhereas the direct inactiva-
tion of virus under the name oonditione xas characterised by a decrease of the titre by 0 .3k log units only .
Thin reanlt supports the interpretation that in
addition to direct vitae inactivation inhibition of virus growth took p1aCe in this aa~periseat . ~e-mode oY action of the used agents is connected xith active ~i groups of en$ymee or some other importgut compounds .
~e suspected mode of action,
summarised on Scheme 1, is represented by substitution of the aulthydryl hydrogen by a small molecule 1n the case of NaIAc, H~ and FAS (reaction a), or by the wddation of parallel sH groups to disulfide bridges in the case of IOB (reaction b) . enayme -3H
en$yme-,SR
eng
enzy~ee
-R
-3
-C~I 2 COONa
IjN-C2H5 -~ ~
(a)
(b)
(NaIAc) (NHM)
2
-s-~
Ns
sa~
1
Zhe Reactiona of Sulfhydryl t}roupe of Nnaymes xith iodoacetate (NaIAn), N-ethylealeimide (NBi+i) and for~eamidinedianlfide (PDS) (reaction a) and xith o-iodoaobenaoate (reaction b) .
lfo . 8
Acriv$ a
a svr~s~aaL c
s n~ vn~s x~DQCria~
NaUc inhibits the enzyme trioaophoaphatedehydrogenaae, various other p~roteina having free SH groupa .
583
bnt it reacts also with
NBIS is reported to bdnd the NS
groups of histidine and of peptidic linkages (8) .
FDS inhibits papain, messe,
transamidinase and transaminaaes (9) . The inhibitory effect of NaIAc on virus groxth further supporta the visor that provided that glucose serves as a substrats, the glycolytic pathoray is important for virus reproduction regardless of the type of energy metabolism of the cell .
Hoorever, the inhibitory effects of the other SH reagents on virus
grarth xithovt demonstrable impairment of glycolyais suggest that is addition to glycolysia other, st present un]marn, metabolic activities requiring active SH groupa are involved in virus grwth . References l.
R .L . THCd~SON, J . Zmisunol . ~, 31S (19117) .
2.
Y . BEC~t, N . (Hi033QiICZ and H . ~7, 77 (1958) .
3.
J . POIdTNZCä and H . L . BICHRA~, Froc . Soc, eut . Biol . Died . 10
4.
D .N . PLANT~t03S,
5.
J . ZF~A, Life Sci , in press (1963) .
6.
H . LIBI~PA, Acta virol . ~, 387 (1961) .
7.
L. PHILIPSON and P . (~iOPPIN, J . e~p . Died, u 2
8,
D .Q . S?RTH, A . NA(~AMATSII and J .S . FRDTON, J . Amer . Chem . Soc . ~ 4600 (1960) . -
9.
J .H . iTAL~R and H .S . VAhâSR ., Arch . Biocham . Bionhus . s 8 6 , 80 (1960) .
BRIiNEOPF, Pros . Soc . eue . Hiol . Ked . 601 (1960) .
Biochim . Hiophsa . Acts ~, 186 (1961) .
455 (1960) .