- Email: [email protected]
Observations on the inactivation of viruses by sulfhydryl reagents
VIROLOGY
17, 176-183 (1962)
Observations
on the Inactivation
of Viruses
by Suifhydryl
Reagents
A. C. ALLISON National Institute
for Medical Accepted
Research, February
London,
N.W.7,
England
6, 1962
The mode of inactivation of vaccinia and fowl plague viruses by thiol reagents has been studied. Inactivation appearsto follow first-order kinetics, cne rates varying with different reagents and proportional to their concentration. Inactivation by mercuric chloride, p-chloromercuribenzoate and merthiolate is largely reversed by addition of L-cysteine or reduced glutathione, whereas inactivation by iodoacetamide and n-ethylmaleimide is not so reversed. Sulfhydryl reagents do not affect the primary attachment of these viruses to host cells, but indirect evidence suggests that they may prevent uncoating of the virus which precedes multiplication. Cells were infected with vaccinia untreated and treated with mercuric chloride. After incubation of cells at 4” and 37” antibody was added. At the higher temperature very little neutralization took place, a resuh suggesting that the virus may have been taken up by the cells and become inaccessibleto antibody.
INTRODUCTION Evidence that the infectivity of many viruses is diminished when they are treated with sulfhydryl reagents has been summarized by Allison et al. (1962). Since this reaction can be used to obtain further information on the superficial structure of viruses and the early stages of virus multiplication-about’ which very little is known -the opportunity was taken to study it in more det,ail. Two viruses that can be obtained in relatively pure suspensions and titrated accurately by plaque techniques, vaccinia and fowl plague, were chosen for analysis. After completion of this work a paper by Choppin and Philipson (1961) appeared, in which similar experiments on ECHO viruses are described. Their results are very similar to those that will be rcported here, although there are some differences in detail. It seems that the conclusions which are drawn may be applicable to a wide range of viruses. MATERIAL
AND
METHODS
Vaccinia virus. The Lister Institute rabbit-adapt,ed st,rain of virus was used. This was propagated in rabbit skin and purified by three cycles of extraction with the fluoro-
carbon Arcton 63 (Gessler et al., 1956), followed by flocculation with M sodium chloride (Dawson and McFarlane, 1948) and brief ultrasonic dispersion in Gey’s solution without bicarbonate, buffered at pH 8.0 with 0.05 M tris (hydroxymethyl) aminomethane (Tris). Virus was titrated by the plaque technique using Tris-buffered overlay as described by Porterfield and Allison (1960). Fowl plague vir[Ls. The Bonn strain was propagated in the allantois of embryonated eggs and harvested after 24 hours’ incubation at 35”. Virus was adsorbed to and eluted from fowl cells as described by Frommhagen and Knight (1959)) submitted to differential centrifugation as described by Ada and Perry (1956)) and suspended in Tris-buffered Gey’s solution. Virus was titrated by plaque counts in chick embryo cell cultures using the method of Porterfield and Allison (1960). Sulfhydryl reagen,ts. A sample of pure pchloromercuribeneoic acid was kindly provided by Dr. L. A. Stocken, Oxford, and made up in an equivalent of sodium hydroxide. Samples of n-ethylmaleimide and iodoacetamide were kindly provided by Dr. R. Cecil, Oxford, and dissolved in Tris
176
SULFHYDRYL
REAGENTS
buffer. Solutions were not kept for longer than 1 week at 4”. Sulfhydryl compounds. L-Cysteine and reduced glu tathione (Nutritional Biochemicals, Inc.) were dissolved in Tris buffer and the pH was adjusted to 8.0 for immediate use. This pH was chosen because virus is stable and reactions with thiol reagents take place readily while there is only a low rate of spontaneous oxidation of thiols to disulfides (Boyer, 1959; Cecil and McPhee, 1959).
AND
VIRUSES
177
-0 -I .e
.-
-2
a
-3
j
-4
-5
OBSERVATIONS
Rate of Inactivation of Infectivity hydryl Reagents
by Sulf-
Preliminary experiments had shown that vaccinia virus and NDV lose infectivity when exposed to sulfhydryl reagents for 1 hour at 18” (Allison et aZ., 1962). In order to examine the rates of inactivation it was necessary to stop the reaction, and this was done in two ways. The first was to add an amount of L-cysteine equivalent to the amount of thiol reagent present; the reaction of sulfhydryl reagents with cysteine is very much faster than that with virus. Although excess cysteine reactivates virus, it was found experimentally that in the absence of excess cysteine significant reactivation did not occur. The second method was to allow the reaction to proceed with 1O-4 M thiol reagents and then dilute one thousandfold before dialyzing out the excess reagent for 6 hours at 4” and titrating the virus by the plaque bechnique. Results obtained by the two techniques were comparable; a representative experiment is shown in Fig. 1. It will be seen that with two reagents the reaction appears to follow first-order kinetics; plots of the logarithms of residual infective virus against time of exposure to the reagents gave straight lines over the greater part of the curves. However, the slopes of the curves varied considerably with the two reagents and were proportional to the concentrations of the reagents when they were tested at different dilutions. Mercuric chloride inactivated the virus much more rapidly than p-chloromercuribenzoate, and, in agreement with Micklem
+0-
120 Time of exposure in minutes
FIG. 1. Infectivity
of vaccinia virus (loglo) relative to controls after exposure for various times at 18°C to p-chloromercuribenzoate at concentrations of 10e4 M (curve A) and 3 X 10m4M (curve B) and to mercuric chloride at concentrations of 10m4M (curve C) and 3 X low4 (curve D).
I +0
I I I 2 Time of exposurein days
Fro. 2. Infectivity of vaccinia virus (log,,) tive to control after exposure to merthiolate concentration of 3 X 10e4 M at 18°C.
I 3 relaat a
and Kaplan (1958), the reaction with merthiolate (thiomersalate) was very slow (Fig. 2). The lower rate of inactivation at the end of curve D might possibly be due to a low rate of reactivation by thiol-containing compounds at the cell surfaces. The
178
ALLISON
slowness of the reaction with merthiolate suggested that virus inactivation might have been brought about by some decomposition product of merthiolate rather than by the compound itself. However, preincubation of the merthiolate solution for 48 hours at 37” did not affect the rate of reaction, so this interpretation seems unlikely. Results with fowl plague virus are given in Fig. 3. The rates of inactivation are somewhat greater than those of vaccinia virus with the same reagents. Again, mercuric chloride inactivated fowl plague virus more rapidly than did p-chloromercuribenzoate. Reversal of Inactivation Reagents
by Sulfhydryl
An important check on the specificity of the inactivation reaction with mercurials is its reversibility in the presence of other sulfhydryl compounds. After the virus had been partially inactivated by sulfhydryl reagents at a concentration of lo-” M for 2 hours at 18” and the excess reagent dialyzed away at 4”, L-cysteine or reduced glutathione, in Tris-buffered Gey’s solution, was added to give a final concentration of 1O-3 M. After 2 hours at 18”, excess cysteine and glutathione were removed by dialysis for 6 hours at 4”, the pH was adjusted to 7.2, and virus
-5 t I +0
I
I
I
I
30
60
90
120
Time of exposure in minutes FIG. 3. Infectivity of fowl plague virus (log,) relative to control after treatment at 18°C with p-chloromercuribenzoate (curve A) and mercuric chloride (curve B) at a conrentration of 3 X lo-’
M.
was titrat’ed. The results are shown in Table 1. It is apparent that both cysteine and reduced glutathione bring about considerable reactivation of infectivity of virus treated with mercurials, but no reactivation of virus treated with iodoacetamide or n-ethylmaleimide. The reactivation is somewhat more efficient with mercuric chloride than with p-chloromercuribenzoate. Mode of Inactivation hydryl Reagents
of Viruses by Sulf-
Experiments on adsorption of radioactively labeled vaccinia and fowl plague viruses have been reported elsewhere (Allison and Valentine, 1960). The rates of attachment of these viruses to chick embryo cell monolayers were not affected by pchloromercuribenzoate, iodoacetamide, or n-ethylmaleimide at concentrations as high as lo-:’ M. Such experiments exclude the possibility that the sulfhydryl reagents inhibit primary attachment of these viruses to host cells to a significant extent, so that the reduction of infectivity must be brought about in some other way. It is widely believed that an early stage in virus replication is the uncoating of the virus particle and that this is responsible for the so-called “eclipse phase,” the stage of low infectivity which precedes the appearance of newly synthesized virus. In an attempt to ascertain whether sulfhydryl reagents prevent the uncoating of the virus, experiments were carried out to see whether virus treated with these reagents goes into eclipse. Vaccinia virus was treated with mercuric chloride at a concentration of 5 x 1O-4 M for 1 hour at 18”; this reduced infectivity to approximately lop5 that of the original preparation. After removal of excess mercuric chloride by dialysis, the treated virus and untreated control virus were added to monolayers of chick embryo cells in petri dishes. After adsorption of virus for 4 hours at 4”, the fluid was removed and the cells were washed three times with cold Tris-buffered Gey’s solution, pH 7.4. Samples of cells were taken at this (zero) time and other samples were taken after incubating the cells at 37” for 3 hours. The cells were then released by incubation
SULFHYDRPL
REAGEXTS TABLE
AND
179
VIRUSES
1
INACTIVATION OF VIRUS INFECTIVITY BY SULFHYDRYL REAGENTS (10WM FOR 1 HOUR AT 18%) AND REACTIVATION BY CYSTEINE AND REDUCED GLUTATHIONE (GSH) (10--3M)” Virus Vaccinia
Fowl plague
Initial titer 7.2 x
8.8 x
Inactivating agent 107
107
a Titers given in plaque-forming
Postinactivation titer
p-Chloromercuribenzoate p-Chloromercuribenzoate Mercuric chloride Mercuric chloride Iodoacetamide Iodoacetamide n-Ethylmaleimide n-Ethylmaleimide p-Chloromercuribenzoate p-Chloromercuribenzoate Mercuric chloride Mercuric chloride Iodoacetamide Iodoacetamide
Reactivating agent
Postreactivation titer
1.2 x
104
Cysteine
7.8 x
1.2 x
10”
GSH
5.0 x 106
1.8 1.8 9.8 9.8 6.8 6.8
X X x X x x
lOa lo1 lo5 1Oj 105 106
Cysteine GSH Cysteine GSH Cysteine GSH
2.4 6.6 9.0 1.2 7.0 6.2
3.8 x
104
Cysteine
5.8 X 106
3.8 x
10’
GSH
9.1 x
106
8.7 8.7 2.2 2.2
103 lo3 105 105
Cysteine GSH
5.8 x 9.1 x
106 106
Cysteine
9.7 x 104
GSH
3.8 x
X X x x
X X x x x X
105
10’ 106 105 106 105 105
10”
units per milliliter.
in 0.025% trypsin for 10 minutes, an equivalent of trypsin inhibitor (Worthington) was added, and the suspension was given a brief (15 seconds) ultrasonic dispersion. One half of the homogenates were treated with 1O-3 M reduced glutathione for 1 hour at 18”; the others were kept in suspension for the same time at, the same temperature. The results of this experiment are sumInarized in ‘Fable 2. It will be seen that on incubation of both untreated viruses with cells for 3 hours there is a marked fall in titer, in other words, a well-defined eclipse. However, on incubation of the mercuric chloride-treated virus with cells the fall in virus titer recoverable by glutathione reactivation is much less marked. Similar results were consistently obtained although the magnitude of the effect varied. Effect of Antibody on T zruses Znactivated by Thiol Reagents ” Vaccinia virus was treated with 5 x 10F1 M mercuric chloride for 1 hour, and the latter was removed by dialysis at 4”. To the virus was added a neutralizing dose of hightiter rabbit antibody. The complex was
treated with lo-” M cysteine or reduced glutathione, but no restoration of infectivity was obtained. Vaccinia virus thus inactivated with mercuric chloride and untreated control virus were allowed to adsorb to chick embryo cell monolayers at 4” as described in the previous section. The cells were washed with cold Tris-buffered Gey’s solution, and half of the monolayers were allowed to remain at 4” for 3 hours while the others were incubated at 37” for 3 hours. Antibody was then added to some of the monolayers and washed off after l/2 hour at 4”. The cells were resuspended by incubation at 37” with 0.025% trypsin and, after addition of trypsin inhibitor, homogenized. To one half of the preparations lo-” M reduced glutathione was added before assay of infectivity. The results of this experiment are shown in Table 3. When antibody was added to t,he cells, the recoverable virus titer was decreased much more when the cells had stood at 4” than when they had been incubated at 37”. These results suggest that at 4” the virus remains at the cell surface in a position readily accessible to
180
ALLIS03 TABLE
2
E;FFECTS OF SULFHYDRYL REAGENTS (5 X lo1 Af FOR 1 HOUR AT 18”) ON THE ECLIPSE OF VIRCSEF
Virus
Vaccinia
Fowl plague
Preliminary treatment
2% g ‘i 3 .; 0
Virus titer at different times 3 Hours
0
None None Mercuric chloride Mercuric chloride
No Yes No
7.1 x 104 4.3 x 103 9.7 x 10” 2.6 X lo3 9.2 x 10’ 3.1 x 10’
Yes
6.4 X 10% 2.1 x 103
None None Mercuric chloride Mercuric chloride
No Yes No
2.2 x 105 9.7 x 102 9.5 x 10’ 3.2 X lo3 7.3 x 102 2.5 X lo*
Yes
8.2 X lo3
1.2 x 103
a Treated and untreated viruses were added to chick embryo cells; the infective virus titer (PFU/ ml) determined at zero time and after incubation for 3 hours at 37”. Some homogenates were reactivated with 10m3M reduced glutathione (GSH).
antibody, whereas at 37” the virus enters the cell and is no longer accessible. Hence it would seem that vaccinia virus inactivated by thiol reagents is taken up by cells, but may not undergo the uncoating that usually follows within the cell. Lack of Effect of Sulfhydryl iveuraminidase Activity
Reagents on
Whether neuraminidase activity plays any part in adsorption of myxoviruses is uncertain. Treatment with p-chloromercuribenzoate (1O-3 M for 1 hour) was found not to have any effect on hemagglutination by fowl plague virus, in agreement with the findings of Klein et al. (1948)) Philipson and Choppin (1960)) and Buckland (1960) with other myxoviruses. The neuraminidase activities of fowl plague and influenza A viruses, tested on fowl erythrocytes as described by Burnet and Stone (1947), were also unaffected by sulfhydryl reagents. DISCUSSION
It has been shown that the infectivity of vaccinia and fowl plague viruses is con-
siderably reduced by several sulfhydryl reagents. There is little doubt that virus sulfinvolved,, . since groups are hydryl considerable reactivation of infectivity follows treatment with excess cysteine or reduced glutathione. A well-known property of mercaptides formed by the combination of sulfhydryl groups with heavy metals is the reversal of the reaction by other sulfhydryl compounds (Boyer, 1959; Cecil and McPhee, 1959). On the other hand, as a result of the reaction of sulfhydryl groups with iodoacetamide and n-ethylmaleimide, stable thioether bonds are formed, and these are not broken by addition of excess thiol. Hence no reactivation of infectivity after treatment with these reagents would be expected, and none was found. One of the conclusions that can be drawn from these experiments and those of Choppin and Philipson (1961) and Allison et al. (1962) is that sulfhydryl groups form an accessible part of the surface structure of many viruses. Too little is known about the surfaces of most viruses (e.g., poxviruses and myxoviruses) for this information to be related to specific structures. However, enteroviruses, adenoviruses, and many plant viruses are known to have a regular arrangement of surface subunits (Klug and Caspar, 1960)) and the possibility that these are held together by disulfide bonds deserves consideration. Thus, the surface subunits or capsomeres of poliovirus have a molecular weight of approximately 80,000 (Klug and Caspar, 1960) and a cysteine content of about 0.7% (Levintow and Darnell, 1960). Each subunit would therefore have about five cysteine residues, some of which might be concerned with bonding subunits together. Reaction with sulfhydryl reagents might prevent the separation of subunits which is necessary for emergence of nucleic acid in the early stages of replication. It is worth recalling that Kozloff et al. (1957) have adduced evidence suggesting that, in the coliphage T2, tail fibers are bound to the tail sheath, and possibly to one another, by thiol ester bonds. The protection afforded by cystine against thermal inactivation of a temperature-sensitive poliovirus mutant (Pohjanpelto, 1958) very probably involves thiol groups. Possibly the formation of cysteine mercaptides by disul-
SULFHYDRYL
REAGENTS
TABLE 3 EFFECT OF ANTIBODY ADDITION TO CELLS PREVIOUSLY INFECTED WITH VACCINIA VIRUS UNTREATED AND TREATED WITH MERCURIC CHLORIDE (5 X 1OF M FOR 1 HOUR AT 18”)a
-
TemPrelimiperanary treat- ture o ment of incuvirus bation (“(I) -None
_----
4 4 4 4 37 37 37 37 --.-
Lddition of anti- Additior of GSH body
Virus titer in final homogenate
No No Yes Yes No No Yes Yes __--
5.3 6.7 4.8 6.2 3.2 3.1 2.7 4.8
No Yes No Yes No Yes No Yes _--~
x X x X X x X X
10’ lo4 10’ 10’ lo3 103 lo2 lo*
3.8 X 10L No No 4 2.3 X lo1 Yes No 4 4 4 Yes No 1.4 x 10’ Yes 4 Yes 2.7 X lo2 No 37 No 1.1 x 101 Yes 37 No 1.2 x 102 Yes No 37 8.6 x 102 Yes Yes 37 u After removal of antibody and washing of the cells they were homogenized and half the homogenates were treated with 1O-3 M reduced glutathione (CSH) for 1 hour at 4” before titration.
and S are their concentrations, r and m the radius and mass of the respective particles indicated by the subscripts, Ic is the Boltzmann constant, and T is the absolute temperature. This has been calculated for pchloromercuribenzoate and vaccinia (radius 115 rnp, mass 3.6 x 10-l” g), and it is apparent that less than one collision in lo7 results in inactivation of the virus. The low efficiency of the reaction is unlike mercaptide formation with simple thiols (Cecil and McPhee, 1959)) a result which suggests that breakage of disulfide bonds may be involved. Nevertheless, the kinetics indicate that inactivation is brought about by the attachment, of a single molecule of sulfhydryl reagent to a critical site on the virus particle. The reaction can be expressed
Mercuric chloride
fide interchange prevents the oxidation, e.g., to sulfinic or sulfonic acids, of thiol groups partially exposed in the mutant but not in the parent virus. One of the unexpected findings in this investigation has been the apparent firstorder kinetics of inactivation of viruses by sulfhydryl reagents. Similar results have been obtained by Choppin and Philipson (1961) for inactivation of enteroviruses, so the nature of the reaction seems to be quite general. The reaction is, however, very inefficient; the number of collisions between sulfhydryl reagents and virus particles can be calculated from the Trauts (1916) equation v”S = VS(r,
+ r,)’ d&T(l/m,
+ l/m,)
where v%’ is the number of collisions/cm3/ set between the two types of particles, V
181
AND VIRUSES
V+Ri
VR
where V is the infective virus, original concentration [V,,], R is the reagent, VR is the inactivated complex and Ic is the rate constant of the reaction. Then d[V]/dt
= [V] [R] k
and for excess [RI, [R] log [I’]
constant,
= log [V,] -
k [R] t
The slope of the graph is - k [RI, and is thus proportional to the concentration of the sulfhydryl reagent. It is possible that a single critical thiol group is present in each virus particle. Thus, the capsomeres might have to come apart near the end of the nucleic acid chain in order that the latter can uncoil and initiate infection, whereas opening of the capsomeres anywhere else might not allow escape of the nucleic acid. However, the data do not exclude the existence of many thiol groups in each virus particle, each having finite probabilities on the one hand of being necessary for multiplication and on the other hand of being inactivated by a single molecule of sulfhydryl reagent. The need for more than one molecule of reagent for inactivation of the site is excluded. The situation is reminiscent of inactivation of virus by excess antibody, which also follows first-order kinetics (Dulbecco et al., 1956).
ALLISOK
182
Inactivation of a critical virus site by the attachment of one molecule of sulfhydryl reagent is more readily understandable if the initial reaction initiates a series of disulfide interchanges of the type described by Jensen (1959) in some proteins, e.g. R-S-S-RI + PCMB
PCMB I + RS
Re-S-S-R3 R2
S-R, I S-R,
SH
where PCMB stands for p-chloromercuribenzoate. This would happen infrequently with divalent mercury, the following being the usual reaction: R-S’
R-S-S-R,
Hg
+ Hg++ + R-S-S-R*
Rz-S-S-R,
‘S-R,
by a mechanism analogous to pinocytosis and there undergo disruption. Fazekas de St. Groth (1948) has presented arguments in support of the latter view, but these are disputed by Rubin and Franklin (1957). Since Rubin and Franklin were concerned with red blood cells their results may not be applicable to other cells. Thus, the recent electron micrographs of Dales and Siminovitch (1961) suggest that vaccinia virus particles are taken up by L cells by pinocytosis. Experiments which have been presented in this paper, and similar experiments with poliovirus briefly mentioned by Hirst (1961)) indicate that’ when cells are kept at low temperatures virus remains in a superficial position readily accessible to cysteine, glutathione, and antibody. On the other hand, at 37” the virus penetrates into the cell and becomes inaccessible to added antibody, even though after treatment with thiol reagents the virus does not go into eclipse in the normal way. Thus, unlike the situation in phage, the stages of cell penetration and uncoating may be distinct. The first stage has a high temperature coefficient and seems to resemble pinocytosis, which has been described by Holter (1959) and others in a wide range of cell types.
Hence a higher probability of restoring the original structure would be expected after inactivation by mercurials divalent toward thiol than after inactivation by mercurials univalent toward thiol; and this has been observed. More evidence is required, however, before it can be accepted that a disulfide interchange reaction takes place. In our experiments sulfhydryl reagents did not affect the rate of attachment of vaccinia REFERENCES and fowl plague viruses to host cells. This is not always true: hemagglutination by a ADA, G. L., and PERRY, B. T. (1956). Influenza virus nucleic acid : relationship between biological number of viruses is prevented by exposing characteristics of the virus particle and propthem to sulfhydryl reagents (Philipson and erties of the nucleic acid. J. Gen. Microbial. 14, Choppin, 1960; Buckland, 1960) and at623633. tachment of ECHO 7 virus to monkey kidALLISON, A. C., &JCKL~SD, F. E., and ANDREWES, ney cells is also reduced by such treatment C. H. (1962). Effects of sulfhydryl reagents on (Choppin and Philipson, 1961). The marked infectivity of some viruses. Virology 17, 171reduction of infectivity of vaccinia and fowl 175. plague viruses by sulfhydryl reagents is ALLISON, A. C., and VALENTINE, R. C. (1969). Virus mediated by another mechanism, and some particle adsorption. III. Adsorption of viruses evidence has been obtained that after treatby cell monolayers and effects of some variables on adsorption. Bioehim. et Biophys. Acta 40, ment with mercurials the uncoating of the 400-410. virus prior to initiation of infection may not BOYER, P. D. (1959). Sulfhydryl and disulfide occur. groups of enzymes. In “The Enzymes” (P. D. The observations have some bearing on Bayer, H. Lardy, and L. Myrbiick, eds.), 2nd ed., the problem whether infecting animals virus Vol. 1, pp. 511-562. Academic Press, New York. particles remain at or near the host cell BUCKLAND, F. E. (1960). Inactivation of virus surface, only infectious nucleoprotein penehaemagglutinins by para-chloromercuribenzoate. trating into the cell, as in coliphage, or Nature 188, 768. whether the part,icles are taken into the cell BURSET, F. M., and STONE, J. D. (1947). The re-
SULFHYDRYL
REAGENTS
ceptor destroying enzyme of V. cholerae. Australian .I. Exptl. Biol. Med. Sci. 25, 227-238. CECIL, R., and MCPHEE, J. R. (1959). The sulphur chemistry of proteins. Advances in Protein Chem.
14,255-389. CHOPPIN, P. W., and PHILIPSON, L. (1961). The
inactivation. of enterovirus activity by the sulfJ. hydryl reagent, p-chloromercuribenzoate. Exptl. Med. 113, 713-734. DALES, S., and SIMINOVITCH, L. (1961). The devel-
opment of vaccinia virus in Earle’s L strain cells as examined by electron microscopy. J. Biophys. Biochem.
Cytol.
10, 475-598.
DAWSON, I. M., and MCFARLANE, A. S. (1948). Structure of an animal virus. Nature 161, 464466. DULBECCO, R., VOGT,M., and STRICKLAND,A. G. R.
(1956). A study of the basic aspects of neutralization of two animals viruses, western equine encephalitis virus and poliomyelitis virus. Virology 2, 162-205. FAZEKAS DE ST. GROTH, S. (1948). Viropexis, the mechanism of influenza virus infection. Nature
162,294-295. FROMYHAGEN, L. H., and KNIGHT, C. A. (1959). Column purification of influenza virus. Virology 8, 198-208.
GESSLER,A. El., BENDER,C. E., and PARKINSON,M. C. (1956). A new rapid method for isolating viruses by selective fluorocarbon deproteinization. Trans. N. Y. Acad. Sci. 18, 701-703. HIRST, G. K. (1961). Reversible inactivation of poliovirus through exposure to sulfhydryl compounds, In “Poliomyelitis,” pp. 14-15. Lippincott, Philadelphia. HOLTER, H. (1959). Pinocytosis. Intern. Rev. Cytol. 8,481-504.
AND
VIRUSES
183
JENSEN, E. V. (1959). Sulfhydryl-disulfide
interchange. Science 130, 1319-1322. KLEIN, M., BREWER, J. H., PEREZ, J. E., and DAY, B. (1948). The inactivation of influenza virus by mercurials and the reactivation by sodium thioglycollate and BAL. J. Zmmunol. 59, 135140. KLUG, A., and CASPAR,D. L. D. (1960). The structure of small viruses. Advances in Virus Rcsearch 7,225-325. KOZLOFF, L. W., LUTE, M. and HENDERSON, K. (1957). Viral invasion. I. Rupture of thiol ester bonds in the bacteriophage tail. J. Biol. Chem. 228,511-528. LEVINTOW, L., and DARNELL, J. E., JR. (1960). A
simplified procedure for purification of large amounts of poliovirus : characterization and amino acid analysis of type 1 poliovirus. J. Biol. Chem. 235,70-73. MICKLEM, L. R., and KAPLAN, C. (1958). The influence of thiomersalate on vaccinia virus. ViroZogy ,6,775-777. PHILIPSON, L., and CHOPPIN, P. W. (1960). On the
role of virus sulfhydryl groups in the attachment of enteroviruses to erythrocytes. J. Exptl. Med.
112,455478. POHJANPELTO, P. (1958). Stabilization of poliovirus by cystine. Virology 6, 472487. PORTERFIELD, J. S., and ALLISON, A. C. (1960).
Studies with poxviruses by an improved plaque technique. Virology 10, 233-244. RUBIN, H., and FRANKLIN, R. M. (1957). On the mechanism of Newcastle disease virus neutralization by immune serum. Virology 3, 84-95. TRAUTZ, M. (1916). Das Gesetz der Reaktionsgeschwindigkeit und der Gleichgewichte in Gasen. 2. anorg. Chem. 96, l-28.
17, 176-183 (1962)
Observations
on the Inactivation
of Viruses
by Suifhydryl
Reagents
A. C. ALLISON National Institute
for Medical Accepted
Research, February
London,
N.W.7,
England
6, 1962
The mode of inactivation of vaccinia and fowl plague viruses by thiol reagents has been studied. Inactivation appearsto follow first-order kinetics, cne rates varying with different reagents and proportional to their concentration. Inactivation by mercuric chloride, p-chloromercuribenzoate and merthiolate is largely reversed by addition of L-cysteine or reduced glutathione, whereas inactivation by iodoacetamide and n-ethylmaleimide is not so reversed. Sulfhydryl reagents do not affect the primary attachment of these viruses to host cells, but indirect evidence suggests that they may prevent uncoating of the virus which precedes multiplication. Cells were infected with vaccinia untreated and treated with mercuric chloride. After incubation of cells at 4” and 37” antibody was added. At the higher temperature very little neutralization took place, a resuh suggesting that the virus may have been taken up by the cells and become inaccessibleto antibody.
INTRODUCTION Evidence that the infectivity of many viruses is diminished when they are treated with sulfhydryl reagents has been summarized by Allison et al. (1962). Since this reaction can be used to obtain further information on the superficial structure of viruses and the early stages of virus multiplication-about’ which very little is known -the opportunity was taken to study it in more det,ail. Two viruses that can be obtained in relatively pure suspensions and titrated accurately by plaque techniques, vaccinia and fowl plague, were chosen for analysis. After completion of this work a paper by Choppin and Philipson (1961) appeared, in which similar experiments on ECHO viruses are described. Their results are very similar to those that will be rcported here, although there are some differences in detail. It seems that the conclusions which are drawn may be applicable to a wide range of viruses. MATERIAL
AND
METHODS
Vaccinia virus. The Lister Institute rabbit-adapt,ed st,rain of virus was used. This was propagated in rabbit skin and purified by three cycles of extraction with the fluoro-
carbon Arcton 63 (Gessler et al., 1956), followed by flocculation with M sodium chloride (Dawson and McFarlane, 1948) and brief ultrasonic dispersion in Gey’s solution without bicarbonate, buffered at pH 8.0 with 0.05 M tris (hydroxymethyl) aminomethane (Tris). Virus was titrated by the plaque technique using Tris-buffered overlay as described by Porterfield and Allison (1960). Fowl plague vir[Ls. The Bonn strain was propagated in the allantois of embryonated eggs and harvested after 24 hours’ incubation at 35”. Virus was adsorbed to and eluted from fowl cells as described by Frommhagen and Knight (1959)) submitted to differential centrifugation as described by Ada and Perry (1956)) and suspended in Tris-buffered Gey’s solution. Virus was titrated by plaque counts in chick embryo cell cultures using the method of Porterfield and Allison (1960). Sulfhydryl reagen,ts. A sample of pure pchloromercuribeneoic acid was kindly provided by Dr. L. A. Stocken, Oxford, and made up in an equivalent of sodium hydroxide. Samples of n-ethylmaleimide and iodoacetamide were kindly provided by Dr. R. Cecil, Oxford, and dissolved in Tris
176
SULFHYDRYL
REAGENTS
buffer. Solutions were not kept for longer than 1 week at 4”. Sulfhydryl compounds. L-Cysteine and reduced glu tathione (Nutritional Biochemicals, Inc.) were dissolved in Tris buffer and the pH was adjusted to 8.0 for immediate use. This pH was chosen because virus is stable and reactions with thiol reagents take place readily while there is only a low rate of spontaneous oxidation of thiols to disulfides (Boyer, 1959; Cecil and McPhee, 1959).
AND
VIRUSES
177
-0 -I .e
.-
-2
a
-3
j
-4
-5
OBSERVATIONS
Rate of Inactivation of Infectivity hydryl Reagents
by Sulf-
Preliminary experiments had shown that vaccinia virus and NDV lose infectivity when exposed to sulfhydryl reagents for 1 hour at 18” (Allison et aZ., 1962). In order to examine the rates of inactivation it was necessary to stop the reaction, and this was done in two ways. The first was to add an amount of L-cysteine equivalent to the amount of thiol reagent present; the reaction of sulfhydryl reagents with cysteine is very much faster than that with virus. Although excess cysteine reactivates virus, it was found experimentally that in the absence of excess cysteine significant reactivation did not occur. The second method was to allow the reaction to proceed with 1O-4 M thiol reagents and then dilute one thousandfold before dialyzing out the excess reagent for 6 hours at 4” and titrating the virus by the plaque bechnique. Results obtained by the two techniques were comparable; a representative experiment is shown in Fig. 1. It will be seen that with two reagents the reaction appears to follow first-order kinetics; plots of the logarithms of residual infective virus against time of exposure to the reagents gave straight lines over the greater part of the curves. However, the slopes of the curves varied considerably with the two reagents and were proportional to the concentrations of the reagents when they were tested at different dilutions. Mercuric chloride inactivated the virus much more rapidly than p-chloromercuribenzoate, and, in agreement with Micklem
+0-
120 Time of exposure in minutes
FIG. 1. Infectivity
of vaccinia virus (loglo) relative to controls after exposure for various times at 18°C to p-chloromercuribenzoate at concentrations of 10e4 M (curve A) and 3 X 10m4M (curve B) and to mercuric chloride at concentrations of 10m4M (curve C) and 3 X low4 (curve D).
I +0
I I I 2 Time of exposurein days
Fro. 2. Infectivity of vaccinia virus (log,,) tive to control after exposure to merthiolate concentration of 3 X 10e4 M at 18°C.
I 3 relaat a
and Kaplan (1958), the reaction with merthiolate (thiomersalate) was very slow (Fig. 2). The lower rate of inactivation at the end of curve D might possibly be due to a low rate of reactivation by thiol-containing compounds at the cell surfaces. The
178
ALLISON
slowness of the reaction with merthiolate suggested that virus inactivation might have been brought about by some decomposition product of merthiolate rather than by the compound itself. However, preincubation of the merthiolate solution for 48 hours at 37” did not affect the rate of reaction, so this interpretation seems unlikely. Results with fowl plague virus are given in Fig. 3. The rates of inactivation are somewhat greater than those of vaccinia virus with the same reagents. Again, mercuric chloride inactivated fowl plague virus more rapidly than did p-chloromercuribenzoate. Reversal of Inactivation Reagents
by Sulfhydryl
An important check on the specificity of the inactivation reaction with mercurials is its reversibility in the presence of other sulfhydryl compounds. After the virus had been partially inactivated by sulfhydryl reagents at a concentration of lo-” M for 2 hours at 18” and the excess reagent dialyzed away at 4”, L-cysteine or reduced glutathione, in Tris-buffered Gey’s solution, was added to give a final concentration of 1O-3 M. After 2 hours at 18”, excess cysteine and glutathione were removed by dialysis for 6 hours at 4”, the pH was adjusted to 7.2, and virus
-5 t I +0
I
I
I
I
30
60
90
120
Time of exposure in minutes FIG. 3. Infectivity of fowl plague virus (log,) relative to control after treatment at 18°C with p-chloromercuribenzoate (curve A) and mercuric chloride (curve B) at a conrentration of 3 X lo-’
M.
was titrat’ed. The results are shown in Table 1. It is apparent that both cysteine and reduced glutathione bring about considerable reactivation of infectivity of virus treated with mercurials, but no reactivation of virus treated with iodoacetamide or n-ethylmaleimide. The reactivation is somewhat more efficient with mercuric chloride than with p-chloromercuribenzoate. Mode of Inactivation hydryl Reagents
of Viruses by Sulf-
Experiments on adsorption of radioactively labeled vaccinia and fowl plague viruses have been reported elsewhere (Allison and Valentine, 1960). The rates of attachment of these viruses to chick embryo cell monolayers were not affected by pchloromercuribenzoate, iodoacetamide, or n-ethylmaleimide at concentrations as high as lo-:’ M. Such experiments exclude the possibility that the sulfhydryl reagents inhibit primary attachment of these viruses to host cells to a significant extent, so that the reduction of infectivity must be brought about in some other way. It is widely believed that an early stage in virus replication is the uncoating of the virus particle and that this is responsible for the so-called “eclipse phase,” the stage of low infectivity which precedes the appearance of newly synthesized virus. In an attempt to ascertain whether sulfhydryl reagents prevent the uncoating of the virus, experiments were carried out to see whether virus treated with these reagents goes into eclipse. Vaccinia virus was treated with mercuric chloride at a concentration of 5 x 1O-4 M for 1 hour at 18”; this reduced infectivity to approximately lop5 that of the original preparation. After removal of excess mercuric chloride by dialysis, the treated virus and untreated control virus were added to monolayers of chick embryo cells in petri dishes. After adsorption of virus for 4 hours at 4”, the fluid was removed and the cells were washed three times with cold Tris-buffered Gey’s solution, pH 7.4. Samples of cells were taken at this (zero) time and other samples were taken after incubating the cells at 37” for 3 hours. The cells were then released by incubation
SULFHYDRPL
REAGEXTS TABLE
AND
179
VIRUSES
1
INACTIVATION OF VIRUS INFECTIVITY BY SULFHYDRYL REAGENTS (10WM FOR 1 HOUR AT 18%) AND REACTIVATION BY CYSTEINE AND REDUCED GLUTATHIONE (GSH) (10--3M)” Virus Vaccinia
Fowl plague
Initial titer 7.2 x
8.8 x
Inactivating agent 107
107
a Titers given in plaque-forming
Postinactivation titer
p-Chloromercuribenzoate p-Chloromercuribenzoate Mercuric chloride Mercuric chloride Iodoacetamide Iodoacetamide n-Ethylmaleimide n-Ethylmaleimide p-Chloromercuribenzoate p-Chloromercuribenzoate Mercuric chloride Mercuric chloride Iodoacetamide Iodoacetamide
Reactivating agent
Postreactivation titer
1.2 x
104
Cysteine
7.8 x
1.2 x
10”
GSH
5.0 x 106
1.8 1.8 9.8 9.8 6.8 6.8
X X x X x x
lOa lo1 lo5 1Oj 105 106
Cysteine GSH Cysteine GSH Cysteine GSH
2.4 6.6 9.0 1.2 7.0 6.2
3.8 x
104
Cysteine
5.8 X 106
3.8 x
10’
GSH
9.1 x
106
8.7 8.7 2.2 2.2
103 lo3 105 105
Cysteine GSH
5.8 x 9.1 x
106 106
Cysteine
9.7 x 104
GSH
3.8 x
X X x x
X X x x x X
105
10’ 106 105 106 105 105
10”
units per milliliter.
in 0.025% trypsin for 10 minutes, an equivalent of trypsin inhibitor (Worthington) was added, and the suspension was given a brief (15 seconds) ultrasonic dispersion. One half of the homogenates were treated with 1O-3 M reduced glutathione for 1 hour at 18”; the others were kept in suspension for the same time at, the same temperature. The results of this experiment are sumInarized in ‘Fable 2. It will be seen that on incubation of both untreated viruses with cells for 3 hours there is a marked fall in titer, in other words, a well-defined eclipse. However, on incubation of the mercuric chloride-treated virus with cells the fall in virus titer recoverable by glutathione reactivation is much less marked. Similar results were consistently obtained although the magnitude of the effect varied. Effect of Antibody on T zruses Znactivated by Thiol Reagents ” Vaccinia virus was treated with 5 x 10F1 M mercuric chloride for 1 hour, and the latter was removed by dialysis at 4”. To the virus was added a neutralizing dose of hightiter rabbit antibody. The complex was
treated with lo-” M cysteine or reduced glutathione, but no restoration of infectivity was obtained. Vaccinia virus thus inactivated with mercuric chloride and untreated control virus were allowed to adsorb to chick embryo cell monolayers at 4” as described in the previous section. The cells were washed with cold Tris-buffered Gey’s solution, and half of the monolayers were allowed to remain at 4” for 3 hours while the others were incubated at 37” for 3 hours. Antibody was then added to some of the monolayers and washed off after l/2 hour at 4”. The cells were resuspended by incubation at 37” with 0.025% trypsin and, after addition of trypsin inhibitor, homogenized. To one half of the preparations lo-” M reduced glutathione was added before assay of infectivity. The results of this experiment are shown in Table 3. When antibody was added to t,he cells, the recoverable virus titer was decreased much more when the cells had stood at 4” than when they had been incubated at 37”. These results suggest that at 4” the virus remains at the cell surface in a position readily accessible to
180
ALLIS03 TABLE
2
E;FFECTS OF SULFHYDRYL REAGENTS (5 X lo1 Af FOR 1 HOUR AT 18”) ON THE ECLIPSE OF VIRCSEF
Virus
Vaccinia
Fowl plague
Preliminary treatment
2% g ‘i 3 .; 0
Virus titer at different times 3 Hours
0
None None Mercuric chloride Mercuric chloride
No Yes No
7.1 x 104 4.3 x 103 9.7 x 10” 2.6 X lo3 9.2 x 10’ 3.1 x 10’
Yes
6.4 X 10% 2.1 x 103
None None Mercuric chloride Mercuric chloride
No Yes No
2.2 x 105 9.7 x 102 9.5 x 10’ 3.2 X lo3 7.3 x 102 2.5 X lo*
Yes
8.2 X lo3
1.2 x 103
a Treated and untreated viruses were added to chick embryo cells; the infective virus titer (PFU/ ml) determined at zero time and after incubation for 3 hours at 37”. Some homogenates were reactivated with 10m3M reduced glutathione (GSH).
antibody, whereas at 37” the virus enters the cell and is no longer accessible. Hence it would seem that vaccinia virus inactivated by thiol reagents is taken up by cells, but may not undergo the uncoating that usually follows within the cell. Lack of Effect of Sulfhydryl iveuraminidase Activity
Reagents on
Whether neuraminidase activity plays any part in adsorption of myxoviruses is uncertain. Treatment with p-chloromercuribenzoate (1O-3 M for 1 hour) was found not to have any effect on hemagglutination by fowl plague virus, in agreement with the findings of Klein et al. (1948)) Philipson and Choppin (1960)) and Buckland (1960) with other myxoviruses. The neuraminidase activities of fowl plague and influenza A viruses, tested on fowl erythrocytes as described by Burnet and Stone (1947), were also unaffected by sulfhydryl reagents. DISCUSSION
It has been shown that the infectivity of vaccinia and fowl plague viruses is con-
siderably reduced by several sulfhydryl reagents. There is little doubt that virus sulfinvolved,, . since groups are hydryl considerable reactivation of infectivity follows treatment with excess cysteine or reduced glutathione. A well-known property of mercaptides formed by the combination of sulfhydryl groups with heavy metals is the reversal of the reaction by other sulfhydryl compounds (Boyer, 1959; Cecil and McPhee, 1959). On the other hand, as a result of the reaction of sulfhydryl groups with iodoacetamide and n-ethylmaleimide, stable thioether bonds are formed, and these are not broken by addition of excess thiol. Hence no reactivation of infectivity after treatment with these reagents would be expected, and none was found. One of the conclusions that can be drawn from these experiments and those of Choppin and Philipson (1961) and Allison et al. (1962) is that sulfhydryl groups form an accessible part of the surface structure of many viruses. Too little is known about the surfaces of most viruses (e.g., poxviruses and myxoviruses) for this information to be related to specific structures. However, enteroviruses, adenoviruses, and many plant viruses are known to have a regular arrangement of surface subunits (Klug and Caspar, 1960)) and the possibility that these are held together by disulfide bonds deserves consideration. Thus, the surface subunits or capsomeres of poliovirus have a molecular weight of approximately 80,000 (Klug and Caspar, 1960) and a cysteine content of about 0.7% (Levintow and Darnell, 1960). Each subunit would therefore have about five cysteine residues, some of which might be concerned with bonding subunits together. Reaction with sulfhydryl reagents might prevent the separation of subunits which is necessary for emergence of nucleic acid in the early stages of replication. It is worth recalling that Kozloff et al. (1957) have adduced evidence suggesting that, in the coliphage T2, tail fibers are bound to the tail sheath, and possibly to one another, by thiol ester bonds. The protection afforded by cystine against thermal inactivation of a temperature-sensitive poliovirus mutant (Pohjanpelto, 1958) very probably involves thiol groups. Possibly the formation of cysteine mercaptides by disul-
SULFHYDRYL
REAGENTS
TABLE 3 EFFECT OF ANTIBODY ADDITION TO CELLS PREVIOUSLY INFECTED WITH VACCINIA VIRUS UNTREATED AND TREATED WITH MERCURIC CHLORIDE (5 X 1OF M FOR 1 HOUR AT 18”)a
-
TemPrelimiperanary treat- ture o ment of incuvirus bation (“(I) -None
_----
4 4 4 4 37 37 37 37 --.-
Lddition of anti- Additior of GSH body
Virus titer in final homogenate
No No Yes Yes No No Yes Yes __--
5.3 6.7 4.8 6.2 3.2 3.1 2.7 4.8
No Yes No Yes No Yes No Yes _--~
x X x X X x X X
10’ lo4 10’ 10’ lo3 103 lo2 lo*
3.8 X 10L No No 4 2.3 X lo1 Yes No 4 4 4 Yes No 1.4 x 10’ Yes 4 Yes 2.7 X lo2 No 37 No 1.1 x 101 Yes 37 No 1.2 x 102 Yes No 37 8.6 x 102 Yes Yes 37 u After removal of antibody and washing of the cells they were homogenized and half the homogenates were treated with 1O-3 M reduced glutathione (CSH) for 1 hour at 4” before titration.
and S are their concentrations, r and m the radius and mass of the respective particles indicated by the subscripts, Ic is the Boltzmann constant, and T is the absolute temperature. This has been calculated for pchloromercuribenzoate and vaccinia (radius 115 rnp, mass 3.6 x 10-l” g), and it is apparent that less than one collision in lo7 results in inactivation of the virus. The low efficiency of the reaction is unlike mercaptide formation with simple thiols (Cecil and McPhee, 1959)) a result which suggests that breakage of disulfide bonds may be involved. Nevertheless, the kinetics indicate that inactivation is brought about by the attachment, of a single molecule of sulfhydryl reagent to a critical site on the virus particle. The reaction can be expressed
Mercuric chloride
fide interchange prevents the oxidation, e.g., to sulfinic or sulfonic acids, of thiol groups partially exposed in the mutant but not in the parent virus. One of the unexpected findings in this investigation has been the apparent firstorder kinetics of inactivation of viruses by sulfhydryl reagents. Similar results have been obtained by Choppin and Philipson (1961) for inactivation of enteroviruses, so the nature of the reaction seems to be quite general. The reaction is, however, very inefficient; the number of collisions between sulfhydryl reagents and virus particles can be calculated from the Trauts (1916) equation v”S = VS(r,
+ r,)’ d&T(l/m,
+ l/m,)
where v%’ is the number of collisions/cm3/ set between the two types of particles, V
181
AND VIRUSES
V+Ri
VR
where V is the infective virus, original concentration [V,,], R is the reagent, VR is the inactivated complex and Ic is the rate constant of the reaction. Then d[V]/dt
= [V] [R] k
and for excess [RI, [R] log [I’]
constant,
= log [V,] -
k [R] t
The slope of the graph is - k [RI, and is thus proportional to the concentration of the sulfhydryl reagent. It is possible that a single critical thiol group is present in each virus particle. Thus, the capsomeres might have to come apart near the end of the nucleic acid chain in order that the latter can uncoil and initiate infection, whereas opening of the capsomeres anywhere else might not allow escape of the nucleic acid. However, the data do not exclude the existence of many thiol groups in each virus particle, each having finite probabilities on the one hand of being necessary for multiplication and on the other hand of being inactivated by a single molecule of sulfhydryl reagent. The need for more than one molecule of reagent for inactivation of the site is excluded. The situation is reminiscent of inactivation of virus by excess antibody, which also follows first-order kinetics (Dulbecco et al., 1956).
ALLISOK
182
Inactivation of a critical virus site by the attachment of one molecule of sulfhydryl reagent is more readily understandable if the initial reaction initiates a series of disulfide interchanges of the type described by Jensen (1959) in some proteins, e.g. R-S-S-RI + PCMB
PCMB I + RS
Re-S-S-R3 R2
S-R, I S-R,
SH
where PCMB stands for p-chloromercuribenzoate. This would happen infrequently with divalent mercury, the following being the usual reaction: R-S’
R-S-S-R,
Hg
+ Hg++ + R-S-S-R*
Rz-S-S-R,
‘S-R,
by a mechanism analogous to pinocytosis and there undergo disruption. Fazekas de St. Groth (1948) has presented arguments in support of the latter view, but these are disputed by Rubin and Franklin (1957). Since Rubin and Franklin were concerned with red blood cells their results may not be applicable to other cells. Thus, the recent electron micrographs of Dales and Siminovitch (1961) suggest that vaccinia virus particles are taken up by L cells by pinocytosis. Experiments which have been presented in this paper, and similar experiments with poliovirus briefly mentioned by Hirst (1961)) indicate that’ when cells are kept at low temperatures virus remains in a superficial position readily accessible to cysteine, glutathione, and antibody. On the other hand, at 37” the virus penetrates into the cell and becomes inaccessible to added antibody, even though after treatment with thiol reagents the virus does not go into eclipse in the normal way. Thus, unlike the situation in phage, the stages of cell penetration and uncoating may be distinct. The first stage has a high temperature coefficient and seems to resemble pinocytosis, which has been described by Holter (1959) and others in a wide range of cell types.
Hence a higher probability of restoring the original structure would be expected after inactivation by mercurials divalent toward thiol than after inactivation by mercurials univalent toward thiol; and this has been observed. More evidence is required, however, before it can be accepted that a disulfide interchange reaction takes place. In our experiments sulfhydryl reagents did not affect the rate of attachment of vaccinia REFERENCES and fowl plague viruses to host cells. This is not always true: hemagglutination by a ADA, G. L., and PERRY, B. T. (1956). Influenza virus nucleic acid : relationship between biological number of viruses is prevented by exposing characteristics of the virus particle and propthem to sulfhydryl reagents (Philipson and erties of the nucleic acid. J. Gen. Microbial. 14, Choppin, 1960; Buckland, 1960) and at623633. tachment of ECHO 7 virus to monkey kidALLISON, A. C., &JCKL~SD, F. E., and ANDREWES, ney cells is also reduced by such treatment C. H. (1962). Effects of sulfhydryl reagents on (Choppin and Philipson, 1961). The marked infectivity of some viruses. Virology 17, 171reduction of infectivity of vaccinia and fowl 175. plague viruses by sulfhydryl reagents is ALLISON, A. C., and VALENTINE, R. C. (1969). Virus mediated by another mechanism, and some particle adsorption. III. Adsorption of viruses evidence has been obtained that after treatby cell monolayers and effects of some variables on adsorption. Bioehim. et Biophys. Acta 40, ment with mercurials the uncoating of the 400-410. virus prior to initiation of infection may not BOYER, P. D. (1959). Sulfhydryl and disulfide occur. groups of enzymes. In “The Enzymes” (P. D. The observations have some bearing on Bayer, H. Lardy, and L. Myrbiick, eds.), 2nd ed., the problem whether infecting animals virus Vol. 1, pp. 511-562. Academic Press, New York. particles remain at or near the host cell BUCKLAND, F. E. (1960). Inactivation of virus surface, only infectious nucleoprotein penehaemagglutinins by para-chloromercuribenzoate. trating into the cell, as in coliphage, or Nature 188, 768. whether the part,icles are taken into the cell BURSET, F. M., and STONE, J. D. (1947). The re-
SULFHYDRYL
REAGENTS
ceptor destroying enzyme of V. cholerae. Australian .I. Exptl. Biol. Med. Sci. 25, 227-238. CECIL, R., and MCPHEE, J. R. (1959). The sulphur chemistry of proteins. Advances in Protein Chem.
14,255-389. CHOPPIN, P. W., and PHILIPSON, L. (1961). The
inactivation. of enterovirus activity by the sulfJ. hydryl reagent, p-chloromercuribenzoate. Exptl. Med. 113, 713-734. DALES, S., and SIMINOVITCH, L. (1961). The devel-
opment of vaccinia virus in Earle’s L strain cells as examined by electron microscopy. J. Biophys. Biochem.
Cytol.
10, 475-598.
DAWSON, I. M., and MCFARLANE, A. S. (1948). Structure of an animal virus. Nature 161, 464466. DULBECCO, R., VOGT,M., and STRICKLAND,A. G. R.
(1956). A study of the basic aspects of neutralization of two animals viruses, western equine encephalitis virus and poliomyelitis virus. Virology 2, 162-205. FAZEKAS DE ST. GROTH, S. (1948). Viropexis, the mechanism of influenza virus infection. Nature
162,294-295. FROMYHAGEN, L. H., and KNIGHT, C. A. (1959). Column purification of influenza virus. Virology 8, 198-208.
GESSLER,A. El., BENDER,C. E., and PARKINSON,M. C. (1956). A new rapid method for isolating viruses by selective fluorocarbon deproteinization. Trans. N. Y. Acad. Sci. 18, 701-703. HIRST, G. K. (1961). Reversible inactivation of poliovirus through exposure to sulfhydryl compounds, In “Poliomyelitis,” pp. 14-15. Lippincott, Philadelphia. HOLTER, H. (1959). Pinocytosis. Intern. Rev. Cytol. 8,481-504.
AND
VIRUSES
183
JENSEN, E. V. (1959). Sulfhydryl-disulfide
interchange. Science 130, 1319-1322. KLEIN, M., BREWER, J. H., PEREZ, J. E., and DAY, B. (1948). The inactivation of influenza virus by mercurials and the reactivation by sodium thioglycollate and BAL. J. Zmmunol. 59, 135140. KLUG, A., and CASPAR,D. L. D. (1960). The structure of small viruses. Advances in Virus Rcsearch 7,225-325. KOZLOFF, L. W., LUTE, M. and HENDERSON, K. (1957). Viral invasion. I. Rupture of thiol ester bonds in the bacteriophage tail. J. Biol. Chem. 228,511-528. LEVINTOW, L., and DARNELL, J. E., JR. (1960). A
simplified procedure for purification of large amounts of poliovirus : characterization and amino acid analysis of type 1 poliovirus. J. Biol. Chem. 235,70-73. MICKLEM, L. R., and KAPLAN, C. (1958). The influence of thiomersalate on vaccinia virus. ViroZogy ,6,775-777. PHILIPSON, L., and CHOPPIN, P. W. (1960). On the
role of virus sulfhydryl groups in the attachment of enteroviruses to erythrocytes. J. Exptl. Med.
112,455478. POHJANPELTO, P. (1958). Stabilization of poliovirus by cystine. Virology 6, 472487. PORTERFIELD, J. S., and ALLISON, A. C. (1960).
Studies with poxviruses by an improved plaque technique. Virology 10, 233-244. RUBIN, H., and FRANKLIN, R. M. (1957). On the mechanism of Newcastle disease virus neutralization by immune serum. Virology 3, 84-95. TRAUTZ, M. (1916). Das Gesetz der Reaktionsgeschwindigkeit und der Gleichgewichte in Gasen. 2. anorg. Chem. 96, l-28.