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Herpes simplex virus proteins: DNA-binding proteins in infected cells and in the virus structure
VIROLOGY
68,
124-134 (1975)
Herpes Simplex
Virus Proteins:
Infected
Virology
Proteins
in
Cells and in the Virus Structure
GARY J. BAYLISS, M.R.C.
DNA-Binding
HOWARD
S. MARSDEN,
AND
JOHN
HAY
Unit, Urziuemity of Glasgou’, Glasgow Gf 1 5JR, Scotland Accepted June 10, 1975
DNA-binding proteins have been isolated from BHKZl/C13 cells infected with herpes simplex virus type 1 strain 17 syn+ by native and denatured DNA affinity chromatography. Sixteen proteins have been identified as specific to the infected cell on SDS polyacrylamide gradient gel electrophoresis and their molecular weights range from 145 x lo3 to 10.5 x 103. After infection with herpes simplex virus many of the mock infected cell specific DNA binding proteins are no longer produced and this “switch off’ of host cell protein synthesis is rapid. The virus-induced DNA-binding proteins fall into two quantitative classes and there is evidence for temporal control of their synthesis. One of the virus-induced DNA-binding proteins has a molecular weight identical to a polypeptide found in full but not empty particles and another binds preferentially to denatured DNA.
Four years ago, Alberts and Herrick (1970) introduced a convenient method that allowed separation from general cell proteins of those proteins that bind to DNA. Since then, several investigators have examined a range of cell types and confirmed the presence of DNA-binding proteins (Salas and Green, 1971; Fox and Pardee, 1971; Tsai and Green, 1973a). Alberts and Herrick (1970) also reported that infection of bacterial cells with viruses induced the formation of new DNA-binding proteins and some of these were shown to be virus coded. The most extensively studied example of these proteins is the gene 32 product of bacteriophage T4 whose role in viral DNA synthesis is probably to weaken the structure of double-stranded DNA and allow replication to proceed (Alberts and Frey, 1970). In African Green Monkey cells infected with adenovirus, two virus induced DNAbinding proteins (48,000 and 72,000 daltons) have been found, and at least one of these proteins appears to be essential for virus DNA synthesis (Van der Vliet and Levine, 1973). Herpes simplex virus (HSV) possesses a
genome of double stranded DNA with a molecular weight of about 100 x lo6 and this considerable protein specifying potential has been shown to be realised in a complex pattern of induction of at least 50 proteins in the infected cell (Honess and Roizman, 1974; Marsden and SubakSharpe, 1975). Genetic studies on several viruses of the herpes group suggest that about half the viral genes are directly or indirectly concerned with viral DNA synthesis (Brown, Ritchie, and Subak-Sharpe, 1973; Schaffer, Aron, Biswal, and Benyesh-Melnick, 1973; Halliburton and Timbury, 1972; Pringle, Howard, and Hay, 1973) and it is possible that the products of at least some of those genes may possess DNA-binding properties. In addition, one or more proteins of the virus structure may be associated with nucleic acid in the particle (Gibson and Roizman, 1972) and it seems likely that others also may be involved in the organisation of capsid substructures around a core of DNA. For the above reasons, and also to isolate a subpopulation of operationally defined proteins from the complex herpes virus124
Copyright@ 1975 by Academic Press. Inc. All rights of reproduction
in any form reserved.
HSV DNA BINDING
infected cell mixture, a study of DNAbinding proteins in herpes virus-infected cells was undertaken. MATERIALS
Cells. Monolayers of BHK21 Cl3 cells (Macpherson and Stoker, 1962) were grown in 80-0~ rotating bottles in Eagle’s medium supplemented with 10% (v/v) tryptose phosphate broth (Difco) and 10% (v/v) calf serum. Virus. Glasgow strain 17 syn+ herpes simplex virus type 1 was grown into stocks by infecting BHK21 Cl3 monolayers at an input multiplicity of 1 PFU/300 cells and harvesting the virus after 3 days of incubation at 31”. Infectivity was assayed using the method of Russell (1962). Label@ of cellular and viral proteins. Cell sheets were used when they were near confluency. The cell sheet was washed with methionine-free Eagle’s medium and then infected at a multiplicity of infection of 10 PFU/cell with virus suspension in 10 ml of Eagle’s medium containing one-fifth the normal concentration of methionine and supplemented with 2% calf serum (E (met/5) C2). After 60 min of adsorption at 37”, 40 ml of E (met/5) C2 were added (this is considered 0 hr after adsorption). [“S lmethionine (100-400 pCi/ml) was added from O-5 hr, 5-24 hr, or O-24 hr postadsorption. Mock infected cultures were treated identically, except that virus was omitted in the initial 10 ml of innoculum. Preparation of cell extracts. Cells were harvested by scraping into the medium and centrifuged at 800 g for 10 min at 4”. The resulting pellet was washed with 0.15 M NaCl, 0.02 M Tris-HCl (pH 8.2), resuspended in buffer 1 (1.7 M NaCl, 1 mM EDTA, 1 mM /3-mercaptoethanol, 20 mM Tris-HCl, pH 8.2) and treated in an ultrasonic bath for 60 sec. Following clarification as before, 10% (w/w) polyethylene glycol (6000) was added to the supernatant and, after standing for 30 min at O”, the precipitate was removed by centrifugation at 10,000 g for 30 set at 4”. This final supernatant was then dialysed against buffer 2 (50 mA4 NaCl, 1 mM EDTA, 1 mM
PROTEINS
125
B-mercaptoethanol, 20 mM Tris-HCl, pH 8.2, 10% (v/v) glycerol) and could be stored at -70” if required. Purification of virus. Virus was labelled from 5 to 24 hr postinfection as described by Marsden and Subak-Sharpe (1975) and purified by the method of Spear and Roizman (1972). of DNA columns. Native Preparation calf thymus DNA-cellulose columns were prepared as described by Alberts and Herrick (1970) and denatured DNA-sepharose columns according to the method of Poonian, Schlaback, and Weissbach (1971). DNA was denatured by boiling for 15 min followed by rapid cooling in iced water. SDS polyacrylamide gel electrophoresis. Tube gels (discontinuous) were prepared according to the method of Laemli (1970). Following electrophoresis, the gels were sliced longitudinally, dried, and set up for autoradiography as below. In experiments carrying low levels of radioactivity, proteins were precipitated (see Fig. 2) prior to electrophoresis. This often resulted in the formation of high molecular weight aggregates. Slab gels (discontinuous, gradient) were prepared as described by Marsden and Subak-Sharpe (1975). The gels were stained with Coomassie Brilliant Blue and set up for autoradiography (Marsden and
FIG. 1. Native DNA cellulose chromatography of proteins extracted from mock-infected BHK21 Cl3 cells (W---m) and cells that had been infected with herpes simplex type 1 strain 17 s.sn+ (A-A).
126
BAYLISS.
MARSDEN
ml o-z4hrs.
AND HAY
Subak-Sharpe, 1975). Kodak BB54 film was used for autoradiography of tube gels and Kodak KD54T for slab gels. The protein samples were denatured by boiling for 2 min in the presence of 0.05 M Tris-HCl pH 6.8, 2% (w/v) SDS, 0.5% P-mercaptoethanol (with bromophenol blue included as a front marker) and applied to the gel after cooling. Enzyme assays. DNA polymerase was assayed using the high-salt herpes-specific conditions of Keir et al. (1966) and thymidine kinase by the method of Hay et al. (1971). RESULTS
Native DNA cellulose chromatography of infected and uninfected BHK21 Cl3 cell proteins, Mock-infected and HSV-1 infected BHK21 Cl3 cells were labelled with [,% Jmethionine from 0 to 24, 0 to 5, or 5 to 24 hr after infection. After harvesting and extraction as described in the Methods section, samples of the dialysed final supernatants were applied to native DNA cellulose columns. As shown in Fig. 1, both infected and mock-infected cell extracts bound to the column and then eluted in a similar manner. We find that the great majority of labelled protein (9%99%) passes through the column. The bound protein was eluted stepwise with buffer 2 containing respectively 0.15 and 0.60 M NaCI. Very little additional material is released from the column by buffer 2 containing 2 M NaCl, and analysis of this (not shown) reveals the presence only of proteins eluted at the lower salt concentrations. If a sample of DNA cellulose from the column is counted after elution, a small and variable amount of the originally bound protein will be FIG. 2. Densitometer traces of autoradiographs found to be uneluted (about l-3% of the prepared from longitudinally sliced and dried polyacrylamide tube gels of the 0.60 M NaCl fraction of originally bound protein). DNA-binding proteins prepared according to the The first series of experiments analysing protocol given in the Methods section, concentrated fractions from the DNA binding column prior to electrophoresis by precipitation with 10 volwas carried out on 7% polyacrylamide tube umes of acetone at -40’ for 16 hr. (a) Mock-infected gels (Methods section). Fig. 2 shows the BHK21 Cl3 cells labelled from 0 to 24 hr (0.7 x lo5 analysis of 0.60 M fraction proteins eluted cpm applied to gel). (b) HSV 1 17 syn+ infected BHK21 Cl3 cells labelled from O-5 hr postinfection. (1 x 10’ cpm applied to gel). (c) HSV 1 17 syn+ infected BHK21 cells labelled from 5-24 hr postinfection. (1 x lo5 cpm applied to gel). Approximate
molecular weights of the major viral peaks are shown in parentheses as daltons x 10m3.The autoradiograph was exposed for 60 days prior to development.
HSV DNA BINDING
PROTEINS
127
from a native DNA cellulose column (the 0.15 and 0.60 M fractions are qualitatively similar). Two aspects emerge. First, the rather complex pattern of DNA-binding proteins (B.P.‘s) from the mock-infected cell extract is replaced with a profile containing four or five major bands. The bands have been numbered according to the pattern found in the slab gel analysis (see Fig. 3). We are able to identify four major bands found in the tube gel analysis with four of the five major bands found in the slab gel analysis (B.P.‘s 2, 3, 4, and 6) since the molecular weights are in reasonable agreement (within 12%). (The peak migrating close to the dye front in the tube gel may be B.P. 12 but may contain more than one polypeptide. This possibility prevents an accurate estimate of the molecular weight for this polypeptide). Approximate molecular weights for the polypeptides were calculated by reference to standard proteins of known molecular weight (those used in the slab gel analysis) and these calculated values (as daltons x 10-3) are shown in parentheses in Fig. 2. The molecular weights of the DNA-binding proteins as analysed on slab gels are given in Table 1.
FIG. 3. Autoradiograph of DNA-binding proteins eluted from native and denatured DNA cellulose columns in the 0.15 and 0.60 M NaCl fractions and electrophoresed in an SDS, 7-15s polyacrylamide gradient slab gel. The DNA-binding proteins were prepared from mock-infected (mi) and herpes simplex virus strain 17 syn+ infected (17) cells labelled from 0 to 24 hr postinfection with [SsS]methionine. The four right-hand positions show the polypeptides that bound to and eluted from native DNA cellulose, while the four left-hand positions show the polypeptides that bound to and eluted from denatured DNA sepharose. The proteins eluting from denatured DNA had been passed through native DNA cellulose prior to their application to the denatured DNA column. Samples
were applied to the gel without concentration. Ten microliters of each sample were used except for the mock-infected DNA binding proteins that had been eluted from the native DNA cellulose column. Here, the counts were appreciably lower than all other samples and 15 hl of the 0.15 A4 fraction and 25 ~1 of the 0.6 M fraction were applied to the gel. The cpm applied were as follows: (a) for those proteins eluted from denatured DNA sepharose; MI 0.15 M, 12.8 x 10s; infected 0.15 M, 10.2 x 103; MI 0.6 M, 9.6 x lOa; info.6 M, 9.6 x 103; and (b) for those proteins eluting from native DNA cellulose; MI 0.15 M, 8.6 x lo3 (in 15 r.d); infected 0.15 M, 10.1 x 10’; MI 0.6 M, 5.2 x lo3 (in 25 ~1); infected 0.6 M, 8.1 x 103. The autoradiograph shown was produced by an exposure of 67 days. The number on the right-hand side of the gel is the given DNA-binding protein identification number (B.P.) for virus-induced polypeptides. A small square ( n ) is placed to the left of each of these polypeptide bands to assist in identification among samples (e.g., B.P. I occurs only in the 17 s.yn+ infected cell native DNA 0.60 M fraction while B.P. 2 occurs in each of the four 17 syn+ infected cell fractions).
128
BAYLISS, TABLE
DNAbinding protein
Molecular weight x 10-3
1 2
145 136
3 4 5 6
87 62 59 43
7 8 9
42 41 40
10 11 12 13 14 15 16
32 31 21 19 17 12 10.5
MARSDEN
1 Additional characterisation
Major species; comigrates with V,,136. Major species Major species Major species; comigrates with V,,43.
Major species from denatured DNA
Major species
The second aspect to emerge from Fig. 2 is that B.P. 2 and B.P. 6 reach their peak of synthesis during the 0- to 5-hr labelling period (Fig. 2B) whereas B.P. 3 and 4 reach their peak of synthesis during 5 to 24 hr (Fig. 2C). Thus, there appears to be some measure of temporal control in the synthesis of the DNA-binding proteins. Infection and labelling of serum-starved “resting cells” (Howard, Hay, Melvin, and Durham, 1974) gave essentially the same results as shown in Fig. 2, but in the resting cell controls, the host DNA-binding proteins were present in reduced amounts, about one-half to one-third of Fig. 2. The protein profiles in a series of repeat experiments employing tube gels were qualitatively identical to those of Fig. 2 but showed some quantitative variation. Subsequent analyses were performed on gradient polyacrylamide slab gels that have superior resolution over a wide range of molecular weights (Marsden and SubakSharpe, 1975). Analysis of native DNA-binding proteins on gradient polyacrylamide slab gel electrophoresis. Proteins eluted from the native DNA-cellulose column as described above were denatured with SDS and /3-mercaptoethanol and electrophoresed on
AND HAY
7-15s acrvlamide gradients (see Methods section). Fig. 3 shows an autoradiograph of the analysis of [3”S]methionine-labelled DNA-binding proteins obtained from strain 17 svn+ infected and mock-infected cell extracts. The increased resolution of this gel system reveals a much more detailed profile than that of the tube gel system (Fig. 2). HSV-induced DNA-binding proteins were identified either (a) by their occurrence in infected cell extracts and absence in the corresponding mockinfected cell extracts, or (b) by their presincreased ence in very significantly amounts in the infected cell extract compared to the mock-infected cell extract. Their position on the autoradiograph is indicated by a small square to the left of the identified band and the numbering system we have adopted for these proteins is given to the right of the autoradiograph. Densitometer traces of these autoradiographs are illustrated in Fig. 4. In the profile of proteins from mock-infected BHK cells eluted at 0.15 M NaCl (Fig. 4A) a complex pattern of proteins is seen while at 0.60 M NaCl the profile is more simple (Fig. 4C). However, the 0.60 M NaCl eluate contains several proteins found in the 0.15 M NaCl fraction in addition to polypeptides that appear only at the higher salt concentration. A dramatic change in the pattern of synthesis of DNA-binding proteins occurs after infection (Figs. 4C and 4D), in agreement with the earlier results (Fig. 2). The DNA-binding proteins of the mock-infected BHK cell have largely disappeared and a spectrum of new species appears in both the 0.15 and 0.60 M NaCl eluates. There is a variation in strength of binding within the population of virusinduced DNA-binding proteins, as evidenced by the relative amounts of polypeptides eluted at the different salt concentrations. Proteins binding to denatured DNA. The proteins that had failed to bind to native DNA were subsequently applied to a denatured DNA column, eluted and analysed as described in the Methods section. Fig. 5 (A, B, C, D) shows densitometer traces of the four left-hand profiles given in the autoradiograph of Fig. 3.
HSV DNA BINDING mioraa
129
PROTEINS
c
mioa
1 I ‘1
FIG. 4. Densitometer traces of Mock-infected cells: 0.15 M eluate 0.15 M eluate from a native DNA cellulose column. (D) HSV 1 strain
the four right-hand positions of the from a native DNA cellulose column. cellulose column. (C) Mock-infected 17 syn+ infected cells: 0.60 M eluate
The mock-infected profiles are complex with little similarity between the populations of proteins eluting at 0.15 and 0.60 M NaCl or with the patterns of native DNAbinding proteins. Following infection many of the host proteins disappear and are replaced by virus-induced proteins. All of these virus-induced proteins are found in the native DNA-binding population (Figs. 4C and 4D), but it is striking that protein B.P. 9, a very minor component of the native DNA pattern, is the major species in the denatured DNA fraction eluted at 0.15 M NaCl (Fig. 3B). This is the behaviour expected of proteins with properties similar to those of bacteriophage T4 gene 32 product (Alberts and Frey, 1970). DNA-binding proteins as components of the uirus structure. Herpes simplex virus strain 17 syn+ was purified by the method of Spear and Roizman (1972) after incuba-
autoradiograph presented in Fig. 3. (A) (B) HSV 1 strain 17 syn+ infected cells: cells: 0.60 M eluate from a native DNA from a native DNA cellulose column.
tion with [3”S]methionine between 5 and 24 hr to minimise labelling of host cell proteins. Virus structural proteins from this preparation were then fractionated on a gradient slab gel side by side with DNAbinding proteins. Fig. 6 shows the autoradiogram of such a gel. One structural protein (V,,43) runs with a mobility identical to B.P. 6, the major virus-induced protein binding to native DNA. As already observed from Fig. 2, B.P. 6 is an early protein as is V,,43 (Marsden and SubakSharpe, 1975), thus providing further evidence for their identity. V,,43 is a minor structural protein of the virion but a major protein of the infected cell (Marsden and Subak-Sharpe, 1975). There may also be identity between V,,136 and B.P. 2. Virus-induced enzymes as DNA-binding proteins. After herpes simplex virus infection DNA polymerase and pyrimidine
BAYLISS. MARSDEN AND HAY
130 h !
mi
045M
C mi
0.6M
:I 1 ;’ ? :
I
r
3 I
h
h
FIG, 5. Densitometer traces of the four left-hand positions of the autoradiograph presented in Fig. 3. (A) Mock-infected cells: 0.15 M eluate from a denatured DNA sepharose column. (B) HSV 1 strain 17 syn+ infected cells: 0.15 M eluate from a denatured DNA sepharose column. (Cl Mock-infected cells: 0.60 M eluate from a denatured DNA sepharose column. (D) HSV 1 strain 17 syn+ infected cells: 0.60 M eluate from a denatured DNA sepharose column. deoxynucleoside kinase activities are induced and appear to be virus-specified
(Keir et al., 1966; Hay et al., 1971). Infected cells were extracted 6 hr after infection and were assayed for these activities. The active extracts were applied to a native DNA cellulose column. Thymidine kinase activity did not bind to the column and was recovered in the void volume and preliminary washings. However, the virus-
specific DNA polymerase measured by the high salt a&say specific for the viral enzyme (Keir et al., 19661 was retained by the column; it consistently eluted largely at 0.60 M KC1 (Fig. 7), which indicates that a relatively strong association exists between the enzyme and DNA. Herpes-specific DNA polymerase similarly binds to and elutes from denatured DNA cellulose. KC1 is used as the eluant since NaCl is inhibi-
HSV DNA BINDING
131
PROTEINS
tory to the enzyme activity while, under the assay conditions used no DNA polymerase activity from uninfected cells can be detected in the eluates from either column. Molecular weight determination on DNA-binding proteins. The mobility of DNA-binding proteins has been correlated with that of 18 standard polypeptides of known molecular weight (220 x lo3 to 5.7 x 103) (Marsden and Subak-Sharpe, 1975) which were coelectrophoresed in adjacent positions in gradient slab gel electrophoresis. Table 1 lists the estimated molecular weights of the herpes simplex virusinduced DNA-binding proteins and gives a summary of their observed properties. As yet, we are unable to fit the DNA polymerase activity into the pattern of molecular weights, as the molecular weight of the purified enzyme has not been accurately determined under denaturing conditions. However, preliminary evidence from studies with viral ts mutants suggests that a molecular weight in excess of 130,000 is likely (Hay, J. and Marsden, H. S., unpublished observations). The estimated molecular weights of several of the DNA-binding proteins correspond with the molecular weights of HSV-1 strain 17-induced nonstructural polypeptides (Marsden and Subak-Sharpe, 1975). Specifically these are: B.P. 8 (MW = 41 x 103); B.P. 9 (MW = 40 x 103); B.P. 13 (MW = 19 x 103) and B.P. 14 (MW = 17 x i03) However, since DNA-binding proteins
Fm. 6. Autoradiograph of electrophoresed proteins from purified herpes simplex virus type 1 17 syn+ (PV) and native DNA-hinding proteins eluting from the DNA matrix at 0.15 M NaCl (0.15) and at 0.60 M NaCl (0.6). For this gel, the samples were diluted to give approximately 20,000 cpm per sample, which was applied in a volume of 20 ~1. The exposure time for the autoradiograph was 67 days. The given DNA-binding protein identification number is shown on the right-hand side of the autoradioeram and a small square (W) is placed to the left of each of these polypeptide hands.
IO Fraction Number
20
30
FIG. 7. Elution of DNA polymerase activity from native DNA cellulose. KC1 is used as eluant since NaCl has an inhibitory effect in the DNA polymerase assay. Fractions were assayed at 0.15 M KC1 according to Keir et al. (1966). The 0.15 M KC1 elution extends from fractions 1 to 16 and the 0.60 M KC1 elution from fractions 17 to 30.
132
BAYLISS,
MARSDEN
AND HAY
amounts as the host proteins they replace. This explanation does not seem likely to us, but until further evidence is obtained to the contrary (e.g., tryptic mapping) the possibility cannot be excluded. The herpes simplex virus-induced DNAbinding proteins appear to fall into two quantitive classes: (1) those produced in large amounts (B.P. 2, 3, 4, 6, 9, and la), and (2) the remainder, produced in relatively small amounts. The distinction appears surprisingly clearcut and might be a clue to their function. Those falling into the former category might be expected to fullfil some function that requires stoichiometric amounts while those in the latter class might have purely catalytic functions. It has not been possible in this study to DISCUSSION distinguish effectively between weakly and From the data presented it can be seen, strongly binding proteins, as many of the that after infection of BHK21 Cl3 cells by proteins elute from the DNA matrix at herpes simplex virus type 1 strain 17 syn+, both the lower and higher salt concentrathe population of DNA-binding proteins is tion used. For example, in Figs. 4 and 5, markedly changed. The complex pattern of B.P. 9 would appear to bind more weakly than B.P. 12; however, in replicate experithe uninfected cell, in which over 40 bands ments, some B.P. 9 has been seen in the can be counted (Fig. 5) is simplified; many of the host proteins are reduced in quantity 0.60 M fraction and some B. P. 12 in the will or absent and have been replaced by 16 0.15 M fraction. Further purification virus-specified or induced proteins. be required before a critical assessment of The “switch off” of synthesis of many the relative binding strengths of individual host DNA-binding proteins must have oc- proteins can be made. curred within 1 hr after infection, as the Of the four major species of virusinfected cells were labelled immediately induced DNA-binding proteins shown in after a 1-hr absorption period. An example Fig. 3, two are synthesised early (B.P. 2 of this is the host polypeptide labelled 0 and B.P. 6) and two late (B.P. 3 and B.P. (MW = 47 x 103) in Figs. 5A and B. On the 4). B.P. 2 and B.P. 6 are found in the virion other hand, some host proteins appear to in very small quantities (Fig. 6 and Heine be synthesised in undiminished amounts et al., 1974), but major species are found in after infection, e.g., polypeptide h (MW = the infected cell which have identical molecular weights to B.P. 2 and B.P. 6 and 49 x 103) of Fig. 5, and this suggests that the virus is selective in its “switch off” of peak early (3 hr after absorption) (Marsden host protein synthesis. Such a class of host and Subak-Sharpe, 1975). B.P. 6 (V,,43), proteins, whose synthesis continues after which is equivalent to VP 21 in the numinfection, may be either required to mainbering scheme of Spear and Roizman tain the cell in a state capable of support(1972) (see Marsden and Subak-Sharpe, ing virus growth (e.g., as part of energy 1975) is particularly interesting, in that VP supply systems) or be involved actively in 21 is found in full, but not empty virus the growth of the virus (e.g., ribosomal capsids (Gibson and Roizman, 1972), and proteins, enzymes of RNA synthesis). In thus, one might have predicted that it addition, there may be virus-induced would have DNA-binding properties. It DNA-binding proteins that have the same may be significant that the distribution of mobilities and are synthesised in the same B.P. 6 closely resembles that of viral DNA have not been coelectrophoresed with infected cell extracts this correspondence should not be interpreted as identity. For similar reasons, the lack of nonstructural polypeptides of molecular weights corresponding to B.P. 4, B.P. 5, B.P. 7 or B.P. 11 should not be interpreted as precluding identity between some of those previously described nonstructural polypeptides and these DNA-binding proteins. A critical evaluation will involve coelectrophoresis and possibly a comparison of other properties such as the kinetics of synthesis. B.P.‘s 15 and 16 have molecular weights lower than any previously described virusinduced polypeptide; no doubt their low intensity prevented detection in whole infected cells.
HSV DNA BINDING
in that most is retained in the host cell and is not incorporated into virions (Wilkie, 1973). One class of protein that might be expected to be present in a DNA-binding fraction would have properties similar to the gene 32 product of bacteriophage T4 (Alberts and Frey, 1970). We have not found a protein in herpes-infected cells that binds exclusively to denatured DNA, but B.P. 9 is a major binding protein on denatured DNA columns and only a very minor one on native DNA columns. Thus, B.P. 9 is a candidate for the role of a herpes virus specific gene 32 type product although this speculation presently lacks any other supporting evidence. Purification of B.P. 9 is now under way to establish whether or not it has DNA “unwinding” properties. In these experiments, we have used calf thymus DNA as it contains a large number of possible binding sequences. However, in herpes virus DNA, there is likely to be present a different set of sequences and further experiments using HSV DNA cellulose columns will have to be undertaken to analyse infected cells for proteins which (a) bind only to cellular DNA, or (b) bind only to viral DNA, and which, in the present study, would not have been detected. ACKNOWLEDGMENTS We thank Prof. J. H. Subak-Sharpe for his interest throughout this study and Mr. Graham Hope for assistance in running the slab gels. G. J. Bayliss was a recipient of a Medical Research Council grant for training in Research Techniques. Note added in proof. Gel filtration of fractions of DNA binding proteins indicates that large aggregates or complexes are present in the sample. This may indicate that these complexes exist in uiuo and that some of the virus-induced DNA binding proteins are in fact present in the fraction because of the formation of complexes with proteins which bind to DNA rather than having DNA binding properties themselves. REFERENCES ALBERTS, B., and FREY, L. (1970). T4 bacteriophage gene 32-a structural protein in the replication and recombination of DNA. Nature (London) 227, 1313-1318. ALBERTS, B., and HERRICK, G. (19701. DNA cellulose
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chromatography. In “Methods in Enzymology (Nucleic Acids)” (L. Grossman and M. Moldave, Eds.), Vol. 23, pp. 198-217. Academic Press, New York. York. BROWN, S. M., RITCHIE, D., and SUBAK-SHARPE, J. H. (1973). Genetic studies with herpes simplex virus type l-the isolation of temperature-sensitive mutants, their arrangement into complementation groups and recombination analysis leading to a linkage map. J. Gen. Viral. 18, 329-346. Fox, S. O., and PARDEE, A. B. (1971). Proteins made in the Mammalian Cell Cycle. J. Biol. Chem. 246, 6159-6165. GIBSON, W., and ROIZMAN, B. (1972). Proteins specified by herpes simplex virus. VIII. Characterization and composition of multiple capsid forms in subtypes I and II. J. Viral. 10, 1044-1052. HAI.LIBCRTON, I. W., and TIMBCHY, M. C. (1973). Characterization of temperature sensitive mutants of herpes simplex virus type 2-growth and DNA synthesis. Virology 54, 60-68. HAY, J., PERERA, P. A. J., MORRISO?;, J. M., GENTRY, G. A., and SUBAK-SHARPE, J. H. (1971). Herpes virus-specified proteins. In “Strategy of the Viral Ciba Foundation Symposium, pp. Genome,” 355-372. Churchill Livingstone, London. HEINE, J. W., HONESS, R. W., CASSAI, E., and ROIZMAN, B. (1974). Proteins specified by herpes simplex virus. XI. Identification and relative molar rates of synthesis of structural and nonstructural herpes virus polypeptides in the infected cell. J. Viral. 12, 1347-1365. HOWARD, D. J., HAY, J., MELVIN, W. T., and DURHAM, J. P. (1974). Changes in DNA and RNA synthesis and associated enzyme activities after the stimulation of serum-depleted BHK21/C13 cells by the addition of serum. Exptl. Cell Res., 86, 31-42. KEIR, H. M., HAY, J., MORRISON, J. M., and SUBAKSHARPE, J. H. (1966). Altered properties of DNA nucleotidyl transferase after infection of mammalian cells with herpes simplex virus. Nature (Lendon) 210, 369-371. LAEMLI, U. K. (1970). Cleavage of structural proteins during assembly of the head of bacteriophage T4. Nature (London) 227, 680-685. MACPHERSON, I., and STOKEK, M. (1962). Polyoma transformation of hamster cell clones. An investigation of genetic factors affecting cell competence. Virology 16, 1477151. MAKSDEN, H. S., and SUBAK-SHARPE, J. H. (1975). Control of protein synthesis in herpes virus infected cells, submitted for publication. POONIAN, M. S., SCHLABACH, A. J.. and WEISSBACH, A. (19711. Covalent attachment of nucleic acids to agarose for affinity chromatography. Biochemistr> 10, 424-427. PRIN~LF., C. R., HOWARD, D. K., and HAY, J. (1973). Temperature-sensitive mutants of pseudorabies
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124-134 (1975)
Herpes Simplex
Virus Proteins:
Infected
Virology
Proteins
in
Cells and in the Virus Structure
GARY J. BAYLISS, M.R.C.
DNA-Binding
HOWARD
S. MARSDEN,
AND
JOHN
HAY
Unit, Urziuemity of Glasgou’, Glasgow Gf 1 5JR, Scotland Accepted June 10, 1975
DNA-binding proteins have been isolated from BHKZl/C13 cells infected with herpes simplex virus type 1 strain 17 syn+ by native and denatured DNA affinity chromatography. Sixteen proteins have been identified as specific to the infected cell on SDS polyacrylamide gradient gel electrophoresis and their molecular weights range from 145 x lo3 to 10.5 x 103. After infection with herpes simplex virus many of the mock infected cell specific DNA binding proteins are no longer produced and this “switch off’ of host cell protein synthesis is rapid. The virus-induced DNA-binding proteins fall into two quantitative classes and there is evidence for temporal control of their synthesis. One of the virus-induced DNA-binding proteins has a molecular weight identical to a polypeptide found in full but not empty particles and another binds preferentially to denatured DNA.
Four years ago, Alberts and Herrick (1970) introduced a convenient method that allowed separation from general cell proteins of those proteins that bind to DNA. Since then, several investigators have examined a range of cell types and confirmed the presence of DNA-binding proteins (Salas and Green, 1971; Fox and Pardee, 1971; Tsai and Green, 1973a). Alberts and Herrick (1970) also reported that infection of bacterial cells with viruses induced the formation of new DNA-binding proteins and some of these were shown to be virus coded. The most extensively studied example of these proteins is the gene 32 product of bacteriophage T4 whose role in viral DNA synthesis is probably to weaken the structure of double-stranded DNA and allow replication to proceed (Alberts and Frey, 1970). In African Green Monkey cells infected with adenovirus, two virus induced DNAbinding proteins (48,000 and 72,000 daltons) have been found, and at least one of these proteins appears to be essential for virus DNA synthesis (Van der Vliet and Levine, 1973). Herpes simplex virus (HSV) possesses a
genome of double stranded DNA with a molecular weight of about 100 x lo6 and this considerable protein specifying potential has been shown to be realised in a complex pattern of induction of at least 50 proteins in the infected cell (Honess and Roizman, 1974; Marsden and SubakSharpe, 1975). Genetic studies on several viruses of the herpes group suggest that about half the viral genes are directly or indirectly concerned with viral DNA synthesis (Brown, Ritchie, and Subak-Sharpe, 1973; Schaffer, Aron, Biswal, and Benyesh-Melnick, 1973; Halliburton and Timbury, 1972; Pringle, Howard, and Hay, 1973) and it is possible that the products of at least some of those genes may possess DNA-binding properties. In addition, one or more proteins of the virus structure may be associated with nucleic acid in the particle (Gibson and Roizman, 1972) and it seems likely that others also may be involved in the organisation of capsid substructures around a core of DNA. For the above reasons, and also to isolate a subpopulation of operationally defined proteins from the complex herpes virus124
Copyright@ 1975 by Academic Press. Inc. All rights of reproduction
in any form reserved.
HSV DNA BINDING
infected cell mixture, a study of DNAbinding proteins in herpes virus-infected cells was undertaken. MATERIALS
Cells. Monolayers of BHK21 Cl3 cells (Macpherson and Stoker, 1962) were grown in 80-0~ rotating bottles in Eagle’s medium supplemented with 10% (v/v) tryptose phosphate broth (Difco) and 10% (v/v) calf serum. Virus. Glasgow strain 17 syn+ herpes simplex virus type 1 was grown into stocks by infecting BHK21 Cl3 monolayers at an input multiplicity of 1 PFU/300 cells and harvesting the virus after 3 days of incubation at 31”. Infectivity was assayed using the method of Russell (1962). Label@ of cellular and viral proteins. Cell sheets were used when they were near confluency. The cell sheet was washed with methionine-free Eagle’s medium and then infected at a multiplicity of infection of 10 PFU/cell with virus suspension in 10 ml of Eagle’s medium containing one-fifth the normal concentration of methionine and supplemented with 2% calf serum (E (met/5) C2). After 60 min of adsorption at 37”, 40 ml of E (met/5) C2 were added (this is considered 0 hr after adsorption). [“S lmethionine (100-400 pCi/ml) was added from O-5 hr, 5-24 hr, or O-24 hr postadsorption. Mock infected cultures were treated identically, except that virus was omitted in the initial 10 ml of innoculum. Preparation of cell extracts. Cells were harvested by scraping into the medium and centrifuged at 800 g for 10 min at 4”. The resulting pellet was washed with 0.15 M NaCl, 0.02 M Tris-HCl (pH 8.2), resuspended in buffer 1 (1.7 M NaCl, 1 mM EDTA, 1 mM /3-mercaptoethanol, 20 mM Tris-HCl, pH 8.2) and treated in an ultrasonic bath for 60 sec. Following clarification as before, 10% (w/w) polyethylene glycol (6000) was added to the supernatant and, after standing for 30 min at O”, the precipitate was removed by centrifugation at 10,000 g for 30 set at 4”. This final supernatant was then dialysed against buffer 2 (50 mA4 NaCl, 1 mM EDTA, 1 mM
PROTEINS
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B-mercaptoethanol, 20 mM Tris-HCl, pH 8.2, 10% (v/v) glycerol) and could be stored at -70” if required. Purification of virus. Virus was labelled from 5 to 24 hr postinfection as described by Marsden and Subak-Sharpe (1975) and purified by the method of Spear and Roizman (1972). of DNA columns. Native Preparation calf thymus DNA-cellulose columns were prepared as described by Alberts and Herrick (1970) and denatured DNA-sepharose columns according to the method of Poonian, Schlaback, and Weissbach (1971). DNA was denatured by boiling for 15 min followed by rapid cooling in iced water. SDS polyacrylamide gel electrophoresis. Tube gels (discontinuous) were prepared according to the method of Laemli (1970). Following electrophoresis, the gels were sliced longitudinally, dried, and set up for autoradiography as below. In experiments carrying low levels of radioactivity, proteins were precipitated (see Fig. 2) prior to electrophoresis. This often resulted in the formation of high molecular weight aggregates. Slab gels (discontinuous, gradient) were prepared as described by Marsden and Subak-Sharpe (1975). The gels were stained with Coomassie Brilliant Blue and set up for autoradiography (Marsden and
FIG. 1. Native DNA cellulose chromatography of proteins extracted from mock-infected BHK21 Cl3 cells (W---m) and cells that had been infected with herpes simplex type 1 strain 17 s.sn+ (A-A).
126
BAYLISS.
MARSDEN
ml o-z4hrs.
AND HAY
Subak-Sharpe, 1975). Kodak BB54 film was used for autoradiography of tube gels and Kodak KD54T for slab gels. The protein samples were denatured by boiling for 2 min in the presence of 0.05 M Tris-HCl pH 6.8, 2% (w/v) SDS, 0.5% P-mercaptoethanol (with bromophenol blue included as a front marker) and applied to the gel after cooling. Enzyme assays. DNA polymerase was assayed using the high-salt herpes-specific conditions of Keir et al. (1966) and thymidine kinase by the method of Hay et al. (1971). RESULTS
Native DNA cellulose chromatography of infected and uninfected BHK21 Cl3 cell proteins, Mock-infected and HSV-1 infected BHK21 Cl3 cells were labelled with [,% Jmethionine from 0 to 24, 0 to 5, or 5 to 24 hr after infection. After harvesting and extraction as described in the Methods section, samples of the dialysed final supernatants were applied to native DNA cellulose columns. As shown in Fig. 1, both infected and mock-infected cell extracts bound to the column and then eluted in a similar manner. We find that the great majority of labelled protein (9%99%) passes through the column. The bound protein was eluted stepwise with buffer 2 containing respectively 0.15 and 0.60 M NaCI. Very little additional material is released from the column by buffer 2 containing 2 M NaCl, and analysis of this (not shown) reveals the presence only of proteins eluted at the lower salt concentrations. If a sample of DNA cellulose from the column is counted after elution, a small and variable amount of the originally bound protein will be FIG. 2. Densitometer traces of autoradiographs found to be uneluted (about l-3% of the prepared from longitudinally sliced and dried polyacrylamide tube gels of the 0.60 M NaCl fraction of originally bound protein). DNA-binding proteins prepared according to the The first series of experiments analysing protocol given in the Methods section, concentrated fractions from the DNA binding column prior to electrophoresis by precipitation with 10 volwas carried out on 7% polyacrylamide tube umes of acetone at -40’ for 16 hr. (a) Mock-infected gels (Methods section). Fig. 2 shows the BHK21 Cl3 cells labelled from 0 to 24 hr (0.7 x lo5 analysis of 0.60 M fraction proteins eluted cpm applied to gel). (b) HSV 1 17 syn+ infected BHK21 Cl3 cells labelled from O-5 hr postinfection. (1 x 10’ cpm applied to gel). (c) HSV 1 17 syn+ infected BHK21 cells labelled from 5-24 hr postinfection. (1 x lo5 cpm applied to gel). Approximate
molecular weights of the major viral peaks are shown in parentheses as daltons x 10m3.The autoradiograph was exposed for 60 days prior to development.
HSV DNA BINDING
PROTEINS
127
from a native DNA cellulose column (the 0.15 and 0.60 M fractions are qualitatively similar). Two aspects emerge. First, the rather complex pattern of DNA-binding proteins (B.P.‘s) from the mock-infected cell extract is replaced with a profile containing four or five major bands. The bands have been numbered according to the pattern found in the slab gel analysis (see Fig. 3). We are able to identify four major bands found in the tube gel analysis with four of the five major bands found in the slab gel analysis (B.P.‘s 2, 3, 4, and 6) since the molecular weights are in reasonable agreement (within 12%). (The peak migrating close to the dye front in the tube gel may be B.P. 12 but may contain more than one polypeptide. This possibility prevents an accurate estimate of the molecular weight for this polypeptide). Approximate molecular weights for the polypeptides were calculated by reference to standard proteins of known molecular weight (those used in the slab gel analysis) and these calculated values (as daltons x 10-3) are shown in parentheses in Fig. 2. The molecular weights of the DNA-binding proteins as analysed on slab gels are given in Table 1.
FIG. 3. Autoradiograph of DNA-binding proteins eluted from native and denatured DNA cellulose columns in the 0.15 and 0.60 M NaCl fractions and electrophoresed in an SDS, 7-15s polyacrylamide gradient slab gel. The DNA-binding proteins were prepared from mock-infected (mi) and herpes simplex virus strain 17 syn+ infected (17) cells labelled from 0 to 24 hr postinfection with [SsS]methionine. The four right-hand positions show the polypeptides that bound to and eluted from native DNA cellulose, while the four left-hand positions show the polypeptides that bound to and eluted from denatured DNA sepharose. The proteins eluting from denatured DNA had been passed through native DNA cellulose prior to their application to the denatured DNA column. Samples
were applied to the gel without concentration. Ten microliters of each sample were used except for the mock-infected DNA binding proteins that had been eluted from the native DNA cellulose column. Here, the counts were appreciably lower than all other samples and 15 hl of the 0.15 A4 fraction and 25 ~1 of the 0.6 M fraction were applied to the gel. The cpm applied were as follows: (a) for those proteins eluted from denatured DNA sepharose; MI 0.15 M, 12.8 x 10s; infected 0.15 M, 10.2 x 103; MI 0.6 M, 9.6 x lOa; info.6 M, 9.6 x 103; and (b) for those proteins eluting from native DNA cellulose; MI 0.15 M, 8.6 x lo3 (in 15 r.d); infected 0.15 M, 10.1 x 10’; MI 0.6 M, 5.2 x lo3 (in 25 ~1); infected 0.6 M, 8.1 x 103. The autoradiograph shown was produced by an exposure of 67 days. The number on the right-hand side of the gel is the given DNA-binding protein identification number (B.P.) for virus-induced polypeptides. A small square ( n ) is placed to the left of each of these polypeptide bands to assist in identification among samples (e.g., B.P. I occurs only in the 17 s.yn+ infected cell native DNA 0.60 M fraction while B.P. 2 occurs in each of the four 17 syn+ infected cell fractions).
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BAYLISS, TABLE
DNAbinding protein
Molecular weight x 10-3
1 2
145 136
3 4 5 6
87 62 59 43
7 8 9
42 41 40
10 11 12 13 14 15 16
32 31 21 19 17 12 10.5
MARSDEN
1 Additional characterisation
Major species; comigrates with V,,136. Major species Major species Major species; comigrates with V,,43.
Major species from denatured DNA
Major species
The second aspect to emerge from Fig. 2 is that B.P. 2 and B.P. 6 reach their peak of synthesis during the 0- to 5-hr labelling period (Fig. 2B) whereas B.P. 3 and 4 reach their peak of synthesis during 5 to 24 hr (Fig. 2C). Thus, there appears to be some measure of temporal control in the synthesis of the DNA-binding proteins. Infection and labelling of serum-starved “resting cells” (Howard, Hay, Melvin, and Durham, 1974) gave essentially the same results as shown in Fig. 2, but in the resting cell controls, the host DNA-binding proteins were present in reduced amounts, about one-half to one-third of Fig. 2. The protein profiles in a series of repeat experiments employing tube gels were qualitatively identical to those of Fig. 2 but showed some quantitative variation. Subsequent analyses were performed on gradient polyacrylamide slab gels that have superior resolution over a wide range of molecular weights (Marsden and SubakSharpe, 1975). Analysis of native DNA-binding proteins on gradient polyacrylamide slab gel electrophoresis. Proteins eluted from the native DNA-cellulose column as described above were denatured with SDS and /3-mercaptoethanol and electrophoresed on
AND HAY
7-15s acrvlamide gradients (see Methods section). Fig. 3 shows an autoradiograph of the analysis of [3”S]methionine-labelled DNA-binding proteins obtained from strain 17 svn+ infected and mock-infected cell extracts. The increased resolution of this gel system reveals a much more detailed profile than that of the tube gel system (Fig. 2). HSV-induced DNA-binding proteins were identified either (a) by their occurrence in infected cell extracts and absence in the corresponding mockinfected cell extracts, or (b) by their presincreased ence in very significantly amounts in the infected cell extract compared to the mock-infected cell extract. Their position on the autoradiograph is indicated by a small square to the left of the identified band and the numbering system we have adopted for these proteins is given to the right of the autoradiograph. Densitometer traces of these autoradiographs are illustrated in Fig. 4. In the profile of proteins from mock-infected BHK cells eluted at 0.15 M NaCl (Fig. 4A) a complex pattern of proteins is seen while at 0.60 M NaCl the profile is more simple (Fig. 4C). However, the 0.60 M NaCl eluate contains several proteins found in the 0.15 M NaCl fraction in addition to polypeptides that appear only at the higher salt concentration. A dramatic change in the pattern of synthesis of DNA-binding proteins occurs after infection (Figs. 4C and 4D), in agreement with the earlier results (Fig. 2). The DNA-binding proteins of the mock-infected BHK cell have largely disappeared and a spectrum of new species appears in both the 0.15 and 0.60 M NaCl eluates. There is a variation in strength of binding within the population of virusinduced DNA-binding proteins, as evidenced by the relative amounts of polypeptides eluted at the different salt concentrations. Proteins binding to denatured DNA. The proteins that had failed to bind to native DNA were subsequently applied to a denatured DNA column, eluted and analysed as described in the Methods section. Fig. 5 (A, B, C, D) shows densitometer traces of the four left-hand profiles given in the autoradiograph of Fig. 3.
HSV DNA BINDING mioraa
129
PROTEINS
c
mioa
1 I ‘1
FIG. 4. Densitometer traces of Mock-infected cells: 0.15 M eluate 0.15 M eluate from a native DNA cellulose column. (D) HSV 1 strain
the four right-hand positions of the from a native DNA cellulose column. cellulose column. (C) Mock-infected 17 syn+ infected cells: 0.60 M eluate
The mock-infected profiles are complex with little similarity between the populations of proteins eluting at 0.15 and 0.60 M NaCl or with the patterns of native DNAbinding proteins. Following infection many of the host proteins disappear and are replaced by virus-induced proteins. All of these virus-induced proteins are found in the native DNA-binding population (Figs. 4C and 4D), but it is striking that protein B.P. 9, a very minor component of the native DNA pattern, is the major species in the denatured DNA fraction eluted at 0.15 M NaCl (Fig. 3B). This is the behaviour expected of proteins with properties similar to those of bacteriophage T4 gene 32 product (Alberts and Frey, 1970). DNA-binding proteins as components of the uirus structure. Herpes simplex virus strain 17 syn+ was purified by the method of Spear and Roizman (1972) after incuba-
autoradiograph presented in Fig. 3. (A) (B) HSV 1 strain 17 syn+ infected cells: cells: 0.60 M eluate from a native DNA from a native DNA cellulose column.
tion with [3”S]methionine between 5 and 24 hr to minimise labelling of host cell proteins. Virus structural proteins from this preparation were then fractionated on a gradient slab gel side by side with DNAbinding proteins. Fig. 6 shows the autoradiogram of such a gel. One structural protein (V,,43) runs with a mobility identical to B.P. 6, the major virus-induced protein binding to native DNA. As already observed from Fig. 2, B.P. 6 is an early protein as is V,,43 (Marsden and SubakSharpe, 1975), thus providing further evidence for their identity. V,,43 is a minor structural protein of the virion but a major protein of the infected cell (Marsden and Subak-Sharpe, 1975). There may also be identity between V,,136 and B.P. 2. Virus-induced enzymes as DNA-binding proteins. After herpes simplex virus infection DNA polymerase and pyrimidine
BAYLISS. MARSDEN AND HAY
130 h !
mi
045M
C mi
0.6M
:I 1 ;’ ? :
I
r
3 I
h
h
FIG, 5. Densitometer traces of the four left-hand positions of the autoradiograph presented in Fig. 3. (A) Mock-infected cells: 0.15 M eluate from a denatured DNA sepharose column. (B) HSV 1 strain 17 syn+ infected cells: 0.15 M eluate from a denatured DNA sepharose column. (Cl Mock-infected cells: 0.60 M eluate from a denatured DNA sepharose column. (D) HSV 1 strain 17 syn+ infected cells: 0.60 M eluate from a denatured DNA sepharose column. deoxynucleoside kinase activities are induced and appear to be virus-specified
(Keir et al., 1966; Hay et al., 1971). Infected cells were extracted 6 hr after infection and were assayed for these activities. The active extracts were applied to a native DNA cellulose column. Thymidine kinase activity did not bind to the column and was recovered in the void volume and preliminary washings. However, the virus-
specific DNA polymerase measured by the high salt a&say specific for the viral enzyme (Keir et al., 19661 was retained by the column; it consistently eluted largely at 0.60 M KC1 (Fig. 7), which indicates that a relatively strong association exists between the enzyme and DNA. Herpes-specific DNA polymerase similarly binds to and elutes from denatured DNA cellulose. KC1 is used as the eluant since NaCl is inhibi-
HSV DNA BINDING
131
PROTEINS
tory to the enzyme activity while, under the assay conditions used no DNA polymerase activity from uninfected cells can be detected in the eluates from either column. Molecular weight determination on DNA-binding proteins. The mobility of DNA-binding proteins has been correlated with that of 18 standard polypeptides of known molecular weight (220 x lo3 to 5.7 x 103) (Marsden and Subak-Sharpe, 1975) which were coelectrophoresed in adjacent positions in gradient slab gel electrophoresis. Table 1 lists the estimated molecular weights of the herpes simplex virusinduced DNA-binding proteins and gives a summary of their observed properties. As yet, we are unable to fit the DNA polymerase activity into the pattern of molecular weights, as the molecular weight of the purified enzyme has not been accurately determined under denaturing conditions. However, preliminary evidence from studies with viral ts mutants suggests that a molecular weight in excess of 130,000 is likely (Hay, J. and Marsden, H. S., unpublished observations). The estimated molecular weights of several of the DNA-binding proteins correspond with the molecular weights of HSV-1 strain 17-induced nonstructural polypeptides (Marsden and Subak-Sharpe, 1975). Specifically these are: B.P. 8 (MW = 41 x 103); B.P. 9 (MW = 40 x 103); B.P. 13 (MW = 19 x 103) and B.P. 14 (MW = 17 x i03) However, since DNA-binding proteins
Fm. 6. Autoradiograph of electrophoresed proteins from purified herpes simplex virus type 1 17 syn+ (PV) and native DNA-hinding proteins eluting from the DNA matrix at 0.15 M NaCl (0.15) and at 0.60 M NaCl (0.6). For this gel, the samples were diluted to give approximately 20,000 cpm per sample, which was applied in a volume of 20 ~1. The exposure time for the autoradiograph was 67 days. The given DNA-binding protein identification number is shown on the right-hand side of the autoradioeram and a small square (W) is placed to the left of each of these polypeptide hands.
IO Fraction Number
20
30
FIG. 7. Elution of DNA polymerase activity from native DNA cellulose. KC1 is used as eluant since NaCl has an inhibitory effect in the DNA polymerase assay. Fractions were assayed at 0.15 M KC1 according to Keir et al. (1966). The 0.15 M KC1 elution extends from fractions 1 to 16 and the 0.60 M KC1 elution from fractions 17 to 30.
132
BAYLISS,
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amounts as the host proteins they replace. This explanation does not seem likely to us, but until further evidence is obtained to the contrary (e.g., tryptic mapping) the possibility cannot be excluded. The herpes simplex virus-induced DNAbinding proteins appear to fall into two quantitive classes: (1) those produced in large amounts (B.P. 2, 3, 4, 6, 9, and la), and (2) the remainder, produced in relatively small amounts. The distinction appears surprisingly clearcut and might be a clue to their function. Those falling into the former category might be expected to fullfil some function that requires stoichiometric amounts while those in the latter class might have purely catalytic functions. It has not been possible in this study to DISCUSSION distinguish effectively between weakly and From the data presented it can be seen, strongly binding proteins, as many of the that after infection of BHK21 Cl3 cells by proteins elute from the DNA matrix at herpes simplex virus type 1 strain 17 syn+, both the lower and higher salt concentrathe population of DNA-binding proteins is tion used. For example, in Figs. 4 and 5, markedly changed. The complex pattern of B.P. 9 would appear to bind more weakly than B.P. 12; however, in replicate experithe uninfected cell, in which over 40 bands ments, some B.P. 9 has been seen in the can be counted (Fig. 5) is simplified; many of the host proteins are reduced in quantity 0.60 M fraction and some B. P. 12 in the will or absent and have been replaced by 16 0.15 M fraction. Further purification virus-specified or induced proteins. be required before a critical assessment of The “switch off” of synthesis of many the relative binding strengths of individual host DNA-binding proteins must have oc- proteins can be made. curred within 1 hr after infection, as the Of the four major species of virusinfected cells were labelled immediately induced DNA-binding proteins shown in after a 1-hr absorption period. An example Fig. 3, two are synthesised early (B.P. 2 of this is the host polypeptide labelled 0 and B.P. 6) and two late (B.P. 3 and B.P. (MW = 47 x 103) in Figs. 5A and B. On the 4). B.P. 2 and B.P. 6 are found in the virion other hand, some host proteins appear to in very small quantities (Fig. 6 and Heine be synthesised in undiminished amounts et al., 1974), but major species are found in after infection, e.g., polypeptide h (MW = the infected cell which have identical molecular weights to B.P. 2 and B.P. 6 and 49 x 103) of Fig. 5, and this suggests that the virus is selective in its “switch off” of peak early (3 hr after absorption) (Marsden host protein synthesis. Such a class of host and Subak-Sharpe, 1975). B.P. 6 (V,,43), proteins, whose synthesis continues after which is equivalent to VP 21 in the numinfection, may be either required to mainbering scheme of Spear and Roizman tain the cell in a state capable of support(1972) (see Marsden and Subak-Sharpe, ing virus growth (e.g., as part of energy 1975) is particularly interesting, in that VP supply systems) or be involved actively in 21 is found in full, but not empty virus the growth of the virus (e.g., ribosomal capsids (Gibson and Roizman, 1972), and proteins, enzymes of RNA synthesis). In thus, one might have predicted that it addition, there may be virus-induced would have DNA-binding properties. It DNA-binding proteins that have the same may be significant that the distribution of mobilities and are synthesised in the same B.P. 6 closely resembles that of viral DNA have not been coelectrophoresed with infected cell extracts this correspondence should not be interpreted as identity. For similar reasons, the lack of nonstructural polypeptides of molecular weights corresponding to B.P. 4, B.P. 5, B.P. 7 or B.P. 11 should not be interpreted as precluding identity between some of those previously described nonstructural polypeptides and these DNA-binding proteins. A critical evaluation will involve coelectrophoresis and possibly a comparison of other properties such as the kinetics of synthesis. B.P.‘s 15 and 16 have molecular weights lower than any previously described virusinduced polypeptide; no doubt their low intensity prevented detection in whole infected cells.
HSV DNA BINDING
in that most is retained in the host cell and is not incorporated into virions (Wilkie, 1973). One class of protein that might be expected to be present in a DNA-binding fraction would have properties similar to the gene 32 product of bacteriophage T4 (Alberts and Frey, 1970). We have not found a protein in herpes-infected cells that binds exclusively to denatured DNA, but B.P. 9 is a major binding protein on denatured DNA columns and only a very minor one on native DNA columns. Thus, B.P. 9 is a candidate for the role of a herpes virus specific gene 32 type product although this speculation presently lacks any other supporting evidence. Purification of B.P. 9 is now under way to establish whether or not it has DNA “unwinding” properties. In these experiments, we have used calf thymus DNA as it contains a large number of possible binding sequences. However, in herpes virus DNA, there is likely to be present a different set of sequences and further experiments using HSV DNA cellulose columns will have to be undertaken to analyse infected cells for proteins which (a) bind only to cellular DNA, or (b) bind only to viral DNA, and which, in the present study, would not have been detected. ACKNOWLEDGMENTS We thank Prof. J. H. Subak-Sharpe for his interest throughout this study and Mr. Graham Hope for assistance in running the slab gels. G. J. Bayliss was a recipient of a Medical Research Council grant for training in Research Techniques. Note added in proof. Gel filtration of fractions of DNA binding proteins indicates that large aggregates or complexes are present in the sample. This may indicate that these complexes exist in uiuo and that some of the virus-induced DNA binding proteins are in fact present in the fraction because of the formation of complexes with proteins which bind to DNA rather than having DNA binding properties themselves. REFERENCES ALBERTS, B., and FREY, L. (1970). T4 bacteriophage gene 32-a structural protein in the replication and recombination of DNA. Nature (London) 227, 1313-1318. ALBERTS, B., and HERRICK, G. (19701. DNA cellulose
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chromatography. In “Methods in Enzymology (Nucleic Acids)” (L. Grossman and M. Moldave, Eds.), Vol. 23, pp. 198-217. Academic Press, New York. York. BROWN, S. M., RITCHIE, D., and SUBAK-SHARPE, J. H. (1973). Genetic studies with herpes simplex virus type l-the isolation of temperature-sensitive mutants, their arrangement into complementation groups and recombination analysis leading to a linkage map. J. Gen. Viral. 18, 329-346. Fox, S. O., and PARDEE, A. B. (1971). Proteins made in the Mammalian Cell Cycle. J. Biol. Chem. 246, 6159-6165. GIBSON, W., and ROIZMAN, B. (1972). Proteins specified by herpes simplex virus. VIII. Characterization and composition of multiple capsid forms in subtypes I and II. J. Viral. 10, 1044-1052. HAI.LIBCRTON, I. W., and TIMBCHY, M. C. (1973). Characterization of temperature sensitive mutants of herpes simplex virus type 2-growth and DNA synthesis. Virology 54, 60-68. HAY, J., PERERA, P. A. J., MORRISO?;, J. M., GENTRY, G. A., and SUBAK-SHARPE, J. H. (1971). Herpes virus-specified proteins. In “Strategy of the Viral Ciba Foundation Symposium, pp. Genome,” 355-372. Churchill Livingstone, London. HEINE, J. W., HONESS, R. W., CASSAI, E., and ROIZMAN, B. (1974). Proteins specified by herpes simplex virus. XI. Identification and relative molar rates of synthesis of structural and nonstructural herpes virus polypeptides in the infected cell. J. Viral. 12, 1347-1365. HOWARD, D. J., HAY, J., MELVIN, W. T., and DURHAM, J. P. (1974). Changes in DNA and RNA synthesis and associated enzyme activities after the stimulation of serum-depleted BHK21/C13 cells by the addition of serum. Exptl. Cell Res., 86, 31-42. KEIR, H. M., HAY, J., MORRISON, J. M., and SUBAKSHARPE, J. H. (1966). Altered properties of DNA nucleotidyl transferase after infection of mammalian cells with herpes simplex virus. Nature (Lendon) 210, 369-371. LAEMLI, U. K. (1970). Cleavage of structural proteins during assembly of the head of bacteriophage T4. Nature (London) 227, 680-685. MACPHERSON, I., and STOKEK, M. (1962). Polyoma transformation of hamster cell clones. An investigation of genetic factors affecting cell competence. Virology 16, 1477151. MAKSDEN, H. S., and SUBAK-SHARPE, J. H. (1975). Control of protein synthesis in herpes virus infected cells, submitted for publication. POONIAN, M. S., SCHLABACH, A. J.. and WEISSBACH, A. (19711. Covalent attachment of nucleic acids to agarose for affinity chromatography. Biochemistr> 10, 424-427. PRIN~LF., C. R., HOWARD, D. K., and HAY, J. (1973). Temperature-sensitive mutants of pseudorabies
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