A Comparison of the Acid-soluble Polypeptides of Five Herpesviruses

Transcription

1 J. gen. Virol. (1985), 66, Printed in Great Britain 2243 Key words: herpesvirus/ HSV/ acid-soluble proteins/ D NA-binding proteins A Comparison of the Acid-soluble Polypeptides of Five Herpesviruses By I. W. HALLIBURTON* AND M. J. FREEMAN MRC Herpesvirus Research Group, Department of Microbiology, The University of Leeds, Leeds LS2 9JT, U.K. (Accepted 4 July 1985) SUMMARY The polypeptides soluble in 0.25 M-HC1 were extracted from the nuclei of BHK cells infected with herpes simplex virus type 1 or type 2 and separated by SDS-PAGE. Seventeen polypeptides were detectable in each extract of which 10 type 1 and nine type 2 polypeptides were reproducibly effectively extracted. In cells infected with bovine mammillitis virus, pseudorabies virus or equine herpesvirus type 1, at least 12, 13 and eight polypeptides respectively were acid-soluble. In addition to histones, three other cellular polypeptides were present in sizeable quantities in the acid extracts and could obscure other acid-soluble viral polypeptides. Possible relationships between some polypeptides of the five herpesviruses are discussed. During productive infection, herpes simplex viruses specify at least 50 polypeptides (Honess & Roizman, 1973; Powell & Courtney, 1975 ; Marsden et al., 1976), a large number of which are thought to be involved in DNA synthesis. One approach which has been adopted to select and allow characterization of such proteins has been to isolate and identify DNA-binding proteins, as many as 23 of which have been reported (Bayliss et al., 1975 ; Powell & Purifoy, 1976; Wilcox et al., 1980; Bookout & Levy, 1980). These DNA-binding proteins are likely to be basic in character and therefore soluble in dilute mineral acid. Acid-soluble polypeptides have been identified in herpes simplex virus type 1 (HSV-1)- and type 2 (HSV-2)-infected cells (Halliburton, 1972; Halliburton & Timbury, 1973) and at least six acid-soluble polypeptides have been identified in pseudorabies virus (PRV)-infected cells (Stevens et al., 1969 ; Chantler & Stevely, 1973; Tyler et al., 1974) but in all of these studies, insensitive electrophoretic methods for separating polypeptides were used and it seems likely that the numbers were underestimated. In this study, we have therefore examined the acid-soluble nuclear proteins not only in cells infected with HSV-1, HSV-2 and PRV but also in cells infected with bovine mammillitis virus (BMV) and equine herpesvirus type 1 (EHV-1). BHK cell monolayers were infected at a m.o.i, of about 10 with HSV-1 (strain HFEM) or HSV-2 (strain 3345) or were mock-infected. Polypeptides were labelled from 3 to 19 h postinfection with [3SS]methionine, aliquots being removed for electrophoresis of whole cell polypeptides and the remaining parts processed for acid extraction. Cells were lysed by homogenization in lysis buffer (0.01 M-Tris HC1 ph 7.4 containing 0.01 M-NaC1, M- MgC12, M-2-mercaptoethanol) and nuclei obtained by centrifugation at 1000g for 10 min. Nuclei were washed twice with 10 times their volume of 0.5% Triton X-100 in lysis buffer to remove adhering cytoplasmic material, the purity of the nuclei being monitored microscopically by staining aliquots with 0.5% crystal violet. The detergent was removed by washing the nuclei once in lysis buffer and once in M-saline. Acid-soluble proteins were then extracted from the washed nuclei by stirring with 0.25 M-HC1 at 4 C for 1 h followed by centrifugation at g for 20 min. The pellet was re-extracted with HCI, the extracts pooled and dialysed against 0-01 M-H2SO 4. The acid-extracted proteins were then precipitated at -20 oc with 6 vol. acetone, O 1985 SGM

2 2244 Short communication I Mock 1 2 HSV- 1 I I HSV ICP t ' a 21 A B E 34/35 39/ Fig. 1. A utoradiograms of total cell polypeptides and nuclear acid-soluble polypeptides from HSV-1- or HSV-2-infected BHK cells or from mock-infected cells. The ceils were labelled with [35S]methionine (3 BCi/ml) from 3 to 19 h post-infection, half then being solubilized for electrophoresis on an 8.5% SDScontaining gel and half extracted with 0.25 M-HCI as described in the text. Lanes 1, infected cell polypeptides (ICP); lanes 2, acid-soluble polypeptides (ASP). A, B and C indicate the positions of acidsoluble cellular polypeptides. ICP numbers identify the infected cell polypeptides according to the numbering system of Honess & Roizman (1973). The molecular weights were determined relative to coelectrophoresed marker proteins. pelleted by centrifugation at 2000 g for 10 min and dissolved in 0.05 M-Tris-HC1 ph 7.0 containing 2% SDS, 5% 2-mercaptoethanol for SDS-PAGE. Fig. 1 shows autoradiograms of the polypeptides in mock-infected and HSV-infected cells and of acid-soluble polypeptides extracted from the nuclei of the same cells. With regard to mockinfected cells, the most readily acid-extracted polypeptides are the histones (which migrated with the SDS front in this gel) but three more polypeptides (labelled A, B and C in Fig. 1) are almost totally acid-soluble and clearly a number of others are either partially soluble in acid or are very minor components quantitatively. The estimated molecular weights for polypeptides A, B and C are 59000, and and they are not related to the histones since they incorporate tryptophan and their synthesis is not coupled to DNA synthesis (data not shown). Polypeptides with the same molecular weight as A and C were extracted by dilute mineral acid from HeLa, MDCK, RK 13, human amnion, KB, human embryo lung and HEp-2 cells as well as from BHK cells but a polypeptide with the same mobility as B was found in high molarity only in acid extracts of MDCK and BHK cells. With regard to HSV-infected cell extracts, autoradiograms of HSV-1- or HSV-2-infected cell polypeptides show at least 30 separate bands with some areas where clear band differentiation was not possible (Fig. 1). Numbering of these bands followed the scheme of Honess & Roizman (1973). At least 17 differences in electrophoretic mobility can be identified between the HSV- 1 (HFEM)- and HSV-2(3345)-infected cell polypeptides during the 3 to 19 h labelling period with

3 Short communication 2245 Table 1. Characteristics of acid-soluble nuclear polypeptides of HS V-l- and HS V-2-infected BHK cells HSV-I HSV-2 Relative acid Relative acid ICP* VP~" Mol. wt. x 10-3 solubility~t ICP* Mol. wt. x 10 -a solubility:~ / / / a * ICP, Infected cell polypeptide. t VP, Virion polypeptide. :~ Relative solubility in 0.25 M-HCI estimated from the data of Fig. 1. Two pluses signify that a greater proportion of the polypeptide is soluble in the acid. Probably host polypeptide. [35S]methionine. One advantage of acid extraction is that it is possible to increase the concentration of acid-soluble polypeptides at the expense of the less acid-soluble ones without overloading the gel. The acid extracts shown in Fig. 1 were from tenfold more cells than the infected cell extracts. The identity, molecular weight and relative solubility in acid of all HSVl(HFEM) and HSV-2(3345) polypeptides that can be seen on the original autoradiogram is shown in Table 1. Thus, at least 17 HSV-1 and 17 HSV-2 polypeptides are acid-soluble to greater or lesser extents. For HSV-l-infected cells, ten of these are easily seen and are ICPs 5, 8, 21a, 21, 26, 31, 39/40, 44 and 45 plus three others which co-electrophorese with the major acid-soluble host proteins A, B and C. In HSV-2-infected cells, the major acid-soluble components are ICPs 5, 8, 21, 31, 36, 39, 40 and 45 plus bands co-migrating with A, B and C. In HSV-l-infected cells, ICP 21a and 21 are related polypeptides functionally equivalent to the one polypeptide ICP 21 in HSV-2-infected cells. ICP 26 and ICP 44 of strain HFEM are apparently acid-soluble whereas the corresponding polypeptides in strain 3345-infected cells are not. Conversely, ICP 36 of strain 3345 is acid-soluble whereas that of strain HFEM does not appear to be so. These may, however, be strain-specific characteristics. The acid-soluble polypeptides from HSV-1- or HSV-2-infected cells therefore form a subset of the total HSV-infected cell polypeptides; they are a specific subset and are not simply polypeptides that are readily soluble and leak easily out of the nuclei since, as stated above, the crude nuclei were washed twice in lysis buffer containing Triton X- 100 once in buffer and once in saline before acid extraction. An additional wash with phosphatebuffered saline did not extract the polypeptides shown in Fig. 1. This compares favourably with the numbers of polypeptides found in the nuclei of infected cells following fractionation of cells into nuclei and cytoplasm (data not shown) when there is only a small reduction in the number of polypeptides in either fraction compared with that found in whole cells (Fenwick et al., 1978). It is well established that cellular histones are basic proteins which are associated with DNA as nucleosomes and these basic proteins are acid-soluble. It is of interest, therefore, to wonder whether or not a link can be found between those polypeptides which are acid-soluble in HSV-

4 2246 Short communication ICP ASP II "7, ~ "7, " rq " A B C 39/4O Fig. 2. Autoradiogram of polypeptides and acid-soluble nuclear polypeptides of cells infected with each of five herpesviruses or mock-infected BHK ceils (M) labelled with [' 4C]arginine (l ~tci/ml),which was found to be a particularly suitable label for these basic polypeptides, and separated by electrophoresis on a 9.25 ~ SDS-containing gel. The numbers on the left identify some of the HSV-1 polypeptides and A, B and C identify host polypeptides. Acid-soluble polypeptides of BMV-, PRV- and EHV-l-infected cells are located by dots to the left of the appropriate lane. Closed circles on the left of each infected cell polypeptide lane locate the major capsid polypeptide according to Killington et al. (1977). Open circles to the left of the same lanes locate the major cross-reacting DNA-binding protein of cells infected with each virus. infected cells and those which can be shown to bind D N A. Accurate comparisons of existing data are however virtually impossible largely due to the lack of a universally agreed protein nomenclature. Despite this, identification of a few selected polypeptides from different studies is possible. Thus, Powell & Purifoy (1976) and H a y & H a y (1980) found that the immediate early polypeptide I C P 4 ( mol. wt.) was a D N A - b i n d i n g protein whereas Bayliss et al. (1975) and Wilcox et al. (1980) did not. F r e e m a n & Powell (1982), however, have shown that I C P 4 binds to D N A only via a component of uninfected cells. I C P 4 would not be detectable in the present studies but following release from a cycloheximide block, I C P 4 is acid-soluble (I. W. Halliburton, unpublished observation). The major capsid polypeptide of molecular weight to has been shown to be a D N A - b i n d i n g protein by Powell & Purifoy (1976), Wilcox et al. (1980) and La Thangue & C h a n (1984) but was not detected as such by Bayliss et al. (1975) or by Bookout & Levy (1980). This polypeptide is weakly acid-soluble. The major D N A binding protein (ICP 8, Powell et al., 1981) is certainly acid-soluble (Fig. 1 and 2). Thereafter, some 15 or more polypeptides ranging in molecular weight from to are both soluble in dilute mineral acid and bind D N A (Bayliss et al., 1975; Powell & Purifoy, 1976; Wilcox et al., 1980; Bookout & Levy, 1980; La Thangue & C h a n, 1984) but specific correlations require further characterization of individual polypeptides.

5 Short communication 2247 In order to examine the solubility of polypeptides from some other herpesviruses, BHK cells were infected with either HSV-I(HFEM), HSV-2(3345), BMV (Castrucci et al., 1972), PRV (isolated by Dr F. Dekking) or EHV-1 (Mayr et al., 1965) at a m.o.i, of 10 to 15. Cells were labelled with [14C]arginine from 4.5 to 11-5 h post-infection and were either processed as whole cell extracts or for acid extraction of nuclei. Fig. 2 shows the resulting autoradiogram. The infected cell polypeptide profiles show considerable variation in polypeptide distribution. Acid extraction of HSV-l-infected cells produced the acid-soluble polypeptides 5, 8, 21a, 21, 26, 39/40, 44 and 45. Extraction of HSV-2-infected cells with 0.25 M-HC1 produced ICP 5, 8, 21, 36, 39, 40 and 45. These results compare with at least 12 major acid-extractable polypeptides which do not co-migrate with cellular polypeptides for BMV, 13 for PRV and eight for EHV-1 (indicated with dots to the left of the appropriate band of acid-soluble polypeptide in Fig. 2). The bands co-migrating with host cell polypeptides A, B and C are apparent with each virus except for HSV-2 where a more effective switch off of cell protein synthesis appears to have occurred and another polypeptide (ICP 29) migrating between A and B is apparent. The major capsid polypeptide of each of the five viruses has been identified as having molecular weights of to for HSV-2 and BMV, for EHV-1 and for PRV relative to for ICP 5 of HSV-1 by Killington et al. (1977) which correspond to polypeptides marked with a large filled dot to the left of the appropriate infected cell polypeptide band in Fig. 2. If these assignments are correct, then the BMV and EHV-1 major capsid polypeptides are completely insoluble in 0.25 M-HC1 whereas the PRV major capsid polypeptide is similar in solubility to that of HSV-1 ICP 5. Structural polypeptides analogous to ICP 21 (VP 14) of HSV-1 or HSV-2 do not seem apparent with BMV or PRV and there is only a faint band of similar mobility for EHV-1. Capsid polypeptides analogous to ICP 39/40 (VP 22/23) of HSV-1 as identified by Killington et al. (1977) could well be acid-soluble with BMV, PRV and EHV-1 since there are bands of similar mobility but one cannot be sure which are the equivalent polypeptides. VP 22 of HSV-1 and HSV-2 is, however, a relatively arginine-rich polypeptide (Halliburton, 1972). The major DNA-binding protein of HSV-1 and HSV-2 has been identified as ICP 8 (Bayliss et al., 1975; Powell & Purifoy, 1976; Purifoy & Powell, 1976; Knipe et al., 1982). Furthermore, Yeo et al. (1981) have shown by immunoprecipitation that a polypeptide of molecular weight about is a major cross-reacting polypeptide induced by HSV- 1, HSV-2, BMV, PRV and EHV-1, and Littler et al. (1981) using tryptic peptide analysis have shown that this cross-reacting DNA-binding protein of all five viruses has structural similarities. ICP 8 polypeptides of HSV-1 and HSV-2 are both readily extractable by dilute acid (Fig. 1 and 2) and polypeptides of molecular weight , and (marked to the left of the appropriate infected cell polypeptide band with an open circle in Fig. 2) are apparent in cells infected with BMV, PRV and EHV-1 respectively and readily apparent in acid-soluble extracts of these cells (Fig. 2) although the PRV polypeptide is a more minor component in the acid extract. The major cross-reacting DNA-binding protein of these five viruses would therefore seem to be acid-soluble. When more is known about the polypeptides specified by BMV, PRV and EHV-1 it should be possible to extend these comparisons to other polypeptides. In the meantime, the use of extraction of nuclei with dilute mineral acid may well be a useful additional approach which can be used to compare and to suggest relationships between polypeptides of related viruses. We would like to thank Mrs C. I. Moorhouse for typing the manuscript. This work was supported by a Medical Research Council project grant. REFERENCES BAYLISS, G. J., MARSDEN, H. S. & HAY, J. (1975). Herpes simplex virus proteins: DNA-binding proteins in infected cells and in the virus structure. Virology 68, BOOKOUT, J. B. & LEVY, C. C. (1980). Comparative examination of the polypeptides of herpes simplex virus: types 1 and 2. Virology 101, CASTRUCC[, G., PEDINI, B., CILLI, V. & ARANCIA, G. (1972). Characterization of a viral agent resembling bovine herpes mammillitis virus. Veterinary Record 90,

6 2248 Short communication CHANTLER, J. K. & STEVELY, W. S. (1973). Virus-induced proteins in pseudorabies-infected cells. Journal of Virology 11, FENWICK, M. L., WALKER, M. J. & PETKEVICH, J. M. (1978). On the association of virus proteins with the nuclei of cells infected with herpes simplex virus. Journal of General Virology 39, FREEMAN, M. J. & POWELL, K. L. (1982). DNA-binding properties of a herpes simplex virus immediate early protein. Journal of Virology 44, HALLIBURTON, I. W. (1972). Biochemical comparisons of type 1 and type 2 herpes simplex viruses. In Oncogenesis and Herpesviruses 2, pp Edited by P. M. Biggs, G. de Th~ & L. N. Payne. Lyon : IARC Scientific Publications. HALLIBURTON, I. W. & TIMBURY, M. C. (1973). Characterization of temperature-sensitive mutants of herpes simplex virus type 2. Growth and DNA synthesis. Virology 54, HAY, R. T. & HAY, J. (1980). Properties of herpesvirus-induced 'immediate early' polypeptides. Virology 104, HONESS, R. W. & ROIZMAN, B. (1973). 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. Journal of Virology 12, KILLINGTON, R. A., YEO, J., HONESS, R. W., WATSON, D. n., DUNCAN, B. E., HALLIBURTON, I. W. & MUMFORD, J. (1977). Comparative analyses of the proteins and antigens of five herpesviruses. Journal of General Virology 37, KNIPE, D. M., Q'dI~qLAN, M. 1'. & SPANG, A. E. (1982). Characterization of two conformational forms of the major DNA-binding protein encoded by herpes simplex virus 1. Journal of Virology 44, LA THANGUE, N. B. & CHAN, W. L. (1984). The characterization and purification of DNA binding proteins present within herpes simplex virus infected cells using monoclonal antibodies. Archives of Virology 79, LITTLER, E., YEO, J., KILLINGTON, R. A., PURIFOY, D. J. M. & POWELL, K. L. (1981). Antigenic and structural conservation of herpesvirus DNA-binding proteins. Journal of General Virology 56, MARSDEN, H. S., CROMBIE, I. K. & SUBAK-SHARPE, J. H. (1976). Control of protein synthesis in herpesvirus-infected cells: analysis of the polypeptides induced by wild-type and sixteen temperature-sensitive mutants of HSV strain 17. Journal of General Virology 31, MAYR, A., B()HM, H. O., BRILL, J. & WOYCIECHOWSKA, S. (1965). Charakterisierung eines Stutenabortvirus aus Polen und Vergleich mit bekannten Rhinopneumonitisvirus Stfimmen des Pferdes. Archly fi~r die gesamte VirusJbrschung 17, POWELL, K. L. & COURTNEY, R. J. (1975). Polypeptides synthesized in herpes simplex virus type 2 infected HEp-2 cells. Virology 66, POWELL, K. L. & PUR1FOY, O. J. M. (1976). DNA-binding proteins of cells infected by herpes simplex virus type 1 and type 2. Intervirology 7, POWELL, K. L., LITTLER, E. & PURIFOY, D. J. M. (1981). Non-structural proteins of herpes simplex virus I I. The major virus-specific DNA binding protein. Journal of Virology 39, PURIFOY, D. J. M. & POWELL, K. L. (1976). DNA-binding proteins induced by herpes simplex virus type 2 in HEp-2 cells. Journal of Virology 19, STEVENS, J. G., KADO-BOLL, G. J. & HAVEN, C. B. (1969). Changes in nuclear basic proteins during pseudorabies virus infection. Journal of Virology 3, TYLER, S. J., HAY, J. & STEVELY, W. S. (1974). Nuclear protein synthesis in hamster kidney cells: the effect of pseudorabies virus infection. Archly flit die gesamte Virusjbrschung 44, 84-9 l. WILCOX, K. W., KOHN, A., SKLYANSKAYA, E. & KOIZMAN, B. (19go). Herpes simplex virus phosphoproteins. I. Phosphate cycles on and off some virus polypeptides and can alter their affinity for DNA. Journal of Virology 33, YEO, J., KILLINGTON, R. A., WATSON, D. H. & POWELL, K. L. (1981). Studies on cross-reactive antigens in the herpesviruses. Virology 108, (Received 29 May 1985)