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J. gen. Virot. (198o), 48, 297-3Io Printed in Great Britain 297 Glycoproteins with Type Common and Type Specific Antigenic Sites Excreted from Cells Infected with Herpes Simplex Virus Types 1 and 2 By R. E. R A N D A L L , * R. A. K I L L I N G T O N AND D. H. W A T S O N Department o f Microbiology, University o f Leeds, Leeds LS2 9JT (Accepted 7 January 1980) SUMMARY Agar immunodiffusion tests demonstrated that BHK 2I cells infected with either HSV I or HSV 2 release only a few HSV-specified antigens into the extracellular fluid (infected cell released polypeptides-ICRP). Neutralization blocking experiments showed that the majority of antigens/(including the Band II common antigen) involved as target sites in antibody-mediated virus neutralization are present in the ICRP of both HSV I and HSV 2. SDS-PAGE identified six regions of virus-specified proteins in the ICRP from both HSV I- and HSV 2-infected BHK cells. All these specifically released proteins are glycosylated, although to varying degrees. The SDS-PAGE profiles of HSV I and HSV 2 ICRP are different but do show some similarities, the most notable being a highly glycosylated protein with an estimated tool. wt. of 5oooo to 540oo in HSV I 1CRP and 52ooo to 5600o in HSV z ICRP. Immune precipitation demonstrated that these two proteins contain the Band II antigenic site. Similar studies showed that the major type t specific antigenic site, which is involved as a target site in the neutralization of virus infectivity, is located in the highest mol. wt. glycoprotein region of HSV I ICRP and has a similar mobility to the VP7/8 region of purified enveloped virus. INTRODUCTION Antigenic analysis o f HSV I and HSV 2 has shown the presence of both type common, i.e. cross reacting, and type specific antigenic sites involved in the interaction of virus with neutralizing antibody (Sim & Watson, t 973). These antigenic sites have been characterized further in that an antiserum has been raised in rabbits to the type common Band II antigen of an HSV I (HFEM) virus isolate (Watson & Wildy, i969). This antiserum to Band II, however, still retains some type I specific neutralizing activity after absorption with excess type 2 antigen which removes the predominant type common neutralizing antibody (Sim & Watson, I973). Antiserum to the major glycoprotein region (VP7/8) of HSV I purified enveloped particles also has type I specific neutralizing activity (Powell et al. I974). However, it was shown that the type I specific activity of this antiserum differed from the type I specific activity found in antiserum to Band II (Halliburton et al. I977). A number of workers have shown that cells infected with herpes viruses release only a few virus-specified antigens into the extracellular fluid (Ben-Porat & Kaplan, I97o; Pennington & McCrae, I977; Norrild & Vestegaard, I979). Furthermore, Kaplan et al. (I975) claimed that the virus-specified proteins released by herpes simplex virus (HSV)-infected cells elicit mainly a type specific immune response in rabbits. * Present address: National Institute for Medical Research, Mill Hill, London NW7 IAA. oo22-I3317/8o/oooo-3968 $o2.oo • 198o SGM Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Tue, 02 May 2017 14:08:23 298 R.E. RANDALL, R. A. KILLINGTON AND D. H. WATSON In this paper we have studied the proteins released by cells infected with HSV using a variety of antisera in immunodiffusion, neutralization blocking and immune precipitation tests. METHODS Cells and virus. BHK 2I cells were grown as described by Watson et al. 0966), in the Glasgow modification of Eagle's medium containing IO °/o (v/v) tryptose phosphate broth and 5 % (v/v) calf serum (ETC). Two HSV t strains, H F E M (Watson et al. 1966) and MPI7 (Brown et al. I973), and one HSV 2 strain, HG52 (Timbury, 197I ), were used. MPI7 and HG52 are non-syncytial, while H F E M is syncytial in plaque morphology on B H K cells. Production and radioactive labelling of polypeptides released from infected cells. Monolayers of BHK 21 cell cultures in 8o oz Winchester bottles were infected at an input multiplicity of 5 to to p.f.u./cell. After adsorption for I h at 37 °C, the inoculum was decanted, the monolayer washed and fresh ETC at 37 °C was added. At 4"5 h after addition ofinoculum (p.i.), the ETC was discarded and the monolayer washed with Glasgow modified Eagle's medium (GMEM) lacking calf serum and tryptose phosphate broth. The cells were then incubated for a further 2o h with radioactively labelled or unlabelled G M E M (5o ml). The medium was collected and centrifuged at I6OOO rev/min for I h. The supernatant fluid was concentrated to approx. IO to 15 ml by polyethylene glycol dialysis and then to 2 to 3 ml by vacuum dialysis. The resulting concentrate was centrifuged at IOOOOOg for I h in a Christ Omega Il ultracentrifuge and dialysed extensively against phosphate buffered saline (PBS). This fraction is termed the infected cell released polypeptide (ICRP) fraction. The average number of BHK 2I cells per confluent monolayer in an 80 oz Winchester bottle was estimated at 5 x io 8 cells. Thus there are approx. 2 × Io 8 cell equivalents per I ml of ICRP fraction. The cells from such an experiment were resuspended in 5 ml of PBS and disrupted with an ultrasonic probe (M.S.E.). This suspension of disrupted cells was termed the total cell antigenic fraction. Labelling medium contained either 35S-methionine (2/~Ci/ml in G M E M with I/5th the normal concentration of methionine) or x4C-glucosamine (sp. act. 52 mCi/mmol, o'33/~Ci/ ml in G M E M with I/2oth the normal concentration of glucose). Purification of radioactively labelled enveloped virus particles. Purified radioactively labelled virus was prepared from the cytoplasm of H F E M infected radioactively labelled (3~S-methionine) BHK 2t cells by sedimentation through Dextran T~o gradients as described by Killington et al. (t977). Antisera. HSV general antiserum was prepared by repeated immunization of rabbits with extracts of HSV 1 (HFEM) or HSV 2 (3345) infected RK13 cells (Watson et al. I966). Type t and type 2 specific antisera were prepared by absorption of general antiserum with excess heterologous antigen (Sim & Watson, ~973); that is, I ml of general antiserum was incubated for 24 h at 4 °C with extract of Io 9 cells infected with heterotogous virus, heatinactivated for 3o min at 56 °C, centrifuged at IOOOOOg for I h in a Christ Omega Ultracentrifuge and the supernatant fluid concentrated to its original volume by vacuum dialysis. Antiserum to Band I1 (HSV l) was prepared by inoculation of rabbit foot pads with the appropriate precipitin line from agar-gel immunodiffusion plates as described by Watson & Wildy (I969). Antiserum to the VP7/8 region of HSV I virus (HFEM) was prepared by immunization of rabbits with polyacrylamide gel slices containing the appropriate polypeptide region as described by Powell et al. (I974). Neutralization blocking experiments. The antigenicity of the ICRP was studied by their ability to absorb the neutralizing activity of the above antisera. Undiluted antisera (40 #1) Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Tue, 02 May 2017 14:08:23 Antigens excreted by HSV-infected cells 299 were incubated overnight at 4 °C with increasing amounts (zo, 8o or I6o #1, i.e. 4 × Ioe, 16 x Io 6 or 32 × Io ~ cell equivalents) of I C R P and made to the same vol. (2oo #1) with mock ICRP. HSV I and HSV 2 general antisera were also diluted to I / 5 and I / I o concentration of the original antisera (as indicated in parentheses in the Results) and absorbed with ICRP. The absorbed antisera were heat-inactivated for 3o rain at 56 °C and tested for residual neutralizing activity in a plaque reduction test in which the indicator viruses, H F E M (syn) and HG52 (syn+), were artificially mixed prior to the test. The virus (approx. 2 × lo 4 p.f.u./ ml each) were incubated for 2 h with an equal vol. ofantisera. Surviving virus was assayed by the suspension method of Russell 0962). Survival of infectious virus after incubation with immune sera (v) is expressed as a percentage of that surviving incubation with pre-immune sera (Vo), i.e., V/Vo × Ioo. Agar gel immunodiffusion tests were performed in 2 to 3 m m layers of 1 % agar in the manner of Watson et al. 0966). Immune precipitation. Immune precipitates were obtained by incubating antisera with HSV I or HSV 2 I C R P fractions in appropriate proportions (usually o'z ml of I C R P fraction with o.2 ml antisera) in highly tapered glass tubes for I8 to 24 h at 4 °C (Killington et al. I978). The tubes were centrifuged at 4ooo rev/min for I5 rain, the supernatant fluid removed and the pellet carefully rinsed three times with cold PBS. The final pellet was resuspended in a small volume (o-Iz ml) of disruption mixture (o'o5 ~-tris-HCl, pH 7"0, 5 % sucrose, 2 % SDS and 5 % mercaptoethanol) by disruption with a M.S.E. ultrasonic probe. The amount of radioactivity present in the resuspended pellet was determined by T C A precipitation on a glass fibre disc and counting in a Beckman LS-23o scintillation counter. An appropriate volume of disrupted precipitate was then electrophoresed on an SDS-polyacrylamide gel. Polyaerylamide gel eleetrophoresis. Polypeptides were separated by SDS polyacrylamide slab gel electrophoresis, followed by fixation, staining and autoradiography (Halliburton et al. I977). HSV polypeptides of known tool. wt. (e.g. VPs 5, 16, I9c and 2z; Heine et al. ~974) were used as calibration protein markers for the estimation of the tool. wt. of unknown polypeptides. RESULTS Immunodiffusion tests on polypeptides released from infected cells HSV I and HSV 2 total cell antigen and I C R P fractions were prepared as described in Methods and reacted against HSV I and HSV z general antisera in agar gel immunodiffusion plates (Fig. 0- Although the I C R P fractions are three times more concentrated in terms o f cell equivalents than the total cell antigen fractions, it is clear that certain antigens in the total cell fractions are either not present or are in undetectable amounts in the I C R P fractions. HSV I and HSV z I C R P share one major type common antigen as detected in agar immunodiffusion (Fig. I c) and this precipitin line was shown to have a line of identity when the I C R P were reacted with antiserum to Band II. In fact the type common Band II antigen is the predominant component of HSV z ICRP, although two minor antigens can be detected when HSV 2 I C R P are reacted against homologous antiserum (Fig. I b). The Band lI antigen is also a major constituent of HSV I ICRP. However, two other antigens are easily detected by an immunodiffusion test. Both of these give better precipitin lines when reacted against homologous rather than heterologous antiserum (Fig. Ta and c). Also, two further minor precipitin lines are usually detected when HSV l I C R P react against homologous general antiserum. Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Tue, 02 May 2017 14:08:23 300 R . E . R A N D A L L , R. A. K I L L I N G T O N AND D. H. W A T S O N iN 0 \ Fig. ~. HSV i and HSV 2 total cell antigen (T1 and T2 respectively) and ICRP (rl and I~) reacted in immunodiffusion tests with HSV [ and HSV z general antiserum (g-a/st and g-a/sz respectively). The Band II type common immune precipitin line is arrowed. Electrophoretic characterization of polypeptides released by infected cells A c o m p a r i s o n was m a d e o f the e l e c t r o p h o r e t i c b e h a v i o u r o f the I C R P with those o f infected cell p o l y p e p t i d e s (Fig. 2). B H K ceils were infected with H F E M and labelled f r o m 4"5 to z4 h p.i. with either 85S-methionine or 14C-glucosamine, a n d t o t a l cell p r o t e i n s were e l e c t r o p h o r e s e d on a 9"25 % S D S p o l y a c r y l a m i d e gel. H F E M I C R P a n d proteins released f r o m uninfected cells ( m o c k I C R P ) labelled with 14C-glucosamine o r aSS-methionine were also co-electrophoresed. In a d d i t i o n , H F E M I C R P labelled with 14C-glucosamine or 35SDownloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Tue, 02 May 2017 14:08:23 Antigens excreted by HSV-infected cells I HSVI ICRP HSV 1 total cell protein I ( 3sS-met ) ( ( x4C-gluc 3oi I MocklCRP i ) 3ss met ,,ff HSV l HSV 1 ICP ICRP 8 9 10 21 26 • 39 40 i rl precipitated by excess HSV 1 g-a/s Fig. 2. Autoradiograms of SDS-polyacrylamide slab gels showing the polypeptides of HSV i (HFEM)-infected BHK cells (slots [ and 2) and the polypeptides released (ICRP) by these and mockinfected BHK cells (slots 5 and 6 respectively). The precipitates formed when the ICRP were reacted with excess homologous general antiserum were also disrupted and subjected to SDS electrophoresis (slots 3 and 4). The cells were labelled with x4C-glucosamine or asS-methionine from 4"5 to 24 h p.i.N.B. No mock ICRP was precipitated by HSV general antisera (g-a/s). m e t h i o n i n e p r e c i p i t a t e d w i t h excess H S V I general a n t i s e r u m were d i s s o c i a t e d a n d electrop h o r e s e d o n the S D S - p o l y a c r y l a m i d e gel. general antisera. It is possible to i d e n t i f y six b r o a d mol. wt. r e g i o n s o f p r o t e i n s (labelled a to f) t h a t are specifically released b y H S V I - i n f e c t e d B H K cells (Fig. 2 a n d 3). A l l six r e g i o n s label w i t h Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Tue, 02 May 2017 14:08:23 R. E. R A N D A L L , 302 R. A. K I L L I N G T O N AND HSV 1 ICRP HSV 1 VP D. H. W A T S O N HSV 2 ICRP 5 a al ~ 87 8.5 , a2 ~ 1 1314 2 16 19 c 22 3 bl b b2 m O c 5 d 23 HFEM purif, virus MPI 7 bet HFEM horn MP17 horn HG52 her HFEM het MPI7 het HG52 horn Fig. 3. Autoradiograms o f SDS-polyacrylamide slab gels o f 35S-methionine labelled polypeptides o f HSV I ( H F E M and M P I 7 ) and HSV 2 (HG52) I C R P immune precipitated with homologous (horn) or heterologous (bet) general antisera at an antiserum to antigen ratio o f I : I (v/v). Polypeptides were labelled from 4"5 to 24 h p.i. H F E M purified (purif.) virus particles, labelled with 35Smethionine, were also co-electrophoresed to give marker virion polypeptides. 14C-glucosamine (Fig. 2), although some proteins are more highly glycosylated than others, e.g. band b is poorly glycosylated compared with bands a and c. Many of these regions are complex, with more than one component being identified in certain regions. For instance, regions a and b of MPI7 ICRP can be seen to contain a minimum of two components (Fig. 3, slot 4)In tool. wt. estimations of H F E M ICRP, region a has an estimated mol. wt. between I24ooo and ~38ooo and corresponds to the major glycoprotein (VP7/8) region of H F E M purified virus (Fig. 3). Region b has an estimated mol. wt. between 59ooo and 65ooo and is analogous in size to a glycoprotein region (VPI7/t8) of H F E M purified virus (Fig. 3). The other four regions do not co-migrate with any glycoproteins as yet identified in purified HSV I virus. Their estimated mol. wt. are 50000 to 54000 (c), 43000 to 45000 (d), 30000 to 35000 (e) and 2IOOO to 22000 (f). When similar experiments were carried out by infecting various cell lines (Chang conjunctival, rabbit kidney, baby hamster kidney and HEp2) with HFEM, it was shown that, while a similar pattern of | C R P was obtained, the mobility of the high mol. wt. region on SDS-PAGE varied. This demonstrates that the host cell plays a role in the post translational processing of these proteins (Fig. 4)HSV 2 ICRP were studied following their immune precipitation with homologous and heterologous general antisera. Fig. 3 shows the SDS-PAGE analyses of the immune preDownloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Tue, 02 May 2017 14:08:23 Antigens excreted by HSV-infected cells HSV 1 ICRP 303 HSV 1 VP 14 BHK Chang HEp2 RK conj. Fig. 4. Autoradiogram of an SDS-polyacrylamide gel showing the 3~S-metbionine labelled region a of HSV I (HFEM) ICRP from BHK 2I, Chang conjunctival, HEp2 and rabbit kidney (RK) infected cells, immune precipitated with homologous general antiserum. cipitates formed when HSV 2 ICRP, labelled with 35S-methionine, were precipitated with excess homologous and heterologous general antisera. Six protein regions (labelled from I to 6) released from HG5z infected ceils are identified in this manner and, like HSV t ICRP, all six regions are glycosylated. Region I has an estimated tool. wt. of between 86ooo and IOOOOO. Regions 2 and 3 have an estimated mol. wt. of 7oooo to 76ooo and 66ooo to 69ooo respectively. Unlike region b of H F E M ICRP, they label efficiently with 14C-glucosamine. Band 4 has an estimated mol. wt. of 52ooo to 56ooo compared with 5oooo to 54ooo for region c of HSV I ICRP and, like region c, is efficiently labelled with ~4C-glucosamine. Although not so easily distinguishable in HG52 ICRP, two diffuse regions (5 and 6) can be detected which co-migrate in the region of d and e of HSV I ICRP. The two bands labelled x and y in Fig. 3 have not been detected in other immune precipitates of HG5z ICRP. The antigenic nature of both HSV I and HSV 2 ICRP were characterized further by their immune precipitation with antisera of differing specificities, and by their ability to block the neutralizing activities of these antisera. Immune precipitation of H S V ICRP HS V general an tisera Although all HSV I ICRP could be precipitated with excess heterologous general antiserum (Fig. 6), a number of ICRP were more efficiently precipitated than others at limiting antiserum concentration. Thus regions a2 and c are precipitated more efficiently than a a and b by heterologous general antiserum (Fig. 3; compare slots 3 and 6 and 4 and 7)- This pattern of precipitation could not be achieved by simple dilution of homologous general antiserum, region al being precipitated relatively more efficiently than b or c in the latter case. Precipitation of HSV z ICRP by homologous and heterologous general antisera produced similar profiles (Fig. 3, compare slots 5 and 8). Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Tue, 02 May 2017 14:08:23 304 R.E. RANDALL, R. A. KILLINGTON AND D. H. WATSON Antiserum to Band II (HSV l) Antiserum to Band II contains all the antibodies important in the cross neutralization of HSV, as well as some of the antibodies involved in type ~ specific neutralization. Fig. 5 shows the precipitation of ICRP by antiserum to Band II. Regions c, d and e of HSV 1 ICRP are precipitated by this antiserum (although d cannot be detected in the photograph), whereas regions a and b are not. A threefold increase in the antiserum : antigen ratio did not alter the pattern of precipitation. Similarly, region 4 of HSV z ICRP is clearly precipitated by the antiserum, whereas regions I, z and 3 are not. Although it is very difficult to detect regions 5 and 6 of HSV z ICRP (they cannot be seen in Fig. 5), a number of experiments have shown that these proteins are also precipitated by antiserum to Band II. Antiserum to VP7/8 (HSV ~) Antiserum to VP7/8, as stated earlier, is prepared by the inoculation of the high mol. wt. glycoprotein region of H F E M purified virus into rabbits. The resulting serum is type 1 specific in neutralization activity. This antiserum has not previously been used successfully, either in reacting to give precipitin lines in an agar gel immunodiffusion test or in immune precipitation with HSV ~ soluble antigen fractions. However, when used in immune precipitation with H F E M ICRP, the high tool. wt. glycoprotein region a is precipitated. Increasing the ratio of antiserum: antigen fails to precipitate any of the other regions identified in HSV I ICRP (Fig. 6). It is not clear whether region a2 is precipitated by this antiserum, since, when present, Band al usually obscures a2. Type I specific antiserum Type r specific antiserum (which contains all the type specific antibodies present in HSV I general antiserum) was also used in the immune precipitation of HSV I ICRP. Fig. 6 shows that the only HSV I ICRP precipitated by this antiserum was region a. However, in this experiment it was not clear whether the plateau level for precipitation had been reached. Thus an increased antiserum:antigen ratio may result in precipitation of further HSV I ICRP regions. Neutralization (serum) blocking tests on proteins released by HSV-infected cells Antisera used in the immune precipitation experiments were also tested for their ability to neutralize HSV, both before and after absorption with HSV I or HSV 2 ICRP. Absorption of antiserum to VP7/8, type I-specific, antiserum to Band II, HSV I general ( r / I o dilution) and HSV z general antisera with HSV I ICRP completely removed the ability of these antisera to neutralize HSV I. Thus in a plaque reduction test the unabsorbed antisera reduced HSV I infectivity to < 2% survival, while after adsorption of 4o/~1 of these antisera with approx. I6 × lO6 cell equivalents of HSV t ICRP (see Methods), there was > 8o % (not significantly different from Ioo%) survival. Absorption of 4o/~1 of HSV I general (I/5 dilution) and antiserum to Band II with approx. 4 × Io6 cell equivalents of HSV 2 ICRP reduced the ability o f these antisera to neutralize HSV z virus from I"7 and I8 % survival, respectively, to IOO% survival. However, absorption of HSV z general antiserum 0 / 5 dilution) with I6 × 1o6 cell equivalents of HSV z ICRP, only reduced the neutralizing activity of this antiserum from 8 % to 61% survival of HSV z, and similarly of type ~ specific antiserum from 6 % to 78 % survival. Doubling the amount of blocking antigen used to absorb these two antisera did not alter the results significantly (6I % and 84% survival respectively). In these experiments it was possible to absorb out the cross neutralizing activity of HSV Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Tue, 02 May 2017 14:08:23 Antigens excreted by HSV-infected cells I HSV 2 ICRP 1 HSV I ICRP 3o5 [ precipitated by pre I. a/s to pre I. a/s to a/s Bll a/s BII ~ C d Fig. 5. Autoradiogram of an SDS 9'25 ~ acrylamide gel of asS-methionine labelled HSV ~ (HFEM) and HSV 2 (HG52) ICRP precipitated by antiserum to Band II (a/s to Band II) and pre-lmmune (pre I.) antiserum). Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Tue, 02 May 2017 14:08:23 306 R. E. R A N D A L L , R. A. K I L L I N G T O N AND D. H. W A T S O N HSV-1 ICRP < d Ratio of antisera to antigen hom het het het 1:1 6:1 3:1 1:1 1 6:1 anti VP 7/8 sera 3:1 1:1 I type 1 specific 1:1 pre I. 1:1 Fig. 6. Autoradiograms of SDS 9'25 ~?/oacrylamide gels of H S V I ( H F E M ) I C R P precipitated with increasing amounts of heterologous (het) general antiserum and anti VP7/8 antiserum. The precipitates formed by reaction o f H F E M I C R P with homologous (horn) general antiserum, and type I specific antiserum, at an antiserum to antigen ratio of I : I, are also shown. general antiserum with a smaller a m o u n t of I C R P than that required to absorb out the type specific neutralizing activity. Thus, incomplete absorption of HSV I general antiserum with HSV I I C R P produced an absorbed antiserum which still reduced HSV t infectivity to 33 O~/o of its original titre in a plaque reduction test, but failed to neutralize HSV 2 0 o 4 ~,') survival). The original unabsorbed antiserum reduced both HSV I and HSV z infective virus to < I °/o survival in the same experiment. Similar results were obtained by absorption o f H S V 2 general antiserum with HSV 2 I C R P , i.e. an incompletely absorbed antiserum still neutralized HSV 2 (28 ~'o survival) but failed to neutralize HSV I. Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Tue, 02 May 2017 14:08:23 Antigens excreted by HSV-infected cells 3o7 DISCUSSION Only a small number of HSV-specified antigens present in infected cells are released into the culture medium (Fig. i). SDS-PAGE of proteins released from HSV I, HSV 2 and uninfected (mock) cells, and analysis of precipitates formed when these ICRP were reacted with HSV general antisera identified six protein regions present in both HSV ~ (regions a to f) and HSV z (regions I to 6) ICRP that were not present in mock ]CRP (Fig. z and 3). Region a of HSV 1 ICRP has a similar electrophoretic mobility on S D S - P A G E to the VP7/8 region of HSV I infected cells and purified virus. Similarly, region I of HSV 2 ICRP may be equivalent to the glycoprotein with a tool. wt. of between 80o0o and 9oooo found in HSV 2 purified virus particles (Cassai et ctl. I975). However, we did not identify infected cell counterparts of other ICRP. Thus, no major glycoprotein was identified in the infected cell which co-migrated with glycoprotein c of HSV I ICRP (Fig. z) or 4 of HSV z ICRP. Such ICRP are probably processed forms or breakdown products of their counterparts in the infected cells. The SDS-PAGE patterns of proteins released from cells infected with either HSV I strain H F E M or MPI 7 are very similar but there are slight differences; for example, the MPI7 region a~, migrates faster than the a2 region of H F E M ICRP (Fig. 3, slot 6 and 7)However, there are a number of marked differences between HSV I and HSV 2 ICRP. Thus in HSV I ICRP there is no protein with a similar tool. wt. to region r of HSV 2, and these in turn have no high tool. wt. glycoprotein which co-migrates with region a of HSV I ICRP. This latter case is surprising, as in both HSV z-infected cells and purified virus there is a glycoprotein region of similar tool. wt. to the VP7/8 region of HSV I (Cassai et al. I975). The major region of equivalence between HSV I and HSV z ICRP is in regions c and 4 respectively. These proteins are both highly glycosylated, have a similar mol. wt. and behave analogously in their precipitation with a variety of antisera. Comparison of proteins released from BHK 2I, HEp2, Chang conjunctival and rabbit kidney cells infected with HSV I show that the host cell influences the processing or glycosyIation of virus-specified proteins. Thus, the electrophoretic mobility of the high tool. wt. glycoprotein (region a) depends on the cell line infected (Fig. 4)- Keller et al. (I97O) also showed that the host cell affects the degree of glycosylation of HSV proteins. They demonstrated differences in the glycosylation of membrane proteins in African green monkey (Vero) and human (HEpz) cells infected with the HSV i strains F or MP. Whether the host cell can also alter the antigenicity of these virus proteins by modifying the processing of glycoproteins has, unfortunately, not been studied. The precipitation of HSV I and HSV 2 ICRP by a variety of antisera and their ability to absorb out the neutralizing activity of these antisera demonstrated that HSV I and HSV 2 ICRP contain the Band II common antigen and the majority, if not all, of the type specific antigens involved in antibody mediated virus neutralization. The apparent inability of HSV 2 ICRP to remove all the neutralizing activity of HSV 2 general antiserum against HSV 2 virus may be because the HSV z VP7/8 region is not represented in HSV 2 ICRP. However, some of the ICRP may be breakdown products of this region. Similarly, regions d and e o f H S V I ICRP and regions 5 and 6 o f H S V 2 ICRP may be breakdown products of regions c and 4, respectively. Thus no antisera used in these experiments precipitated regions c and 4 independently of regions d and e, and 5 and 6 respectively. Another possibility is that these proteins are found in small aggregates in the ICRP preparations and that the minor low mol. wt. components are host proteins. However, regions a, b and c o f H S V t ICRP behave independently of each other in their precipitation with various antisera, demonstrating that they are distinct HSV antigens and physically independent in the ICRP Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Tue, 02 May 2017 14:08:23 308 R.E. RANDALL, R. A. KILLINGTON AND D. H. WATSON fraction. For similar reasons it is unlikely that regions T, 2 or 3 are antigenically related to the low mol. wt. regions of HSV 2, although they could be related to one another. These results also strongly suggest that the main type specific antigenic sites of HSV I ICRP are to be found in the high tool. wt. glycoprotein region al and the main type common antigenic sites in the low tool. wt. glycoprotein region c. This conclusion is based on the fact that region al is inefficiently precipitated by heterologous general antiserum but preferentially by type I specific and anti-VP7/8 sera. On the other hand region c (and region 4 of HSV 2 ICRP) is comparatively efficiently precipitated by heterologous general antiserum and antiserum to Band II. It is therefore likely that the main type I specific antigen, important in neutralization, will also be found in the high mol. wt. glycoprotein region and the type common in the low tool. wt. glycoprotein regions. However, as all ICRP are precipitated by heterologous general antisera, even those proteins with type specific antigenic determinants must have some type common antigenic sites as well. Presumably, the type specific determinants are located on the outside of the envelope, while most type common determinants are embedded within the envelope. If all these type common determinants were exposed on the surface of the virus particle there would probably be more type common antigens than the Band II antigens involved as target sites for heterologous antibody mediated virus neutralization. The results presented here are therefore in general agreement with those of Kaplan et al. (r975) and Norrild & Vestergaard 0979), who also showed that only a few virus proteins are released into the extracellular fluid of cells infected with HSV. These authors showed that the type specific antigenic determinant is the major constituent of proteins released by HSV-infected rabbit kidney and HEp2 cells respectively. However, our results clearly show that the type common Band II antigen is a major constituent of polypeptides released by both HSV I- and HSV z-infected BHK cells. Certainly the major type specific antigen is present in a soluble form in higher concentrations in the ICRP fractions than in total cell antigen fraction, probably being intimately associated with cellular membranes in the latter case. In contrast, the type common Band II antigen is present in a soluble form in both the ICRP and total cell antigen fractions (Fig. I). Norrild & Vestegaard (1979), however, did detect a type common antigenic determinant in proteins released from HSV-infected HEp2 cells. Also, in the SDS polyacrylamide gel profiles of released polypeptides from HSV infected rabbit kidney cells presented by Chen et al. (I978), it is likely that their released virus polypeptide C is equivalent to our HSV I ICRP region c and HSV 2 ICRP region 4, and contains the Band II type common antigenic site. Cohen et al. 0978), studying the antigen CP-I, which was shown to be equivalent to the Band II antigen in that it contained the cross-reacting antigenic determinants (Watson & Wildy, I969; Cohen et al. I972; Powell & Watson, I975), indicated that a protein (gp52) with a mol. wt. of 52ooo and CP-I antigenic activity was the probable precursor of the low tool. wt. glycoprotein, gP59, found in purified enveloped particles. Cohen et al. (I978) suggested that gp52 corresponded to Spear's glycoprotein pD (Spear, I976); gp52 and pD are also probably antigenically equivalent to a glycoprotein with an estimated tool. wt. of 47ooo which was precipitated from the soluble antigen fraction of HSV x-infected BHK cells by antiserum to Band II (Honess & Watson, I974). However, Cohen et al. 0978) identified another glycoprotein in membranes of HSV-infected KB cells which also had CP-I antigenic activity and a tool. wt. of 52ooo. They could not rule out that this glycoprotein was a breakdown product or a further processed form of gP59 and it is this latter possibility that the work presented here would favour. Thus we would like to suggest that glycoprotein c of HSV r ICRP and 4 of HSV 2 ICRP are equivalent to the glycoprotein identified by Cohen et al. (r978) in the plasma membrane of HSV-infected KB cells. It Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Tue, 02 May 2017 14:08:23 Antigens excreted by HSV-infected cells 309 seems unlikely that the highly glycosylated ICRP c or 4 could be a precursor of the gP59 identified in purified enveloped particles. The ICRPs contain most of the antigens important in eliciting neutralizing antibodies to HSV infectivity and therefore they may provide a source of virus glycoproteins free from infective virus with which to test the potential of subunit vaccines in preventing primary HSV infections. If subunit vaccines prove efficient in protecting against primary HSV infections, similar subunit vaccines may prove successful in immunization against other important herpes infections such as those caused by cytomegalovirus and Epstein-Barr virus. We are indebted to the Medical Research Council for the award of a project grant in support of this work (DHW) and to the Science Research Council for a research studentship (RER). REFERENCES BEN-PORAT, T. & KAPLAN, A. S. (I970). Synthesis o f proteins in infected cells with herpesvirus. V. Viral glycoproteins. Virology 4x, 265-273. BROWN, S. M., RITCHIE, D. A. & SUBAK-SHARPE, J. H. (I973). Genetic studies with herpes simplex type I. 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(Received zr September I 9 7 9 ) Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Tue, 02 May 2017 14:08:23