VIROLOGY
98, 255-260 (1979)
Proteins of Herpesvirus Type 2. V. Isolation and Immunologic Characterization Viral Proteins in a Virus-Specific Antigenic Fraction
CYNTHIA The Johns
Hopkins
C. SMITH
AND LAURE
AURELIAN
Medical Institutions, Division of Comparative Medicine, Department Biophysics, 720 Rutland Avenue, Baltimore, Maryland 21206 Accepted
July
of Two
of
Biochemistry
and
6, 1979
Previous reports from our laboratory have shown that “crude” AG-e, a type-common HSV antigen, induces an antigen-driven cell-mediated immune response in patients with HSV cervicitis or cervix cancer. Anti-crude AG-e sera give rise in crossed immunoelectrophoresis against total viral soluble antigenic mixtures (HSV-2 (G) SAM) to one precipitin band (“pure” AGe). Pure AG-e is immunologically identical to crude AG-e as determined by immunodiffusion and it resolves into two infected cell proteins, ICP 12 (MW: 140,000) and ICP 14 (MW: 130,000) upon SDS-acrylamide gel electrophoresis. ICP 12 and ICP 14 are purified to radiochemical homogeneity by SDS-acrylamide gel electrophoresis. Antisera to ICP 12 react with HSV-2 (G) SAM in crossed immunoelectrophoresis and both anti-ICP 12 and ICP 14 sera stain HSV-2 (G) infected cells. ICP 12 and ICP 14 appear to be envelope proteins as evidenced by: (i) the absorption of the reactivity of anti-ICP 12 and ICP 14 sera with HSV-2 (G) virions but not with “mock-virus” preparations, (ii) the ability of the anti-ICP 12 and ICP 14 sera to neutralize HSV-2 in presence of anti-IgG, and (iii) the resolution of a protein with the relative mobility of ICP 14 in virions the surface of which was iodinated with lactoperoxidase.
Patients with cervix cancer or herpetic cervicitis develop an antigen-driven cellmediated immune response measured by an in vitro lymphokine assay against a virusspecific, type-common antigenic fraction designated AG-e. Control subjects are nonresponsive (1). In this report we describe the purification of AG-e, and demonstrate that its immunogenic proteins (ICP 12 and ICP 14) comigrate on SDSacrylamide gel electrophoresis with two proteins located within the virion envelope. Soluble antigenic mixtures (SAM), prepared as previously described (1, 2) from extracts of HEp-2 cells exposed to 5 PFU/cell of HSV-2 (G) for 24 hr at 3’7”, were cleared of virions by two cycles of centrifugation at 100,000 g for 2 hr and fractionated on brushite columns by stepwise elution with increasing concentrations of phosphate buffer, pH ‘7.0, containing 0.1 M NaCl. The 0.4 M phosphate buffer eluate (“crude” AG-e) was concentrated with XM-50 Amicon membrane filters, emulsified in com255
plete Freund’s adjuvant, and used to inoculate rabbits as described (1). Duplicate samples, dialyzed against 0.18 M Tris0.06 M barbital buffer (pH 8.6) with 1% Triton X-100, were further purified by crossed immunoelectrophoresis (CIE) performed against anti-crude AG-e sera in 0.038 M barbital-O.11 N NaCl buffer with 0.0025% Triton X-100 (pH 8.2) (3). A single precipitin band was discerned by both Coomassie blue staining and autoradiography using either crude AG-e or HSV-2 (G) SAM as antigen (Fig. la). Agarose segments containing this precipitin band henceforth designated “pure” AG-e, were cut, washed, emulsified in complete Freund’s adjuvant and used to inoculate rabbits (10 segments/animal). Antisera thus prepared gave rise to one band of identity with antisera to crude AGe when assayed against HSV-2 (G) SAM in immunodiffusion (Fig. lb) and their reactivity could be absorbed with pelleted HSV-2 (G) virions but not with “mock” virus prepared identi0042-6822/79/130255-06$02.06/O Copyright All rights
Q 1979 by Academic Press. Inc. of reprcductmn in any form reserved.
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FIG. 1. (a) Autoradiograms of crossed immunoelectrophoretic analysis of L-[35S]methionine-labeled HSV-2 (G) SAM (20 pg protein) against anti-pure AG-e serum. Identical results are obtained with antisera to crude AG-e or ICP 12 and with crude AG-e antigen. (b) One line of identity between antisera to crude and pure AG-e assayed against HSV-2 (G) SAM in double gel immunodlffusion in 0.7% A37 agarose. (c) Absence of immunoprecipitation between HSV-2 (G) SAM and anti-pure AG-e serum adsorbed with HSV-2 (G) virions pelleted by centrifugation at 100,000 g for 1 hr. Sera (0.3 ml) were exposed three times to virions (2600 pg protein) for 1 hr at 37”. They were cleared of virions by centrifugation at 100,000 g for 2 hr prior to use in immunodiffusion. Note presence of reactivity in sera adsorbed with “mock” virus prepared identically but from HEp-2 cells exposed to PBS instead of virus. (d-f) Indirect immunofluorescent staining with fluorescein-conjugated goat anti-IgG (-r-chain specific). HEp-2 cells infected with HSVB for 24 hr were stained with antiserum to pure AG-e (d), anti-ICP 14 serum (e), or anti-ICP 14 serum adsorbed with pelleted HSV-2 (G) virions (f).
tally but from uninfected HEp-2 cells series of experiments, agarose segments (Fig. 1~). containing the precipitin band from the In order to identify the proteins in AG-e CIE of HSV-2 (G) SAM and anti-crude antigen, two series of experiments were AG-e sera (Fig. la) were dissociated and performed. In the first series, immunoelectrophoresed on 8.5% SDS-acrylamide precipitates resulting from the reaction of gels. Only two proteins with electrophoretic extracts of HSV-2 (G) infected cells labeled mobilities of ICP 12 and ICP 14 and COwith L(-35S)methionine 4-16 hr p.i., with migrating with virion proteins VP 5 and optimal concentrations (4) of anti-crude VP 6 were resolved. AG-e sera and antiglobulin were dissociated ICP 12 and ICP 14 were purified as and electrophoresed on 8.5% SDS-acrylpreviously described (5) from extracts of amide gels as previously described (4, 6). HSV-2 (G) infected HEp-2 cells labeled with Two proteins (Fig. 2d) with electrophoretic L-[35S]methionine lo-16 hours p.i. by mobilities of ICP 12 and ICP 14 (Fig. 2c) electrophoresis on 8.5% acrylamide gels in and comigrating with virion proteins VP 5 presence of 0.1% SDS for 15 hr, a time and VP 6 (Fig. 2b) were resolved in the interval six times longer than normal (6). precipitates. The reaction is virus specific Under these conditions, most of the proas evidenced by the observation that pro- teins migrate off the bottom of the gel and teins were not precipitated by the pre- excellent resolution is obtained between immune rabbit sera (Fig. 2e). In the second the high molecular weight proteins which
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FIG. 2. Densitometric scans of autoradiograms of 8.5% SDS-acrylamide gels oE (a) HSV-2 (G) virions purified by dextran gradient centrifugation (6) and iodinated by exposure to 1 pglml of lactoperoxidase and Na12”I (0.67 mCi/g I) in presence of 2 ~1 of 1 mkf H,O, (9). Free iodide was removed by chromatography on Sephadex G-75 equilibrated with 0.01 M Tris-HCl and 25 mg BSA. (b) HSV-2 (G) virions labeled with L-[Wlmethionine and purified by dextran gradient centrifugation (6). (c) Soluble extracts of HSV-2 (G) infected HEp-2 cells labeled with L+Y??]methionine 4-16 hr p.i. (d) Precipitates resulting from the reaction of extracts as in (c) with anti-crude AG-e serum. Antigen in 0.02 M Tris-HCl, pH 7.4, with 0.1 M NaCl and 0.001 M EDTA (TEN) was cleared of aggregates by three cycles of centrifugation at 3000 9 for 10 min. It was exposed (25 ~1) for 1 hr at 37” to optimal concentration (114) of antiserum and for an additional hour to optimal amounts of goat anti-rabbit IgG quantitated by precipitation (4). Precipitates, washed in TEN buffer for 10 min, were dissociated in 0.05 M Tris-HCl, pH 7.0,2% SDS, 5% 2p-mercaptoethanol for gel electrophoresis (5, 6). (e) Precipitates obtained as in (d) but with preimmune rabbit serum.
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remain on the gels. Representative gels containing infected or “mock” infected cell extracts were stained, sliced longitudinally, and used for autoradiography. The remaining gels were stored at -80”. Autoradiograms revealed six major proteins in the infected cell extracts including two with the relative mobilities of ICP 12 and ICP 14. Bands of similar relative mobilities were absent in extracts of uninfected cells. The relative mobilities of ICP 12 and ICP 14 were used as a guide for slicing segments of frozen gels. For each group of gels run at one time, a segment containing ICP 12 and one containing ICP 14 were autoradiographed in order to monitor the accuracy of the slicing procedure. The ICP 12 and ICP 14 containing segments were homogenized in complete Freund’s adjuvant and used to inoculate rabbits or dissociated and electrophoresed on 8.5% SDS-acrylamide gels. Proteins in these segments migrate on 8.5% SDS-acrylamide gels as single bands with respective electrophoretic mobilities of ICP 12 or ICP 14. The immunologic reactivity of the antisera to pure AG-e, ICP 12 and ICP 14 was studied. Four assays were used in these series: CIE, immunodiffusion (ID), indirect immunofluorescence (FA), and antiglobulin-enhanced neutralization (5). In the latter assay, increasing dilutions of antisera were incubated with HSV-2 (G) (3 x lo5 PFU/ml) for 1 hr at 37” and with goat anti-rabbit IgG (y-chain specific) for an additional hour. Survivors were assayed on HEp-2 cells. Sera were assayed simultaneously with the same virus stocks in order to enable comparison of the neutralizing potentials (7, 8). Anti-pure AG-e sera gave rise to a monoprecipitin band when assayed against HSV-2 (G) SAM in CIE (Fig. la) and immunodiffusion (Fig. lb) and they stained HSV-2 (G) infected cells in immunofluorescence (Fig. Id). Staining was predominantly located in the cytoplasm. The immunofluorescent titer, expressed as the reciprocal of the highest dilution still staining 25% of the cells, was 12. On the other hand, antisera to ICP 12 and ICP 14 did not react with HSV-2 (G) SAM in immunodiffusion and only anti-ICP 12 sera reacted in CIE. However, both antisera stained the cytoplasm of HSV-2 (G) infected
cells (Fig. le) displaying titers of8. Antisera to crude or pure AG-e, ICP 12, and ICP 14 neutralized HSV-2 (G) but only in presence of antiglobulin. Under the experimental conditions used in these studies, the neutralizing potential of the sera is reflected by the constant of neutralization (K) calculated from the formula: log v/v, = -0.43 Kc where v/v, is the proportin of survivors and c equals the antibody concentration expressed in terms of the amount of original serum per unit volume of the reaction mixture (5, 7, 8). The K (X100) values reproducibly obtained for antisera to crude AG-e, pure AG-e, ICP 12, and ICP 14 were 32 + 6, 35 + 2, 3.2 + 0.5, and 18 + 1, respectively. The reactivity of all three antisera was virus-specific. Thus: (i) uninfected cells, SAM or AG-e prepared from “mock” infected cells and “mock” virus preparations, as well as preimmune sera, were nonreactive in CIE, ID, and FA, (ii) preimmune sera did not neutralize virus infectivity, and (iii) the reactivity of the antisera in all four assays was abrogated by their absorption with HSV-2 (G) but not with “mock” virus preparations (Figs. lc and f). The virus neutralizing potential of the anti-ICP 12 and ICP 14 sera and the observation that their reactivity is absorbed with HSV-2 (G) virions are consistent with the comigration of ICP 12 and ICP 14 on SDS-acrylamide gels, with two virion proteins designated VP 5 and VP 6 (Fig. 2b). We have previously shown that VP 5 and VP 6 are resolved in SDS-acrylamide gels of solubilized envelope fractions prepared from dextran gradient purified HSV-2 (G) virions by treatment with 0.1% NP-40 (5). To further inquire into the virion location of VP 5 and VP 6, cytoplasmic HSV-2 (G) virions purified by dextran gradient centrifugation (6) were iodinated with lactoperoxidase (9), a method that labels the virion surface, and electrophoresed on SDS-acrylamide gels. VP 6 but not VP 5 was resolved in the protein profiles of these vii-ions (Fig. Za), an observation consistent with the better neutralizing potential of the anti-ICP 14 sera. AG-e, a biologically significant, typecommon HSV antigen, induces an antigendriven cell-mediated immune response
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in patients with herpetic cervicitis or cervix cancer (1). Chromatographic procedures having been relatively unsuccessful in purifying undissociated HSV antigens (2,10); we chose to purify “crude” AG-e by CIE, an immunogic assay of relatively high resolving power (3). Although eight proteins were resolved by SDS-acrylamide gel electrophoresis in crude AG-e fractions (unpublished), only one precipitin band consisting of ICP 12 and ICP 14 is observed in CIE of anti-crude AG-e sera and HSV-2 (G) SAM. This observation may reflect the relative insensitivity of the CIE assay. Alternatively, it may result from the absence of antibody in anti-crude AG-e sera, against all proteins other than ICP 12 and ICP 14. Such an immunogenic restriction, previously reported for HSV-1 proteins (11) appears to be a function of the presentation to the immune system of different proteins in an antigenic mixture (12). Although it cannot be excluded that proteins other than ICP 12 and ICP 14 have not been resolved by the procedures employed in these studies, the data indicate that ICP 12 and ICP 14 are radiochemically homogeneous on SDS-gel electrophoresis. As previously reported (5, 13) for other HSV proteins, ICP 12 and ICP 14 purified by SDS-acrylamide gels maintain their immunogenicity. Based on estimates of the percentage of protein in densitometric scans from gels of 16-hr infected cell extracts, we conclude that rabbits were inoculated with approximately 10 pg of ICP 12 protein. Similar calculations are not possible for ICP 14 due to its relatively low abundance in these extracts. These interpretations suggest that ICP may be superior immunogens, reflecting the possibility that animals mount a good response when inoculated with minute quantities of a single protein (12) or that SDS-unfolded proteins have more antigenie sites exposed (5,LT). On the other hand, our data demonstrate that anti-ICP 12 and ICP 14 sera do not react in immunodiffusion and have lower neutralizing potentials than antisera to pure AG-e. These observations are amenable to two interpretations not mutually exclusive. First, it is possible that SDSisolated ICP induce a special antibody with restricted serologic reactivity. Alterna-
259
tively, the dissociation of AG-e antigen results in a reduction andlor modification of antigenic reactivity (14). VP 5 and VP 6, proteins that comigrate with ICP 12 and ICP 14, are located in the virion envelope (5). However, consistent with the better neutralizing potential of the anti-ICP 14 sera, only VP 6 appears to be located on the surface of the virions (Fig. 2a). Although comigration of VP 5 and VP 6 with ICP 12 and ICP 14, respectively, cannot be accepted as evidence of their identity, two observations are consistent with an envelope location for ICP 12 and ICP 14: (i) the immunologic reactivity of the anti-ICP sera with HSV-2 (G) virus, and (ii) the absorbing potential of HSV-2 (G) virions for the reactivity of the anti-ICP sera. It has been suggested that antiglobulin-mediated neutralization results from the attachment of antibody to sites on the virion envelope that are noncritical to the initiation of infection (15). According to this interpretation, AG-e is such a noncritical site, a determinant(s) of which (ICP 14) is located on the virion surface. Consistent with the partial removal of VP 5 from NP-40 treated virions (5), and its absence from protein profiles of lactoperoxidase-iodinated virions, the relatively lower neutralizing potential of the anti-ICP 12 sera may reflect the partial subsurface location of this envelope ICP. Absolute evidence that ICP 12 and ICP 14 are virus coded can be obtained only by using purified viral DNA in a coupled in vitro transcription, translation system. In the absence of an adequate in vitro transcription system this is an as yet unfeasible experiment. However, alternative interpretations of the nature of ICP 12 and ICP 14, such as the possiblity that they are host proteins induced by viral infection or synthesized at higher rates post infection, are unlikely since: (i) ICP 12 and ICP 14 are not detected in virions grown in prelabeled HEp-2 cells (Strnad and Aurelian, unpublished), (ii) host protein synthesis is turned off by 4 hr p.i. (16), and (iii) ICP 12 and ICP 14 are incorporated into the virions. Studies now in progress in our laboratory are designed to determine the kinetics and regulation of the synthesis of ICP 12 and
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ICP 14, and whether they undergo modification upon incorporation into virions (17). ACKNOWLEDGMENTS This work was supported in part by Grant AI 14341 from the National Institute of Allergy and Infectious Diseases, and in part by Grant CA 16043 from the National Cancer Institute, NIH, PHS. We acknowledge helpful discussions with Dr. Robert B. Bell. REFERENCES
1. BELL,
R. B., AURELIAN, L., and COHEN, G. H., Cell. Immunol. 41, 86-102 (19’78). 8. COHEN, G. H., PONCE DELEON, M. P., and NICHOLS, C., J. Viral. 10, 1021-1030 (1972). 3. NORRILD, B., and VESTERGAARD, B. F., J. viroz. 22, 113-117 (1977). I. CAMPBELL, D. H., GARVEY, J. S., CREMER, N. E., and SUSSDORF, D. H., “Methods in Immunology,” p. 246. Benjamin, New York, 1970. 5. STRNAD, B. C., and AURELIAN, L., Virology 87, 401-415 (1978).
6. STRNAD,
B. C., and ARELIAN, L., Virology 69, 438-452 (1976). 7. DULBECCO, R., VOGT, M., and STRICKLAND, A. G. R., Virology 12, 162-205 (1965). 8. AUREWAN, L., ROYSTON, I., and DAVIS, H. J., J. Nat. Cancer Inst. 45, 455-464 (1970). W. L., and AUGUST, J. T., 9. MCLELLAN,
J. Viral. 20, 627-636 (1976). B. C., and AURELIAN, L., Virology 10. STRNAD, 73, 244-258 (1976). (1976). 11. SPEAR, P. G., J. Viral. 17, 991-1008 12. DAVIS, B. D., DULBECCO, R., EISEN, H. N., GINSBERG, H. S., and WOOD, B. W., “Microbiology,” pp. 464-467,480-482. Harper&Row, Hagerstown, Md., 1973. 1s. FLANNERY, V. L., COURTNEY, R. J., and SCHAFFER, P. A., J. Viral. 21,2&i-291 (1977). B., and UNANUE, E. R., 14. BENACERRAF, “Textbook of Immunology,” pp. 20-23, Williams & Wilkins, Baltimore, Md. 1979. K., HASHIMOTO, M., and SHINKAI, 15. YOSHINO, K., Microbial. Zmmunol. 21, 231-241 (1977). R. J., and ROIZMAN, B., Virology 16. SYDISKIS, 32, 678-686 (1967). 17. PEREIRA, L., WOLFF, M. H., FENWICK, M., and ROIZMAN, B., Virology 77,733-749 (1977).
98, 255-260 (1979)
Proteins of Herpesvirus Type 2. V. Isolation and Immunologic Characterization Viral Proteins in a Virus-Specific Antigenic Fraction
CYNTHIA The Johns
Hopkins
C. SMITH
AND LAURE
AURELIAN
Medical Institutions, Division of Comparative Medicine, Department Biophysics, 720 Rutland Avenue, Baltimore, Maryland 21206 Accepted
July
of Two
of
Biochemistry
and
6, 1979
Previous reports from our laboratory have shown that “crude” AG-e, a type-common HSV antigen, induces an antigen-driven cell-mediated immune response in patients with HSV cervicitis or cervix cancer. Anti-crude AG-e sera give rise in crossed immunoelectrophoresis against total viral soluble antigenic mixtures (HSV-2 (G) SAM) to one precipitin band (“pure” AGe). Pure AG-e is immunologically identical to crude AG-e as determined by immunodiffusion and it resolves into two infected cell proteins, ICP 12 (MW: 140,000) and ICP 14 (MW: 130,000) upon SDS-acrylamide gel electrophoresis. ICP 12 and ICP 14 are purified to radiochemical homogeneity by SDS-acrylamide gel electrophoresis. Antisera to ICP 12 react with HSV-2 (G) SAM in crossed immunoelectrophoresis and both anti-ICP 12 and ICP 14 sera stain HSV-2 (G) infected cells. ICP 12 and ICP 14 appear to be envelope proteins as evidenced by: (i) the absorption of the reactivity of anti-ICP 12 and ICP 14 sera with HSV-2 (G) virions but not with “mock-virus” preparations, (ii) the ability of the anti-ICP 12 and ICP 14 sera to neutralize HSV-2 in presence of anti-IgG, and (iii) the resolution of a protein with the relative mobility of ICP 14 in virions the surface of which was iodinated with lactoperoxidase.
Patients with cervix cancer or herpetic cervicitis develop an antigen-driven cellmediated immune response measured by an in vitro lymphokine assay against a virusspecific, type-common antigenic fraction designated AG-e. Control subjects are nonresponsive (1). In this report we describe the purification of AG-e, and demonstrate that its immunogenic proteins (ICP 12 and ICP 14) comigrate on SDSacrylamide gel electrophoresis with two proteins located within the virion envelope. Soluble antigenic mixtures (SAM), prepared as previously described (1, 2) from extracts of HEp-2 cells exposed to 5 PFU/cell of HSV-2 (G) for 24 hr at 3’7”, were cleared of virions by two cycles of centrifugation at 100,000 g for 2 hr and fractionated on brushite columns by stepwise elution with increasing concentrations of phosphate buffer, pH ‘7.0, containing 0.1 M NaCl. The 0.4 M phosphate buffer eluate (“crude” AG-e) was concentrated with XM-50 Amicon membrane filters, emulsified in com255
plete Freund’s adjuvant, and used to inoculate rabbits as described (1). Duplicate samples, dialyzed against 0.18 M Tris0.06 M barbital buffer (pH 8.6) with 1% Triton X-100, were further purified by crossed immunoelectrophoresis (CIE) performed against anti-crude AG-e sera in 0.038 M barbital-O.11 N NaCl buffer with 0.0025% Triton X-100 (pH 8.2) (3). A single precipitin band was discerned by both Coomassie blue staining and autoradiography using either crude AG-e or HSV-2 (G) SAM as antigen (Fig. la). Agarose segments containing this precipitin band henceforth designated “pure” AG-e, were cut, washed, emulsified in complete Freund’s adjuvant and used to inoculate rabbits (10 segments/animal). Antisera thus prepared gave rise to one band of identity with antisera to crude AGe when assayed against HSV-2 (G) SAM in immunodiffusion (Fig. lb) and their reactivity could be absorbed with pelleted HSV-2 (G) virions but not with “mock” virus prepared identi0042-6822/79/130255-06$02.06/O Copyright All rights
Q 1979 by Academic Press. Inc. of reprcductmn in any form reserved.
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FIG. 1. (a) Autoradiograms of crossed immunoelectrophoretic analysis of L-[35S]methionine-labeled HSV-2 (G) SAM (20 pg protein) against anti-pure AG-e serum. Identical results are obtained with antisera to crude AG-e or ICP 12 and with crude AG-e antigen. (b) One line of identity between antisera to crude and pure AG-e assayed against HSV-2 (G) SAM in double gel immunodlffusion in 0.7% A37 agarose. (c) Absence of immunoprecipitation between HSV-2 (G) SAM and anti-pure AG-e serum adsorbed with HSV-2 (G) virions pelleted by centrifugation at 100,000 g for 1 hr. Sera (0.3 ml) were exposed three times to virions (2600 pg protein) for 1 hr at 37”. They were cleared of virions by centrifugation at 100,000 g for 2 hr prior to use in immunodiffusion. Note presence of reactivity in sera adsorbed with “mock” virus prepared identically but from HEp-2 cells exposed to PBS instead of virus. (d-f) Indirect immunofluorescent staining with fluorescein-conjugated goat anti-IgG (-r-chain specific). HEp-2 cells infected with HSVB for 24 hr were stained with antiserum to pure AG-e (d), anti-ICP 14 serum (e), or anti-ICP 14 serum adsorbed with pelleted HSV-2 (G) virions (f).
tally but from uninfected HEp-2 cells series of experiments, agarose segments (Fig. 1~). containing the precipitin band from the In order to identify the proteins in AG-e CIE of HSV-2 (G) SAM and anti-crude antigen, two series of experiments were AG-e sera (Fig. la) were dissociated and performed. In the first series, immunoelectrophoresed on 8.5% SDS-acrylamide precipitates resulting from the reaction of gels. Only two proteins with electrophoretic extracts of HSV-2 (G) infected cells labeled mobilities of ICP 12 and ICP 14 and COwith L(-35S)methionine 4-16 hr p.i., with migrating with virion proteins VP 5 and optimal concentrations (4) of anti-crude VP 6 were resolved. AG-e sera and antiglobulin were dissociated ICP 12 and ICP 14 were purified as and electrophoresed on 8.5% SDS-acrylpreviously described (5) from extracts of amide gels as previously described (4, 6). HSV-2 (G) infected HEp-2 cells labeled with Two proteins (Fig. 2d) with electrophoretic L-[35S]methionine lo-16 hours p.i. by mobilities of ICP 12 and ICP 14 (Fig. 2c) electrophoresis on 8.5% acrylamide gels in and comigrating with virion proteins VP 5 presence of 0.1% SDS for 15 hr, a time and VP 6 (Fig. 2b) were resolved in the interval six times longer than normal (6). precipitates. The reaction is virus specific Under these conditions, most of the proas evidenced by the observation that pro- teins migrate off the bottom of the gel and teins were not precipitated by the pre- excellent resolution is obtained between immune rabbit sera (Fig. 2e). In the second the high molecular weight proteins which
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FIG. 2. Densitometric scans of autoradiograms of 8.5% SDS-acrylamide gels oE (a) HSV-2 (G) virions purified by dextran gradient centrifugation (6) and iodinated by exposure to 1 pglml of lactoperoxidase and Na12”I (0.67 mCi/g I) in presence of 2 ~1 of 1 mkf H,O, (9). Free iodide was removed by chromatography on Sephadex G-75 equilibrated with 0.01 M Tris-HCl and 25 mg BSA. (b) HSV-2 (G) virions labeled with L-[Wlmethionine and purified by dextran gradient centrifugation (6). (c) Soluble extracts of HSV-2 (G) infected HEp-2 cells labeled with L+Y??]methionine 4-16 hr p.i. (d) Precipitates resulting from the reaction of extracts as in (c) with anti-crude AG-e serum. Antigen in 0.02 M Tris-HCl, pH 7.4, with 0.1 M NaCl and 0.001 M EDTA (TEN) was cleared of aggregates by three cycles of centrifugation at 3000 9 for 10 min. It was exposed (25 ~1) for 1 hr at 37” to optimal concentration (114) of antiserum and for an additional hour to optimal amounts of goat anti-rabbit IgG quantitated by precipitation (4). Precipitates, washed in TEN buffer for 10 min, were dissociated in 0.05 M Tris-HCl, pH 7.0,2% SDS, 5% 2p-mercaptoethanol for gel electrophoresis (5, 6). (e) Precipitates obtained as in (d) but with preimmune rabbit serum.
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remain on the gels. Representative gels containing infected or “mock” infected cell extracts were stained, sliced longitudinally, and used for autoradiography. The remaining gels were stored at -80”. Autoradiograms revealed six major proteins in the infected cell extracts including two with the relative mobilities of ICP 12 and ICP 14. Bands of similar relative mobilities were absent in extracts of uninfected cells. The relative mobilities of ICP 12 and ICP 14 were used as a guide for slicing segments of frozen gels. For each group of gels run at one time, a segment containing ICP 12 and one containing ICP 14 were autoradiographed in order to monitor the accuracy of the slicing procedure. The ICP 12 and ICP 14 containing segments were homogenized in complete Freund’s adjuvant and used to inoculate rabbits or dissociated and electrophoresed on 8.5% SDS-acrylamide gels. Proteins in these segments migrate on 8.5% SDS-acrylamide gels as single bands with respective electrophoretic mobilities of ICP 12 or ICP 14. The immunologic reactivity of the antisera to pure AG-e, ICP 12 and ICP 14 was studied. Four assays were used in these series: CIE, immunodiffusion (ID), indirect immunofluorescence (FA), and antiglobulin-enhanced neutralization (5). In the latter assay, increasing dilutions of antisera were incubated with HSV-2 (G) (3 x lo5 PFU/ml) for 1 hr at 37” and with goat anti-rabbit IgG (y-chain specific) for an additional hour. Survivors were assayed on HEp-2 cells. Sera were assayed simultaneously with the same virus stocks in order to enable comparison of the neutralizing potentials (7, 8). Anti-pure AG-e sera gave rise to a monoprecipitin band when assayed against HSV-2 (G) SAM in CIE (Fig. la) and immunodiffusion (Fig. lb) and they stained HSV-2 (G) infected cells in immunofluorescence (Fig. Id). Staining was predominantly located in the cytoplasm. The immunofluorescent titer, expressed as the reciprocal of the highest dilution still staining 25% of the cells, was 12. On the other hand, antisera to ICP 12 and ICP 14 did not react with HSV-2 (G) SAM in immunodiffusion and only anti-ICP 12 sera reacted in CIE. However, both antisera stained the cytoplasm of HSV-2 (G) infected
cells (Fig. le) displaying titers of8. Antisera to crude or pure AG-e, ICP 12, and ICP 14 neutralized HSV-2 (G) but only in presence of antiglobulin. Under the experimental conditions used in these studies, the neutralizing potential of the sera is reflected by the constant of neutralization (K) calculated from the formula: log v/v, = -0.43 Kc where v/v, is the proportin of survivors and c equals the antibody concentration expressed in terms of the amount of original serum per unit volume of the reaction mixture (5, 7, 8). The K (X100) values reproducibly obtained for antisera to crude AG-e, pure AG-e, ICP 12, and ICP 14 were 32 + 6, 35 + 2, 3.2 + 0.5, and 18 + 1, respectively. The reactivity of all three antisera was virus-specific. Thus: (i) uninfected cells, SAM or AG-e prepared from “mock” infected cells and “mock” virus preparations, as well as preimmune sera, were nonreactive in CIE, ID, and FA, (ii) preimmune sera did not neutralize virus infectivity, and (iii) the reactivity of the antisera in all four assays was abrogated by their absorption with HSV-2 (G) but not with “mock” virus preparations (Figs. lc and f). The virus neutralizing potential of the anti-ICP 12 and ICP 14 sera and the observation that their reactivity is absorbed with HSV-2 (G) virions are consistent with the comigration of ICP 12 and ICP 14 on SDS-acrylamide gels, with two virion proteins designated VP 5 and VP 6 (Fig. 2b). We have previously shown that VP 5 and VP 6 are resolved in SDS-acrylamide gels of solubilized envelope fractions prepared from dextran gradient purified HSV-2 (G) virions by treatment with 0.1% NP-40 (5). To further inquire into the virion location of VP 5 and VP 6, cytoplasmic HSV-2 (G) virions purified by dextran gradient centrifugation (6) were iodinated with lactoperoxidase (9), a method that labels the virion surface, and electrophoresed on SDS-acrylamide gels. VP 6 but not VP 5 was resolved in the protein profiles of these vii-ions (Fig. Za), an observation consistent with the better neutralizing potential of the anti-ICP 14 sera. AG-e, a biologically significant, typecommon HSV antigen, induces an antigendriven cell-mediated immune response
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in patients with herpetic cervicitis or cervix cancer (1). Chromatographic procedures having been relatively unsuccessful in purifying undissociated HSV antigens (2,10); we chose to purify “crude” AG-e by CIE, an immunogic assay of relatively high resolving power (3). Although eight proteins were resolved by SDS-acrylamide gel electrophoresis in crude AG-e fractions (unpublished), only one precipitin band consisting of ICP 12 and ICP 14 is observed in CIE of anti-crude AG-e sera and HSV-2 (G) SAM. This observation may reflect the relative insensitivity of the CIE assay. Alternatively, it may result from the absence of antibody in anti-crude AG-e sera, against all proteins other than ICP 12 and ICP 14. Such an immunogenic restriction, previously reported for HSV-1 proteins (11) appears to be a function of the presentation to the immune system of different proteins in an antigenic mixture (12). Although it cannot be excluded that proteins other than ICP 12 and ICP 14 have not been resolved by the procedures employed in these studies, the data indicate that ICP 12 and ICP 14 are radiochemically homogeneous on SDS-gel electrophoresis. As previously reported (5, 13) for other HSV proteins, ICP 12 and ICP 14 purified by SDS-acrylamide gels maintain their immunogenicity. Based on estimates of the percentage of protein in densitometric scans from gels of 16-hr infected cell extracts, we conclude that rabbits were inoculated with approximately 10 pg of ICP 12 protein. Similar calculations are not possible for ICP 14 due to its relatively low abundance in these extracts. These interpretations suggest that ICP may be superior immunogens, reflecting the possibility that animals mount a good response when inoculated with minute quantities of a single protein (12) or that SDS-unfolded proteins have more antigenie sites exposed (5,LT). On the other hand, our data demonstrate that anti-ICP 12 and ICP 14 sera do not react in immunodiffusion and have lower neutralizing potentials than antisera to pure AG-e. These observations are amenable to two interpretations not mutually exclusive. First, it is possible that SDSisolated ICP induce a special antibody with restricted serologic reactivity. Alterna-
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tively, the dissociation of AG-e antigen results in a reduction andlor modification of antigenic reactivity (14). VP 5 and VP 6, proteins that comigrate with ICP 12 and ICP 14, are located in the virion envelope (5). However, consistent with the better neutralizing potential of the anti-ICP 14 sera, only VP 6 appears to be located on the surface of the virions (Fig. 2a). Although comigration of VP 5 and VP 6 with ICP 12 and ICP 14, respectively, cannot be accepted as evidence of their identity, two observations are consistent with an envelope location for ICP 12 and ICP 14: (i) the immunologic reactivity of the anti-ICP sera with HSV-2 (G) virus, and (ii) the absorbing potential of HSV-2 (G) virions for the reactivity of the anti-ICP sera. It has been suggested that antiglobulin-mediated neutralization results from the attachment of antibody to sites on the virion envelope that are noncritical to the initiation of infection (15). According to this interpretation, AG-e is such a noncritical site, a determinant(s) of which (ICP 14) is located on the virion surface. Consistent with the partial removal of VP 5 from NP-40 treated virions (5), and its absence from protein profiles of lactoperoxidase-iodinated virions, the relatively lower neutralizing potential of the anti-ICP 12 sera may reflect the partial subsurface location of this envelope ICP. Absolute evidence that ICP 12 and ICP 14 are virus coded can be obtained only by using purified viral DNA in a coupled in vitro transcription, translation system. In the absence of an adequate in vitro transcription system this is an as yet unfeasible experiment. However, alternative interpretations of the nature of ICP 12 and ICP 14, such as the possiblity that they are host proteins induced by viral infection or synthesized at higher rates post infection, are unlikely since: (i) ICP 12 and ICP 14 are not detected in virions grown in prelabeled HEp-2 cells (Strnad and Aurelian, unpublished), (ii) host protein synthesis is turned off by 4 hr p.i. (16), and (iii) ICP 12 and ICP 14 are incorporated into the virions. Studies now in progress in our laboratory are designed to determine the kinetics and regulation of the synthesis of ICP 12 and
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ICP 14, and whether they undergo modification upon incorporation into virions (17). ACKNOWLEDGMENTS This work was supported in part by Grant AI 14341 from the National Institute of Allergy and Infectious Diseases, and in part by Grant CA 16043 from the National Cancer Institute, NIH, PHS. We acknowledge helpful discussions with Dr. Robert B. Bell. REFERENCES
1. BELL,
R. B., AURELIAN, L., and COHEN, G. H., Cell. Immunol. 41, 86-102 (19’78). 8. COHEN, G. H., PONCE DELEON, M. P., and NICHOLS, C., J. Viral. 10, 1021-1030 (1972). 3. NORRILD, B., and VESTERGAARD, B. F., J. viroz. 22, 113-117 (1977). I. CAMPBELL, D. H., GARVEY, J. S., CREMER, N. E., and SUSSDORF, D. H., “Methods in Immunology,” p. 246. Benjamin, New York, 1970. 5. STRNAD, B. C., and AURELIAN, L., Virology 87, 401-415 (1978).
6. STRNAD,
B. C., and ARELIAN, L., Virology 69, 438-452 (1976). 7. DULBECCO, R., VOGT, M., and STRICKLAND, A. G. R., Virology 12, 162-205 (1965). 8. AUREWAN, L., ROYSTON, I., and DAVIS, H. J., J. Nat. Cancer Inst. 45, 455-464 (1970). W. L., and AUGUST, J. T., 9. MCLELLAN,
J. Viral. 20, 627-636 (1976). B. C., and AURELIAN, L., Virology 10. STRNAD, 73, 244-258 (1976). (1976). 11. SPEAR, P. G., J. Viral. 17, 991-1008 12. DAVIS, B. D., DULBECCO, R., EISEN, H. N., GINSBERG, H. S., and WOOD, B. W., “Microbiology,” pp. 464-467,480-482. Harper&Row, Hagerstown, Md., 1973. 1s. FLANNERY, V. L., COURTNEY, R. J., and SCHAFFER, P. A., J. Viral. 21,2&i-291 (1977). B., and UNANUE, E. R., 14. BENACERRAF, “Textbook of Immunology,” pp. 20-23, Williams & Wilkins, Baltimore, Md. 1979. K., HASHIMOTO, M., and SHINKAI, 15. YOSHINO, K., Microbial. Zmmunol. 21, 231-241 (1977). R. J., and ROIZMAN, B., Virology 16. SYDISKIS, 32, 678-686 (1967). 17. PEREIRA, L., WOLFF, M. H., FENWICK, M., and ROIZMAN, B., Virology 77,733-749 (1977).
