Polypeptides Electrophoretically Separated and Transferred

INFECTION AND IMMUNITY, Feb. 1981, p. 660-667 0019-9567/81/020660-08S02.00/0

Vol. 31, No. 2

Immunological Reactivity of Herpes Simplex Virus 1 and 2 Polypeptides Electrophoretically Separated and Transferred to Diazobenzyloxymethyl Paper B. NORRILD,' B. PEDERSEN,' AND B. ROIZMAN2* Institute of Medical Microbiology, University of Medical Microbiology, DK-2100 Copenhagen, Denmark,1 and Marjorie B. Kovler Viral Oncology Laboratories, University of Chicago, Chicago, Illinois 606372

In this paper we report that viral polypeptides from herpes simplex virus 1 (HSV-1) and 2 (HSV-2)-infected cells electrophoretically separated in sodium dodecyl sulfate-polyacrylamide-agarose gels and transferred to diazobenzyloxymethyl paper can react with rabbit hyperimmune sera, both polyvalent and prepared against specific antigens. The polyvalent hyperimmune sera against HSV-1 reacted with 17 HSV-1 polypeptide bands and 8 HSV-2 polypeptide bands. Concordantly, polyvalent sera against HSV-2 reacted with at least 16 HSV-2 polypeptide bands and 8 HSV-1 polypeptide bands. The antisera prepared against the specific antigens reacted with a smaller number of polypeptide bands. Preimmune sera and immune sera did not react with electrophoretically separated polypeptides from infected and uninfected cells, respectively. The immune localization of separated antigens test provides a powerful technique for identification of immunogenic viral polypeptides, especially those which are normally insoluble and therefore unavailable for immunological reactivity in immune precipitation tests.

Herpes simplex viruses 1 and 2 (human herpesvirases 1 and 2; HSV-1, HSV-2) are genetically and immunologically related. Their genomes share 50% of the nucleotide sequences, with good matching of base pairs (7, 11-13, 21), and numerous studies have shown that cells infected with either one of the viruses contain antigens which react with antisera produced against both the homologous and heterologous virus (3-6, 8, 9, 23-25, 29). However, detailed analysis of the virus-specific infected cell polypeptides (ICPs) has been restricted to only a subset of the approximately 50 polypeptides specified by each virus in the course of productive infection. Predominant among these are the glycoproteins gA, gB, gC, gD, and gE, which are readily solubilized in nonionic detergents, and a small number of other polypeptides which are also soluble and therefore available for immune precipitation (2-5, 17, 18, 26-28). With respect to the glycoproteins, the results obtained to date show that HSV-1 gC reacts with antisera to HSV-1 only, whereas all other glycoproteins of HSV-1 are precipitable by antisera to both HSV1 and HSV-2 (26-28). Similar analyses have not been extended to other virus-specific ICPs, mainly because a large fraction of the ICPs are insoluble under conditions appropriate for immunological reactivity and therefore their immunological specificity cannot be readily measured.

In this paper we show that a large fraction of the ICPs transferred from sodium dodecyl sulfate (SDS)-polyacrylamide-agarose gels to diazobenzyloxymethyl (DBM) paper according to the procedure of Renart et al. (20) retain their immunological reactivity. This technique, which we shall designate as immunological localization of separated antigens, can be used to study the immunological specificity of HSV-1- and HSV2-specific ICPs. (A preliminary account of this study was presented at the International Conference on Human Herpesviruses, Atlanta, Ga., March 1980.) MATERIALS AND METHODS Virus strains. HSV-1 strain F [HSV-1(F)] and HSV-2 strain G [HSV-2 (G)] were propagated in HEp2 cells grown in Eagle minimal essential medium supplemented with 1 % fetal calf serum (22). The mutant of HSV-1, HSV-1(HFEM)tsB5, was obtained from A. Buchan, Department of Microbiology, University of Birmingham, Birmingham, England (14). Infection of cells and labeling with radioisotopes. Vero cells were propagated as monolayers in 25-cm2 tissue culture flasks in Eagle minimal essential medium with 10% fetal calf serum. The cells were infected with HSV-1(F), HSV-2(G), or HSVl(HFEM)tsB5 in 0.5 ml at a multiplicity of infection of 10 plaque-forming units per cell. After adsorption at 34°C for 1 h, the inoculum was replaced with 5 ml of Eagle minimal essential medium supplemented with 1% fetal calf serum. 660

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HSV POLYPEPTIDES ON DBM PAPER

For preparation of labeled viral glycoprotein, infected cells were incubated at 340C for 4 h, the medium was then aspirated, and 2.5 ml of fresh minimal essential medium, supplemented with 1 % fetal calf serum and D-["4C]glucosamine (2 #Ci/ml; Amersham, England; specific activity, 60.8 mCi/mmol), was added. HSV-1(HFEM)tsBs-infected cells were incubated from the end of the adsorption period at either 34 or 390C as indicated in the text. Labeling of viral proteins with L-[`S]methionine (New England Nuclear, Dreieich, West Germany; specific activity, 756.86 Ci/mmol) was done in a medium containing 1/10 of the amount of methionine contained in Eagle minimal essential medium. The labeling of viral proteins was from 4 to 18 h postinfection in all experiments. Unlabeled viral proteins were prepared by extraction of cells 18 h postinfection as described (15). SDS-polyacrylamide-agarose gel electrophoresia. HSV-infected cell extracts were separated in 9.25% (wt/vol) acrylamide gels cross-linked with 0.24% (wt/vol) N,N-diallyltartamide (Bio-Rad Laboratories, Richmond, Calif.) and containing in addition 1% (wt/vol) agarose (Litex, Copenhagen, Denmark) as described by Renart et al. (20). The stacking gel was 3% (wt/vol) acrylamide-0.08% (wt/vol) N,N-diallyltartardiamide. The buffer conditions and the procedures for staining, drying, and autoradiography were as previously described (15). Before electrophoresis, the cells were disrupted with 2% (wt/vol) SDS and 5% (vol/ vol) 2-mercaptoethanol. The ICPs were numbered according to the nomenclature of Morse et al. (15), and the glycoproteins were numbered according to the nomenclature adapted at the 1979 Herpesvirus Workshop held at Cold Spring Harbor, N.Y. Transfer of HSV proteins to DBM paper. The DBM paper was freshly prepared for each experiment from aminobenzyloxymethyl paper as described by Alwine et al. (1). The SDS-polyacrylamide agarose gel was processed for the transfer of proteins largely as described by Renart et al. (20). Briefly, the gel was treated twice with 100 ml of 2% periodic acid for 30 min at room temperature, followed by a 15- to 30-min treatment with 0.5 M sodium phosphate buffer (pH 7.5). Finally the gel was washed twice with 0.05 M sodium phosphate buffer (pH 7.5). Extraction of the proteins from the gel and immobilization by transfer to the DBM paper were done according to the methods described by Southern for transfer of deoxyribonucleic acid (25). The transfer was done for 20 h at room temperature; after transfer the paper was soaked at 370C for 2 h in a buffer containing 0.1 M tris-

cells was not demonstrable by any of the tests. Serum 0 was obtained from nonimmunized animals. Serum 11 was a hyperimmune serum reactive with glycoproteins gA and gB; it was produced by immunizing rabbits with immunoprecipitates of antigen fraction 11 (Ag-il) of HSV-1 cut out of agarose gels (29). The serum neutralized both HSV-1 and HSV-2, and in crossed immunoelectrophoretic tests it reacted with both HSV-1 and HSV-2. Serum 8 was a hyperimmune serum reactive with glycoprotein gD; it was produced by immunizing rabbits with immunoprecipitates of Ag-8 of HSV-1 cut out of agarose gels (29). The antiserum neutralized HSV-1 and HSV-2 and caused lysis of both HSV-1and HSV-2-infected cells in immune cytolytic tests. In immune precipitation tests only gD and its precursors of both HSV-1 and HSV-2 were precipitated by this serum (16). Serum 6 was a hyperimmune serum reactive with glycoprotein gC of HSV-1; it was produced by the immunization of rabbits with Ag-6 of HSV-1. This serum neutralized HSV-1 but not HSV-2. The specificity of the serum for HSV-1 was also demonstrable in immunoprecipitation and in immunocytolytic tests (16, 28). Serum 4 was obtained from a rabbit immunized with antigen Ag-4 of HSV-2; it neutralized both HSV1 and HSV-2, but the neutralization of the homologous HSV-2 was stronger. In crossed immunoelectrophoretic tests the serum reacted only with Ag-4 of HSV2 (29). In antibody-dependent cell-mediated cytolysis the serum mediated the lysis of both HSV-1- and HSV-2-infected cells. After extensive absorption with HSV-1-infected cells in monolayer culture, the serum mediated the lysis of HSV-2-infected cells only. This absorbed preparation was used in the present study. None of these rabbit sera reacted with uninfected host cell antigens in any of the control tests done in parallel with tests designed to measure the reactivity of the sera with infected cell preparations. Identification of HSV proteins immobilized by binding to DBM paper. The paper strips corresponding to individual slots from the SDS-polyacrylamideagarose gels were incubated in 10 ml of 1:10 dilutions of the different rabbit hypernmmune sera as detailed in legends to figur. The sera were diluted in a 0.05 M tris(hydroxymethyl)aminomethane-hydrochloride buffer (pH 7.4) containing 0.15 M NaCl, 0.005 M ethylenediaminetetraacetate, 0.25% (wt/vol) gelatin, and 0.05% (vol/vol) Triton X-100. The strips were incubated at 37°C for 5 to 6 h, then washed in 50 ml of the same buffer for 18 h and incubated for 2 h in the same buffer containing 0.25 yCi of 125I-labeled protein A at 37°C (Amersham, England; specific activity, 30 mCi/mg of protein). The final wash of the strips was done for 2 h in the same buffer supplemented with 1 M NaCl and 0.4% (vol/vol) Sarkosyl. Autoradiographic images were developed on Kodak XPR-1 film. Exposure was at 40C for 3 to 7 days.

(hydroxymethyl)aminomethane-hydrochloride (pH

9.0)-10% (vol/vol) ethanolamine-0.25% (wt/vol) gelatin (19). Antisera. Rabbit sera were used throughout the study. Sera 1 and 2 were hyperimmune to HSV-1 and HSV-2, respectively. The animals were injected with either HSV-1- or HSV-2-infected rabbit cornea cells grown in rabbit serum. The sera neutralized both HSV-1 and HSV-2 viruses, but neutralized the homologous virus more strongly (28). These sera precipitated viral proteins in crossed immunoelectrophoresis. and lysed both HSV-1- and HSV-2-infected cells in immune cytolytic tests (16). Activity against uninfected

RESULTS Transfer of HSV proteins to DBM paper. Figure 1 shows the autoradiographic images of polypeptides labeled in HSV-1-infected cells

662

INFECT. IMMUN.

NORRILD, PEDERSEN, AND ROIZMAN

A

B

C

D

5

6-

gC gB gA

>8 w110 1 ^

_ i

_ -

=.l

-

15

:

_

lo

s

not shown).

17

Immunological reactivity of HSV-1 polypeptides after transfer to DBM paper. Unlabeled HSV-1 proteins were electrophoretically

20

9D

from 4 to 21 h postinfection, electrophoretically separated in SDS-polyacrylamide gels, then transferred to DBM paper as described in Materials and Methods. Comparisons of slots A, B, and C of Fig. 1, representing the autoradiographic images of polypeptides separated in a polyacrylamide-agarose gel and of the polypeptides transferred to DBM paper after electrophoretic separation, indicate that with few exceptions the polypeptides transferred to DBM paper reflected the molar abundance in the polyacrylamide agarose gels and that the separation of the polypeptides not significantly imof the polyacrylpaired Analyses by the transfer.was after transfer indicated that gels of the polypeptides in each amide-agarose r more than 50% no band were transferred to the DBM paper (data

separated in SDS-acrylamide-agarose gels, * ;:transferred to the DBM paper, and then reacted IXwith HSV-1 serum (no. 1) as described in Ma[2 5 ^ s terials and Methods. As shown in Fig. 1, slot D, 8_w the antiserum reacted with most of the HSV polypeptides immobilized on DBM paper. The autoradiographic image shown in Fig. 1 was 29 _ produced with dilutions of antisera as high as 1: x ! 200. Of particular interest is the observation that among the polypeptides strongly reactive with the antiserum to HSV-1 were ICPs 5, 6, 8, 25, 3 and 36, none of which is a surface-glycosylated polypeptide. It is noteworthy also that no immunological reactivity was observed with ICPs 10 and 20. It should be emphasized that the specificity of the immune reaction was tested as follows. (i) Polypeptides from mock-infected cells were sep44 arated in SDS-polyacrylamide-agarose gels, transferred to DBM paper, and incubated with : tI HSV-1 antibodies. No binding to host cell poly45wlF peptides was demonstrable (data not shown). (ii) 35 3 5 - 125 Binding ofpreimmune rabbit serum to the HSVS S1 polypeptides was not demonstrable (Fig. 2, slot .

14c

Me Prot-A Me Iu FIG. 1. Autoradiographic images of electrophoretically sep aratedpolypeptides from cells infected with

HSV-1(F)). Slots A, B, and C are controls: (A) cell lysate fro)m infected cells labeled with D-['4CJgluco-

samine from 4 to 18 h postinfection; (B) cell lysate from infected cells labeled with L-[35SJmethionine from 4 to 18 h postinfection; (C) as (B) except that after electrophoresis in SDS-polyacrylamide-agarose gels the polypeptides were transferred to DBM paper. (D) Cell lysate from unlabeled cells harvested 18 h postinfection, subjected to electrophoresis in SDSpolyacrylamide-agarose gels, transferred to DBMpaper, and then reacted with serum no. 1, followed by

D). Reactivity of HSV-1 and HSV-2 immobilized polypeptides with rabbit hyperimmune sera. Labeled and unlabeled HSV-1 and HSV-2 polypeptides from infected cells were binding of 125I-labeled protein A. Slots A and B represent autoradiograms of the original SDS-polyacrylamide-agarose gel. The autoradiograms of slots C and D represent images from polypeptides bound to DBM paper. The polypeptides were numbered as described in the text. Note that the pattern in slot C is a mirror image of the pattern shown in slot B.

VOL. 31, 1981 Labeled


Unlabeled Polypeptides

Polxpeptides

Original Gel A B

663

-*-

C

D

Transferred F E

G

H

I

J

WA

.... -....

gC B

..

gA

44 45-

48

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4-

1251- Protein

A

Me Glu Me FIG. 2. Autoradiographic images of electrophoretically separated polypeptides from cells infected with HSV-1(F). Slots A, B, and C represent controls as described in Fig. 1. Slots D to Jrepresentpolypeptides from unlabeled cells harvested at 18 h postinfection, electrophoretically separated on SDS-polyacrylamide-agarose gels, transferred to DBM paper, and reacted with sera and then with "LI-labeled protein A. The sera were as follows: (D) serum 0; (E) serum 1; (F) serum 2; (G) sera 8 and 11; (H) serum 8; (I) serum 6; and (J) serum 4.

solubilized, electrophoretically separated on SDS-polyacrylanide-agarose gels, and transferred to DBM paper. The DBM-paper strips

with unlabeled polypeptides were then incubated with various preparations of hyperimmune rabbit sera as specified in the legends to

664 NORRILD, PEDERSEN, AND ROIZMAN the figures. The results shown in Fig. 2 and 3 were as follows. The hyperimmune rabbit serum to HSV-1 (serum no. 1) reacted with approximately 17 HSV-1 polypeptide bands but only with 8 HSV-2 polypeptide bands (Fig. 2, slot E; Fig. 3, slot E). Concordantly, the hyperimmune rabbit serum to HSV-2 proteins (serum no. 2) reacted with 16 polypeptide bands of HSV-2 and 8 polypeptide bands of HSV-1 (Fig. 2, slot F; Fig. 3, slot F). Of interest is the observation that the HSV-1 CPs 5, 6, 8, 11, 25, 29, 39 or 40, and 48 reacted with both HSV-1 and HSV-2 antisera, and therefore these polypeptides contained both HSV-1 and HSV-2 antigen determinant sites. In contrast, HSV-1 ICPs 15, 17, 36, and 37 bound antibody to HSV-1 (serum 1) only, and therefore these polypeptides may contain HSV-1 antigenic determinant sites only. The HSV-2 ICPs 11, 18, 20, 25, 36, 39, and 44 bound HSV-2 antiserum (serum 2) only, and therefore these polypeptides may contain HSV-2 antigenic deterninant sites only. The major glycoproteins of HSV-1 and HSV2 were identified among the immobilized proteins by the incubation of the paper strips with rabbit hyperimmune sera produced to the individual glycoproteins gD and gC and the mixture of gA and gB. The resolution of glycoproteins gA, gB, and gC in the gel system was analyzed by separation of ["4C]glucosamine-labeled glycoproteins extracted from cells infected with the mutant HSV-1(HFEM)tsB5. This mutant is temperature sensitive for the accumulation of glycoprotein gB (14). Comparison of the electrophoretic profile of proteins labeled at the permissive temperature (Fig. 4, slot A) with that of proteins labeled at the nonpermissive temperature (Fig. 4, slot B) indicates that gA, gB, and gC were well separated. The results of the immune reactions were as follows. Serum no. 8 reacted strongly with glycoprotein gD of HSV-1 (Fig. 2, slot H). Three polypeptide bands were visible, and the polypeptide binding the antibodies most strongly was the polypeptide ICP 29, which previously has been shown to correspond to gD. The serum reacted less strongly with the band containing the polypeptides with a lower apparent molecular weight than that of ICP 29, but these polypeptides might be precursor molecules to ICP 29. The relationship to glycoprotein gD of the polypeptide with a higher molecular weight than that of ICP 29 is unknown. Incubation of the electrophoretically separated polypeptides transferred to DMB-paper strips with a mixture of sera 11 and 8 resulted in the visualization of glycoproteins gA and gB in addition to glycoprotein gD (ICP 29), as shown in Fig. 2, slot G. The reaction with gA was stronger than that with gB. In addition, the sera

INFECT. IMMUN.

Labeled PolypeptidelUnlabeled Original Gel Transferred A

B

D

C

E

F

A.*.

6.

gBm

9A

39

..

44

.....

45

14C-

35S

35 _ 1251

Me Me kIu Protei n A FIG. 3. Autoradiographic images of electrophoretically separated polypeptides from cells infected with HSV-2(G). Slots A, B, and C represent controls as described in Fig. 1. Slots D to F represent polypeptides from unlabeled cells harvested at 18 h postinfection, electrophoretically separated on SDS-polyacrylamide-agarose gels, transferred to DBM paper, and reacted with sera and then with '251-labeled protein A. The sera were as follows: (D) serum 0; (E) serum 1; (F) serum 2.


VOL. 31, 1981

A

B

gC

B9A

gB

9E

gE

gD

665

after electrophoresis and transfer to DBM paper (serum 6) (Fig. 2, slot I). Other batches of antiglycoprotein C sera also failed to react with HSV-1 polypeptides transferred to DBM paper. However, other studies with hybridoma antibodies to gC (19) showed that gC can be transferred and can react. Studies of the hybridoma antibodies to various glycoproteins have been published elsewhere (19). The reason for the failure of the serum used in this study to react to gC is not clear. The reactivity of transferred HSV-1 and HSV2 polypeptides with antiserum to the HSV-2 antigen Ag-4 (serum 4) is shown in Fig. 2, slot J and in Fig. 5, slot E. The two bands of HSV-2 which react with this serum correspond in electrophoretic mobility to the location of HSV-2 ICP 8 and HSV-2 ICP 18-20, respectively. Very likely, however, the antiserum reacts with glycoproteins which comigrate with these polypeptide bands. It is uncertain whether the highmolecular-weight polypeptide corresponds to gC of HSV-2. No binding of serum 4 to HSV-1 polypeptides was demonstrable. It should be noted that antibodies present in serum 2 bound to polypeptides comigrating with ICP 18 and ICP 20 of HSV-2; the electrophoretic mobility of these bands corresponded to that of partially and fully glycosylated forms of glycoprotein E, respectively.

DISCUSSION As noted in the introduction, analyses of immunological specificity of HSV-1 and HSV-2 proteins have been limited by the observation that a large fraction of the viral proteins are insoluble under the physiological conditions appropriate for immunological tests (2, 17, 18, 26, 27, 30). Only a subset of the viral proteins have therefore been available for immunoprecipitation. In this paper we show that the HSV-1 and HSV-2 polypeptides can be identified by the immunological localization of separated antigens test according to the procedure described by Renart et al. (20). Specifically, several polyspecific antisera have been shown to react with HSV-1 and HSV-2 polypeptides separated in FIG. 4. Autoradiographic images of polyeptides from cells infected with HSV-I(HFEM)tsB5, infected and maintained at permissive (A) and nonpermissive temperature (B). Cells were labeled with D-114C]glucosamine from 4 to 28 h postinfection. Note that gB did not accumulate in cells maintained at the non-

permissive temperature.

reacted with ICP 15, a probable precursor to gA or gB (26, 27). In the experiments done with the rabbit antisera, the glycoprotein gC of HSV-1 did not react

SDS-polyacrylamide-agarose gels and covalently bound to DBM paper. Several aspects of these studies should be noted. (i) Experiments with radioactively labeled viral polypeptides show that most of the viral polypeptides can be transferred from SDSpolyacrylamide-agarose gel to DBM paper (Fig. 1, slots B and C). In our experiments, approximately 50% of the labeled polypeptides present in each band were transferred. (ii) At least 17 HSV-1 and 16 HSV-2 polypeptides appeared to retain their immunological

666

NORRILD, PEDERSEN, AND ROIZMAN L a be e d O rig inal

Polypeotides

UUnlabeled Tr a n sfe r red

Gel

A

B

-'NN-.

mm

D

C

.#

4

.

E

.: 4:

46., .,r

..

!P. .:

5 6 gC B

11

gA

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3 6*

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45

1 4C -

Glu

35S Me

35S Me

1251 _ Protein

A

FIG. 5. Autoradiographic images of electrophoretically separatedpolypeptides from cells infected with HSV-2(G). Slots A, B, and C represent controls as described in Fig. 1. Slots D and E represent polypeptides from unlabeled cells harvested at 18 h postinfection, electrophoretically separated on SDS-polyacrylamide-agarose gels, transferred to DBM paper, and reacted with sera and then with '251-labeled protein A. The sera were as follows: (D) serum 0; (E) serum 4.

reactivity after electrophoretic separation in SDS-polyacrylamide gels and transfer to DBM paper (Fig. 2, slot E; Fig. 3, slot E). The failure

INFECT. IMMUN.

of other polypeptides to react with the homologous hyperimmune sera 1 and 2 may be due to lack of corresponding antibodies in the polyspecific sera or to loss of immunological specificity as a consequence of the denaturation of the polypeptides. We should note that none of the antisera in use reacted to electrophoretically separated and DBM-bound polypeptides from uninfected cells. Furthermore, none of the viral polypeptides reacted with the preimmune rabbit sera. In addition, the observation that most if not all of the viral polypeptides that reacted with antisera were readily identified by electrophoretic mobility also argues for the specificity of the reaction of the hyperimmune sera with immobilized viral polypeptides. (iii) This study demonstrated that several of the HSV-1 and HSV-2 polypeptides reacted with both homotypic and heterotypic antisera (ICPs 5, 6, 8, 11, 25, 29, 39 or 40, and 48 of HSV-1 [Fig. 2, slots E and F] and ICPs 5, 6, 8, 29, 39 or 40, and 48 of HSV-2 [Fig. 3, slots E and F]), whereas other polypeptides reacted only with homotypic antisera (ICPs 15, 17, 36, and 47 of HSV-1 and ICPs 11, 18, 20, 25, 36, 39, and 44 of HSV-2 [Fig. 1, slot E, and Fig. 2, slot F]). It is likely that the polypeptides reactive with both homotypic and heterotypic antisera indeed contain type-specific as well as type-common antigen determinant sites. With respect to the polypeptides reactive with homotypic sera, additional studies will have to be done to establish whether heterotypic antigenic sites have been lost or whether the antisera used in this study for unknown reasons did not contain antibodies to heterotypic antigenic sites. The immunological localization of separated antigens test has several advantages compared to immunoprecipitation tests. First, the test specifically identifies the polypeptides carrying the antigen sites. This is of particular interest in the case of proteins consisting of several different polypeptides. Second, the test identifies both the precursor and products as well as immunologically related polypeptides differing in electrophoretic mobility. Furthermore, sensitivity of the test compares favorably with immune precipitation tests inasmuch as it does not require an antibody excess. For example, positive reactions were obtained with sera diluted several hundred-fold. A major disadvantage of the test is that some polypeptides might lose their immunological reactivity. The immunological localization of separated antigens test has many obvious applications, especially in the identification of viral antigens to which antibodies are produced in the course of human and experimental infections with HSV.


VOL. 31, 1981 ACKNOWLEDGMENTS Studies done at the University of Copenhagen were supported by a grant from the NOVO Foundation. The studies done at the University of Chicago were supported by grants from the National Cancer Institute (Public Health Service grants CA-08494-15 and CA 19264-05) and the American Cancer Society (MV-2-P). LITERATURE CITED

16.

17.

1. Alwine, J. C., D. J. Kemp, and G. R. Stark. 1977. Method for detection of specific RNA's in agarose gels by transfer to diazobenzyloxymethyl paper and hybrid-

ization with DNA probes. Proc. Natl. Acad. Sci. U.S.A. 74:5350-5354. 2. Baucke, B., and P. G. Spear. 1979. Membrane proteins specified by herpes simplex viruses. V. Identification of an Fc-binding glycoprotein. J. Virol. 32:779-789. 3. Cohen, G. H., M. Katze, C. Hydrean-Stern, and R. J. Eisenberg. 1978. Type-common CP-1 antigen ofherpes simplex virus is associated with a 59,000-molecularweight envelope glycoprotein. J. Virol. 27:172-181. 4. Courtney, R. J., and K. L Powell. 1975. Immunological and biochemical characterization of polypeptides induced by herpes simplex virus types 1 and 2. IARC Sci. Publ. 11:63-73. 5. Eisenberg, R. J., C. Hydrean-Stern, and G. H. Cohen. 1979. Structural analysis of precursor and product forms of type-common envelope glycoprotein D (CP-1 antigen) of herpes simplex virus type 1. J. Virol. 31:608620. 6. Glorioso, J. C., L A. Wilson, T. W. Fenger, and J. W. Smith. 1978. Complement mediated cytolysis of HSV1 and HSV-2 infected cells: plasma membrane antigens reactive with type-specific and cross-reactive antibody. J. Gen. Virol. 40:443-454. 7. Goodheart, C. R., G. Plummer, and J. L Waner. 1968. Density differences of DNA of human herpes simplex viruses types 1 and 2. Virology 35:473-475. 8. Honess, R. W., K.- L Powell, D. J. Robinson, C. Sim, and D. H. Watson. 1974. Type specific and type common antigens in cells infected with herpes simplex virus type 1 and on the surfaces of naked and enveloped particles of the virus. J. Gen. Virol. 22:159-169. 9. Honess, R. W., and D. H. Watson. 1974. Herpes simplex virus-specific polypeptides studied by polyacrylamide gel electrophoresis of immune precipitates. J. Gen. Virol. 22:171-185. 10. Honess, R. W., and D. H. Watson. 1977. Review article. Unity and diversity in the herpesviruses. J. Gen. Virol. 37:15-37. 11. Kieff, E. D., S. L Bachenheimer, and B. Roi2man. 1971. Size, composition and structure of the DNA of subtypes 1 and 2 herpes simplex virus. J. Virol. 8:125132. 12. Kieff, E. D., B. Hoyer, S. L Bachenheimer, and B. Roizman. 1972. Genetic relatedness of type 1 and type 2 herpes simplex viruses. J. Virol. 9:738-745. 13. Ludwig, H. O., N. Biswal, and M. Benyesh-Melnick. 1972. Studies of the relatedness of herpesviruses through DNA-DNA hybridization. Virology 49:95-101. 14. Manservigi, R., P. G. Spear, and A. Buchan. 1977. Cell fusion induced by herpes simplex virus is promoted and suppressed by different viral glycoproteins. Proc. Natl. Acad. Sci. U.S.A. 74:3913-3917. 15. Morse, L. S., L. Pereira, B. Roizman, and P. A. Schaf-

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fer. 197& Anatomy of herpes simplex virus (HSV) DNA. X. Mapping of viral genes by analysis of polypeptides and functions specified by HSV-1 x HSV-2 recombinants. J. Virol. 26:389-410. Norrild, B., S. L Shore, and A. J. Nahmias. 1979. Herpes simplex virus glycoproteins: participation of individual herpes simplex type 1 glycoprotein antigens in immunocytolysis and their correlation with previously identified glycopolypeptides. J. Virol. 32:741-748. Norrild, B., and B. F. Vestergaard. 1977. Polyacrylamide gel electrophoretic analysis of herpes simplex virus type 1 immunoprecipitates obtained by quantitative immunoelectrophoresis in antibody-containing agarose gel. J. Virol. 22:113-117. Norrild, B., and B. F. Vestergaard. 1979. Immunoelectrophoretic identification and purification of herpes simplex virus antigens released from infected cells in tissue culture. Intervirology 11:104-110. Pereira, L, T. Klassen, and J. R. Baringer. 1980. Type-common and type-specific monoclonal antibody to herpes simplex virus type 1. Infect. Immun. 29:724-

732. 20. Renart, J., J. Reiser, and G. R. Stark. 1979. Transfer of proteins from gels to diazobenzyloxymethyl-paper and detection with antisera: a method for studying antibody specificity and antigen structure. Proc. Natl. Acad. Sci. U.S.A. 76:3116-3120. 21. Roizman, B., and D. Furlong. 1974. The replication of herpesviruses. Compr. Virol. 3:229-403. 22. Roizman, B., and P. G. Spear. 1971. Herpes virus antigens on cell membranes detected by centrifugation of membrane-antibody complexes. Science 171:298-300. 23. Schneweis, K. E., and A. J. Nahmias. 1971. Antigens of herpes simplex virus type 1 and 2-immunodiffuLsion and inhibition passive hemagglutination studies. Z. Immunitaetsforsch. Immunbiol. 141:471-487. 24. Shore, S. L, C. M. Black, F. M. Melewicz, P. A. Wood, and A. J. Nahmia8. 1969. Antibody-dependent cellmediated cytotoxicity to target cells infected with type 1 and type 2 herpes simplex virus. J. Immunol. 116: 194-201. 25. Southern, E. M. 1975. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J. Mol. Biol. 98:503-517. 26. Spear, P. G. 1975. Glycoproteins specified by herpes simplex virus type 1: their synthesis, processing and antigenic relatedness to HSV-2 glycoproteins. IARC Sci. Publ. 11:49-61. 27. Spear, P. G. 1976. Membrane proteins specified by herpes simplex viruses. I. Identification of four glycoprotein precursors and their products in type 1-infected cells. J. Virol. 17:991-1008. 28. Vestergaard, B. F., and B. Norrild. 1978. Crossed immunoelectrophoresis of a herpes simplex virus type 1specific antigen: immunological and biochemical characterization. J. Infect. Dis. 138:639-643. 29. Vestergaard, B. F., and B. Norrild. 1979. Crossed immunoelectrophoretic analysis and viral neutralizing activity of five monospecific antisera against five different herpes simplex virus glycoproteins. IARC Sci. Publ. 24: 225-234. 30. Wilcox, K. W., A. Kohn, E. Sklyanskaya, and B. Roizman. 1980. Herpes simplex virus phosphoproteins. I. Phosphate cycles on and off some viral polypeptides and can alter their affinity for DNA. J. Virol. 33:167182.