studies on herpes simplex virus and cancer...

[CANCER RESEARCH 36, 845-856, February 1976]

Studies on Herpes Simplex Virus and Cancer I

Joseph L. Melnick, 2 Richard J. Courtney, Kenneth L. Powell, Priscilla A. Schaffer, Matilda Benyesh- Melnick, Gordon R. Dreesman, Takashi Anzai, and Ervin Adam

Department of Virology and Epidemiology, Baylor College of Medicine, Houston, Texas 77025

Summary

Virus-induced polypeptides of cells infected by herpes simplex virus (HSV) types 1 and 2 were investigated by analysis on polyacrylamide gels and by determination of their antigenicity. Some polypeptides, VP154 and VP134, had immunological reactivity common to both virus types, while others (VP175 and VP123) were type specific. Only the glycosylated polypeptides were able to induce neutralizing antibody. The expression of viral genetic information was studied in newborn mice infected with wild-type and ts

mutant viruses; some mutants had become attenuated and had lost pathogenicity for newborn mice while others had not. From induction experiments in HSV-transformed hamster cells, it appears that detection of enhanced replication of ts mutants in human cancer cells would be an indication of resident HSV genetic information.

Sera obtained from cancer patients were examined for antibodies to early proteins synthesized in HSV-infected cells. The method used was an indirect radioimmune precipitation test followed by polyacrylamide gel electrophoretic analysis of immune precipitates. Cervical cancer patients had sera with a higher reactivity to early nonstructural polypeptides than to breast cancer patients or to matched healthy women. In contrast to the results with early polypeptides, little difference was detectable between the matched sera in their reactivity with the major capsid polypeptide, which is synthesized late in the infectious cycle.

Immunological Characterization of Individual P01ypeptides Induced by HSV-1 :~ and HSV-24

Little information is presently available concerning a di- rect immunological comparison of the polypeptides induced by HSV-1 and HSV-2. However, it has been well established that type-specific as well as type-common antigens are detectable upon immunological analysis of the virus-induced proteins (17, 20, 21). The objective of this report is to describe the immunological characterization of purified HSV-induced polypeptides. This approach will con- tribute towards: (a) a more precise understanding of the

Presented at the symposium "Immunological Control of Virus-associated Tumors in Man: Prospects and Problems," April 7 to 9, 1975, Bethesda, Md. Supported by Research Contract CP 53526 within the Virus Cancer Pro- gram and by Research Grant CA 10,893 from the National Cancer Institute.

2 Presenter. 3 The abbreviations used are: HSV-1, herpes simplex virus type 1 ; HSV-2,

herpes simplex virus type 2; HSV, herpes simplex virus; SDS, sodium dodecyl sulfate; ts, temperature sensitive; WT, wild type; NT, neutralizing; i.c., intra- cranial; TPDs0, 50% tumor-producing dose.

4 With Richard J. Courtney and Kenneth L. Powell.

antigenic relatedness of HSV-1- and HSV-2-induced antigens; (b) the eventual establishment of a functional role for each polypeptide; (c) the eventual identification of which polypeptides are present within HSV-transformed cells; and (d) identification of specific polypeptides that may preferen- tially react with sera from patients with cervical cancer.

The general procedure used for the isolation of the individual polypeptides and the preparation of antipolypeptide sera has been described previously (5) and is outlined in Chart 1. Briefly, virus-induced polypeptides obtained from HSV-infected cells were solubilized in 1~ SDS, 0.5 M urea, and 1% mercaptoethanol at 100 ° for2 min. The SDS polypeptides were then separated by discontinuous preparative polyacrylamide gel electrophoretic analysis. The stacking gel (pH 6.7) contained 2.9~ acrylamide and the separating gel contained 7.0~ acrylamide. Following the elution of the separated polypeptides from the preparative gel, the peak fractions containing the desired polypeptide were pooled and concentrated. These fractions were then subjected twice to electrophoresis on 0.65- x 10-cm cylindrical gels for further purification. After locating the polypeptide band fol lowing the 2nd electrophoresis on cylindrical gels, the region of the gel containing the polypeptide band was cut out with a razor blade and frozen at - 7 0 ° . For immuniza- tion, the gel sections were mixed with Freund's complete adjuvant and inoculated into the hind footpads of rabbits, fol lowed 2 weeks later with a 2nd dose injected i.m.

Four polypeptides, VP175, VP154, VP134, and VP123, were chosen for this study and are designated in the left portion of Chart 2 containing the autoradiogram of a polyacrylamide slab gel. Th is autoradiogram shows the polypeptides obtained from the cytoplasmic fraction of human embryonic lung cells infected with HSV-1 t sB2 (a ts mutant) and HSV-1 KOS (a WT virus). The incubation temperature was 39 ° (nonpermissive temperature for tsB2) for both cell cultures, and they were labeled with 14C-labeled amino acids (3/~Ci/ml) for 4 to 24 hr after infection.

Each of these 5 polypeptide regions was fractionated by preparative polyacrylamide gel electrophoresis as described above and previously (5, 6), and the general properties of the antipolypeptide sera prepared are shown on the right-hand portion of Chart 2. Antisera to VP175, an early nonstructural polypeptide of HSV-1 that accumulates in large amounts in tsB2-infected cells at the nonpermissive temperature, demonstrated type specificity when measured by immunofluorescence of HSV-1- and HSV-2-infected cells. Using anti-VP175, HSV-l-infected cells showed a strong nuclear immunofluorescence reaction (5). Antisera to VP154, the major nucleocapsid potypeptide, and to VP134, an early nonstructural polypeptide, exhibited strong

FEBRUARY 1976 845

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J. L. Meln ick et al.

HSV-INFECTED CELLS [24 Hr P.I.)

1% SDS

O. 5 M Urea

1% ME

2 Min 100 ~

PREPARATIVE SDS-PAGE

8 i o

x 6 t,i

4

~u

-t-

10 20 30 40 50 ~,,, bO 70 80

+ Fraction Number J - l

J �9 ,. ( ANALYTICAL SDS-PAGE

_ I:: I

O, 5 mm Slices 'illi l wl =- IMMUNIZE Ira,, El~e and Pool Peak Fractions "I RABBITS

ANTI-HSV POLYPEPTIDE + + SE RA

Chart 1. A general outline of the procedure used for the preparation of antisera to individual HSV-induced polypeptides. Briefly, the HSV-infected cell extracts were solubilized and subjected to electrophoresis on preparative SDS-polyacrylarnide gel electrophoresis (PAGE); the peak fractions were then pooled and concentrated. These fractions were then subjected to electrophoresis twice on analytical (cylindrical) SDS-polyacrylamide gel electrophoresis. After the 2nd electrophoresis, the portion of the gel containing the polypeptide band was cut out and used to immunize rabbits. P.I., postinfection; ME, rnercaptoethanol.

nuclear immunofluorescence reactions and varying de- grees of cytoplasmic immunofluorescence reactivity. Nei- ther of these sera demonstrated type specificity in the immunofluorescence reaction. In addition, none of the above sera would neutralize infectious virus. In contrast, type- specific neutralization was demonstrated with anti-VP123, a serum produced against the major envelope glycoprotein of HSV-l- infected cells. A similar type-specific serum prepared against the major envelope glycoprotein of purif ied virus particles has also been reported (16). In addit ion, antisera to the altered glycosylated polypeptide (VP123') produced in the presence of 2-deoxy-D-glucose (7) also demonstrated type-specif ic neutralization. Recently, antisera to the major envelope glycoproteins isolated from HSV-2-infected cells or from HSV-2-purified virions has also been shown to neutralize infectious virus, s

In order further to demonstrate the use of this procedure and the relative purity of these fractionated polypeptides isolated from the preparative polyacrylamide gel electrophoresis, the fol lowing analyses were conducted. Cultures were infected with HSV-1 and labeled from 4 to 24 hr after infection. The nuclear fraction of the infected cells was isolated

s R. J. Courtney and K. L. Powell, in preparation.

and then fractionated by SDS-preparative polyacrylamide gel electrophoresis. The preparative polyacrylamide gel electrophoretic profi le of the separated polypeptides is shown in Chart 3. As can be seen, the separation of VP154 from VP134 in preparative polyacrylamide gel electrophoresis is excellent, especially when compared to their separation by analytical gels (see left-hand portion of Chart 2). The 2 peak fractions of each of the 3 peaks were pooled, concentrated, and then again subjected to electrophoresis on a polyacrylamide slab gel. An autoradiogram of the slab gel was made, and the peaks were traced on a densitome- ter. As shown in Chart 4, the pooled peak fractions are relatively free of neighboring polypeptides. These purified polypeptides are proving to be of great benefit for the further biochemical and immunological characterization of HSV-1 and HSV-2 proteins and glycoproteins.

In this report, the value of characterizing the individual polypeptides isolated from HSV-infected cells has been outlined. Certain polypeptides (VP154 and VP134) are type common, while others (VP175 and VP123) are type specific in their immunological reactivity. Only the glycosylated polypeptides were able to induce neutralizing antibody. With the ability to purify relatively large amounts of any individual polypeptide or glycosylated polypeptide, the application of these methods towards the purif ication of antigenic sub- units of HSV for vaccine purposes appears promising.

Effect of Partial Expression of the HSV Genome on Attenuation of HSV Infection in Mice and Tumorigenicity and Other Properties of HSV-transformed Hamster Cells 6

Pathogenicity and oncogenicity of viruses are complex properties that depend upon the function of a number of cistrons. While pathogenicity presumably depends upon the expression of all (or nearly all) viral cistrons to produce lytic infection, tumorigenici ty appears to depend upon the expression of only a limited amount of viral information. Studies of HSV genetics using ts mutants have led us to examine the expression of viral information fol lowing infection of newborn mice with WT and ts mutant viruses, and fol lowing induction in hamsters with HSV-transformed cells.

Pathogenicity of ts Mutants of HSV-1. The biochemical alterations exhibited by 22 ts mutants of HSV-1 with defects in 15 cistrons at the nonpermissive temperature are cur- rently being studied. Phenotypic properties that have been examined include the synthesis of viral DNA, DNA polymer- ase, thymidine kinase, and virus-specific polypeptides, as well as virion thermal stability, kinetics of growth, and the ability of mutants to synthesize virus particles as determined by electron microscopic observation of thin sections. Results of these tests have demonstrated that mutants defective in different cistrons exhibit different phenotypic properties (18).

Since ts mutants are, by definit ion, defective in cistrons essential for virus replication, they theoretically express all but the ts gene during infection at high nonpermissive

6 With Priscilla A. Schaffer and Matilda Benyesh-Melnick.

846 CANCER RESEARCH VOL. 36



A N T I S E R A TO P O L Y P E P T I D E S O F HSV-1 AND HSV-2

HSV and Cancer

tsB2 39

w

i

i

WT 39

III I

M o l e c u l a r P o l y p e p t i d e w e i g h t S o u r c e F u n c t i o n

I V P 1 7 5 (4-6) 175 ,000 S

I gl

pVP154 (7) 154, 000 g l f

] J /

/ S S

~ s . . V P 1 3 4 (9) 134 ,000

N e u t r a l i z a t i o n of Immunofluorescence

H S V - I HSV-2 H S V - I HSV-2

HSV-1 E a r l y n o n - - - + (N)

tsB2 structural

H S V - I - - + (C, N) + (C, N) M a j o r c a p s i d

HSV-2 - - + (C, N) + (C, N)

HSV-1 E a r l y n o n - - - + (PN, N) + (PN, N)

HSV-2 s t r u c t u r a l - - + (PN, N) + (N)

M a j o r . . . . . . . VP123 (13-15) 123 ,000 HSV-1 e n v e l o p e , + (1:32) - + (C, PN) + (PN)

g l y c o s y l a t e d

M o d i f i e d H S V - I

V P I 2 3 v (C5') 94, 000 0 .1% dglc enve lope , + (1:8) - + (C, PN) • (PN) g l y c o a y l a t e d

Chart 2. A summary of the properties of antisera prepared to the polypeptides of HSV-1 and HSV-2. Left, an autoradiogram obtained from a slab gel of the polypeptides (labeled 4 to 24 hr postinfection with '4C-labeled amino acids) synthesized in WT (KOS) and tsB2-infected cells at 39 ~ dglc, 2-deoxy-o-glucose; N, nuclear; C, cytoplasmic; PN, paranuclear.

PREPARATIVE PAGE

'o 6 VP 98 VP 154

X ~E

U ' 4

2

J I I I I 20 40 60 80

F R A C T I O N NUMBER

Chart 3. A preparative polyacrylamide gel electrophoresis (PAGE) profile of a nuclear fraction of HSV-l-infected cells labeled from 4 to 24 hr postinfection with '4C-labeled amino acids.

temperatures. It was of interest, therefore, to examine the correlat ion in vivo between pathogenic i ty and defects in part icular viral cistrons using ts mutants as models. Al- though a correlat ion between temperature-sensi t ive and altered pathogenic i ty has been noted for a variety of animal viruses, inc luding HSV (23), the HSV mutants were not

suff ic ient ly well character ized phenotyp ica l ly to at tr ibute these al terat ions to defects in specif ic c istrons.

Representative mutants with ts defects in each of 15 HSV- 1 cistrons and the WT virus from which the mutants were derived (18) were tested for pathogenic i ty in vivo. Al though permissive and nonpermissive temperatures for these mu-

FEBRUARY 1976 847



J. L. Me ln ick et al.

VP 154

VP 134

L.

VP 911

Chart 4. Densitometric tracing of an autoradiogram of a slab gel on which the preparative polyacrylamide gel electrophoresis fractions were analyzed. The 2 peak fractions of each peak (VP154, VP134, and VP98) were pooled and again subjected to electrophoresis on the slab gel. Bottom, an example of the polypeptide content of a similar sample prior to fractionation on preparative polyacrylamide gel electrophoresis.

tants in in vitro studies were 34 and 39 ~ respectively, it was expected that the ts gene product would also be defective at an intermediate temperature (3T, mammal ian body temperature) since ts lesions are usually expressed as a continu- ously variable funct ion of temperature.

The degree of virulence of each virus was tested by inocu- lating serial 10-fold di lut ions of virus i.p. into 8 newborn mice/virus di lut ion (Table 1). Viruses were tested from 2 to 4 t imes with similar results. Compared with the WT virus, 3 mutants (tsC7, H10, and I l l ) were maximal ly attenuated and 3 mutants (tsF17, M19, and N20) were minimal ly attenuated. The remaining mutants were of intermediate virulence. The increased latent period before the observations of 1st and

last mouse deaths for mutants tsC4, tsE5, tsG3, and tsN20 may reflect attenuation, although none of the maximally attenuated mutants or 1 minimal ly attenuated mutant was in this group.

Attempts were made to isolate ts and ts § virus from brains and livers of dead animals in order to ascertain whether mutant or WT revertant virus was the cause of death. As seen in Table 1, no correlat ion was observed between virulence and the nature of the virus isolated. Revertant, ts + virus was isolated from mice that died fo l lowing inoculation with 4 moderately attenuated mutants.

Sera of surviving mice were tested for the presence of NT antibody as a determinat ion of whether survivors had indeed been infected and as a reflection of the immunogenic- ity of each defective virus. Results of these tests are shown in Table 1, Column 7. Such a determinat ion does not strictly reflect the immunogenic i ty of each mutant since different doses of each virus were used as inocula; however, the relative abil i ty of mutants to induce NT ant ibody was ascer- tained. Of the maximally attenuated mutants, none of the mice surviving inoculat ion with ts111 made NT antibody; only one-third of those inoculated with tsC7 and one-half inoculated with tsH10 made NT antibody, even when mice had been inoculated with 106 plaque-forming units of each mutant. These data indicate that maximally attenuated mutants were also poorly immunogenic. On the other hand, sera of all survivors of infection with minimal ly attenuated tsF17 contained NT antibody. None of the sera of survivors of infections with minimal ly attenuated tsM19 and N20 contained NT ant ibody to HSV, indicating that they had not been infected. Thus, generally speaking, virulent mutants were immunogenic whi le attenuated mutants were only weakly so.

Mice surviving infection with maximally attenuated mutants tsB2 and tsC7 and moderately attenuated mutants tsD9 and tsE6 were challenged 6 weeks post infect ion by i.c. inoculat ion of 100 i.c. 50% lethal doses of WT virus (Table 2). Although the mutants tested varied in their degree of attenuation and in their abil i ty to induce NT ant ibody in newborn mice, the abil ity of " immun ized" mice to resist challenge with WT virus was nearly the same for all 4 of the mutants tested. These data suggest that factors other than humoral immuni ty (e.g., cellular immunity) determine the ability of mice to resist challenge. Consequently, the expression by the mutant of gene products that st imulate humoral immuni ty (viral envelope proteins and glycoproteins) may be of less signif icance than viral funct ions that stimulate cellular immuni ty in determining the outcome of challenge.

A comparison of the in vivo pathogenici ty and immuno- genicity of ts mutants with in vitro biochemical propert ies is shown in Table 3 for 4 maximally, 2 moderately, and 3 minimal ly attenuated ts mutants and also for the WTvirus.

The data obtained in pathogenici ty experiments demonstrated that some mutants had lost, or nearly lost, their pathogenici ty for newborn mice whi le other mutants did not dif fer signif icantly from the WT virus in this regard. Since the mutants tested exhibited ts defects in di f ferent cistrons, we may assume that mutat ions in some cistrons resulted in attenuation, whi le others did not. Thus, general ly speaking, the data in Table 3 indicate that the less pathogenic mutants




Table 1 Virulence of WT virus and ts mutants of HSV-1 for newborn mice

Day of mouse Isolation of virus from death dead mouse" Presence of NT an-

i.p. 50% le- tibody in sera of Virus thai dose/ml First Last Brain Liver survivors b

WT 6.1 2 8 WT WT 2/2 (101) c tsA15 2.5 2 7 WT WT 2/3 (10~), 2/3 (104) tsB2 1.8 4 7 ts + WT ts 5/5 (10 ~) tsC4 2.0 6 10 ts 0 3/4 (1 (P) tsC7 <1.0 2/6 (106) tsD9 3.5 3 8 ts ts 3/8 (104) tsE6 2.3 5 9 ts ts 1/3 (104) tsF17 6.1 2 9 0 0 1/1 (10=), 2/2 (10 =) tsG3 1.8 6 8 0 0 6/6 (10 s) tsH10 <1.0 3/6 (106) ts111 <1.0 0/8 (106) tsJ12 1.5 3 7 ts ts 3/8 (105) tsK13 4.1 3 8 'WT 0 1/3 (1 0=) tsL14 4.8 2 8 ts 0 1/1 (10=), 1/2 (10=) tsM19 6.3 2 9 WT 0 0/5 (10 ~ tsN20 6.2 5 7 ts ts 0/2 (101) tsO22 4.1 3 10 WT ts 3/3 (10 :~)

" Extracts of 5~. suspensions of mouse brain and liver were assayed simultaneously at 34 and 39 ~ Brains and livers of 2 to 4 mice were examined for each mutant.

b Presence of HSV NT antibody was assessed using 1:20 dilution of serum. Sera that neutralized ->60% of 100 WT plaques were considered to be positive. Fractions, number of sera positive/number of sera tested.

" Numbers in parentheses, virus dose (plaque-forming units) used for inoculation.

Table 2 Resistance of mice to challenge with WT virus"

HSV a n d C a n c e r

WT tsB2 tsC7 tsD9 tsE6 Virus dose Ab/S b RCh/Ab Ab/S RCh/Ab Ab/S RCh/Ab Ab/S RCh/Ab Ab/S RCh/Ab

1 6/8 ND 0/8 NA 0/8 NA 0/8 NA 1/8 0/1 101 2/2 ND 0/8 NA 0/8 NA 0/8 NA 8/8 0/8 10 = ND 8/8 0/8 5/8 0/5 5/8 1/5 8/8 1/8 10 ~ ND 8/8 3/8 5/8 0/5 1/1 1/1 8/8 5/8 104 N D 8/8 5/8 8/8 3/8 6/6 3/6 105 N D 4/4 4/4 8/8 4/8 4/4 4/4 10 ~ ND 8/8 8/8 1/1 1/1

Total 8/10 28/44 12/28 34/56 15/34 6/25 2/6 36/43 14/36

% 80 63 42 61 44 24 33 84 39

a Survivors of mutant inoculations were challenged 6 weeks postinfection with 100 i.c. 50~ lethal doses of WT virus. Ab/S, no. with antibody/no, of survivors; RCh/Ab, no. resistant to challenge/no, with antibody; ND, not done; NA,

not applicable.

were able to synthesize fewer virus part icles, which proba- bly reflects defects in po lypept ide synthesis exhib i ted by some of these mutants. No corre lat ion was observed between pa thogen ic i t y and viral DNA phenotype. Also, however, most mutants were less pa thogen ic than the WT virus was. A more complete descr ipt ion of the pathogenic i ty of ts

mutants fo r newborn mice wil l appear elsewhere. 7 Expression of HSV Genetic Information in Transformed

Cells. S ince the lack of expression of 1 essential HSV cis- tron could affect pathogenic i ty , the correlat ion between

7 p. A. Schaffer, M. Benyesh-Melnick, and I. Wimberly, in preparation.

tumor igen ic potent ia l and the express ion of HSV genet ic in format ion in HSV-t ransformed cel ls was examined.

Most at tempts to t ransform cells with HSV in v i t ro have, of necessity, involved inact ivat ion of lyt ic genes by UV irradia- t ion or some other t reatment. In the process of inact ivat ion, it is l ikely that t ransforming genes are also inact ivated. Also, certain genes involved in the HSV repl icat ive cycle, inact i- vated in this case, may also be involved in induc ing transformation. Fur thermore, no 2 inact ivated genomes would be likely to contain the same comp lement of b io log ica l ly active genet ic in format ion. In order to obta in a more un i fo rm populat ion of noncyto ly t ic HSV for t ransformat ion, at tempts to t ransform cells with ts mutants at the nonpermiss ive

FEBRUARY 1976 849



J. L. Me ln i c k et al.

Table 3 Comparison of pathogenicity with phenotypic properties of ts mutants of HSV-1

Virus

Phenotypic properties in vitro, 39 ~ Abilityto in- Virus parti-

- Log 50~ le- duce NT Viral Thermal cle produc- Kinetics of thai dose/ml antibody DNA" Viral polypeptides b stability" tion c (% WT) growth (34 ~

WT 6.1 + + - - 100

tsF17 6.1 + + = WT = WT 90 = WT tsM19 6.3 ? + =WT =WT 15 =WT tsN20 6.2 9 + = WT = WT 90 = WT

tsD9 3.5 _+ - ~VP154 =WT 4 =WT tsE6 2.3 ___ + = WT < WT 30 = WT

tsB2 1.8 + - 1'VP175, ~VP154, 80 =WT 0 =WT tsC7 <1.0 _ - ~VP154, 80 = WT 6 Retarded" tsH10 <1.0 _+ + =WT =WT 2 =WT ts111 <1.0 - + =WT =WT 8 =WT

" From Schaffer et al. (18). t, From Courtney et al., in preparation.

From Schaffer et al. (19). ' Growth of tsC7 at 34 ~ lagged 2 hr behind the growth of the WT virus regarding time of 1st virus increase and time of

maximum titer.

temperature have also been conducted (13, 14, 22). Since only the ts gene is nonfunct ional at high temperature, all other genes are presumably intact; yet, by virtue of the ts lesion, virus repl icat ion and consequent ly damage to the cell are minimized. However, if t ransformat ion occurs following infection of cells wi th ts mutants at the nonpermissive temperature, the ts gene would not be involved in the induct ion or maintenance of the t ransformed state since the gene would be nonfunct ional . In order to establish the role of a single viral gene in the t ransformat ion process, it would be necessary first to inactivate (e.g., UV irradiation) the mutant, t ransform at permissive temperature, and observe cul tures for a ts- t ransformed phenotype. Al though a l imited number of t ransformat ion attempts have been made using this technique, no HSV-transformed cell line which is ts for t ransformat ion has been reported.

Since t ransformat ion, to date, has not been achieved with a ful ly funct ional genome, it is perhaps not surprising that attempts to induce the genome in the transformed cells available have been unsuccessful. The question then arises as to how much HSV genetic in format ion is present in HSV- t ransformed cells and how much is expressed. Indeed, only a segment of the HSV genome may be incorporated dur ing t ransformat ion and even less may actual ly be expressed. At tempts to quanti fy the amount of viral DNA present in cells t ransformed in vi tro with HSV by nucleic acid hybridizat ion techniques have been unsuccessful .

Two ways to detect expression of resident HSV information have thus far been successful: (a) directly, by observa- t ion of t ransformed cells for the presence of viral gene products, i.e., by immunof luorescence; and (b) indirectly, by enhancement of growth of HSV ts mutants in transformed cells at the nonpermissive temperature by complementat ion mechanisms (12).

At tempts in this laboratory to t ransform hamster embryo f ibroblast cells with HSV-1 and HSV-2 using a variety of

techniques have resulted in the establ ishment of 8 oncogenically t ransformed cell lines (13). A list of the t ransforming agents used and of the cell lines obtained is shown in Table 4. In view of the variety of t ransforming agents and transformation procedures used in these studies, it is not surprising that the result ing tumor cell lines derived from init ial ly transformed cells are phenotypical ly dif ferent with regard to the expression of HSV-specif ic funct ions. A summary of the virus-specif ic funct ions expressed by 6 of the 8 transformed cell lines is shown in Table 5.

It can be seen from Table 5 that all 6 lines expressed HSV- 2 surface antigens whi le only 3 of the 6 lines expressed the early, nonstructural viral polypeptide VP134. In view of the fact that all tumor lines were surface antigen posit ive, the

Table 4 Hamster embryo fibroblast lines established fol lowing

transformation with HSV-1 and HSV-2"

Transforming agent Transformed cell

lines

UV-irradiated HSV-2 strain 186

UV-irradiated HSV-2 strain 333 (hamster embryo fibroblast cells preirradiated for4 and 6 sec with UV)

UV-irradiated HSV-2 strain 333 (hamster embryo fibroblast cells pretreated for 48 hr with 5-bromodeoxyuridine, 20/~g/ml)

ts mutants of HSV-1 strain KOS, and HSV-2 strain 186 (infected at nonpermissive temperatures)

U15 U26

U4V-8 U6V-7

B20V-9

HSV-1,864-12

HSV-2, 155-4 HSV-2, 50-14

From Kimura et al. (13).




HSV and Cancer

Table 5 Expression of HSV-specific functions in hamster embryo fibroblasts oncogenically

transformed by HSV-2

Viral antigens detected by immunofluorescence using

Cell line" Anti-HSV-2 t' Anti-VP134 ~

Neutralizing antibody to HSV-2 in weanling hamsters" (no. of sera positive/no, of sera tested)

Hamster embryo fibro- - - 0/12 (0) ~ blast

U15 Tu + + 1/12 (8) U26 Tu + - 3/11 (27) U4V-8 Tu + + 5/12 (42) U6V-7 Tu + - 3/12 (25) B20V-9 Tu + + 4/12 (33) ISS-4 + - 11/12 (92)

" Tumor cell lines (Tu) were established from tumors produced in newborn hamsters by the cell lines described in Table 4.

i, Hyperimmune rabbit antiserum to HSV-2 used to detect surface antigens in unfixed cell preparations.

Hyperimmune rabbit antiserum to HSV-2 viral polypeptide VP134 used to detect internal antigens in fixed preparations.

d Weanling hamsters were inoculated with 105 cells of each tumor line. The presence of neutralizing antibody in sera was determined by the plaque reduction technique. Tumor- bearing sera diluted 1:8 or greater which showed 50~ plaque reduction were considered positive.

Numbers in parentheses, percentage.

presence of NT antibody in sera of hamsters bearing tumors induced by all 6 lines is not surprising. Although the number of animals tested was admittedly small, the differences in the NT ant ibody- inducing capacity among the 6 lines are noteworthy. Whether this variation is due to variation in the expression of HSV-specific surface antigens among the cell lines is not known, since the immunof luorescence technique is insuff ic ient ly sensitive to be used for quantitative purposes. Furthermore, polyacrylamide gel electrophoresis of tumor cell extracts has failed to reveal the presence of virus-specific proteins or glycoproteins above the high background of cellular protein.

A comparison of the phenotypic expression of HSV-2- specific funct ions in the 6 transformed cell lines with their tumorigenic properties is shown in Table 6. The line with the highest TPD~0 also had the longest latent period (B20V- 9). One line with a low TPD.~0 exhibited the shortest latent period. No such correlations were evident between TPDs0 and latent period for the other 4 lines.

Comparison of the data in Tables 5 and 6 suggests the fol lowing: (a) the least tumor igenic line (B20V-9 Tu) expressed both HSV-specific surface and internal antigens and induced moderate levels of NT ant ibody in weanl ing hamsters; (b) the most tumor igenic line (U26-Tu) expressed surface but not internal HSV-specific antigen and induced NT antibodies in tumor-bearing hamsters equally as well as B20V-9. Thus, no clear correlation has yet emerged between the expression of HSV-specific funct ions and degree of tumorigenic i ty in HSV-2-transformed cell lines. When additional markers of the expression of HSV information in transformed cells become available, such a correlation may emerge.

If even a partial genome of HSV were present and functional in HSV-2-transformed cells, yields of ts mutants of HSV-2, which are unable to replicate eff iciently in normal

Table 6 Tumorigenicity of HSV-2-transformed tumor cell lines

TPDs0 in weanling ham- Cell line sters" Latent period t' (wk)

Human embryonic lung

U15 Tu 1022 2 U26 Tu <102"~ 1 U4V-8 Tu 10' ~ 4 U6V-7 Tu 10 ''~ 2 B20V-9 Tu 10:3.7 5 155-4 Tu 101"~ 2

" TPDs0 determined by s.c. inoculation of weanling hamsters with varying numbers (105, 104, etc., cells/0.1 ml) of cultured newborn tumor (Tu) cells.

~' Time to appearance of 1st tumor(s).

cells at the nonpermissive temperature, 38 ~ , might be enhanced in HSV-2-transformed cells at the nonpermissive temperature through complementat ion with resident HSV genes and gene products.

As shown in Table 7 there was signi f icant enhancement of the growth of ts mutants in 2 HSV-2-transformed cell lines (333-8-9 and MS4-1) at the nonpermissive temperature, which suggests that resident, funct ional HSV genetic information assisted the replication of the mutants by complementation mechanisms. Lack of enhancement of the growth of HSV-2 ts mutants in SV40 tumor cells suggests that the presence of a homologous virus genome is necessary for enhancement and that an oncogenical ly transformed cell phenotype per se is insuff ic ient to produce enhancement. An enhancing phenomenon similar to that observed in the present study has been reported recently with an SV40 ts mutant in SV40-transformed cells (4).

In standard complementat ion tests between ts mutant pairs of either HSV-1 or HSV-2 studied in this laboratory (9,

FEBRUARY 1976 851



J. L. M e l n i c k et al.

Table 7 Replicat ion of 8 ts mutants of HSV-2 at 38 ~ in normal, HSV-2-transformed, and SV40-induced tumor

cells See Kimura et al. (12) for details.

Ratios of yields of ts mutants"

Cell type WT tsA1 -~ tsA8- tsB5- tsC2- tsD6 § tsE7 § tsF3 § tsG4 +

HEF 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 333-8-9 1.1 3.5 1.2 3.3 3.9 36.0 3.2 2.1 18.0 MS4-1 1.2 3.5 1.8 2.1 1.7 26.0 1.3 1.8 13.0 SV40/Tu 0.3 1.3 1.7 0.5 1.7 0.7 0.5 0.6 1.1

" Ratios, 38 ~ yield (plaque-forming units) per 105 transformed cells/38 ~ yield (plaque-forming units) per 10 s normal cells, assayed at 34 ~

b _, DNA-negative ts mutants; +, DNA-positive ts mutants.

18) and in other animal virus ts mutant systems (3), complementat ion values of 2 or greater were considered to repre- sent signi f icant complementat ion. The enhancement observed in the present study was substantial ly greater than 2- fold. Of addit ional interest is the fact that the greatest degree of enhancement was observed with mutant t sD6 which has been shown to be thermolabi le at 38 ~ (9). The effect of thermolabi l i ty would be to decrease, rather than increase, the titer of infect ious virus in these experiments.

Furthermore, the 2 HSV-2 ts mutants (tsD6 and tsG4), which exhibited enhanced growth in HSV-2-transformed cells, are located in the same region of the HSV-2 genetic map, whi le nonenhanced mutants are not located in this region (8). This clustering could be coincidental; however, an alternative explanat ion is that this region of the HSV-2 genome codes for gene products involved in the HSV transformation process. The latter possibi l i ty is suggested by the fact that the enhancement occurred in 2 independ- ently derived HSV-2-transformed cell lines, suggesting some specif ic i ty of HSV-2 genetic information present in these 2 HSV-2-transformed cell lines. The lack of enhancement of the other 6 mutants in this study indicated either that the HSV genetic informat ion required for the enhancement of their growth was not present in the HSV-2-transformed cells, or that the informat ion was present but not funct ional.

The resu Its suggest that the detect ion of enhanced replication of ts mutants in human cancer cells would be an indication of resident HSV genetic informat ion in the malig- nant cells.

Whether variation in the amount of HSV genetic information present and expressed in cells transformed in nature by HSV ( i .e., cervical cancer cells) also occurs is presently unknown. Hopeful ly, more sensitive nucleic acid hybridiza- tion techniques, coupled with improved methods for detect- ing HSV-specif ic funct ions, wil l help to answer this question.

Antibody to HSV-2-induced Nonstructural Proteins in Women with Cervical Cancer and in Control Groups"

To investigate a possible et iological role of HSV-2 in the occurrence of cervical cancer, sera have been collected in

s With Gordon R. Dreesman, Takashi Anzai, Richard J. Courtney, and Ervin Adam.

our laboratory from different geographic areas for the past 6 years. These include sera from patients with cervical cancer, patients with breast cancer, and control women matched for race and socioeconomic background. The results obtained from a series of seroepidemiological studies conducted in this laboratory and summarized elsewhere (15) indicate signi f icant differences in the occurrence of neutralizing ant ibodies to HSV-2 between patients with cervical cancer and matched control women and breast cancer patients. However, recent reports have shown that antibodies to HSV-induced antigens detected by complement fixation tests may be differentiated from the neutralizing antibodies in sera of patients with cervical cancer (1,2) and in sera of patients with cancer of dif ferent sites (10).

This study was undertaken to detect ant ibodies to early "nonstructura l " antigens of HSV-2 in sera of patients with cervical cancer and of control groups and also to identify the reactive virus-specif ic nonstructural antigens. For this purpose, an indirect radio immune precipi tat ion test was developed in combinat ion with polyacrylamide gel electrophoretic analysis of immune precipitates (radioimmune pre- c ip i tat ion-polyacrylamide gel electrophoresis test). Sera from patients with cervical cancer, from patients with breast cancer, and from control women, with predetermined levels of neutralizing ant ibodies to HSV-2, were coded and subjected to the rad io immune precipi tat ion-polyacrylamide gel electrophoresis test. The results obtained suggest that sera from patients with cervical cancer possess a higher reactivity to early nonstructural proteins synthesized by HSV-2 as compared to the 2 control groups and that the reactivity is independent of the level of neutral izing ant ibodies in the sera exami ned.

For the present study, matched cervical cancer case- control pairs were selected according to the previously determined presence of neutral izing HSV-2 ant ibody expressed by the II/I threshold index. To assure as much diversity as possible, the levels of HSV-2 ant ibody of controls in some of the matched pairs were identical to those in the cervical cancer cases, while in others the matched controls had a higher or lower II/I threshold index for HSV-2 antibody than the cases.

These sera were caused to react with isotopical ly labeled early HSV-2 antigens. The method used was an indirect radioimmune precipitat ion test fol lowed by polyacrylamide gel electrophoret ic analysis of immune precipitates. The




HSV and Cancer

Diluted human serum . . . . . . . . . . . . 0.05 ml~

Early HSV-2 antigen . . . . . . . . . . . . . . 0.10 ml

Goat anti-human IgG . . . . . . . . . . . . . 0.10 ml

0.05 ml

Radioactivity in ppt.

37 ~ 1 Hr

i 4 ~ Overnight

4 ~ , Overnight

Centrifuge 2500 rpm, 20 min, 4 ~

Wash ppt. twice with phosphate-buffered saline

Solubilize ppt. in 0.25 ml of 1% SDS, 0.5 M urea, and 1% 2-mercaptoethanol (100 ~ 2 min)

0.20 ml

SDS-polyacrylamide gel electrophoresis

Chart 5. Analysis of precipitates from indirect radioimmune precipitation tests by polyacrylamide gel electrophoresis.

reactivity to early HSV-2 antigens and the relative reactivity to the early nonstructural protein VP134 were then determined for each individual serum. As discussed in the 1st part of this report, VP134 is one of the major nonstructural proteins (M.W. 134,000) synthesized in the cell during the early stage of HSV infection, even when the synthesis of new viral DNA is blocked by cytosine arabinoside.

The basic protocol of the radio immune precipitat ion-polyacrylamide gel electrophoresis test is shown in Chart 5. To prepare the antigens, human embryonic lung f ibroblasts were infected with HSV-2 and pulse-labeled with trit iated leucine between 1 and 3 hr after infection. The soluble cytoplasmic fractions were prepared from infected cells at 3 hr after infection and used as early HSV-2 antigens. The tests were carried out at serum di lut ions of 1:4 and 1:8 to ensure that the measurements were performed in ant ibody excess. After incubation at 37 ~ for 1 hr, the reaction mixtures were placed in a refrigerator at 4 ~ overnight; goat anti- human IgG serum was then added to the reaction mixtures to coprecipitate the preformed immune complexes. The amount of goat serum used per rad io immune precipitat ion test had been predetermined by a quantitat ive immune precipitation test (11) and was in a quant i ty suff icient to com- bine in opt imal proport ions with human globul ins present in the primary reaction mixtures. Tests run in the absence of goat ant i-human IgG serum resulted in the precipitat ion of less than 5~ of the total radioactivity. The reaction mixtures were further incubated at 4 ~ overnight and centri fuged at 1600 x g for 20 min at 4 ~ . The immune precipitates obtained were washed with ice-cold 0.1 M phosphate-buffered saline (pH 7.2) and then solubil ized in 0.25 ml of 1% SDS, 0.5 M urea, and 1% 2-mercaptoethanol at 100 ~ for 2 min. Since all of the tests were performed in the zone of antibody excess, zones of antigen excess did not affect the results, for the immune complexes were precipitated at opt imal propor- tions with goat anti-human IgG serum.

The radioactivity of portions (0.05 ml) of the solubil ized immune precipitates was determined. The total radioactivity

precipitated with each individual serum was then calculated and compared with the total radioactivi ty of each ant igen added to the serum.

The antigen preparations used in the radio immune precipitation test and the remaining port ions (0.20 ml) of the solubil ized immune precipitates obtained by the radioimmune precipi tat ion test were then analyzed by polyacrylamide gel electrophoresis. In each polyacrylamide gel electrophoret ic analysis the fo l lowing preparat ions were subjected to electrophoresis in parallel: (a) the starting ant igen preparation; and (b) the immune precipitates obtained with sera from patients with cervical cancer as well as those obtained with sera from matched control women and patients with breast cancer.

The radioactivity of sliced gel fract ions was determined and the electrophoret ic profi les of the antigens and immune precipitates were obtained. The area under each of the protein peaks resolved by polyacrylamide gel electrophoretic analysis of the immune precipitates obtained with each serum was measured and compared with the area under the corresponding protein peaks f rom the gel of the ant igen preparation.

Chart 6 i l lustrates the electrophoretograms of an early HSV-2 antigen used in the rad io immune precipi tat ion test and of immune precipitates obtained by reacting the fo l lowing sera with the antigen: a serum of a patient with cervical cancer, a serum of a patient with breast cancer matched with the cervical cancer patient, and a serum of a normal individual wi thout detectable neutral izing ant ibody to either type of HSV. The antigen preparat ion revealed several dis- t inct peaks, the most prominent of which was that of VP134, one of the major nonstructural early proteins. The amount of protein precipitated by the serum wi thout detectable HSV antibody was negl igible, which attested for the HSV specif ic- ity of the rad io immune precipi tat ion test. The electrophoretic profi les of the immune precipitates of a serum from a patient with cervical cancer and of a serum from a matched breast cancer patient did not di f fer with regard to the hum-

FEBRUARY 1976 853



A

J. L. M e l n i c k et al .

4

'o p

x 3

u

w ! w I w

2 C

1 _ _ _ ~

0 w w i i i w

'J~ i

0 2 4 6 8 10 CM FROM ORIGIN

Chart 6. Polyacrylamide gel electrophoretograms of an early HSV-2 antigen preparation used in the indirect radioimmune precipitation test and of immune precipitates obtained by causing human sera diluted 1:8 to react with the antigen. The antigen was prepared from the cytoplasmic fractions of HSV-2-infected human embryonic lung cells that had been pulse-labeled with [3H]leucine between 1 and 3 hr after infection. Immune precipitates were obtained with human sera that had been caused to react with the antigen by the indirect radioimmune precipitation test. The antigen and immune precipitates were prepared for polyacrylamide gel electrophoretic analysis as described in the text. A, early HSV-2 antigen; B, immune precipitate obtained with serum from a patient with cervical cancer; C, immune precipitate obtained with serum from a control woman matched with the cervical cancer patient; D, immune precipitate obtained with serum from a normal individual without detectable neutralizing antibodies to either type of HSV.

ber of p ro te ins be ing p rec ip i ta ted . However , the quan t i t y of VP134 was m u c h g rea te r in the i m m u n e prec ip i ta te obta ined wi th the se rum f rom a pa t ien t w i th cervical cancer than in the i m m u n e p rec ip i ta te ob ta ined f rom a breast cancer pat ient .

The spec i f i c i t y of these resul ts was tested by react ing the same human sera w i th a cy top lasm ic ex t rac t f rom uninfected cel ls labeled w i th [:~SS]methionine. E lec t ropho re to -

g r a m s of the an t i gen used in the test and of each of the

i m m u n e p rec ip i ta tes ob ta ined are i l lus t ra ted in Char t 7. No s ign i f i can t react iv i ty occu r red wi th normal ce l lu la r ant i - gens.

The rep roduc ib i l i t y of the test resul ts is s h o w n in Char t 8. The sera tested were selected at r a n d o m f rom 3 of the 15 pat ients wi th cerv ical cancer and 3 of the 10 pat ients w i th breast cancer in the s tudy g roups . A l t hough no great d i f ferences were observed in the react iv i ty to the ear ly HSV-2 ant igens, analys is of the i m m u n e prec ip i ta tes by po lyacry l -

35- A

30.

25.

20-

15-

_

1 0 .

8 -

t -

4 -

? o 2-

0 , f w ,

I i i v

i ! i i i i

|1 y I l

O 2 4 6 ! I0 CM FROM ORIGIN

Chart 7. Polyacrylamide gel electrophoretograms of an extract of uninfected human embryonic lung f ibroblasts used in the indirect radio immune precipitat ion test and of immune precipi tates obtained by caus ing human sera diluted 1:8 to react with the uninfected control extract. The extract was p repared from the cy toplasmic fract ions of uninfected human embryonic lung cells which had been pulse-labeled with [35S]methionine for 3 hr. Prepa- ration of the immune precipi ta tes and the p rocedure for polyacrylamide gel e lec t rophore t ic analysis were the s a m e as descr ibed in Chart 6. A, uninfected human embryonic lung extract; B, C, and D, precipitates obtained with sera descr ibed in Chart 6, B, C, and D, respectively.




HSV and Cancer

Table 8

Antibody reactivity to an HSV-2-induced early nonstructural protein

Cervical cancer Breast cancer Controls

Mean reactivity to VP134 No. VP134 positive No. VP134 positive/no. HSV-2 positive No. VP134 positive/no. HSV-2 negative

43.5 _ 8.8" 30.8 +- 9.5 34.5 _+ 8.4 14/15 (93)" 3/10 (30) 6/15 (40) 10/10 (100) 3/10 (30) 6/11 (55) 4/5 (80) 0/0 0/4

a M e a n _ S.D. b Numbers in parentheses, percentage.

A B C D

N O ~

1OU.

Chart 8. Antibody reactivity to an HSV-2-induced early nonstructural protein (VP134) in sera from 3 patients with cervical cancer, 3 patients with breast cancer, and 3 reference sera as measured by the radioimmune precipitation-polyacrylamide gel electrophoresis test. No antibody, serum obtained from a normal individual without detectable neutralizing antibodies to either type of HSV; HSV-2, serum obtained from a male with a past history of a genital infection; HSV-1, serum obtained from a male with recurrent HSV-1 infections of the lip. Sera were tested at a dilution of 1:4 in Experiments A and B and at a dilution of 1:8 in Experiments C and D. In Experiment D, sera from 2 of the patients with cervical cancer and 2 of the patients with breast cancer were exami ned.

amide gel e lect rophores is revealed a s ign i f icant d i f ference in the reactivi ty to VP134. A l though the level of reactivity of each serum varied f rom exper iment to exper iment , a h igher reactivity to VP134 was always observed with sera f rom patients with cervical cancer as compared to the reactivi ty of sera from pat ients with breast cancer and to the reference sera. The reactivity to VP134 of the reference serum for HSV-1 was simi lar to that of the reference serum for HSV-2, and both exceeded the react iv i ty of the reference serum wi thout detectable neutra l iz ing ant ibodies to ei ther type of HSV.

In order to compare the reactivi ty of sera to VP134, the immune precip i tates obtained with 40 sera f rom the 3 study groups were subjected to e lect rophores is on SDS-polyacry l - amide gel e lect rophores is gels, and the reactivity to VP134 was then determined for each indiv idual serum (Table 8). For evaluat ing the occurrence of serum samples with h igher values of react iv i ty to VP134, the mean reactivity of the control group was chosen for the threshold level. Approx i -

mately 90% of sera f rom pat ients with cervical cancer showed h igher react ivi ty to VP134 than the mean of contro l sera, whereas 30% of sera f rom pat ients with breast cancer and 40% of sera f rom control women exceeded the mean value of contro l sera.

Recent results with other sera have not only conf i rmed the above f ind ings with VP134 but also demonstrated that there are a number of other prote ins in the HSV-2 early ant igen preparat ion which preferent ia l ly react with sera of cervical cancer pat ients. In contrast to the results with VP134 and other early po lypept ides, pre l iminary tests have suggested l i t t le or no di f ferences detectable in the reactivi ty of the cervical cancer sera and the control sera with the major capsid po lypept ide (VP154), which is pr imar i ly synthesized late in the infect ious cycle.

Sera obtained f rom 4 control women wi thout detectable neutral iz ing ant ibod ies to HSV-2 fai led to exhib i t an enhanced reactivi ty to VP134 (Table 8, Line 4). In contrast, 4 of 5 patients with cervical cancer, w i thout serological evi- dence for a pr ior HSV-2 infect ion, showed an increased level of react ivi ty to VP134. This f ind ing suggests that the reactivity to VP134 is independent of the level of neutral izing ant ibodies to HSV-2 in sera of patients with cervical cancer. Neutra l iz ing ant ibody is considered to be a measure of the response of the body to the presence of the comple te virus part icle. The above results suggest that some cervical cancer pat ients show a response to an incomplete (or defective) cycle of herpesvirus repl icat ion (that is, only to the early ant igens), whi le others respond both to incomplete and complete viruses (that is, to both early and late antigens). The precise nature of such an immune response and its relat ionship to human cancer needs fur ther study. Al- though the mature infect ious virus may not be produced in some patients for a long per iod of t ime, the presence of a latent persistent infect ion may now be detectable by measur- ing the response to the early nonst ructura l virus prote ins found and character ized in this study.

R e f e r e n c e s

1. Aurelian, L., Shuman, B., Marcus, R. L., and Davis, H. J. Antibody to HSV-2 Induced Tumor Specific Antigens in Serums of Patients with Cervical Carcinoma. Science, 181: 161-164, 1973.

2. Aurelian, L., Strand berg , J. D., and Marcus, R.L. Neutralization, Immuno- fluorescence and Complement Fixation Tests in Identification of Anti- body to a Herpesvirus Type 2-Induced, Tumor-specific Antigen in Sera from Squamous Cervical Carcinoma. Progr. Exptl. Tumor Res., 19: 165- 181,1974.

3. Burge, B. W., and Pfefferkorn, E. R. Complementation between Tempera- ture-sensitive Mutants of Sindbis Virus. Virology, 30: 214-223, 1966.

4. Calothy, G., and Defendi, V. Replication of a Temperature-sensitive Mutant of Simian Virus 40 in Normal and SV40-transformed Monkey

FEBRUARY 1976 855



J. L. Me ln i ck et al.

Cells. Virology, 58: 605-608, 1974. 5. Courtney, R. J., and Benyesh-Melnick, M. Isolation and Characterization

of a Large Molecular Weight Polypeptide of Herpes Simplex Virus Type 1. Virology, 62: 539-551, 1974.

6. Courtney, R. J., and Powell, K. L. Immunological and Biochemical Char- acterization of Polypeptides Induced by Herpes Simplex Virus Types 1 and 2. Proceedings of the Symposium on Oncogenesis and Herpesvi- ruses, in press.

7. Courtney, R. J., Steiner, S. M., and Benyesh-Melnick, M. Effects of 2- deoxy-o-glucose on Herpes Simplex Virus Replication. Virology, 52: 447-455, 1973.

8. Esparza, J. Studies with Temperature-sensitive Mutants of Herpes Simplex Virus Type 2. Ph.D. Dissertation. Baylor College of Medicine, Houston, Texas, 1974.

9. Espar-za, J., Purifoy, D. J. M., Schaffer, P. A., and Benyesh-Melnick, M. Isolation, Complementation and Preliminary Phenotypic Characteriza- tion of Temperature-sensitive Mutants of Herpes Simplex Virus Type 2. Virology, 57: 554-565, 1974.

10. Hollinshead, A. C., Lee, O., Chretien, P. B., Tarpley, J. L., Rawls, W. E., and Adam, E. Antibodies to Herpesvirus Nonvirion Antigens in Squamous Carcinomas. Science, 182: 713-715, 1973.

11. Kabat, E. A., and Mayer, M. M. Experimental Immunochemistry, Ed. 2. Springfield, II1.: Charles C Thomas Publisher, 1961.

12. Kimura, S., Esparza, J., Benyesh-Melnick, M., and Schaffer, P. A. En- hanced Replication of Temperature-sensitive Mutants of Herpes Simplex Virus Type 2 (HSV-2) at the Nonpermissive Temperature in Cells Trans- formed by HSV-2. Intervirology, 3: 162-169, 1974.

13. Kimura, S., Flannery, V. L., Levy, B., and Schaffer, P. A. Oncogenic Transformation of Primary Hamster Cells by Herpes Simplex Virus Type 2 (HSV-2) and an HSV-2 Temperature-sensitive Mutant. Intern. J. Cancer, 15: 786-798, 1975.

14. Macnab, J. C. M. Transformation of Rat Embryo Cells by Temperature- sensitive Mutants of Herpes Simplex Virus. J. Gen. Virol., 24: 143-153, 1974.

15. Melnick, J. L., Adam, E., and Rawls, W. E. The Causative Role of Herpesvirus Type 2 in Cervical Cancer. Cancer, 34: 1375-1385, 1974.

16. Powell, K. L., Buchan, A., Sim, C., and Watson, D. H. Type-specific Protein in Herpes Simplex Virus Envelope Reacts with Neutralizing Anti- body. Nature, 249: 360-361,1974.

17. Savage, T., Roizman, B., and Heine, J. W. Immunological Specificity of the Glycoproteins of Herpes Simplex Virus Subtypes 1 and 2. J. Gen. Virol., 17: 31-48, 1972.

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1976;36:845-856. Cancer Res Joseph L. Melnick, Richard J. Courtney, Kenneth L. Powell, et al. Studies on Herpes Simplex Virus and Cancer

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