Archives of Virology 64, 35--45 (1980) Archives of Virology © by Springer-Verlag 1980

Comparative Analysis of the Virion Polypeptides Specified by Herpes Simplex Virus Type 2 Strains

By

E. CASSAI, O. DI LuoA, 1~. MANSERVIGI, M. TOGNON, and ANTONELLA ROTOLA Istituto di Mierobiologia dell'UniversitY,

Ferrara, Italy

With 4 Figures

Accepted October 24, 1979

Summary

This paper reports on the variability of structural polypeptides of 32 strains of Herpes simplex virus 2 (HSV-2) isolated from various locations in the United States and Italy. Most strains were passaged a limited number of times at low multiplicity outside the human host; a few strains were characterized by numerous passages at variable multiplicities in cell culture. The acrylamide gel electro- phoresis of polypeptides from purified virions revealed very few differences. Con- sidering these differences the tISV-2 strains analysed can be classified into 3 groups according to the variabili ty of three polypeptides. The study of poly- peptides in HSV-2 infected cells revealed more differences than those seen in the structural proteins. Comparisons of the structural proteins specified by HSV-2 and HSV-1 virions, revealed clear variant features of the electrophoretic profiles, especially when pH of main gel was 8.9.

Introduction

Herpes simplex virus type 2 (HSV-2) virion polypeptides have not been studied in detail, although there has been much interest in the type 2 strains be- cause it has been suggested tha t infection with HSV-2 may be linked with cancer of the cervix (7, 16). In the seroepidemiologic studies there are significant dif- ferences in prevalence of antibodies among cases, and also among centre]s, from different geographic areas, while case-control differences are not observed every- where (15). This could be explained by the fact tha t different laboratories used different virus strains as antigens in their tests; but conversely samples of sera from different areas were tested with identical antigens when examined in the same laboratory.

CAssAI et al. (3) have reported a comparison of the structural polypeptides of virions from 5 independent HSV-2 isolates and those from one HSV-1 strain. Other direct studies have included only one strain of HSV-2 (9, 13, 23).

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36 E. CAssAx et al. :

These i n c o m p l e t e d a t a h a v e s e rved as a s t i m u l u s t o w a r d s tud ies of p o l y p e p t i d e

d i f fe rences a m o n g a large n u m b e r of H S V - 2 s t ra ins . I n th is r epor t , we d i r ec t e ly c o m p a r e t h e v i r ion p o l y p e p t i d e s f r o m t h i r t y - t w o

i n d e p e n d e n t H S V - 2 i so la tes a n a l y s e d b y S D S a c r y l a m i d e gel e l ec t rophores i s ; we

h a v e also s t u d i e d p o l y p e p t i d e s i n d u c e d b y fou r d i f f e ren t s t ra ins of H S V - 2 a f t e r

h igh m u l t i p l i c i t y in fec t ion of H E p - 2 cells.

Materials and Methods

Cells

H u m a n epidermoid carc inoma no. 2 (HEp-2) cells were grown in monolayers in Eagle ' s m i n i m u m essentiM med imn (EMEM), supplemented wi th 10 per cent fetM bovine serum, penicill in (150 I .U . /ml ) and s t r ep tomyc in (150 Izg/ml).

Viruses

Two sets of t tSV-2 strains were selected for these studies. The first, des ignated as the l imited-passage strains, consisted of isolates f rom var ious locations in U n i t e d States and I ta ly . All of these s trains were passaged a l imi ted number of t imes in culture, usual ly at re la t ive ly low multiplici t ies. An average of 5 - - 7 passages at low mult ipl ici t ies (0.01 to 0.05 p laque-forming units/cell) were necessary to prepare s tock virus of high t i te r before pur i f ica t ion of enveloped virions. The second set, designated as unl imited-passage strains, consis ted of l abora to ry viruses whose h is tory was ei ther uncer ta in or known to include numerous passages outside the h u m a n host. The passage history, site of isolation, count ry of origin, and the number of independent purifica- tions of the th i r ty - two strains are given in Table 1. All strains were ident i f ied as HSV-2 on the basis of neut ra l iza t ion assays. The procedure for p repara t ion of H S V stocks was as described by Rolzs:rA~ and SPEA~ (20), except t h a t the cells were infected a t low mul t ip l ic i ty .

Solut ions and Chemicals

Cells were infected in phosphate buffered saline (6) supplemented wi th 1 per cent calf se rum (PBS-SV1). Maintenance med ium for infected cells was E M E M supplement- ed wit.h 1 per cent fet, M bovine serum. Label ing med ium consisted of E M E M 1 per cent dia lyzed ca.lf serum containing one t en th the usual concent ra t ion of all amino acids except for arginine, which was present at the usual concentrat ion, and a mix tu re of 16 14C labeled amino acids all wi th specific act ivi t ies of about 57 mCi /mAtom. The concent ra t ion of labeled amino acids ranged f rom 0.5 to 0.6 ~Ci/mt of label ing medium. Labeled amino acids were purchased f rom the Radioehemiea l Center, Amersham, England.

Product ion of Radiolabeled, Enveloped Vir ions

l%oller bo t t le monolayer cultures of H E p - 2 cells, conta ining approx ima te ly 108 cells, were infected wi th an input mul t ip l ic i ty of 5 - -10 P F U (plaque-forming units) of v i rus per cell, then incuba ted at 34 ° C in main tenance medium. The infected cells were labeled in 30 ml of labeling med ium from 6 hours post infection. The cells were harves ted at 24 36 hours post infection.

Puri] ieat ion o / E n v e l o p e d V i rus Particles

Virions were purif ied from cytoplasmic ext rac ts of infected cells essential ly ~s described by SPEAR and ~={OIZMAN (22) and modif ied by H m ~ E et al. (12). All steps in the pur i f icat ion procedure were per formed at 4 ° C. Briefly, cells labeled f rom 6 to 24 hours post infect ion were scraped off t he glass surface on which they were grown and col lected by low-speed eentr i fugat ion. The superna tan t f luid was decanted and the infected-cell pel let was suspended in app rox ima te ly 2 volumes of 1 m ~ phospha te buffer, p H 7.4. Af te r swelling in ice for 30 minutes , the cells were d is rupted wi th t en strokes of a t ight - f i t t ing ])ounce homogenizer . The nuclei were pel le ted by low-speed

S t r u c t u r a l P o l y p e p t i d e s of HtS¥-2 S t ra ins

T a b l e 1. Classi]ication and properties o /HSV-2 strains

37

Group C o u n t r y s t r a in of Site of de s igna t ion origin isola t ion

No. of inde- No. of pend - poly- ent aeryl - Source

Passage pur i - amide a n d a n d flea- gel refer- h i s t o ry t ions runs enee

G r o u p A

82 I T h i g h L P 2 4 0 86 I Th igh L P 2 4 0 118 I T h i g h U P 1 1 00 13 P V I T h i g h L P 2 2 0 174 I G en i t a l a reas U P 2 3 00 15 I Gen i t a l areas L P 2 2 0 6 7 G N U.S.A. Gen i t a l areas L P 2 4 d- 2 3 B D U.S.A. Gen i t a l areas L P 1 2 + H-445 U.S.A. Cerv ix L P 3 3 @ + H - 4 4 5 A Aggreg a t i ng I V a r i a n t s se lected U P 3 3 000 I{ -445P Po lyea r ioey togen ie ~ f rom H-445 L P 1 2 000 S-678 U.S.A. B r a i n L P 1 1 + + MS U.S.A. B r a i n U P 1 1 + + X 79 U.S.A. N e w b o r n U P 2 2 4

G r o u p B

3:33 U.S.A. Gen i t a l a reas U P 1 2 + -~-+ E-304 U.S.A. Gen i t a l areas L P 2 3 T-[ - 9 -GH U.S.A. Gen i t a l areas L P 2 3 -~ G U.S.A. Gen i t a l a reas L P 5 7 o F r e n c h U.S.A. Geni tM areas U P 1 2 -l- q- + Collins U.S.A. Gen i t a l a reas L P 1 2 + 316 U.S.A. Geni ta l a reas LI? 2 3 + -t- + 26 U.S.A. Gen i t a l a reas L P 1 1 + 29 LS U.S.A. Gen i t a l a reas L P 1 2 + 629 I Gen i t a l a reas L P 2 3 000 Segala I G en i t a l a reas L P 2 3 00 6 I Gen i t a l areas L P 1 2 0 H-420 U.S.A. Cervix U P 2 3 -- 6 0 D I U.S.A. Cervix L P 1 1 -~ 783 I Po r t i o L P 2 4 00 147 I Th igh U P 2 2 00 N V U.S.A. F i n g e r L P 2 2 00

Group C

83 I Gen i t a l a reas L P 2 4 0

A b b r e v i a t i o n s : I , I t a l y ; LP , l imi ted passage h i s to ry ; UP , cha rac t e r i zed b y n u m e r o u s passages outs ide t he h u m a n h o s t ; - - , S t r a i n f rom F. I~APP, H e r s h e y P e n n s y l v a n i a U n i v e r s i t y ; ~-, P. BALDUZZI, U n i v e r s i t y of Roches t e r , N .Y. ; + + , A. NAm~IAS, E r n o r y U n i v e r s i t y , A t l a n t a , Ga. ; + +- t~ , W. RAWLS, t t a m i l t o n College Toron to , C a n a d a ; o, B. t~OIZSIA~ T, Univers i t f f of Chicago, Ill . ; 0, G. GER?CA, U n i v e r s i t y of Pav ia , I t a l y ; 00, E . CASSAI, U n i v e r s i t y of F e r r a r a , I t a l y ; 000, M. BASERGA, U n i v e r s i t y of

Bologna , I t a l y

38 E. CASSAI et al. :

centr i fugat ion, and the cytoplasmic ex t rac t f rom 4 × 10 s cells was centr i fuged through a 35 ml dex t ran T10 densi ty gradient (1.04 to 1.09 g/eros prepared in 1 mM phospha te buffer) for 1 hour a t 23,000 rpm in the Beckman SW 27 rotor. The virions (as enveloped nueleoeapsids) were found in a l ight-scat ter ing band a t the middle of the tube. The band was aspira ted by punc tur ing the side of the tube wi th needle and syringe, d i lu ted to 36 ml wi th 1 m~i phosphate buffer, and centr i fuged a t 25,000 rpm for 1 hour in the S~V 27 rotor to pel le t the virions.

The radio labeled virions were resuspended in 0.15 to 0.30 ml of 1 m ~ phospha te buffer, depending on the pel let size, and s tored a t - -80 ° C before solubil izat ion and eleetrophoresis on po lyaery tamide gels.

Labeling o/ Proteins Synthesized by In/ected and Unin/eeted Cells

Confluent H E p - 2 cell monolayers in tissue cul ture flasks (approximate ly 2 × 106 cells per flask) were exposed to 20 P F U of virus per cell (in 1 ml of ma in tenance medium) or were mock infected with 1 ml of this medium. Inocu la ted cultures were incubated wi th constant agi ta t ion for 1 hour, at 36 ° C, and thereaf te r virus or mock inoeula were decanted. The monolayers were then r insed (5.0 ml of ma in tenance med ium per flask), replenished wi th 5.0 ml of ma in tenance med ium per flask, and re incuba ted at 34 ° C. Label ing took place at the t imes indicated in the figures as hours post virus addit ion. Label ing med ium conta ined normal amounts of arginine, 1/lo the concentra- t ion of o ther aminoacids, and 1 per cent fetal bovine serum, t4C-Iabeled aminoae id mix tu re was used a t a concent ra t ion of 5 ~u.Ci/ml. A t the end of the labeling period the cells were rinsed wi th iee-eold phosphate-buffered saline (3 × 5.0 ml/flask) to t e rmina te incorpora t ion ("pulse") and then were ha rves ted by scraping and s tored frozen a t - -80 ° C unt i l subsequent po lyaery lamide gel electrophoresis.

Polyaerylamide Gel Eleetrophoresis

The electrophoret ic , stMning and autoradiographie techniques were as described previous ly (8, 12, 18, 21), employing the discontinuous buffer sys tem (4, 17) modif ied by the inclusion of SDS (5, 14). The solution from which the main gel was polymer ized conta ined 0.1 per cent (w/v) SDS, 0.03 per cent (v/v) T E M E D , 0.035 per cent (w/v) a m m o n i u m persulfate, 8.5 per cent acrylamide, 0.224 per cent N-N ' -d ia t ly l - t a r ta r - diamide, 0.375 ~ Tris-hydrochlor ide p H 8.9 or 8.5. The s tacker gel consisted of 0.125 M Tris-hydrochlor ide p H 7.0, 0.1 per cent (w/v) SDS, 0.03 per cent (v/v) T E M E D , 0.07 per cent (w/v) a m m o n i u m persulfate, 3 per cent aerylaraide, 0.08 per cent N-N ' - d ia l ly l - tar tard iamide . The electrode buffer at p H 8.35 conta ined 0.025 ~ Tris base, 0.192 ~ glycine ~nd 0.1 per cent SDS. I m m e d i a t e l y prior to electrophoresis, the pro- teins were dena tu ra ted and solubilized by the addi t ion of concent ra ted reagents to yield final concentra t ions of 0.05 ~ Tris-hydrochIoride (pH 7.0), 2 per cent, SDS, 5 per cent ~-mercaptoe~hanoI, 3 per cent sucrose and 0.005 per cent b romophenol blue followed by boiling for 2 minutes.

The solubilized proteins in about 50 ~.I volumes were subjected to eleetrophoresis at, a constant current of I0 rnA. The gels were stMned with Coomassie brilliant blue, destained in acetic acid and propanol, and dried. Analyses of radioactive proteins were done by autoradiography with Cronex X-ray film (Dupont).

Results

Variations in the Etectrophoretic Mobility o / H S V - 2 Structural Polypeptides

I n F i g u r e 1 are s h o w n 14C-aminoae id- labe led p ro te ins p r e sen t in v i r ions f r o m

d i f f e r en t H S V - 2 isolates. T h e n u m b e r s ass igned to i n d i v i d u a l p o l y p e p t i d e b a n d s fo l low t h e de s igna t i on ass igned b y CASSAI et al. (3). T h e e l e e t r o p h o r e t i c ana lys i s has s h o w n v e r y few d i f fe rences in t h e s t r u c t u r a l p ro t e ins speci f ied b y t h e t h i r t y t w o

Structural Polypeptides of HSV-2 Strains 39

HSV-2 strains examined. The most significative differences concern the poty- peptide bands designated VP 12 and VP12.8. Every strain examined contained one of these two polypeptides, but no strain contained both. Moreover, comparing the different electrophoretic mobility of VP 14 of HSV-2 83 with VP t4 of all the other isolates, we have clustered the thirty two HSV-2 strains into three groups. The thirty two strains are distributed almost uniformly into two groups (A and B) : exactly fourteen strains in group A (absence of VP 12 and presence of VP12.8), and seventeen in gToup B (presence of VP 12 and absence of VP 12.8). Group C in- eludes only HSV-2 83, which differs from group A strains merely for a faster migration of VP 14. Note that HSV-2 82 and 83 differ with respect to the migra- tion rate of VP 14, although these two strains were isolated from two sexually related partners. Other small variations can be seen in some polypeptides with a lower molecular weight (Fig. 1).

Fig. 1. Autoradiogram of an SDS-potyaerylamide gel slab containing eleetrophoretieally separated polypeptides of representative I-ISV~2 strains. The single letter at the top identifies the group in which the strain was placed. The designation below the letter identifies the strain. The origin and other data eoneerning the strains are listed in

Table 1. The pit of resolving gel used was 8.9

40 E. CASSAI et al. :

Of the thirty two strains examined, fifteen were independently purified and subjected to electrophoresis at least three times and an additional five strains were analyzed in this manner twice; and in each instance, the electrophoretie profiles were identical. As regards phenotypie stability, we can say that the strains characterized by numerons passages outside the human host are similar to those strains with limited passage. Thus no difference was observed between strain H445A (passaged extensively in HEp-2 cells) and parental strain H445 (limited passage history).

Fig. 2. Aut.oradiogram of an SDS-polyaerylamide gel slab. A comparison of the poly- pept, ides induced by 1tSV-2 strains (G, 82) at 1--2, 3--4, 7---8, 11----12 hours after

infection of HEp-2 cells

Comparison o /Polypept ides Induced by H S V - 2 Strains

Since the analysis of structural proteins revealed very little variability we have examined the differences in proteins synthesized in HEp-2 cells after infection with different HSV-2 strains. In this experiment, cells were infected with four different strains of HSV-2 (G and 9 GH from group B; 82 and 86 from group A). Infected or mock infected cells were pulse-labeled with t4C amino acids from 1 to 2, 3 to 4, 7 to 8 and 11 to 12 hours after infection and thenharvesged, solubilized and subjected to electrophoresis on 8.5 per cent acrylamide gels. All four strains of HSV-2 inhibited host protein synthesis within the first two hours after infection as

Structural Potypeptides of HSV-2 Strains 41

already observed by POWELL et al. (19). Some polypeptides (for example 4, 12, 28) were synthesized, only during the first hours post-infection, while other poly- peptides (as 15, 23, 33) were not synthesized until 7--8 hours post infection. The polypeptides of mock-infected HEp-2 cells and those induced by two different strains of HSV-2 (82, G) at various times after infection are shown in Figure 2. The most significant differences among polypeptides synthesized by these two strains and by strains 9 GH and 86 (data not shown) could be seen in ICP 11--18, 23--27 and 27--29.

Fig. 3. Autoradiogram of an SDS-polyacrylamide gel slab showing the 14C amino acid- labeled polypeptides present in virions from five independent isolates of HSV-2 and from four of ttSV-1. The HSV-1 polypeptides designated 5, 12, 22 (4-) do not have identical counterparts in all of the HSV-2 virions analyzed by us. The pH of the main

geI used was 8.5

Comparison o/the Structural Polypeptides o/HSV-2 and HS V-1

I t is apparent from the profiles shown in Figure 3 tha t HSV-2 virions contain approximately the same number of polypeptide species as HSV-1 virions. I t is also apparent tha t some of the polypeptides found in HSV-1 virions are not present in identical form in HSV-1 virions and vice versa. A study of the structural poly- peptides specified by 53 different HSV-1 strains revealed tha t certain polypeptide

¢2 E. CASSAI et al. :

bands with characteristic eteetrophoretie mobilities in SDS gels were detected in all the strains (including I-ISV-1 analyzed by us) (18). Of these bands, at least three (designated VP5, 12 and 22 in Fig. 3) were not detected in identical form in any of the HSV-2 isolates analysed in this study. The positions of these I-ISV-1 poly- peptides are marked by the filled triangles in Figure 3. Our studies show- that VP5 has the same eleetrophoretic mobility in all the 32 HSV-2 isolates, but it is slower than VP5 of HSV-1 (Fig. 3). These results, according with those previously pub- lished by CASSAI et al . (3), were obtained using a resolving gel at pH 8.5 (as de- scribed in Material and Methods). Using a resolving gel at pH 8.9, the polypeptide band designated VP7 was dear ly dissociated into two distinct bands in all the HSV-2 isolates examined, while in the HSV-1 strains analysed by us the poly- peptide band VP 7 migrated always as a single band, using both a resolving gel a t pH 8.9 and at p H 8.5. This behaviour is shown in Figure 1 and 4. In Figure 4, are compared 4 HSV-2 and 4 HSV-I strains belonging to 3 different groups according to the classification made by P~R~IRA et al. (18).

Fig. 4. Autoradiograms of two SDS-polyacrylamide gel slabs containing electro- phoretieMly separated polypeptides of four Iq[SV-2 and four I-ISV-1 strains. The pIKs

of t, he main gels used were 8.9

Discussion

The results presented in this report show that the 32 HSV-2 strains can be classified into three groups according to the presence and the electrophoretie mobility of a few structural polypeptides in SDS-poiyacrylamide gels. In view of

Structural Polypeptides of HSV-2 Strains 43

the observation that the two most numerous groups differ from each other mainly by the presence or absence of VP12 and VP12.8, it is clear that these electro- phoretic analyses reveal very few differences in the structural proteins specified by these 32 HSV-2 isolates.

The results presented here suggest a substantial homogeneity among these HSV-2 isolates. However, it is likely that application of high resolution techniques such as two-dimensional separation of viral structural proteins will make apparent more subtile differences between HSV-2 isolates. In support to this hypothesis, it may be recalled that early analyses on DNAs extracted from different HSV-1 and HSV-2 strains with restriction enzymes (10, 11, 2) could be interpreted to indicate that DNAs from HSV-2 strains were more homogeneous than those from HSV-1 strains; more recent studies (T. G. Bucns~A~ et al., manuscript in preparation) in- dicate that HSV-2 strains are as variable with respect to the location and presence of restriction enzyme cleavage sites as the DNAs of HSV-1 strains.

Although the studies described in this article were not designed to investigate the possible relationship between structural polypeptides and site of localization in the human body, certain trends are readily apparent. Thus, the distribution of strains into groups by country of origin (Table 2) failed to show clear evidence of geographic clustering, but the number of strains was too low to permit a de- finitive conclusion. On the other hand, the clustering of isolates from genitalia in group B and from other parts of the human body in group A (Table 3) could sug- gest that there might be a relationship between the biochemical properties of virus and its location.

Table 2. Distribution of H S V-2 strains according to country of origin,

Frequency distribution of isolates among groups

Country of isolation A B C

lJnited States 6 12 -- Italy 6 5 1

Table 3. Distribution o] H S V - 2 strains according to site o/ isolation on human body

Frequency distribution of isolates among groups

Site of isolation A B C

Genitalia 4 12 Cervix and portio 1 3 Brain 2 Thigh 4 1 Newborn 1 Finger s i

We have isolated this virus from the finger of a researcher who previously had been infected in the same finger by a needle contaminated by ttSV-2 (G). In this table have not been considered H445A and H445P, two variants isolated from ttSV-2 H445

44 E. CASSAI et al. :

From the comparison of proteins synthesized in ItEp-2 cells infected with four different HSV-2 strains, labeled for one hour at different times from infection, the most significant differences have appeared in infected celI polypeptides 1 t through 29. In the analysis of polypeptides of infected ceils we have chosen two HSV-2 strains (G and 9GH) from group B and two ItSV-2 strains from group A (82 and 86). Our design was to determine if the differences in polypeptides from infected cells reflected the differences seen in the structural proteins from purified virions. In Figure 2 it is shown the comparison among polypeptides from cells infected with G and 82 strains. Our results show that there are variations in the eleetrophoretic mobility of infected cell polypeptides isolated from cells infected by HSV-2 strains from different groups. Furthermore, strains showing the same structural polypeptides do not necessarily have the same ICPs (data not reported).

The H S V - 2 isolates could be differentiated from ItSV-1 on the basis of the electrophoretie mobilities of certain polypeptides that appear to be characteristic of each serotype. Specifically, the major eapsid protein of HSV-1, VP5, migrates slightly faster than those of HSV-2. Two prominent HSV-1 polypeptide bands, designated VP12 and VP22, have electrophoretie counterparts in all HSV-1 isolates examined to date, but not in any of the HSV-2 isolates analysed in the study (Fig. 3).

Moreover, in all the HSV-I strains analysed the polypeptide band designated VP7 is always compact and in the same position when pH of main gel is 8.9. On the contrary, in all the HSV-2 strains studied by us VP7 appeared dissociated into two sharp bands. The behaviour of VP7 in HSV-2 and HSV-1 could be due to a different glyeosylation of t.he protein. Since SDS makes negative all the charges of the proteins (1), but does not bind to the carbohydrate groups, a rise in pi t could change the charges of the carbohydrate moieties so that at pH 8.9 it dis- soeiates into two bands that appear as a single band in HSV-2 at pH 8.5. A second hypothesis could be that one of the two bands is formed by a precursor of the fully glyco@ated form. Whatever the correct hypothesis may be, it is pos- sible to say that this different behaviour of VP7 according to pH changes (8.5 or 8.9) is a peculiar characteristic of HSV-2.

Acknowledgments This work was supported by grants from the Consiglio Nazionale delle Ricerche,

Progetto Finalizzato Virus grant 79.00379.84, and from NATO grant 1225.

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Authors' address: Dr. E. CASSAI, Isti tuto di Microbiologia dell' UniversitY, Via Luigi Borsari, 46, 1-44100 Ferrara, Italy.

Received July I0, 1979