Archives of Virology

Archives of Virology 55, 293--304 (1977}

© by Springer-Verlag 1977

Studies on Glyeopeptides of Herpes Simplex Virus Infeeted Cells By S. OLOFSSO~ and J. BLOMBERG Department of Virology, Institute of Medical Microbiology, University of GSteborg, GSteborg, Sweden With 6 Figures Accepted July 13, 1977

Summary Trypsinates from HSV infected cells, radioactively labeled with glucosamine and galactose were further digested by pronase. The digests were subjected to gel filtration, and it was shown that some of the eluted material consisted of saccharides, highly devoid of residual peptide. This material elated between linear dextran markers of 19,000 and 3000 daltons. Only slight differences between uninfected and HSV infected cells in galactose and glucosamine derived radioactivity profiles of the ehromatograms were detected. High molecular weight glucosamine labeled material, present in digest from uninfected cells, was not detectable in digests from HSV infected cells. The concanavalin A adsorbability of Muted fractions was tested. Changes in the relative adsorbability between materials from HSV and uninfected cells were present, especiM]y for the g]ucosamine l~beled saccharides.

Introduetion Herpes simplex virus (HSV) specifies at least four glyeoproteins in HSV infected cells (t3, 17). They are present in the plasma membrane and other parts of the smooth cell membranes (7, 16) and during virus maturation the glycoproteins become integral parts of the viral envelope (t7). Viral glycoproteins conceivably participate in several reactions initiated by the virus such as attachement of virus, intracellular transport and virus assembly (3, 7, 8, 17) and are, in addition, at, least partly responsible for the altered social behaviour of cells in ttSV infected cultures (10). The polypeptide parts of HSV induced glycoproteins are most likely coded for by the viral genome, but as for all oligosaccharides of viral glycoproteins, it is unknown to what extent the structure of the oligosaccharide moieties is virus-specified.

29~

S. O5OFSSON

and J. BLOXBERG:

H o ~ s s a n d ROIZMAN s t u d y i n g the time course of glycosylation of viral glyeoproteins (8) f o u n d a stepwise addition of sugars in the f o r m a t i o n of glycopeptides i n HSV infected cells. BI~]~xA~ et al. (t) showed t h a t the galaetose m e t a b o l i s m is altered u p o n H S V infection. 14C-galaetose label was f o u n d p a r t l y incorporated as glucose in glyeoproteins b u t the glucose p a r t of the label greatly decreased after H S V infection. I n the present report gtycopeptides from the surface of u n i n f e c t e d a n d H S V infected cells were prepared b y t r y p s i n a n d pronase digestions. The molecular weight d i s t r i b u t i o n of glycopeptides labeled ~dth precursor sugars was d e t e r m i n e d b y gel filtration. S u b s e q u e n t l y , the fraetionated glyeopeptides were assayed for their affinity to c o n c a n a v a l i n A (Con A).

Materials and Methods Virus and Cells A I-ISV type 1 strain, F, (supplied by Dr. B. Roizman, Chicago) and an established line of Green Monkey Kidney cells GMK AI-t-1 (6), were used throughout the study. The cells were grown in Eagle's Minimal Essential Medium. Radiochemicals L-(4,5-'~H) Leucine (58 Ci/mmol), D-(L3H) galaetose (5 Ci/mmol), D-(1-3H) galactose (5 Ci/mmol), D-(1-~I-I) glueosamine (3 Ci/mmol), L~(UA4C) leueine (50 mCi/mmol), D-(1-14C)-glueosamine (59 mCi/mmol) and D-(1-14C) galac~ose (60 mCi/mmol) were purchased from the Radiochemical Centre, Amersham. Chemicals P-nitrophenyl glycosides of s-D-glucose, ~-D-glucese, ~-D.gaiactose, ~-D-galactose, ~-D-mannose and ~-N-aeetyl-glueosamine from Sigma Chemical Corp were used for control experiments. The proteolytie enzymes were Protease from Streptomyees griseus (Sigma, type VI), "pronase", and crystalline trypsin (Novo industri AS Kobenhavn). Sephadex G 100, Blue Dextran 2000, Con A-Sepharose, FITC-Dextran 20, (Fluoreseein isoghiocyanate-eonjugated dextran with a mean molecular weight of 19,000) and FITCDextran 3 (mean M.W. 3000 daltons) were purchased from Pharmaeia Fine Chemicals, UppsMa, Sweden. Preparations o/ Trypsinates The trypsinates were prepared essentially as described by BL~e~ et al. (2). Uninfected and HSV in.leered (20 PFU/eell) roller cultures were radioactively labeled from 5 to 20 hours post-infection. Then the cells were washed three times in TBS (0.15 ~I NaCI, 0.02 M Tris HC1, p H 7.5) and 7 ml of trypsin in TBS (1 mg/ml) was added to each bottle. The bottles were incubated in the roller for 15 minutes at 37 ° C. The resulting suspension was transferred to a centrifuge tube, chilled in an ice bath and centrifuged at 1000 × g for t0 minutes. 85--95 per cent of trypsinated infected and uninfected cells excluded t r y p a n blue, which indicated a high degree of viability. The supernatant from this centrifugation is subsequently referred to as the trypsinate. Pronase Digestion The pronase digestion was carried out according to SPIno (18). The reaction mixture consisted of 0.1 ml pronase (10 mg/ml, sterile filtered by Millipore Millex 0.45 ~ filter) dissolved in TBS and 10 ml trypsinate. Antibiotics (penicillin, 40,000 units, and streptomycin sulphate, 40 nag) and one drop of toluene were added to prevent microbial growth. The pronase was allowed to digest for five days at 37 ° C. After three days of digestion another 0.1 ml of pronase was added. The digests were lyophilized and stored at --20 ° C before further analysis.

G l y e o p e p t i d e s of H S V I n f e c t e d Cells

295

To d e t e c t t h e possible p r e s e n c e of glycosidases, 0.1 m l from t h e t r y p s i n a t e s a n d t h e p r e t e n s e s o l u t i o n were a d d e d to 3 m l of e a c h of t h e p a r a n i t r o p h e n y l glycosides m e n t i o n e d u n d e r " C h e m i e M s " as 6 mM s o l u t i o n s i n T B S . T h e r e a c t i o n was m o n i t o r e d a t 410 n m in a B e c k m a n D B s p e c t r o p h o t o m e t e r ( S t a n d a r d s c~-mannosidase, ~-gMactosidase a n d e - g l u e o s i d a s e ; S i g m a C h e m i c a l s corp.) w i t h a l i m i t of d e t e c t a b i l i t y of a b o u t 3 n a n o c a t a l s p e r m l i n t h e 6 m ~ s u b s t r a t e . No glyeosidase a c t i v i t y was d i s c e r n i b l e in t h e t r y p s i n a n d p r o n a s e s o l u t i o n s u s e d in t h e digestions.

Control o/Glycol@id Content o] Pronase Digests A s l i g h t l y m o d i f i e d F o l c h p a r t i t i o n (14) of t h e p r o n a s e digests in c h l o r o f o r m / m e t h a n o l / w a t e r g a v e 4 a n d 9 p e r c e n t of t h e g l u e o s a m i n e a n d g a l a e t o s e c o u n t s r e s p e c t i v e l y i n t h e c h l o r o f o r m p h a s e . T h i n l a y e r c h r o m a t o g r a p h y (TLC) o n Silica gel H (0.25 r a m , 20 × 20 era) i n c h l o r o f o r m . / m e t h a n o ] / w a t e r 6 5 : 2 5 : 4 r e s u l t e d i n m i g r a t i o n of 5 a n d 6 p e r c e n t of t h e c o u n t s , respectively. T h u s t h e p a r t i t i o n a n d T L C e x p e r i m e n t s i n d i c a t e d t h a t n o t m o r e that1 a b o u t 5 a n d 7 p e r c e n t of t h e g l u e o s a m i n e a n d g a l a c t o s e l a b e l r e s p e c t i v e l y were d u e t o t h e p r e s e n c e of t h e c o m m o n glycolipids w i t h less t h a n five s u g a r s i n t h e p r o n a s e digests.

Gel Filtration Analysis 0.2 m l s a m p l e s of r e h y d r a t e d t r y p s i n a t e s or p r o n a s e digests were p l a c e d o n t o p of 800 × 9 m m c h r o m a t o g r a p h y c o l u m n s c o n t a i n i n g S e p h a d e x G 100. I n o r d e r t o o b t a i n good solubilizing a n d d i s s o c i a t i n g c o n d i t i o n s , t h e c o l u m n s were e l u t e d w i t h T B S c o n t a i n i n g 0.1 p e r c e n t 2 - m e r c a p t o e t h a n o l , 0.5 p e r c e n t l a u r o y l s a r e o s i n a t e a n d 0.01 p e r c e n t E D T A (2 m l / h o u r ) . B l u e D e x t r a n w a s u s e d t o define t h e v o i d v o l u m e . F I T C - d e x t r a n s of 19,000 a n d 3000 d a l t o n s , were u s e d as l i n e a r p o l y s a e c h a r i d e m o l e c u l a r w e i g h t m a r k e r s . 0.4 m l f r a c t i o n s were t a k e n a n d subj coted to liquid s c i n t i l l a t i o n d o u b l e isotope a n a l y s i s (11).

Concanavalin A A //inity F o r m e a s u r e m e n t s of t h e p e r c e n t a g e u n a d s o r b e d , r e v e r s i b l y a n d i r r e v e r s i b l y a d s o r b e d m a t e r i a l s , m i n i c o l u m n s were p a c k e d in p a s t e u r p i p e t t e s c o n t a i n i n g one m l of a 50 p e r c e n t s u s p e n s i o n of Con A - S e p h a r o s e in P B S . A f t e r w a s h i n g w i t h 2 m l of P B S (0.14 ~ NaC1, 3 m ~ KC1, 8 m ~ N a H P O 4 , 1 r n ~ K H 2 P 0 4 ) 0.05 t o 0.2 m l of t h e a p p r o p r i a t e l y l a b e l e d p r o n a s e digest, w h i c h h a d b e e n t r e a t e d a t t 0 0 ° C for 10 m i n u t e s in o r d e r to r e m o v e p r o t e o l y t i c a c t i v i t y (see below) was p l a c e d o n t h e c o l u m n . T h e u n a d s o r b e d m a t e r i a l was e l u t e d w i t h 5 m l of P B S . S u b s e q u e n t l y t h e r e v e r s i b l y a d s o r b e d m a t e r i M (of. T a b l e 1) w a s e l u t e d b y 2 m l of 2 p e r c e n t (w/v) M p h a m e t h y l m a n n o s i d e (c~-MM) h~ P B S . T h e r a d i o a c t i v i t y r e m a i n i n g o n t h e c o l u m n was a s s a y e d f r o m a s u s p e n s i o n of t h e gel in 5 m l P B S ( C o l u m n f r a c t i o n of T a b l e 1). As m e n t i o n e d a b o v e , t h e p r o n a s e digests h a d to b e t r e a t e d a t 100 ° C for 10 m i n u t e s to i n a c t i v a t e t h e e n z y m e a c t i v i t y before c o n t a c t w i t h t h e Con A a d s o r b e n t . T h e condit i o n s for t h e p r o n a s e i n a c t i v a t i o n were t e s t e d in s e p a r a t e e x p e r i m e n t s . T r a n s f e I ~ n l a b e l e d b y 14C-acetic a n h y d r i d e was i n c u b a t e d w i t h a p r o n a s e p r e p a r a t i o n w h i c h h a d b e e n h e a t e d a t 70 ° a n d 100 ° C for d i f f e r e n t times. T h e d i a l y z a b l e r a d i o a c t i v i t y r e l a t i v e t o t h a t of c o n t r o l s t r e a t e d w i t h P B S w a s t a k e n as a m e a s u r e of p r o t e o l y t i e a c t i v i t y . C o m p l e t e i n a c t i v a t i o n w a s difficult t o o b t a i n a t 70 ° C, b u t a t 1 0 0 ° C t h e a c t i v i t y d i m i n i s h e d r a p i d l y w i t h a hMf t i m e of a b o u t 1.5 m i n u t e s , a n d n o a c t i v i t y was d e t e c t a b l e a f t e r 8 m i n u t e s . C o n s e q u e n t l y , 10 m i n u t e s of i n a c t i v a t i o n was c h o s e n for t h e f u r t h e r experimentation. To 0.3 mI of e a c h gel tilt,r a t i o n f r a c t i o n 0.5 m l of a s u s p e n s i o n of C o n - A - S e p h a r o s e was a d d e d . T h e a d s o r b e n t t a k e n d i r e c t l y f r o m t h e p a c k a g e w a s u s e d s u s p e n d e d 1 : 8 in P B S . A f t e r occasional s t i r r i n g a t r o o m t e m p e r a t u r e for 30 m i n u t e s , t h e gel f i l t r a t i o n f r a c t i o n s were i n d i v i d u a l l y w a s h e d t h r e e t i m e s w i t h 5 m l P B S o n G F / C glass fiber filters i n a Millipore m u l t i f i l t r a t i o n a p p a r a t u s . A f t e r d r y i n g , t h e filters were c o u n t e d w i t h t h e I n s t a - g e l s c i n t i l I a t i o n c o c k t a i l i n a n I n t e r t e c h n i q u e l i q u i d scintilla%ion c o u n t e r c a l i b r a t e d for d o u b l e isotope a n a l y s i s (11).

296

S. OLOFSSONand J. BLOW,BEInG:

Results

E//icacy o/ the Pronase Digestion Procedure Figure 1 shows results of Sephadex G 100 gel filtrations of aH-leucine labeled trypsinates of infected cells, before and after pronase digestion. I t is seen t h a t in the untreated trypsinate the leucine label is fairly homogenously distributed throughout the ehromatogram, indicating the presence of peptides with molecular weights of up to at least 80,000 daltons. After pronase treatment, however, almost all the leucine radioactivity was found as low molecular m a t t e r in the totally ineluded fraction. T h e rest of the leueine label, 0.5--1.5 per cent, is found in the totally excluded fraction. Similar results were obtained for uninfected cell trypsinates. The results were interpreted as indicating t h a t the material eluted between the void volume and the totally included volume to a high extent was devoid of peptide chains and probably consisted of the oligosaecharide portions of glyeoproteins, with one or only a few amino acid residues attached. This glycopeptide material is in the following referred to as heterosaecharides (19, 8). 3H C )m x I03 108642-

10~ 40 BD

50

60

70

80

9b~ Fractionno. 'I'-R

Fig. 1. Sephadex elution profiles from sI-I-leucine labeled trypsinates of HSV infected cells before ( -- o -- o -- ) and after ( -- o -- o -- ) pronase digestion. BD and (p-R indicates the position of Blue Dextran and phenol red in the two experiments

Gel Filtration o/Pronase Digests From Glucosamine Labeled Cells HSV-infected and uninfected GMK-cells labeled with 14 C-glucosamine a n d 3 Hg h c o s a m i n e , respectively, were trypsinized and further subjected to pronase digestion. The digests were filtered on Sephadex G 100 g e l The elution diagram obtained was divided into four regions ( I - - I V ) , as shown in Figure 2 A. I n the c h r o m a t o g r a m of uninfected cells the first region, incorporating the Blue D e x t r a n marker, demonstrated one prominent peak not observed with HSVinfected cells. Thus, there seemed to be high molecular weight saccharides in uninfected cells not synthetized b y the HSV-infected cells. The second chromatographic region was represented b y material eluting immediately before and after the F I T C - d e x t r a n marker having a molecular weight of 19,000 daltons. The sI-I peak, observed with digests of uninfected cells, h a d a relatively well defined gaussian shape, while the 14C radioactivity graph, of the

297

Glycopeptides of HSV Infected Cells

HSV-infeeted cells appeared more extended, forming a plateau after its maximum. The results suggested that in HSV infected cells glycopeptides of region I I had a more heterogeneous size distribution than glycopeptides of uninfected cells. The third region was located between the two FITC-dextran markers with molecular weights of t9,000 and 3000, respectively. Only minor differences were observed between results with uninfected and HSV infected materials. Both ehromatograms demonstrated in this region one major peak of radioactivity with the same localization. The fourth region of the chromatograms which consisted of the totally included material, probably representing free ghcosamine and sialic acid and their derivatives, revealed no qualitative difference between infected and uninfected cells. 3H CPM x 10.3 3-

3

2-

-2

I

-I

I

I

TT

I

m

I

IK

tB

3

-3

2-

-2

I-

-I

Fraction no.

BD

FI9

Fig. 2. Sephadex G 100 elution profiles of glucosamine (panel A) and galactose labeled (panel B) pronase digests of trypsinates from uninfected (3H label . - - . - e--) and HSV infected (14Clabel -- o -- 0 -- ) GMK cells. BD and ?-t~ indicates the positions of Blue Dextran and phenol red. F 19 and F3 represented the position of the dextran molecular weight markers of 19,000 and 3000 daltons, respectively

298

S. OLOFSSOr¢and J. BLOMBERG:

To avoid misinterpretations due to differences in metabolic fates and isotopic effects of the 14C and ~tt labels in the glyeopeptides, the experiments were carried out also with reversed labels i.e. 14C-glueosamine labeled HSV-infeeted cells and 3H-glueosamine labeled uninfected cells. The results obtained did not differ from those described above. Furthermore, digests of 3H and 14C glueosamine labeled HSV-infeeted as well as a H and 14C glueosamine labeled uninfected cells were subjeered to G 100 gel filtration. Congruent 14C- and 3H-radioactivity profiles were observed in both cases indicating that the results displayed in Figure 2 did not reflect artefacts due to differences in metabolism of aH and 14C in the 1 position of glncosamine.

Gel Filtration o/Pronase Digested Galactose Labeled Cells Galaetose labeled digests were studied chromatographically (Fig. 2B) using the same woeedure as in the experiments described previously. In contrast to the ehromatograms of the glueosamine-labeled materials the first chromatographic region contained both 14C and 3 H radioactivity peaks, i.e. revealed no qualitative differences between digests of uninfected and HSV-infeeted cells. The second region contained a broad plateau of 14C radioactivity of HSV-infected cells but only small amounts of 3H labeled galaetose of uninfected cells. With galaetose labeled cells no radioactivity appeared in region I I I of the ehromatograms. The fourth region again, containing the totally included material, and probably representing free gtaetose and low molecular metabolic derivatives like glucose, nueleosides and nueleotides, revealed no qualitative differences bet,ween HSV-infeeted and uninfected cells. The same type of controls as described above i.e. reversed and double isotope labeling, were performed. No metabolic differences between the a H and 14C galaetose labeled materials were found.

A//inity to Con A Sepharose Table 1 presents the percentages of radioactively labeled glucosamine and galactose which adsorbed to Con A Sepharose. In addition, the relative amounts of reversibly and irreversibly bound oligosaeeharides were estamined by determining the pereentzges of labeled material which could or could not be eluted b y ~-~IM. The inactivation of pronase in the digestion mixtures b y brief boiling m a y cause minor Iosses of terminal siatic acid and fucose. These sugars do not interact with Con A. Preliminary tests of digests made with gel bound pronase before and after heating indicated at most minor changes in the Con A affinity of the labeled glycopeptides. In comparison to results with HSV infected cells, significantly more of the glueosamine label in uninfected materials was adsorbed to the Con A Sepharose. This difference was mainly due to the ]urger proportion of material which was not elutible with ~-~rl. A different picture was observed when digests of galaetose labeled materials were analyzed. Again, more of the label of uninfected than of HSV infected cells material was adsorbed to Con A Sepharose. However, the difference was now found in the relative amounts of material released with ~-515~. Thus, in digests of uninfected cells 58 per cent of the label was adsorbed to Con A Sepharose (52.5 per

Glycopeptides of HSV Infected Cells

299

cent elutibte with :¢-MH and 5.5 per cent non-elutible). Of the HSV infected ceil material 37 per cent of labeled galactose adsorbed to Con A Sepharose, and most of the adsorbed label could be eluted b y ~-MM. I t should be mentioned that a second run of fractions, which had once passed the Con A Sepharose columns unadsorbed, were recovered in 95--98 per cent yields in the unadsorbed fraction both for glucosamine and galactose labels. Table 1. A ]]inity to Concanavalin A o/ glucosamine (3H-GLCN or J4C-GLCN ) and galactose (3H-GAL or 14C-GAL) labeled pronase digested glycoproteins o] unin]ected and H S V injected G M K cells Per cent label (Means

and SEM)

Cells

Label

No. of tests Unadsorbed

Eluted with ~ ~M

Retained in cohunn

Uninfected HSV infected Uninfected HSV infected

8H GLCN 14C GLCN 3H & laC GAL 3I~I & z4C GAL

3 3 6 6

4.3±0.3 4.7±0.3 52.5±2.1 32.5±2.0

1t.3~2.8 2.3±0.3 5.5-kl.2 4.3=k0.6

84.3±2.7 93.6=k0.3 42.3-4-4.7 63.5=k2.2

More detailed information on the Con A binding glycopeptides species was obtained from studying the adsorption of the individual fractions after gel filtration. I n Figures 3 and 4 patterns of uninfected and HSV infected GMK cells labeled with glucosamine are depicted. The Con A binding portions are shown as hatched areas. In fractions of uninfected cell material (Fig. 3), Con A binding material was noted as one peak located immediately behind the void peak (region II). The Stoke's radius would correspond to that of a linear polysaeeharide of about 20,000 daltons, iknother broad peak of low molecular weight (corresponding to a linear oligosaeeharide of 3000--8000 daltons) was present in the valley between the gel filtration pe~ks of regions I I I and IV. I t was, therefore, detectable only b y the Con A adsorption analysis. I n gel filtrations of ItSV infected cell materials (Fig. 4) only low molecular weight Con A binding glyeopeptides were indicated. The broad peak observed seemed to correspond to the one observed with uninfected cell materials. Thus, after HSV infection Con A binding high molecular weight saccharides seemed to disappear. In separate experiments, the material remaining in the columns after ~ - ~ elution was further investigated. 2 ml of 3.4 M ammonium thiocyanate (AMT) added to the columns elutcd in additional 40 per cent of the radioactivity. This occurred irrespective of the type of label used or if the cells were infected or not. The fractions eluted with ~-MM and AMT were then separately subjected to gel chromatography on Sephaclex G 100. The ~-MM fractions were characterized chromatographically b y a void peak and a peak just preceding the totally included material (region IV). The AMT fraction contained an additional peak in region I I . Thus, the Con A binding glucosamine containing glyeopeptides of high affinity, which seemed to decrease after ttSV infection, appeared chromatographically as a peak in region I I with a Stoke's radius corresponding to t h a t of a linear polysaccharide of about 20,000 daltons. Arch. Virol. 55/4

20

300

S. OLOFSSO~

and J. BLOZ~BEBC~:

Figures 5 a n d 6 e x h i b i t the corresponding results ,~dth galaetose labeled materiM. Most of the galactose labeled m a t e r i a l which was adsorbed to Con A was r e c o v e r e d as a p e a k in t h e void fractions. These a p p a r e n t l y high molecular polysaceharides occurred in b o t h u n i n f e c t e d a n d H S V infected cells b u t decreased as a result of t h e infection.

I

II

1

III

l i~-~

3H 30CPIOMxlO-3 2520t5105-

4x BD

40

60

80

4x Fraction ~'Rn °

Fig. 3 14C CPIOMxl0 -2

I

II

Ill

IV

252015lOS-

40

60

BD

80

T Fraction no. "fR

Fig. 4 Fig. 3. Assay of eoneanavalin A adsorbability of ~H glueosamine labeled pronase digests of trypsinates from uninfected GMK cells. The hatched area under the graph with open circles denotes the amount of material binding to Con A Sepharose tested for each Sephadex G 100 fraction. The total elution profile from the gel filtration is shown by the outer curve (-- e - - 8 ) Fig. 4. Assay of concanavMin A adsorbability of 14C glucosamine labeled pronase digests of trypsinates from I~[SV infected GMK cells. The hatched area under the graph with open circles denotes the amount of material binding to Con A Sepharose tested for eaeh Sephadex G 100 fraction. The total elution profile from the gel filtration is shown by the outer curve ( - - , - o -- )

301

Glycopeptides of H S V I n f e c t e d Cells

Regions I, II and III of the elution diagrams thus seem to contain a very high proportion of Con A binding galactose-labeled glycopeptides, before as well as after infection with HSV. The broad peak of low molecular substances does not contain any Con A binding material. The results of Figure 5 and 6 fit well with the results presented in Table I.

1

I

I

3H 60CP10M xl() 3

t

II

1

llI

iV

I

5040302010* l,'0 BD

6b

8'0

,~ Fraction na ~R

Fig. 5 I

I

]

i

II

t

Ill

H

IV

14C 2 CPlOMx103020 10 'b /.0

60

BD

80

~b Fraction no ~R

Fig'. 6 Fig. 5. Assay of concanavalin A adsorbability of SH galactose labeled pronase digests of t r y p s i n a t e s from u n i n f e c t e d G M K cells. The h a t c h e d area u n d e r t h e g r a p h w i t h open circles denotes t h e a m o u n t of m a t e r i a l binding to Con A Sepharose tested for each S e p h a d e x G 100 fraction. T h e t o t a l elution profile from t h e gel filtration is shown b y t h e outer c u r v e ( -- • -- ®-- ) Fig. 6. Assay of c o n c a n a v a l i n A adsorbabitity of 14C labeled pronase digests of trypsinares f r o m H S V infected G M K cells. T h e h a t c h e d area u n d e r t h e g r a p h w i t h open circles denotes t h e a m o u n t of m a t e r i a l binding to Con A-Sepharose t e s t e d for each S e p h a d e x G t00 fraction. The t e r m e]ution profile from t h e get filtration is shown b y t h e o u t e r cl.lrve ( -- ®-- o -- )

Discussion

T h e m e t h o d of p r e p a r i n g h e t e r o s a e e h a r i d e p o r t i o n s of g l y c o p r o t e i n s b y p r e t e n s e d i g e s t i o n was d e s c r i b e d b y SPIBO (18), in s t u d i e s o n t h e c a r b o h y d r a t e 20*

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units of thyroglobulhl. In order to characterize heterosaceharides of plasma membrane glyeoproteins from transformed and normal cells BUCK et al. (2) combined trypsinization of cells with protease digestion. This method was used in the present study reporting observations on surface heterosaccharides of radioactively labeled HSV infected and uninfected GMK-cells. Material released by the treatment was assumed to originate from the cell surface, although the possibility of slight cytoplasmic contamination was not specifically investigated. Gel filtration of surface peptides and gtycopeptides obtained by trypsinization of GMK cells showed marked size heterogenity. Some of the peptides obviously were very large molecules since a peak of radioactivity appeared with the material totally excluded by the gel. The exclusion limit for globular proteins in Sephadex G 100 is over 80,000 daltons. The subsequent pronase digestion of the trypsinates efficiently eliminated peptides as almost all of the leucine label used appeared as low molecu]ar material in G 100 ehromatograms. Therefore, it is reasonable to assume that the material which was partially included in the gel consisted predominantly of the heterosaccharides. The gel filtrations of heterosaeeharides in pronase digested glucosamine and galactosedabeled trypsinates demonstrated some interesting differences between HSV infected and noninfected cells. The most striking of these differences was that after the infection a peak corresponding to glucosamine in region I of the chromatograms almost totally disappeared. This peak corresponded to material eluted before FITC-dextran 20 (MW 19,000 daltons) and with the void volume and thus should comprise high molecular weight cell surface material. It may consist of mucopolysaccharides, proteoglycan or undigested glycopeptide material, as indicated by presence of small amounts of residual leucine label in materials eluted with the void volume. In contrast, region 11 of chromatograms obtained with galactose-labeled materials revealed only minor differences between HSV infected and uninfected cells. In both cases a peak of material was observed merging with the void volume. Thus, the HSV infection seemed to influence the metabolism of glucosamine and galactose in two different ways. The relative incorporation of galactose label into the surface macromolecules seemed only slightly diminished by the HSV infection, while the incorporation of glucosamine label into surface macromolecules was almost totaJly arrested by the infection. The decrease of galactose labeled void saccharides was not as marked as found by BRE:NNA:N et al. (1), reporting a prominent decrease of galaetose label into maeromoleenles in connection with HSV infection. However, these authors used another type of cell (human embryonic lung fibroblasts) and, furthermore, they studied pronase digests of material obtained by acid precipitation of whole cell fractions. A second important difference was the change to a more complex size pattern of heterosaccharides after IISV infection. In the class of heterosaeeharides with Stoke's radius corresponding to linear dextrans of 19,000 daltons an obvious trend to heterogeneous glueosamine-labeled heterosaeeharides occurred. Moreover, galaerose labeled heterosaeeharides of this size seemed to increase as a result of the infection. Smaller heterosaeeharides (detectable in regions I I I and IV of our ehromatograms) did not demonstrate any qualitative differences attributable to the HSV infection. Noticeable was the discrepancy between gIucosamine and galactose

Glyeopeptides of HSV Infected Cells

303

labeled heterosaceharides in HSV- as well as uninfected cells. I n general we seem to obtain larger sugar structures in HSV infected cells than BRENNAZq et aI. (1) and HoNEss and ROIZMAN (8). We believe that this might depend on both differences in cell lines used and t h a t we preferentially have analy~zed the cell surface structures. The glycosylation of hbst cell glycoproteins has been shown to be shut off in Hep-2 cells (8). There is no reason to assume t h a t GMK AH-1 cells are different in this respect. Lectins are useful not only for purification but also for characterization of glycopeptides. Sepharose-bound Coneanavalin A binds mainly mannobiose-eonraining internal sequences of heterosaccharides (12, 15, J. Blomberg, to be published). These are generally linked via N-acetylglneosamine and asparagine on the polypeptide chain. However, for mucopolysaecharides special binding rules seem to exist (4). With these exceptions in mind the results of the affinity chromatography experiments were interpreted as suggesting that some surface heterosaceharides of HSV infected cells contained less amounts of mannobiose-rich sequences sterieally available to Con A than did heterosaccharides from uninfected cells. This finding was relevant for the larger glueosamine labeled saecharides whieh eluted before and together with the F I T C 20 marker. The Concanavalin A affinity chromatography proved to be in itself able to disclose a class of heterosaecharides not differentiated in gel chromatography. Fractions corresponding to the vatley between two peaks in tile gel ehromatogn'am demonstrated Concanavalin A binding material in both HSV- and uninfected cell materials. Finally, in contrast galaetose labeled void saech~rides demonstrated unchanged relative amounts of mannobiose-rich sequences available to Con A after HSV infection of GIVIK ceils. In conclusion the HSV infection of GMK cells seemed to cause some changes in the population of sugar structures on the cell surface. This might indicate synthesis of new viral heterosaccharide species or a seleetion of the preexisting cellular heterosaceharides. To further evaluate this process there is a need for more structural information such as a chemical characterization of the heterosaecharides and a determination of the type of bond between heterosaeeharides and peptide. Such work is in progress and will be reported later. In general, however, the observations of molecular weight distributions of eelI surface saecharides emphasized the several similarities between HSV and uninfected cells, suggesting that in spite of the huge ttSV genome much of the newly synthetized sugar sequences of glyeoproteins and mucopolysaeeharides of the HSV-infeeted cells result from cell coding. Similarly, the genetically relatively small enveloped viruses such as Sindbis (9) and Vesicular stomatitis virus (5) also have been found to utilize the cell battery of glycosyl transferases.

Acknowledflments The excellent technical assistance of I. SjSblom is gratefully appreciated. We thank

Dr. E. Lycke for suppoi~b and constructive criticism. This study was supported by Grant B 75-16X-4511-01 from The Swedish Medical Research Council and by Grant from the Medical Faculty of G6teborg.

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