Archives of Virology

Archives of Virology 57, 97--105 (1978)

(~ by Springer-Verlag 1978

Inactivation of Human Cytomefalovirus by Phytohemaffllutinin* By M. ITO1, LINDA GII~VII%and A. L. BARRO~ Department of Microbiology, State University of New York at Buffalo, Buffalo, New York, U.S.A., and Department of Microbiology and Immunology, University of Arkansas for Medical Sciences, Little Rock, Arkansas, U.S.A. VVith I Figure Accepted October 17, 1977

Summary H u m a n cytomegatovirus (ClVIV) was inactivated b y treatment with phytohemagglutinin (PHA) in contrast to herpes simplex virus (HSV), which was not. Approximately 90 per cent of infectivity was lost following exposure of CMV to PHA. Greater reduction of infectivity, more than 99 per cent, was obtained following pretreatment of ceils with P H A than b y direct mixture of the virus and the leetin. Protection of cells was still observed 48 hours after pretreatment of cells with PI-IA. No difference was found in sensitivity to P H A with 3 strains of CMV tested. P H A preparations from different sources were ahnost equally effective in inactivation of CMV, whereas leucoagglutinin had minimal effect. Our data suggest t h a t the site of action of P H A occurs at the step(s) of penetration and/or uncoating. Resistance of CMV to trypsin was confirmed, and the enzyme had no effect on sensitivity to lectins.

Introduction Interactions of lectins and viruses have been examined for a wide range of enveloped viruses. A number of reports have been published on the effect of concanavMin A (Con A) : Semliki Forest virus (3, 16), Sindbis virus (4), vesicular stomatitis virus (17), fowl plague virus (3), influenza virus (14, 17), SV5 (3), Sendai virus (15), routine oncornaviruscs (6, 7, 12, 19), avian oneornaviruses (8), herpes simplex virus (HSV) types 1 (t0, 11, 15, 18) and 2 (10, 11). Fewer studies * Presented in part at the Annual Meeting, American Society for Microbiology, New York, N.Y., t975. 1 Current address : Department of Medical Viral Ontology, t~oswell Park Memorial Institute, Buffalo, New York, U.S.A. 7 Arch.Virol, 57•2

0304-8608/78/0057/0097/$ 01.80

98

M. I~o, LINDA

GII~VIX, and A. L. BARRON:

on the effect of p h y t o h e m a g g l u t i n i n (PHA) on virus i n f e c t i v i t y h a v e been :reported: ]?Mend mouse l e u k e m i a virus (7), a v i a n oneornaviruses (8), and t t S V (l 1). I n t h e present s t u d y we found t h a t in c o n t r a s t to HSV, h u m a n e y t o m e g a l o v i r u s (CMV), a n o t h e r herpesvirus, was i n a c t i v a t e d b y t r e a t m e n t w i t h P H A . I n this c o m m u n i c a t i o n , we r e p o r t r e d u c t i o n in i n f e c t i v i t y of CMV following p r e t r e a t m e n t of ceils w i t h P H A , as well as i n a c t i v a t i o n following d i r e c t t r e a t m e n t . Possible m e c h a n i s m s i n v o l v e d in P H A i n a c t i v a t i o n of CMV are considered.

Materials and Methods Cells

F T cells, a line of human fibroblasts, were originally received from Dr. B. Wentworth, University of Washington, Seattle, Washington, U.S.A. (22). The cells were grown in Eagle's minimum essential medium (MEM) in Earle's balanced salt solution plus 10 per cent fetal calf serum, 0.075 per cent sodium bicarbonate, penicillin (100 units/ ml), streptomycin (100 ~xg/ml), and/or aureomycin (50 ~g/mI). After virus inoculation, the cells were maintained in MEM containing 3 per cent fetM cMf serum, 0.15 per cent sodium bicarbonate and antibiotics. For infectivity assays, F T cells were cultivated in 35 m m plastic petri dishes, Contur Dish (Lux Scientific Corporation, Thousand Oaks, California--MicrobiologicaI Associates, Bethesda, Maryland) or Linbro multidish disposo-trays, 16 m m (Linbro ChemicM Co., New Haven, Connecticut). Dishes or trays were seeded with F T cells in growth medium containing 0.15 per cent sodium bicarbonate and incubated at 36 ° C in a CO2 incubator (5 per cent, CO2 plus 95 per cent air). Viruses

Three CMV strains were used in the present study: AD-169, Davis, and VeCa (received from Dr. J. DeMarehi). AD-t69 strain had been passaged 6---7 times in F T cells in our laboratory and the titer of the material used was 3 X ]06 P F U / m l . Davis and VeCa had been passaged 5--6 times in F T cells. Titers of preparations of these viruses were 9 x t05 and 8 × 10~ P F U / m l , respectively. HSV type t, MaeIntyre Vt~ 3 strain (9, 10) was passaged once in F T cells and employed in this study. The titer was 5 X 10~ P F U / m l . Virus stocks of CMV were prepared as follows: F T cells grown in 16 oz prescription bottles were inoculated with CMV and incubated at, 36 ° C for severM days. When the cell monolayers showed 85--90 per cent cytopathic effect, cells were scraped into the medium plus 10 per cent dimethyl suIfoxide. The cells were dispersed, and the suspension was distributed into vials and stored at --70 ° C. Immediately before use, the stock preparation was thawed and sonified for 20 seconds using the microtip accessory of a Branson Sonifier Cell Disruptor (Heat Systems, Co., Melville, New York) at an output of around 60 watts. Chemicals

Baeto .phytohemagglutinin-P (PHA-P) and Baeto -phytohemagglutinin-M (PI-IA-M) were obtained from Difco Laboratories, Detroit, Michigan. Phytohemagglutinin-M (PttA-M) and pokeweed mitogen were received from Grand Island BiologicM Co. (GIBCO), Grand Island, New York. These leetins were prepared according to the manufacturers' instructions and undiluted preparations were considered as a 1:1 solution. Leucoagglutinin was purchased from Pharmacia Fine ChemieMs, Piseataway, New Jersey. Other chemicals were: coneanavalin A (Con A), 2 times crystallized, Lot No. 5594 (Nutritional Biochemieals Corp., Cleveland, Ohio), potassium metaperiodate (Matheson, Collman and Bell Co., Norwood, Ohio), and ~-methyl-D-mam~oside (Calbiochem, LaJolla, California). Crude wheat germ hemagglutinin was prepared from wheat germ lipase (Schwarz Mann, Orangeburg, New York) by the method described by AtrB et al. (1). The chemicals listed above were diluted with phosphate buffered sMine (PBS), pI-I 7.2.

I n a c t i v a t i o n of CMV b y PI.IA

99

C M V In/ectivity Assay T h e p l a q u e a s s a y p r o c e d u r e of ~rE~mWO~TH a n d FIgENCH (22) was u s e d w i t h m i n o r m o d i f i c a t i o n s . A n i n o c u l u m of 0.2 m l of CMV d i l u t e d in M E M or l°BS w a s a p p l i e d to e a c h d i s h ( t r a y c u l t u r e s - - 0 . 1 m l p e r well) a n d allowed t o a d s o r b for 1 h o u r a t 3 6 ° C in a COs i n c u b a t o r . A n o v e r l a y was p r e p a r e d b y c o m b i n i n g d o u b l e s t r e n g t h M E M c o n t a i n i n g 4 p e r c e n t f e t a l calf s e r u m , 0.3 p e r c e n t s o d i u m b i c a r b o n a t e , penicillin (200 u n i t s / m ] ) , a n d s t r e p t o m y c i n (200 y.g/ml) w i t h a n e q u a l v o l u m e of 0.6 p e r c e n t a g a r o s e (Seakem, B a u s c h a n d L o m b , R o c h e s t e r , N e w Y o r k ) . A v o l u m e of 1.8 m l of o v e E a y m e d i u m w a s a d d e d to e a c h m o n o l a y e r in dishes ( t r a y c u l t u r e - - 0 . 8 m l p e r well). S e c o n d o v e r l a y of t h e s a m e m e d i u m w a s a p p l i e d 7 d a y s later. T h e cell l a y e r s were fixed 8 - - 1 4 d a y s a f t e r i n o c u l a t i o n w i t h 10 p e r c e n t f o r m a l i n d e p e n d i n g o n t h e s t r a i n . O v e r l a y s were carefully r e m o v e d w i t h o u t d i s t u r b i n g t h e m o n o l a y e r , w h i c h was t h e n s t a i n e d w i t h 0.3 p e r c e n t a q u e o u s m e t h y l e n e blue. T h e d a r k s t a i n e d loci w e r e c o u n t e d u s i n g a d i s s e c t i n g m i c r o s c o p e a t 2 5 × . T h e t o t a l n u m b e r of loci in 3 dishes ( t r a y c u l t u r e - - 3 well) p e r e a c h CMV d i l u t i o n was d e t e r m i n e d a n d P F U / m l (plaque f o r m i n g units) calculated.

H S V In/ectivity Assay I n F T cell c u l t u r e s , t h e i n o c u l u m a n d first o v e r l a y were as d e s c r i b e d for CMV assay. However, the second overlay, which was added 3 days after inoculation, contained Special A g a r N o b l e (Difco) a t a final c o n c e n t r a t i o n of 1.2 p e r c e n t a n d 0.01 p e r c e n t n e u t r a l red. T h e dishes were i n c u b a t e d a t 36 ° C for 4 h o u r s following a p p l i c a t i o n of t h e s e c o n d o v e r l a y . D i s h e s were p l a c e d i n a n a n a e r o b i c j a r (TorbM, Clifton, N e w J e r s e y ) , gassed w i t h 5 p e r c e n t COe plus 95 p e r c e n t air, a n d i n c u b a t e d o v e r n i g h t a t 4 ° C. T h e plaques were counted on the fourth day after inoculation. In later studies, BGM cells (2) grown in Linbro multi-dish disposotrays were also employed. Monolayers in wells received 0.1 ml each of dih~£ed I-ISV. After adsorption for I hour at 360 C, an overlay consisting of MEM, 2 per cent fetal calf serum, 0.7 per cent agarose, and antibiotics was applied to each well (0.5 ml per well). The cultures were incubated for 3--4 day-s prior to staining. Thereafter, an equal volume (0.5 ml) of the second overlay, containing neutral red and Noble agar as described above, was added to each well. The trays were incubated 3--4 hours at 36 ° C before counting plaques. In this ease no ft~rther incubation at 4 ° C was necessary.

Pretreatment o / F T Cells A f t e r r e m o v M of g r o w t h m e d i u m , cells were w a s h e d once w i t h P B S , a n d t h e l e e t i n was a d d e d t o e a c h dish (0.5 ml) or to e a c h well (0.15 ml) of t r a y cultures. T h e c o n t r o l was t r e a t m e n t of c o r r e s p o n d i n g c u l t u r e s w i t h P B S . T h e c u l t u r e s were i n c u b a t e d a t 36 ° C for 1 h o u r a n d w a s h e d 3 t i m e s w i t h P B S t o r e m o v e a n y free l e c t i n solution. The treated cells were then used for infectivity assays. In some experiments, the maintenance medium was added following treatment of cells ~dth PI-IA-P, and the cells were incubated for various time intervals in a COs incubator prior to virus inoculation.

Direct Treatment el C2YlV and H S V ¥ i r u s e s were d i l u t e d to t h e desired c o n c e n t r a t i o n a n d c o m b i n e d w i t h a a e q u a l v o l u m e of l e c t i n s o l u t i o n or a n e q u a l v o l u m e of P B S as c o n t r o l . CMV suspeilsions were r o u t i n e l y sonified before use. T h e m a t e r i a l w a s i n c u b a t e d a t 25 ° C ( r o o m t e m p e r a t u r e ) for 1 h o u r . F u r t h e r d i l u t i o n s were m a d e in M E M (0.15 p e r c e n t b i c a r b o n a t e w i t h o u t s e r u m ) , a n d i n f e c t i v i t y a s s a y s were s u b s e q u e n t l y p e r f o r m e d . Treatment~ w i t h p e r i o d a t e was c a r r i e d o u t a c c o r d i n g to t h e m e t h o d p r e v i o u s l y d e s c r i b e d (10).

Treatment With ~-Methyl-D-Mannoside C M V ( A D - I 6 9 ) i n f e c t e d celt s u s p e n s i o n w a s sonified a n d d i l u t e d in P B S to a c o n c e n t r a t i o n of 4.1 X 10 ~ P F U / m l . T h e v i r u s w a s m i x e d w i t h a n e q u a l v o l u m e of e i t h e r P H A - P (1:4), Con A (100 lzg/ml), or P B S as control. T h e m i x t u r e s were e a c h c o m b i n e d w i t h a n e q u a l v o l u m e of 0.2 ~ ~ - m e t h y L D - m a n n o s i d e (e-MM) i n P B S a n d i n c u b a t e d for 1 h o u r a t 25 ° C before s e n s i t i v i t y w a s a s s a y e d . P B S w a s u s e d as t h e c o n t r o l t r e a t m e n t for ~-MM. 7*

100

M. ITo, LINDA GIRVIN, and A. L. BAt~taON:

Treatment of C¢VIV and 11SV With Trypsin Equal volumes of virus were mixed with either 0.25 per cent trypsin or the corresponding buffer a~d the mixtures were incubated for 1 hour a~ 36° C. After dilution in PBS, trypsin-treated viruses were exposed to either PBS, PI-IA-P, or Con A for 1 hour at 25 ° C. Residual infectivity was assayed by plaque method.

Results

Pretreatment o/ F T Cells With P H A - P W h e n F T cells were p r e t r e a t e d with P I I A - P , the r e d u c t i o n of i n f e c t i v i t y was m u c h greater t h a n b y direct i n a c t i v a t i o n (Table 1). F o r comparison, we performed similar experiments with Con A (data n o t shown). I n this ease, inactivatiort of CMV was greater following direct t r e a t m e n t of the virus w i t h Con A t h a n b y p r e t r e a t m e n t of F T cells with the lectin. A t 1 : 16 d i l u t i o n of P H A , a n effect on cells could be discerned, b u t size a n d shape of CMV plaques was identical to u n t r e a t e d ceils. Table 1. Ef]ect of pretreatment o/tZTcells with P H A - P on the in/ectivity o~CM V (AD-169) :Pretreatment (FT ceils + PHA) PFU~ PI-IA-P 1: 1 4 16 64 256 1024 4096 PBS

~o Reduction

PFUb

~o tl~eduction

99.4 99.7 97.6 93,9 61.3 0

140 280 365 523 810 1080 1010 1160

87.9 75.9 68.5 54.9 30.2 6.9 t2.9 0

Toxic Toxic 17 10 72 183 1170 4096

Direet treatment (PttA + CMV)

a Measured in pretreated F T celt cultures b Determined following treatment with PI-IA

Site o/Action o / P H A - P in Growth Cycle o/ C M V As expected, p r e t r e a t m e n t of F T cells with P H A - P followed b y i n o c u l a t i o n of the virus resulted i n m a r k e d r e d u c t i o n of i n f e c t i v i t y (Fig. 1). Considerable r e d u c t i o n in i n f e c t i v i t y was also observed when a second set of F T cells was inoculated with CMV, i n c u b a t e d a t 4 ° C for 2 hours a n d washed with P B S followed b y a d d i t i o n of P t I A - P . Little r e d u c t i o n of infectivity was o b t a i n e d w h e n cells were inoculated with CMV a n d i n c u b a t e d at 36 ° C for 2 hours prior to t r e a t m e n t with P H A - P . I t could be a s s u m e d t h a t a t 4 ° C CI~IV adsorbed to F T cells b u t did n o t p e n e t r a t e (21) ; the adsorbed virus was sensitive to i n a c t i v a t i o n b y P H A - P . A t 36 ° C the m a j o r i t y of infectious particles p r o b a b l y p e n e t r a t e d a n d were u n coated and, a t this stage, the lectin had no effect.

I n a c t i v a t i o n of CMV b y P ~ A

101

I00

\

q5

,6

\

i4 DILUTION

•--. • • --

2;6

+696

,oi+

PHA-P

Fig. 1. Site of a c t i o n of PI~[A-P i n g r o w t h cycle of CMV (A])-169) • • F T cells p r e t r e a t e d w i t h P H A - P a t 25 ° C for 1 h o u r p r i o r t o inoculation with CMV . - - ~ + + . - - , , F T cells i n o c u l a t e d w i t h CMV, i n c u b a t e d a+~ 4 ° C for 2 h o u r s followed b y a d d i t i o n of P H A - P -® . . . . • . . . . . ® F T cells i n o c u l a t e d w i t h CMV, i n c u b a t e d a t 36 ° C for 2 h o u r s followed b y a d d i t i o n of P H A - P

Duration o/ E//ect o/ Pretreatment o/ Cells With P I J A - P on Reduction o/ C M V In/ectivity F T cells w e r e p r e t r e a t e d w i t h P H A - P f o r 1 h o u r a t 2 5 ° C. O n e s e t of c u l t u r e s w a s w a s h e d a n d i m m e d i a t e l y i n o c u l a t e d w i t h C M V (0 h o u r ) . A s e c o n d s e t w a s w a s h e d a n d i n c u b a t e d i n m a i n t e n a n c e m e d i u m f o r 3 h o u r s a t 36 ° C b e f o r e c h a l l e n g e with CMV. Additional sets were washed and incubated in the maintenance medium f o r 24, 48, 72, a n d 96 h o u r s , r e s p e c t i v e l y , p r i o r t o c h a l l e n g e w i t h C M V ( T a b l e 2). T a b l e 2. Duration of reduction o / C M V (Davis) in/ectivity by pretreatment o/~'T cells

with P H A - P % Reduction infectivity Pretreatment with

Hours

a

P H A -P

0

3

24

48

72

120

1 : 16 64 256 1024 4096

99.2 98.1 88.1 63.6 23.8

90.6 83.4 25.8 t6.4 6.0

86.4 77.1 54.2 46.6 7.5

77.7 61.8 39.8 33.2 17.8

62.0 56.1 46.0 48.0 34.0

59.0 i9.2 27.6 12.5 0

T i m e i n t e r v a l b e t w e e n p r e t r e a t m e n t of F T cells w i t h P H A - P a n d C M V i n o c u l a t i o n

102

M. I~o, LINDA.GIRVlN, and A. L. BAI~aoN:

The greatest reduction in infectivity was observed when the FT cells were inoculated with CMV immediately following pretreatment with PHA-P. However, distinct redaction in infectivity was still detected following incubation of cells for 48 hours at 36 ° C after pretreatment of cells with PItA-P. Inactivation o~ CM V and H S V by Various Lectins and Chemicals As shown in Table 3, direct treatment of CMV with P I t A - P resulted in almost 90 per cent reduction of infectivity, whereas no significant effect on HSV-1 was observed. On the other hand, Con A strongly inactivated both C ~ V and HSV. Wheat germ agglutinin and pokeweed mitogen had no effect on either virus. Treatment with periodate resulted in almost complete inactivation of both viruses. Table 3. E/]ect o] plant lectins and chemicals on the in]ectivity o / C M V (AD-169) and H S V type 1 °//o l~eduetion infectivitya, CMV HSV- 1 P:t-IA-P Con A Wheat germ agglutinin Pokeweed mitogen Periodate

1: 1 25 ~g/ml 10 mg/mI 1: 1 0.01 ~

87.2 99.9 0 O 99.9

t 1.0 95.2 0 0 100

a Infectivity assayed in FT cells in dishes Different eommereiM preparations of PHA, such as P H A - P (Difeo), PHA-M (Difco and GIBCO), and also leucoagglutinin, which contains one pure component of PHA-P, were tested for their effeet on the infectivity of CMV. No striking differences were observed among the various PI-IA-P and -M preparations tested either b y pretreatment of FT cells or by direct inactivation. Only minimal inactivation of CMV was detected following treatment with leucoagglutinin. Three different strains of CMV, AD-t69, Davis, VeCa, were tested for sensitivity to PHA-P, and it was found that all the strains were inactivated by P H A - P with no marked difference observed among the three strains tested. As expected, inactivation of CMV by Con A was inhibited by the addition of ~-MhI since inactivation of HSV by Con A could be specifically inhibited by the addition of c¢-MMor c~-methyl-D-glueoside (10, 15, 18). However, treatment with ~-MM had no effect on P t I A - P inactivation of CMV. Preliminary experiments indicated that N-acetyl-])-galactosamine (5) also failed to inhibit inactivation by P H A - P (M. ITo, unpublished data). Exposure of CMV to 0.25 per cent trypsin for 1 hour at 36 ° C resulted in no decrease of infectivity (Table 4). In contrast to CMV, infectivity of HSV-1 was markedly decreased in agreement with the data reported by KIM and CARP (13). Sensitivity of CMV to inactivation by P H A - P or Con A was not affected by trypsinization of the virus.

Inaet,iva~ion of CMV by PI-IA

103

Table 4. E/Ject o/trypsi~t treatment o] C I ~ V (AD-169) and H S V type 1 on inactivation by lectins

Virus CMV

HSV- i s

Pretreatment a of virus vdth trypsin

Treatmen~

-+

PBS

--

PHA-:P (1 : 16)

--}--

Con A (25 ~xg/ml)

PFU 73,600 77,400

PBS

~/o Reduction infectivity -0

7,290 7,620

90.1 90.2

190 38 2,600,000 370

99.7 99.9 -99.9

a Refer Materials and Met~hods b Infectivity assayed in BGM cells in trays

Discussion I t is well known that CMV has very different biological properties from HSV which include: high degree of cell association (23); marked species specificity (23); long replication cycle (20); and resistance to trypsin (13). Our studies on inactivation of CMV by P H A have demonstrated another biological difference between the two viruses, lgeeently, IsmzAxI and BoI~oGs~st (8) demonstrated that various avian oneornaviruses responded randomly to inactivation by P H A and Con A. Their results indicate a possible subelassification of viruses in the same group according to their sensitivity to lectins. In the present study, marked loss of CMV was observed for as long as 48 hours following pretreatment of FT cells with PHA. This is much longer than the protective period afforded by Con A as reported by OKADA and K I ~ (15) for HSV. The difference might be explained by the presence of Con A inhibitors in the growth medium used in the latter studies, since various substances, such as serum and laetalbumin hydrolysate, contained Con A inhibitors (M. Iwo, unpublished data). Other experiments not reported here have revealed the presence of P I t A on the surface of FT cells 24 hours after treatment, as detected b y hemadsorption using sheep erythroeytes. Pretreatment of cells with PI-IA was more effective in reduction of CNV infectivity than direct interaction with the leetin. While a eytotoxie effect was noted at low dilutions of P I I A following pretreatment, marked reduction in plaque numbers was obtained at higher dilutions where this effect was minimal and did not influence plaque morphogenesis. I n the ease of Con A, a greater reduction in infectivity was found following direct treatment. These observations may have some meaning in interpreting the mode of action of P H A as compared to Con A. P H A m a y act on the surfaces of host cells rather than directly on the virions themselves. Our preliminary data revealed that the major site of action of P H A in CMV replication cycle was at the step(s) of penetration and/or uneoating. Although adsorbed virus was still sensitive to PHA, it is not known if pretreatment of cells

104

M. ][TO, LIND]~ GIJ{wN, and A. L. Bare,oN:

blocks adsorption. Experiments using radiolabeled CMV would be useful in further resolving this question. In this regard, marked reduction in yie]ds of infectious HSV occurred when PHA was added to the medium following adsorption and penetration steps (M. ITO, unpublished data). Thus, it would appear as if leetins m a y inhibit virus r e p l i c a t i o n d u r i n g synthesis as well as p e n e t r a t i o n or uneoating. I t has been r e c e n t l y shown t h a t i n a c t i v a t i o n of t I S V b y Con A could be enhanced b y s e c o n d a r y t r e a t m e n t w i t h P H A (1 i). T h e h y p o t h e s i s has been p r e s e n t e d t h a t the envelope of H S V has receptors for Con A a n d P H A , b u t these r e c e p t o r s differ in their critical role in infectivity. CMV, on t h e o t h e r h a n d , carries r e c e p t o r s on virions for b o t h Con A a n d P H A , a n d in this instance t h e y are b o t h critical for t h e infectious process. I n considering t h e m o d e of a c t i o n of Con A on direct ina c t i v a t i o n of t I S V , considerable a t t e n t i o n was p a i d to e x e h d i n g a g g r e g a t i o n (10). A t present, it is n o t possible to t e s t t h e effect, of P H A on CMV using t h e same e x p e r i m e n t a l a p p r o a c h because of lower t i t e r s of CMV a n d u n a v a i l a b i l i t y of suitable PI-IA inhibitors. lgesistanee of CMV to t r y p s i n (13) has been confirmed in t h e p r e s e n t s t u d y . S e n s i t i v i t y to either P H A or Con A d i d n o t change following t r e a t m e n t of CMV w i t h this enzyme. R e s i s t a n c e to t r y p s i n m a y be a unique p r o p e r t y of CMV since p r e l i m i n a r y studies h a v e shown t h a t a n o t h e r herpesvirus, Herl)esvir,~," 8airrdri, is i n a c t i v a t e d b y t r y p s i n , (N[. ITO, u n p u b l i s h e d data).

Aeknowledfments This work was supported b y General Igesearch Support Grants Rig 05400 (State University of New- York at BuffMo) and Rig 05350 (University of Arkansas for 1}iedieal Sciences) from the General igeseareh Support Branch, Division of Research Igesourees, National Institutes of I-Iealth. We t h a n k Dr. igyotaro Ishizaki, D e p a r t m e n t of Surgery, Duke University Medical Center, for his t]elpful suggestions. The excellent technical assistance of Mrs. Fumiko Ito is gratefully acknowledged.

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i0. ITo, M., BA:aI~0N, A. L. : Inactivation of herpes simplex virus by eoncanavalin A. J. Virol. 13, 1312--1318 (1974). 11. I~o, M., BAI~t~ON,A. L. : E n h a n c e m e n t by phytohaemaggtutinin of inactivation of herpes simplex virus b y concanavalin A. J. gen. ViroI. 33, 259--266 (1976). 12. KATSLY, J. R., FaI~D~A~, H. : Prevention of Friend virus leukemogenesis b y coneanavalin A. I. Detection of dormant virus and role of humoral antibody in protected mice. J. Nat. Cancer Inst. 53, 151--158 (1974). 13. KI~, K. S., CA~P, R. I. : Effect of proteolytie enzymes on the infectivity of a number of herpesviruses. J. inf. Dis. 128, 788--790 (1973). 14. KLEIN, P. A., ADAMS, W. R. : Location of ferritin-labeled concanavalin A binding to influenza virus and tumor cell surfaces. J. Viroh 10, 844--854 (1972). 15. OKADA, Y., KIM, J.: Interaction of eoneanavalin A with enveloped viruses and host cells. Virology 50, 507--515 (1972). 16. OlCAM,J. D., ELLWOOD, D. C., APPLEYARD, G., STANLEY,J. T. : Agglutination of an arbovirus by concanavatin A. Nature (New Biol.) 233, 50--51 (1971). 17. PE~HOE~, E., 0LSE~', C., CAtCI~SOS-,S., LAI~COt~BIEt~E,M., NlCOI~SO~%G. L. : Quantitative interaction of R i c i n u s c o m m u n i s agglutinin and eoneanavalin A with influenza and vesicular stomatitis viruses and virus-infected normal and polyomatransformed cells. Biochem. 13, 3561--3566 (1974). 18. PONCE DE LEON, M., HESSa~E, I-I., COttEX, G. H.: Separation of herpes simplex virus-induced antigens by concanavMin A affinity chromatography. J. Virol. 12, 766--774 (1973). 19. S~EWAR~, M. L., SUMMERS, D. F., SOE~t~O, R., FIE~DS, B. N., MAIZEL, J. V., J~. : Purification of oncornaviruses b y agglutination with concanavalin A. Proc. Nat. Acad. Sci. U.S.A. 70, 1308--1312 (1973). 20. SMITH, J. D., 9E HA~EN, E. : Herpes simplex virus and human cytomegalo~irus replication in WI-38 cells. I. Sequence of viral replication. J. Virol. ]2, 919--930 (1973). M. : Interactions of human cytomegalovirus with 21. VONKA, V., BEI~YESH-MELNICK, human fibroblasts. J. Bacteriol. 91, 213-220 (1966). 22. WENTWORTI~, B. B., FRENCH, L. : Plaque assay of cytomegalovirus strains of human origin. Proc. Soc. exp. Biol. Med. 135, 253-~-258 (1970). 23. WILDY, P. : Classification and nomenclature of viruses. Monographs in Virology 5, 1--81 (1971).

Authors' address : Dr. A. L. BAaao~, Department of Microbiology and Immunology, College of Medicine, University of Arkansas for Medical Sciences, Little Rock, AR 72201, U.S.A. ~eceived September t5, 1977

Inactivation of human cytomegalovirus by phytohemagglutinin.

Archives of Virology Archives of Virology 57, 97--105 (1978) (~ by Springer-Verlag 1978 Inactivation of Human Cytomefalovirus by Phytohemaffllutini...
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